Bukar Alhaji, Mary Ann Amarachi Umeh, Obi Simon Osita, Waziri Gimba, Medugu Jessy Thomus
Department of Haematology, University of Maiduguri Teaching Hospital, Maiduguri,
Anthony Nwobi, Osakue Eguagie Osareniro
Department of Chemical Pathology, Igbinedion University Teaching Hospital, Okada
Olaniyan Matthew Folaranmi
Department of Medical Laboratory Science, Achievers University, Owo
Jeremiah Zaccheaus Awortu.
Department of Medical Laboratory Science, Niger Delta University

All correspondence to: alhajibukar@gmail.com.

ABSTRACT

Haemoparasite,such as Plasmodium and Microfilaria are animal parasite living in the blood of a vertebrate host. The study was aimed to determine the prevalence of Haemoparasite (Plasmodium and Microfilaria) in blood donors attending University of Maiduguri Teaching Hospital. A total of 230 blood donors were recruited for this study using simple random sampling. A semi-structured questionnaire was used to collect data regarding demographic and social profile of the subjects. Giemsa stained thick blood film was used for the detection of malaria parasite while wet preparation was used for the detection of microfilaria. A total of 78 blood donors had malaria parasites while no filarial parasite was recorded showing a prevalence of 33.9% and 0% respectively. The prevalence of malaria parasites in the blood donors was not significantly associated with usage of insecticide and/or insecticide treated net. The prevalence of malaria parasite was however significantly associated with treatment with antimalarial drugs. It is therefore necessary for the government to improve the sanitary condition of Maiduguri which will in turn reduce the availability of breeding sites for mosquitos.

Keywords: Malaria, microfilaria, blood donors.

INTRODUCTION

Blood  transfusion  is  potentially  a  lifesaving therapeutic procedure and a common form of tissue transplantation which is aimed to provide patients  with  blood  components  which  they  are deficient.¹.  Although,blood  transfusion  is  generally believed to save human lives, blood can nonetheless be a route for the transmission of infections generally referred to as transfusion transmissible infections (TTIs). TTI occurs when a patient is infected by the same parasite that was  present in  the   donor’s   blood.   TTIs are broadly classified  into  viral, bacterial, amoebal or parasitic. Haemoparasite is an animal parasite such as a haemoflagellate or filarial worm living in the blood of a vertebrate host². These parasites reside either in the blood cells or in the plasma. Malaria parasite and Babesiaare haemoparasites that resides in the red blood cells, while leishmania and filarial wormsresides in the white blood cells and the plasma respectively³.

In Nigeria, malaria and filariasis are more prevalent and over the years varying prevalence has been recorded among Nigerian blood donors⁴. Haemoparasites constitute a serious threat to human race as they can result in increased morbidity and mortality⁵. Malaria is sporozoan parasite of the genus Plasmodium, its infection is transmitted naturally through the bite of infected female Anopheles mosquitoes⁶. In endemic areas, malaria transmission is so intense that a large proportion of the population is infected but not made ill by these parasites⁷. These carriersharbour low levels of the parasitesand shows no clinical signs of infection as they are immune to parasitic illness but not to the infection and for this reason, blood from such donors contains malaria parasite which can easily be transmitted to recipients by blood transfusion.

Filariasis on the other hand is a parasitic disease that is caused by thread-like nematodes (roundworms) belonging to the superfamily Filarioidea. These parasites are transmitted from host to host by blood feeding arthropods, mainly black flies and mosquitoes.

As adults, the worms can survive and reproduce for up to 7 years within which the worms gradually build-up in the vessels of their host. This interfereswith the lymphatic system’s ability to fight infection and causes lymph fluid to accumulate in the arms, legs, breasts and male genitals leading to welling and disfigurement^{3, 11}.

In all species, sexually mature female worms release microfilariae, which are their pre-larval stages into the bloodstream of their infected host.If, the blood from microfilaraemic individuals is transfused into a patient, the transfused microfilariae may persist in the recipient’s circulation for up to 3 years¹². Recipient of these blood component usually develop post transfusion allergic reactions due to dying microfilariae¹³.

In Nigeria, screening for parasitic infections is not routinely done in blood banks, nor stipulated in the current National Blood Guidelines. This is because transmission of parasitic infections such as malaria through blood transfusion is generally not regarded as a serious problem in adult and adolescent whose level of immunity is thought to be sufficiently effective in combating post transfusion malaria in an endemic area like Nigeria⁶. These parasites are prevalent in Nigeria but the extent to which it currently affects blood donors attending UMTH is unknown, we therefore, considered it necessary to contribute some information on this subject.

A bite from an infected mosquito may cause malaria by introducing as few as 15 parasites while a single parasite identified on a thick film (4ul) is equivalent to approximately 10,000 parasites in 450ml unit thereby causing malaria in transfused patients⁸. Transfusion-transmitted malaria can however have serious consequences, as infection with P. falciparum may prove rapidly fatal when such blood is transfused especially into children under 5 years, pregnant women, trauma victims with acute blood loss and immunosuppressed patients⁹. Malaria destroys red blood cells and converts it to methaemoglobin leading to methemoglobinemia causing illness especially in immune compromised individuals ^{7, 10}

MATERIALS AND METHOD

This study was conducted at the University of Maiduguri Teaching hospital (UMTH) from February 2017 to May 2017. A total of 230blood donors which are negative to HIV 1/2, HBsAg, Syphilis and HCVwere recruited for the study. These donors were recruited using simple random sampling. Two millitres of the donor’s venous blood was collected into an EDTA container. ABO and RhD blood groups of the donors were determined using tile method. Malaria parasite was qualitatively determined by making thick blood films in duplicates for each blood samples on a clean grease free glass slide. these was allowed to air-dry after which it was stained with Giemsa stain. Stained films were examined under x100 objective lens of microscope with Immersion oil for any stage of malaria parasite. A slide is defined as negative if no asexual stage of the parasite is found after counting 100 microscopic fields. For, microfilaria parasite, a drop of anticoagulated blood was dispensed on a cleaned grease-free slide and covered with cover slip. It was examined microscopically using x10 and x 40 magnification for motile microfilaria. Result were analysed using, percentage and SPSS 20.0 statistical package. Chi-square was used to determine if prevalence was dependent on certain factors. A P-value of less than or equal to 0.05 (p= 0.05) was considered as statistically significant.

RESULTS

A total of two hundred and thirty subjects (230) were recruited for the study. The subjects were within the age group of 18-55 years with the age group 20-29 having the highest mode (47.6%, 110/230) and age group 50-59 years with the least (3%, 7/230).

Table I shows the prevalence of malaria parasite and filarial worms in the blood donors attending UMTH.Out of the 230 blood donors studied, 78 donors had malaria parasite giving a prevalence rate of 33.9% while none had filarial worm giving a prevalence rate of 0%.

Table II shows the prevalence of malaria parasite in blood donors attending UMTH in relation to blood group and donation history. Malaria parasite in respect to ABO blood group, group B donors had the highest prevalence rate of 38.1% (16/42) while blood group AB donors had no malaria parasite in their blood. This difference was not statistical significant (x² = 1.513, df= 3, p-value = 0.679). Malaria parasite with relation to Rh D blood group, Rh D- donors had a higher prevalence rate of 37.5% (6/16) while Rh D+ donors had a lower prevalence of 33.6% (72/214). This difference was also not statistically significant (x² =0.99, df =1, p-value = 0.753). Family replacement donors had a higher prevalence of malaria infection 34.2% (77/225) when compared to voluntary donors who had a prevalence rate of 20% (1/4), this difference was also not statistically significant (x² =0.441, df= 1, p=0.506). There was no commercial blood donor in this study. Repeat donors had a higher prevalence rate of malarial infection 36.1% (49/119) while first-time donors had a lower prevalence rate 35.1% (35/111). This prevalence is not statistically significant (x² =0.543, df= 1, p=0.461).

Table III shows the prevalence of malaria parasite in relation to some social factors.Female donors had higher prevalence rate of malaria parasite (36.4%, 4/11) compared to male donors who had a prevalence rate of 33.8% (74/219). This difference is not statistically significant (x²=0.31, df= 1, p=0.860). Donors below 20 years had the highest prevalence of malarial infection 40% (4/10) while those within the age range of 40-49 had the least prevalence.It is not statistically significant (x²=3.994, df= 4, p=0.479).The prevalence is higher among the singles, 40.7% (50/123) while no infection was detected among the divorced donor. It is not statistically significant (x²=5.66, df= 2, p=0.059).

Table IV shows the prevalence of malaria parasite in relation to usage of insecticides and/ or insecticide treated net. Blood donors who neither used insecticides nor insecticide treated net had the highest prevalence rate of 50% (18/18) while those who used both insecticides and insecticide treated net had the least prevalence rate of 23.5% (4/17). This difference is not statistically significant.

Table V shows  the  prevalence of  malaria parasite in relation to treatment with antimalarial drugs. Donors who said to have never been treated with antimalarial drugs had the highest prevalence of 56.7% (17/30) while donors who self-administered antimalarial drug within the last six months had the least prevalence 14.5% (11/76). This difference is statistically significant. No Filarial parasite is found in any of the donors.

Table I: Prevalence of Malaria Parasite and Filarial
Worms among the Blood Donors Attending UMTH 

DISCUSSION

Results obtained from this study showed that 78 blood donors had malaria while none had filarial worm showing a prevalence of 33.9% and 0% respectively. The prevalence of malaria parasitaemia in this study was lower than that reported by Abioye et al.¹⁵ who recorded a prevalence rate of 56% (140/250) in Abuja and was higher than the report of Garba et al.¹⁶ who reported a prevalence of 7.5% (27/360) in Kaduna. These differences in regional prevalence could be attributed to variation in predisposing factors such as present of Anopheles species, environmental conditions, climatic conditions, period of study, the study population and diagnostic test method used. The high prevalence rate may be attributed to current security challenges in Borno which forced people from other villages within other towns of the state to relocate to Maiduguri which in turn increases the population and decreases sanitary condition of the city. The decreased sanitary condition has resulted in increased chocked drainage channels which provide a suitable breeding ground for Anopheles mosquito.However, in relation to filarial worms the result from this study was inconsistent with the report of Bolaji et al.³ who reported a prevalence of 2% with Loa  loa, Brugria Malayi and Wuchereria    Bancrofti in the following proportion; 4 (1.33%), 1 (0.33%) and 1(0.33%) respectively. This difference may be attributed to difference in the number of subjects and geographical locations. Although Ochocerca Volvulus is prevalent in some areas in Bornu such as Hawul¹⁸,it is not prevalent in Maiduguri possibly due to lacks fast flowing water which is a suitable breeding site for its biological vector (Backfly).

This study further revealed that malaria parasitsitaemia is higher among blood group B donors while no malaria parasite was recorded in AB blood donors. This result does not tally with the report of Agboola et al.⁵ who reported a higher prevalence among blood group O donors. This difference may be as a result of chance. The difference in malaria among ABO blood groups in this study was however not statistically significant, indicating that susceptibility to malaria parasite is independent of a person’s ABO blood group. Also, a higher prevalence of parasitaemia among Rh D negative blood donors was reported in this study compared to the Rh D+ blood donors. This finding was not similar to a report  by Bankole  et  al.¹⁹ who reported a higher prevalence among Rh D+ blood donors. The difference between Rh D blood groups in this study was not statistically significant, indicating that susceptibility to malaria parasite is independent of a person’s Rh D blood group. The study clearly suggests that family replacement donors were the major source of blood for transfusion in UMTH. This is consistent with findings from other researchers^{19, 20}, indicating that family replacement donors were the major source of blood for transfusion in most states in Nigeria.There is higher prevalence of malaria parasitaemia in family replacement donors when compared to voluntary blood donors, this result is in line with the report of Olawumi et al.²⁰, however not statistically significant. The lower prevalence recorded in this study indicates that there is reduced risk of transmission ofmalaria when blood productsare derived from voluntary donors. Result from this study shows a higher prevalence of parasitaemia in repeat donors when compared to first-time donors. This result is consistent with the report of Garba et al.¹⁶. This could be as a result of the fact that first-time donors are apprehensive and those having mild symptoms of malaria such as headache are usually excluded to donate blood.
The prevalence of malaria parasite in this study shows high rate among female donors when compared to male donors. The difference in prevalence between the genders may not be conclusive owing to the relatively small number of female donors who participated in the study. Higher prevalence of parasitaemia was found in donors whose age where below twenty and the least prevalence was seen in donors within the age range of 40-49 years of age. This result does not tally with the report of Ekwunife et al.(2011)⁶ who reported the highest prevalence among donors within the range of 25-29 and the least prevalence among donors within the age range of 50-54 years of age. The difference may be due to chance.There is higher prevalence rate in single (unmarried) blood donors while malaria parasite was not detected in the blood of the divorced donor. The result does not tally with the report of Alli et al.,⁴ who reported a higher prevalence among married donors which might be probably by chance.

Overall, there is no significant relationship between the prevalence of malaria infection and the usage of personal protection against mosquitos. This indicatesthat the current prevalence of malaria parasite among the blood donors is not dependent on the use of insecticide and/or insecticide treated nets.

The results also indicated that there is statistically significant relationship between the prevalence of malaria infection and treatment with anti-malaria drugs. This indicates that treatment with antimalarial drugs significantly reduces the prevalence of malaria among blood donors. This coincides with the report of UNICEF ²¹, which states that the two major ways to reduce the spread of malaria are the use of insecticide treated mosquito nets and early diagnosis and prompt treatment with antimalarial medications.

CONCLUSION

In conclusion the result from this study shows a progressive increase in the prevalence of malaria parasite among blood of donors attending UMTH when compared with previous results. This increase is alarming as these donors are apparently healthy subjects indicating an increased risk of transmission of malaria through transfusion in Maiduguri. No filarial worm was recorded in this study. No statistically significant relationship was established betweenmalaria infection and the usage of Insecticide and/ or insecticide treated net. However, statistically significant relationship between the prevalence of malaria parasite and treatment with antimalarial drugs is noted.

Recommendations
Haemoparasites can be transmitted through transfusion of infected blood derived from asymptomatic donors. This may negatively affect patient’s health and increase the duration of their illness. State government should improve the sanitary condition of Maiduguri and environs which will in turn reduce mosquito breeding sites. Screening donors for parasitic infections should be included in the current nation’s transfusion guidelines. Enlighten donors on better ways of preventing infections with haemoparasites. Encourage prompt and effective treatment of infected prospective donors.In additions incentives such as insecticide treated mosquito net, insect repellent and refreshments should be given to donors as this may encourage voluntary donation and as well reduce the prevalence of haemoparasites in the blood of donors.

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The post Prevalence of HaemoParasites (Plasmodium and Microfilia) in Blood Donors Attending University of Maiduguri Teaching Hospital (UMTH) appeared first on Nigerian Biomedical Science Journal.

Akpulu S.P
Department of Human Anatomy,Ahmadu Bello University, Zaria, Kaduna State, Nigeria..
Abdurrahman S.K and Ineji E.A
School of Health Information Management Ahmadu Bello University Teaching Hospital, Zaria.
All Correspondences to: petosw2000@yahoo.com

ABSTRACT

Back ground: Most managers are yet to understand why reward and recognition programs are very necessary, and how they are used to motivate employees which in turn lead to job satisfaction. This study examined the impact of reward and recognition programs on employee’s motivation and satisfaction in General Hospital Makarfi Kaduna. Material and methods: Descriptive survey design was adopted in this study. The target population was 117 employees of the hospital. A sample size of 91 was used for this study. The questionnaire was employed as the research instrument. Descriptive statistics was used to analyzed and present the results. Results: The findings shows that reward and recognition programs gives employees extra motivation and job satisfaction which stimulate productivity. Conclusion:. The study concluded that dissatisfaction and demotivation in the job can cause staff to consider alternative employment; and that payment, promotion, working conditions and personal perception were factors affecting employees work satisfaction and motivation. Recommendation: The study recommend that managers of the hospital should placed more focus on reward and recognition programs, because it leads to motivation, higher job satisfaction and productivity.

Key word: Motivation, satisfaction, productivity and employee.

Reward system is a process that reinforces behavior to hit the target and meet the standards: Deci (1971) as cited by Ali & Ahmed (2008) explained that reward and recognition programs come within the discussion on extrinsically motivated behavior that occurs when an activity is rewarded by incentives not inherent in the task. Njanjaet al (2013) sees reward management as one of the strategies used by human resource managers for attracting and retaining suitable employee as well as facilitating them to improve their performance through motivation and to comply with employment legislation and regulation. Mound (2001) stresses that rewarding employee performance should be an ongoing managerial activity, not just an annual ritual. Rewards can be financial (pay, commission, bonus, profit sharing pensions, employee stockoptions, fringe benefits) or nonfinancial (praise, promotions, status, additional assignments, special awards, commendations, recognition).
Lack of reward and recognition has a negative effect on stress and job satisfaction and could adversely influence employee turnover. According to Torrington et al (2008), burn out comes from being frustrated by trying to make an impact and not being recognized or listened to. Feelings of helplessness in employee germinate in a soil of unappreciative attitudes and bear the fruit of demotivation and dissatisfaction in the work place. (Njanja et al, (2013).
In organizational settings, managers of human resources understand the great gains derived by linking rewards and recognition to their business strategy (Flynn, 1998).
Several studies have been carried out by many researchers
on the impact of rewards and recognition programs on employee’s performance, but non of such has been conducted in Makarfi General Hospital, Kaduna Nigeria. In recent time employee of Makarfi General Hospital have witness low performance and job dissatisfaction and demotivation due to lack of rewards and recognition in their work place.

MATERIALS AND METHODS:

The design used for this study was descriptive studies and the type employed was survey which involves the used of questionnaire. The study population included employees of General Hospital Makarfi, Kaduna State, who had a total population of 117.The sample were determined using the formula of Yaro Yaraune as stated below.
Questionnaire was used as the measuring instrument. The data gathering techniques used included a biographical section and the work satisfaction and motivation questionnaire section. The questionnaire was self-developed and consisted of nine dimensions that impact employee satisfaction and motivation. The nine dimensions include; work content, payment, promotion, recognition, working conditions, benefits, personal, leadership or supervision and general. All the data collected were processed and descriptive statistics was used to analyzed the data (mean and standard deviation). The results were presented using frequency tables.

RESULTS AND DISCUSSION:
Out of 91 questionnaires issued to the respondents, 80 were completed and returned, representing 87.9 percent. The table 1 above shows that the female respondents 45 (56.2%) were more than males 43.8%. the most represented age range was 31-40 years (37.5%) and followed by those from 21-30 years (26.3%). 33.8% of the respondents were holders of national diploma / national certificate, 31.3% had either HND/Degree, 28.7 were holders of SSCE / WASC and only 6.2% of them had higher degrees. Regarding the grade levels in the office, 51(63.7%) of the respondents were of grade level 7-14 and 29 (36.3%) were of grade level 3-6. This shows that majority of the participants were senior staff. Most of the respondents have 11-20 years (46.3%) working experience, 28.7% had 0-10 years, 21.2% had 21-30 years and only 3.8% of them have work above 31 years.

Grand Mean (x) = 3.38. Strongly Agree (SD), Agree (A), Undecided (U), Disagree (D), Strongly Disagree (SD), Frequency (F), Mean ( ), Percentage (%).

Regarding working conditions as work motivation and satisfaction table 3 revealed that 62.5% of the respondents disagree that it was not favourable, and 32.5% agrees the working condition was favourable. It was found that 75% of the respondents agrees staff mix freely with colleagues and have interpersonal relations. The grand mean rating for working conditions was 3.38.

Table 3 indicates that the means for the work content, payment, promotion; recognition, working conditions, benefits, personal, leadership/supervisor, general and rewards and recognition programs ranged from a low of 2.01 to a high of 4.57. it therefore implies that employees in the sample are relatively motivated. However, the mean values for payment, benefits, and working conditions were the lowest. These mean values indicate the areas that employees were most likely to be dissatisfied and demotivated. This revealed that employees in the study were most likely to be motivated due to rewards and recognition programs, general personal, leadership / supervisor, work content and promotion dimensions.
Majority of the respondent were dissatisfied with present salary. This finding was in disagreement with Ali & Ahmed (2008) who found that staff in the sample were relatively motivated by payment. The study’s results indicated that the hospital has definite policy regarding to promotion, and that staff promotion should be based on competitive basis as well as performance. The also indicated that the hospital accords the staff recognition and feedback on good performance. The employees reported that the working conditions in the hospital was not favorable, but however, staff mix freely with colleagues and they have interpersonal relationships. The results shows that employees’ benefits in the hospital such as pension, medical schemes and leave were not satisfactory. The findings revealed that employees shows positive attitude towards their jobs. Therefore, their personal feeling is one of the motivating mechanism in the work place.
The above findings were in line with Ali and Ahmed (2008) who reported that in their study that if rewards or recognition offered to employees were to be altered, then there would be a corresponding change in motivation and satisfaction. They found that employees were less motivated by rewards and recognition than some of the other dimensions of the work satisfaction and motivation survey. By implication, this means that if more focus is placed on rewards and recognition, it could have a
resultant positive impact on motivation and thus result in higher level of job performance (Ali and Ahmed, 2008). All businesses use pay, promotion, bonuses or other types of rewards to encourage high levels of performance (Cameron and Piece, 1977). Recognition gives employees extra motivation and made them put in more efforts in their jobs. Ali and Ahmed (2008) found that there was significant relationships between work content and work motivation and satisfaction.

In conclusion, the study indicated that the mean values for payment, benefits, and working conditions were the lowest, indicating dissatisfaction and demotivation. This implies that employees in the study were most likely to be motivated due to rewards and recognition programs.

Recommendations: The following recommendations were made based on the study’s findings:
1. Managers of the hospitals should emphasized more on rewards and recognition programs, because it leads to positive impact on motivation and higher job satisfaction.

2. The work place should be made conducive and safe to stimulate the staff to work harder.

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Lusa, H.Y.
Department of Medical Microbiology: School of Medical Laboratory Science, AhmaduBello University Teaching Hospital, Zaria.
Mukhtar, M.D
Department of Microbiology, Bayero University Kano. :mukhtardauda@yahoo.com
All Correspondences to: Lusa H.Y Department of Medical Microbiology School of Medical Laboratory Sciences
Ahmadu Bello University Teaching Hospital, Zaria. harunalusayadock@gmail.com

ABSTRACT
Background: Macrolides have been used in treatment of infectious diseases, since the late 1950s. Erythromycin or other Macrolide can be used in treatment of patients who are allergic to penicillin. As with other antibiotics there is increasing global concern regarding the progressive development of bacterial resistance to these agents. Objective: This study was carried out to determine the prevalence of macrolide-resistant enteric bacteria among febrile patients attending selected hospitals in Zaria. Methods: The enteric bacterial pathogens were detected by Culture, Gram technique and Biochemical tests both conventionally and Micro Gen ID. On the basis of the in vitro activity of enteric pathogens, studies of Azithromycin and four commonly used macrolide antibiotics was carried out. Results: Three hundred and fifty (350) febrile patient’s blood samples were cultured, of which 75(21.4%) yielded positive growth. Of the positive samples 37(49.3%) were microbiologically documented as Gram negative-enteric bacteria mainlyEscherichia coli 20.0%, Salmonella typhi 12.0%, Klebsiella pneumoniae 9.3%, Acinetobacter haemolyticus 4.1% and Enterobacter aerogenes 4.1%.The macrolide susceptibility of enteric bacteria from bloodstream of febrile patients in Zaria indicates: Clindamycin 2(5.4%); Erythromycin 4(10.8%); Clarithromycin 5(13.5%);Telithromycin 8(21.6%);Azithromycin 16(43.2%).The rate of macrolide resistant enteric bacteria in Zaria is show Clindamycin 29(78.4%), Clarithromycin 24(64.9%), Erythromycin and Telithromycin 22(59.5%) each and Azithromycin 13(35.1%). Conclusion: The study shows that among the enteric bacterial pathogens macrolide resistant Escherichia coli, Salmonella typhi and Klebsiella pneumoniae were among the leading causes of febrile illness in patients age >15 years. This highlighted the major contribution of resistant Gram-negative bacterial diseases in febrile patients to hospital admissions in BSIs other than Malaria and other Gram-positive bacteria.

Key words: Resistance, Macrolides, Enteric bacteria, Febrile, Blood stream infections, prevalence.

INTRODUCTION

Macrolides are a group of drugs (typically antibiotic) whose activity stems from the presence of a Macrolide ring ,a large macro cyclic lactones ring to which one or more de-oxy sugars, usually, cladinose and desosamine may be attached. The lactones rings are usually 14-, 15-, and 16- membered. Macrolide belong to the polyketolide class of natural products. They are commonly used to treat respiratory tract infections. Three of the most widely used agents in the class of Macrolide are Azithromycin (Pfizer), Clarithromycin (abbot), and Erythromycin (generic).Erythromycin or other Macrolide can be used in treatment of patients who are allergic to penicillin(Jaggi etal.,2007).Felmingham et al., (2002) reported an outbreak of resistant Streptococcuspneumoniae to Erythromycin and Azithromycin. The incidence of BSIs is high in the tropics and the initial treatment is usually with broad spectrum empiric antibiotics. This indiscriminate ‘blind’ treatment has in many instances given rise to ineffective treatment with relapses, complications and development of resistance with increasing number of bacteria becoming resistant to available antibiotics.
Febrile illness is a leading reason for admission to hospital in Africa ( Petersetal,2004).Febrile illness are fuelled by the HIV epidemic (Onankpaetal, 2008). Fever or febrile illness was defined by Hughes etal.(2002) as a single oral temperature of >38.3oc(101oF) or a temperature of >38oc for 1hr.Fever higher than 38.9oc associated with BSIs (Brentetal, 2006; Bellet al,2001;Peters etal, 2004).The in vitro activity of azithromycin against enteric bacterial pathogens show high activity against campylobacterspp, E.coli, Shigellaspp, Salmonellaspp; including Salmonellatyphi.
Malaria has been the primary focus of work on fever-related illness in sub-Saharan, Africa for decades because of its high prevalence and simple laboratory diagnosis, while laboratory capacity to diagnose non-malaria causes of fever has been limited (Reddy et al.,2010).In Nigeria ,the outcome of treatment of neonatal septicaemia has remained poor, with reports of mortality of 33% to41% two tertiary hospitals in the country (Adeleke and Belenwu,2006;Martins et al.,2005; Mokuola et al.,2002;Nwodioha et al.,2010).The results of bacteriological cultures and antibiotics susceptibility tests take 3-7 days (Kavitha et al.,2010).Thus it is a common practice to institute early empirical treatment with broad spectrum antibiotics in patients presenting with symptoms of bacteraemia (Murty and Gyanshwari,2007).This is only possible with knowledge of commonly isolated bacteria and their susceptibility to antibiotics in a given place(Shahlaet al.,2009). Malaria has been the primary focus of work on fever-related illness in sub-Saharan, Africa for decades because of its high prevalence and simple laboratory diagnosis, while laboratory capacity to diagnose non-malaria causes of fever has been limited (Reddy et al.,2010).In Nigeria ,the outcome of treatment of neonatal septicaemia has remained poor, with reports of mortality of 33% to41% two tertiary hospitals in the country (Adeleke and Belenwu,2006;Martins et al.,2005;Mokuola et al.,2002;Nwodioha et al.,2010).The results of bacteriological cultures and antibiotics susceptibility tests take 3-7 days (Kavitha et al.,2010).Thus it is a common practice to institute early empirical treatment with broad spectrum antibiotic, since patients are presenting with symptoms of bacteremia(Murty and Gyanshwari,2007).This is only possible with knowledge of commonly isolated bacteria and their susceptibility to antibiotics in a given place (Shahlaet al., 2009).Blood Stream infections (BSIs) rival malaria as a common cause of morbidity and mortality in sub-Saharan African (Reddy et al, 2010). The most important older members of the group are erythromycin, oleandomycin, triacetyloleandomycin and spiramycin. Newer macrolides are semi-synthetic molecules that differ from the original compounds in the substitution pattern of the lactones ring system; they include Roxithromycin, Clarithromycin, Azithromycin (Stephen et al., 2004).

MATERIALS AND METHODS

Study Area
The study coveredtwo Local Government areas (Zaria,Sabon gari) of Kaduna State, Nigeria.

Sample Size
A sample size of 350 was obtained for the purpose of the study; determined using the WHO (1989) format,with a previous prevalence of 30 %(Felminghametal., 2002)

Subjects
A total of 350 patients were drawn from consecutive febrile patients admitted or attending the selected hospitals ABUTH (Shika,T/Wada,Banzzau); GamboSawaba General Hospital KofanGayan and Major Ibrahim Abdullahi Memorial Hospital SabonGariZaria on account of Blood Stream Infections (BSIs) were sampled.No discrimination was made based on gender, age, ethnicity or religion. Patients having fever due to non-infectious causes such as blood transfusion, drug infusion and patients attending hospital without symptoms of fever were excluded from the study. Inclusion of patients was based on those that volunteered of all sexes and ages with febrile illness.Febrile patients were defined as those with a single
oral temperature of =38.3?C (101? F) or a temperature of =38?C for 1 hr or alternatively, those with fever higher than 38.9?C associated with BSIs.

Collection of Samples
Blood samples (4mls) werecollected through venipuncture from the Median cubital vein in adults and from the suprapubic region under aseptic technique in infants into a pair of Brain-Heart infusion (aerobic) and Thioglycollate (anaerobic) broths. Blood samples were collected before antimicrobial treatment has started. The Samples were properly registered before transport to Microbiology Laboratory Ahmadu Bello University Teaching Hospital, Zaria for incubation and bacteriological analysis. Blood culture bottles were incubated at 37?C for up to 7days and regular subcultures done at 24,72hrs and 7thday.

Sub-Culture
Subcultures of blood culture broths were done on Blood, Chocolate and MacConkey’s agar plates. The tops of the bottles were sterilized with ethanol-ether swab using a sterile needle and a 2ml syringe in each case, about 1ml of the broth culture was withdrawn from each bottle through the rubber liner in the caps .The withdrawn broth cultures was then inoculated on the plates. Blood/chocolate agar plates were incubated in aerobically and in candle extinction jar to provide CO2 while MacConkey’s agar was incubated in air at 37oC for 24hrs. The tops of the culture bottles were sterilized again with ethanol-ether swab and re-incubated after each withdrawal.

Conventional Identification of isolates
The identification of organisms was carried out by Gram reaction and Microscopy, colonial morphology, biochemical and serological characterization of isolates to species level, performed according to recommended guidelines (Cheesbrough, 2005).

Microgen A-ID Characterization of isolates
The tartive enteric bacteria isolates were further sent to a reference Laboratory for confirmation using Microgen A-ID System (Microgen Bio-products, 2003).

Antibiotic Susceptibility test
The resistance pattern of pure bacterial isolates was determined by disc diffusion method.The Macrolides included in the tests were Erythromycin (15µg), Azithromycin (15µg) Clarithromycin (15µg),Telithromycin(15µg) and Clindamycin (2µg) manufactured by Oxoid and Liofilchem Ltd.
A sterile straight wire was used to touch 5 colonies of each isolate and suspended in a sterile Bijou bottle containing 5ml peptone water (Lab M) which was incubated overnight at 37oC.The overnight broth cultures were diluted with sterile saline to 106(Cfu) per mL and comparing the turbidity of the inoculum with 0.5 McFarland turbidity standard (Cheesbrough, 2000). A sterile cotton-tipped applicator is then introduced into standardized inoculums and used to inoculate dried plates of Muller-Hinton agar (oxoid, England). Sterile single antibiotic discs were then aseptically placed on each inoculated plate and incubated for 24hrs aerobically at 37?C. Zone diameters of inhibition of each isolate to the disc was measured and resistant ones Laboratory Standards Institute (CLSI) to determine sensitivity or resistance (Ferraro, 2000).The CLSI quality control values were used to cross check correctness with the new guidelines for antibiotic disc Susceptibility tests.

RESULTS
A total of 350 blood samples were cultured from 5 hospitals in Zaria, Kaduna State. The samples yielded 75(21.4%) isolates; 37 isolates were microbiologically documented as
Gram-negative enteric bacteria including Escherichia coli 15(20.0%),Salmonella enterica typhi 9(12%), Klebsiella pneumoniae 7(9.3%), Acinetobacter haemolyticus 3(4.0%) and Enterobacter aerogenes 3(4%).
Table 1 shows the distribution of bacterial isolates from the study area.Escherichia coli 15(20.0%) and Staphylococcus aureus were the highest recorded isolates while Viridians streptococci 2(2.7%) were the least frequently isolated.

DISCUSSION
Macrolide resistance (MR) has been well documented in several countries and has become clinically significant since the large increases in macrolide usage during the 1980s.Macrolide resistance has also increased dramatically during the last decade (Pallereset al.,2005).There are several case reports of failures of macrolide antimicrobials in treatment of infections leading to major public health problems.

Bacterial infections in febrile patients are a major cause of morbidity and mortality .In thisstudy 75(21.4%) bacterial isolates were microbiologically documented from BSIs with 38(50.7%) Gram-positive organisms and 37(49.3%) Gram-negative enteric pathogens (0.5>p>0.1) as in table1.This rate is slightly higher than that reported by Nwadiohaet al.(2010) in Kano but relatively low compared to 44.9% in Calabar (Martins et al.,2005) and 30.8 in Ilorin(Mukuolaet al.,2002).The probable reason is that this study involves all febrile cases while other studies involved only children suspected of septicaemia.Most studies have reported isolation of more Gram negative organisms(Nwabuisi and Nwafor,2000).However the finding of more Gram positive isolates (50.7%) is a reflection of similar reports and reasons in Europe and USA (Siefert andWisplinghoff,2005;Guilarde et al.,2007).The most predominant enteric bacteria isolate (Table3) is Escherichia coli 15(40.5%),others are;Salmonella typhi9(24.3%),Klebsiella pneumoniae 7(18.9%),Enterobacter aerogenes and Acinetobacter haemolyticus 3(8.1%) each.The frequency of enteric isolates is higher in females aged 15-24 years 7(18.9%) and 6(16.2%) in males, slightly lower than a previous study in Abuja,Nigeria that recorded 22% females and 17.9% males (Iregbuet al., 2006).The reason for this is the anatomy of the female reproductive organ or it is only suggestive that females are more susceptible to bacteraemia than males.

In this research a high resistance of enteric pathogens isolated from blood stream of febrile patients in Zaria was observed (Table 7).Isolates from Shika were 100% resistant to Clarithromycin and Clindamycin (P<0.05 at 95% CI) similar to that reported by Tariq et al., (2003), probably because the hospital is a tertiary hospital.Multi-drug resistance of many bacteria to many antibiotics has been reported in developing countries due to misuse of antibiotics. In this study the phenotypic resistance pattern (Table 5) of Gram negative enteric bacteria showed multiple drug resistance with a number of the macrolide antibiotics ranging from 3-5 with the highest phenotypic resistance pattern showing isolates being resistant to 5 macrolide antibiotics [AZM,CLR,E 89% and TEL,CD 59.3%] the least of the multiple resistance with 3 antibiotics [CLR,CD 43.2%,and E,TEL 21.6%].The high resistance rates to these antibiotics in this study and in recent studies may be a reflection of the abuse and misuse of these agents coupled with the inherent resistance mechanisms of the enteric bacteria as observed by Okeke and Sosa,( 2003).The implication for the presence of these multiple drug resistant organisms on our already strained health care system leads to longer hospital stay,more expensive toxic drugs and higher mortality rates. The highest rates of macrolide resistant bacteria are observed in age group 15-24 years. Clarithromycin and Clindamycin 25% each(Table 8),the lowest resistance is recorded in age group >45years with Azithromycin and Clarithromycin; similar to findings of Surbhiet al .,(2003).The incidence rate of macrolide resistant enteric bacteria show Escherichia coli being more prevalent ;Clindamycin 35.1%,Clarithromycin and Erythromycin 32.4% each,Telithromycin 29.7%,and Azithromycin 18.9% resistant Enterobacter aerogenes has the least occurrences with a rate of 8.1% for all the antibiotics except Azithromycin 5.4%(Fig.1-3).Similar work done by Joshua et al.,(2006).

CONCLUSION
This work shows that among the enteric bacterial pathogens, macrolide resistant Escherichia coli,Salmonella entericatyphi and Klebsilla sp. are the leading causes of febrile illness in patients >15 years, other enteric isolates prevalent in the study area are Acinetobacter haemolyticus and Enterobacter aerogenes. The study also revealed that empiric treatment of febrile patients is significantly associated with outcome of culture and sensitivity test results of suspected bacteraemia due to multi-drug resistant strains recorded. This research also showed that azithromycin can be used as a drug of choice for treatment of bacteraemia due to enteric pathogens. The rate of macrolide resistant enteric bacteria in Zaria is Clindamycin 29(78.4%),Clarithromycin 24(64.9%),Erythromycin and Telithromycin 22(59.5%) respectively, and Azithromycin 13(35.1%). The high macrolide resistance recorded among Gram negative enteric pathogens suggests that if no effort is done to control use of antibiotics, most especially macrolide antibiotics in the communities ,then we are leading for a doom in terms of antibiotic usage.

RECOMMENDATIONS

1. National policies should be formulated to combat development of resistance and control macrolide antibiotic usage.

2. Health institutions should undertake isolation and determination of susceptibility pattern of the prevalent causative organisms of BSIs of their localities periodically to reduce erroneous diagnosis of febrile illness.

3. It should be made mandatory to determine the spectrum of locally prevalent pathogens and their susceptibility to antimicrobials before placing febrile patients on empiric antimicrobial regimens to prevent longer hospital stay, reduce cost of treatment and mortality rates.

4. Further investigations should be carried out in order to find solution to genotypic resistant mechanisms of the enteric bacteria prevalent in our settings.

REFERENCES

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The post Prevalence of Macrolide Resistant Enteric Bacteria in Bloodstream Infections (BSIS) of Febrile Patients Attending Some Selected Hospitals in Zaria Kaduna State. appeared first on Nigerian Biomedical Science Journal.

Lusa H.Y
Department of Medical Microbiology School of Medical Laboratory Sciences AhmaduBello University Teaching Hospital, Zaria.
Olonitola O.S, Ameh J.B., Ado S.A
Department of Microbiology, Faculty of Life Sciences, Ahmadu Bello Univesity, Zaria.
Olayinka A.T
Department of Medical Microbiology, Ahmadu Bello University Teaching Hospital, Zaria.
All Correspondences to: Lusa H.Y Department of Medical Microbiology School of Medical Laboratory Sciences
AhmaduBello University Teaching Hospital, Zaria. harunalusayadock@gmail.com

ABSTRACT
Background: Bacterial infections that are difficult or impossible to treat are becoming increasingly common and are causing a global health crises due to antibiotic resistance ,while reports have indicated that pneumococcal infection is an important public health concern throughout the world with global burden in causing disease and death comparable to that of Human immunodeficiency virus(HIV), Malaria and Tuberculosis(TB). The Penicillin and Macrolides belong to the groups of most commonly used, clinically important antibiotics for treatments of infections caused by Gram-positive bacteria such as S.aureus, S.pneumoniae and S. pyogenes.Some bacterial pathogens have innate resistance against antibiotics and this typically reflects variations in the structure of their cell envelope.Polysaccharide capsule of Streptococcus pneumoniae defines over ninety serotypes, which differ in their carriage prevalence and invasiveness for poorly understood reasons. They also showed there is a link between serotype and growth in-vitro.This study was carried out to determine pneumococcal serotypes and resistance to selected antibiotics among patients with symptoms of respiratory tract infections (RTIs) attending Acute-care health facilities in Kaduna state, Nigeria. Methods: Sputa was collected from patients with suspected cases of RTIs. Culture and sensitivity was conducted, and pneumococci were serotyped. Results: A total of 450 Subjects were enrolled for the study. 207(46.0%) yielded pathogens of which 41(9.1%) were Streptococcus pneumoniae.Pneumococcal isolates were commonly resistant to Cefuroxime 76.2%, Ampicillin 64.4%, erythromycin 45.2 % and Azithromycin 40.5%. Four different pneumococcal serotypes were detected in the study area. Conclusion: This study shows that the pattern of pneumococcal resistance against Penicillin and Erythromycin discourages the common practice of doing sensitivity on one antibiotic and another is used for treatment due to availability or cost implication. The Pneumococcal isolates have shown resistance not only to penicillin and erythromycin but also to alternative antibiotics like Azithromycin, cefuroxime and ceftriaxone. The most common serotypes were 3 and 13.

Key words: Resistance, Sensitivity, Minimum inhibitory concentration (MIC), E-test, serotype Acute-Care Health care facility, Erythromycin, Penicillin, Antibiotics, Pneumococci, RTI, HIV, and TB.

INTRODUCTION

Streptococcus pneumoniae is the most common cause of bacterialacute respiratory infections(ARI)and Pneumococcal resistance is the principal cause of concern regarding treatment failures for ARI (Schrang et al.,2001).Pneumococcus is also the most common cause of community acquired pneumonia (CAP), sporadic bacterial meningitis and bacteremia worldwide. In 2005, WHO estimated that 1.6 million people die of pneumococcal disease every year(Bandettini and Melioli,2012). Nigeria is currently one of the highest pneumococcal disease burdened countries and accounts for 5% of the global burden (Iwalokun et al., 2012; Iliyasu et al., 2014).Thoseexposed to or residing in risk areas such as Acute-care health facilities are at higher risk for invasive pneumococcal disease and death than those residing in communities (Kusteret al.,2014).An association has been established between the type of polysaccharide capsule surrounding the bacteria and harmless colonization versus invasive disease.Antibiotic resistant bacteria that are difficult or impossible to treat are becoming increasingly common and are causing a global health crisis (Abla et al., 2014).Drug resistant infections could kill an extra10 million people across the world every year,if by 2050 they are not tackled they could cost the world around $100 trillion in cost output (AMR Review, 2014).Drug resistance trends are not well documented in most developing countries due to limited laboratory capacity and acute respiratory infections (ARI) are often treated empirically with antibiotics (Schrang et al., 2001). The global emergence of in-vitro antimicrobial resistance and dissemination of penicillin and macrolide resistant Streptococcus pneumoniae strains has become a serious clinical concernworld wide (Mohammad et al., 2012; Alba et al., 2014). Pneumococcal infection is an important public health concern throughout the world with global burden in causing disease and death comparable to that of Human immune-deficiency virus (HIV), Malaria and Tuberculosis (TB) (Iwalokun et al., 2012).Ninety-three capsular serotypes have been identified in Streptococcus pneumoniae, each differing in chemical structure and immunogenicity (Calix and Nahm, 2010).

MATERIALS AND METHODS

Study Area: Kaduna state is located at the Centre of Northern Nigeria, in the Northwest geopolitical zone. The state lies between latitude 90 and 1400 N of the equator with a time of 1 hour ahead of the GMT. The state shares boundaries with Niger state to the west, Zamfara, Katsina and Kano states to the North, Bauchi and Plateau to the east and FCT Abuja and Nasarawa state to the south. It has a population of 6.63 million based on the 2006 census projections. The state is divided into three senatorial zones: Northern, Central, and Southern zones.Kaduna state has 739 Primary health facilities, 29 secondary care facilities and five tertiary hospitals (Abubakar et al., 2013).Kaduna state is administratively divided into 23 local government areas (LGAs) with several districts and wards in each LGA.

Ethical Consideration and Confidentiality Ethical approval was obtained from the Ethical committees of Kaduna State Ministry ofHealth and the targeted facility.Confidentiality was maintained throughout the test analysis.

Sample Collection Sputum samples from 450 patients with suspected cases of RTIs were collected in wide mouth leak-proof containers.Samples were then transported to the laboratory as soon as possible.( CDC; Tests online, 2014).Patients consent was also sort before recruiting them for the study. Purulent part of the sputum samples were inoculated on plates of 5% Blood agar and incubated immediately in presence of 5% CO2 overnight at 37oC(Mohammed, 2013;Iliyasu et al.,2015). Bacterial isolates were identified based on morphological characters, Gram reaction, catalase test, bile solubility, susceptibility to Optochin and hemolysis (Iliyasu et al., 2015).Suspected S.pneumoniae colonies were translucent or mucoid, with raised or having draughtsman appearance and 1-2mm in diameter showing alpha-hemolysis.Microscopic examination was carried out on suspected pneumococcal colonies. The smear was Gram stained. All slides were examined under the oil immersion lens. S.pneumoniaeappear gram-positive, elongated (lanceolate) diplococcus, or forms short chains (Bacteriology-test, 2014). Appearance of gas bubble will indicated positive catalase test (Bacteriology-test, 2014).Evidence of clearing of turbidity in the tube marked test compared with the saline control was considered positive while suspension remaining turbid in both tubes was considered negative solubility to bile (WHO/CDC, 2003). A chemical-impregnated disk containing Optochin was aseptically placed in the center of an inoculated Mueller- Hinton agar surface. The inoculated plate was incubated at 37?C for 24 hours Growth of the lawn up to the margin of the disk indicates the bacterium is resistant to ethylhydrocupreine hydrochloride (optochin). An obvious clear zone around the disk (=14mm in diameter) indicates susceptibility (WHO/CDC, 2003).The susceptibility of S. pneumoniae was determined by the disk diffusion (oxoid, Thermo Fisher, Scientific, Australia) following CLSI guidelines (Wayne, 2006). Breaking points for non-susceptibility and resistance was used to indicate susceptibility of the pneumococci to the antibiotics including:Ampicillin (10µg), erythromycin (15µg), penicillin (10U), Cefuroxime (10µg), Azithromycin (10µg), Ceftriaxone (10µg), Chloramphenicol (30µg) and Tetracycline (30µg).MICs of strains displaying resistance to erythromycin and penicillin from the disk diffusion method were determined by E-test methodology (LiofilChem Diagnostics, Italy). The E-test strips were: Erythromycin (0.016-256µg/mL) and Penicillin G (0.016-256µg/mL). Resistance against the antibiotics wereinterpreted according to MIC test Strip Technical Sheet Streptococcus pneumoniae (MIC Interpretation Guide, 2015). E- Strips(Liofilchem) were applied to an inoculated Mueller-Hinton agar plate E- test and incubated at 37?C for 24 hrs in the presence of 5% CO2. Readings on the scale were considered as the MIC (in µg/mL). Serotyping (PCR Strategy)was carried out. Genomic DNA was extracted from pneumococcal cells of a fresh overnight culture.Amplicons from PCR were analyzed with 1.5% agarose gel electrophoresis (Marcus et al., 2012).The amplicons with expected cps band sizes were purified using the PCR purification kit (Qiagen) according to the manufacturer’s instructions.Sequences were analyzed and viewed with Bionumerics soft ware version5 (Marcus et al.,2012).The amplicon nucleotide sequences were used to interrogate the GenBank data base,and if the highest BLAST bit score gave the serotype.Identification was then considered as a ”match”. Where the query results did not correlate or were not consistent with conventional serotyping methods, the strain/serotype was considered as ”misidentified” (Marcus et al., 2012).

Statistical Analysis The data collected were analyzed using tables and Chi-square tests .Results were considered as significant if the ?2 p-value was ? 0.05 and non-significant if p-value was > 0.05.All statistical analysis were done using IBM/SPSS Software (v20.0).

RESULTS
A total of 450 sputum samples were cultured and isolates included: Streptococcus viridians ‘Group (SVG) 56(27.1%), Staphylococcus aureus 52(25.1%), Streptococcus pneumoniae 41(19.8%), Klebsiella pneumoniae 30(14.5%), Streptococcus pyogenes 20(9.7%), Haemophilus influenzae 5(2.4%), Enterobacter aerogenes 2(1.0%) and Moraxella catarrhalis 1(0.5%).Statistical analysis showed a significant association (P?0.05) with respect to frequency of isolates and geographical distribution as in table 1.
Enterobacter aerogenes 2(1.0%) and Moraxella catarrhalis 1(0.5%).Statistical analysis showed a significant association (P?0.05) with respect to frequency of isolates and geographical distribution as in table 1.

DISCUSSION
Streptococcus pneumoniae is both an aggressive pathogen and a normal part of the human microbiome (Blaschke, 2016).Viridian’s Streptococci bacteria are generally disregarded as the etiologic agent of pneumonia even when isolated from cultures of sterile material.This study show that viridians streptococci should not be ruled out as a cause of pneumonia as reported by Freitas et al. (2006).The high pneumococcal prevalence in this study was likely due to predisposing factors such as; Prior exposure to risk areas such as health care settings and immuno compromised patients or patients with other medical comorbidities (Kusteret al.,2014). Penicillin has been the drug of choice worldwide for the treatment of pneumococcal infections. However penicillin resistant strains have emerged resulting in a shift to the use of other drugs e.g. cefotaxime, chloramphenicol and erythromycin (Dankor et al., 2010).The Pneumococcal isolates have shown resistance to penicillin, erythromycin and other alternative antibiotics as reported byIliyasu in a study carried out in Kano (Iliyasuet al.,2014).Investigations from some parts of Nigeria and other countries have shown high prevalence of resistance to Penicillin and other agents. Penicillin resistant pneumococci (PRP) and Erythromycin resistant pneumococci (ERP) had prevalence of 53.7% and 46.7% respectively, in this study .The prevalence of PRP closely agrees with findings by Akpede et al. (1994) but way below findings’ by Habib et al. (2003), and Kadankai et al. (2009) who recorded prevalence of 85.4% and 93.0% respectively, however rates in this work were higher than that reported by some researchers (Emele, 2000; Oduyebo et al., 2006; Abla et al., 2014) likely due to repeated course of therapy with ß-lactams or macrolide antibiotics (Jorget al.,2016).Erythromycin and other macrolides used in this study as alternatives to ß-lactams for treatment of Respiratory tract infections(RTI) had high resistance rates as compared to works of other researchers (Abla et al., 2014; David et al., 2015), but lower than that recorded by Mohammad et al.,2012. Erythromycin resistance in some Countries were found to be higher than those recorded in this study (Song et al., 2004; Reinert, 2005; Carmargos, 2006; Jenkings, 2008).This difference might be related to the shift in use of other antibiotics in these areas as reported by Dankor et al.(2010). Higherprevalence of Penicillin resistant pneumococci (PRP) and Erythromycin resistant pneumococci (ERP) was obtained among the elderly of age (40-60) years which is in agreement with findings by Nicholas et al. (2013), the reason probably, they are immunocompromised. Year group 20-29 had higher prevalence for penicillin resistance, which is similar to findings by Mohammed, (2013) probably due to nature of exposure to more predisposing factors such as drug abuse, HIV and TB that could lower the immunity. This finding agrees with that reported by Nicholas et al. (2013) in a study carried out in Ghana. Pneumococci are normally exquisitely sensitive to penicillin, which is lethal at concentrations <0.1 µg/mL. In this study high-level (MIC =2µg/ml) PRP and ERP has been associated with bacterial pneumonia which is similar to findings by Mellay et al., (2003);Fukushima et al.,(2008) and Abla et al.,(2014).WHO in 2007 recommended the use of pneumococcal conjugate vaccines (PCV) in all countries, urging that the highest priority for introduction be given to countries with high pneumonia and under-5 mortality.Nigeria only joined other African countries to launch pneumococcal conjugate vaccine (PCV10) as part of its Routine immunization schedule on 22nd December, 2014(WHO, 2015).Accurate serotyping is essential for epidemiologic study of S.pneumoniae [CDC, (nd).The serotypes Sequenced and identified in this study were serotypes 3, 13, 20 and 23. Serotype 3 is included in the 10-valent Pneumococcal Conjugate Vaccine (PCV), the 23F serotype is included in the heptavalent conjugate vaccine. The finding is not in complete agreement with findings of Onyemelukwe and Greenwood,1998 who identified serotypes 1,2 3, and 5 in Zaria and Abla et al., 2013 in Algeria, probably because their subjects comprises both children and adults. Serotypes 13 and 20 are non-vaccine types. Serotypes 3 and 23 are also among the dominant serotypes as reported by WHO, 2007 .The findings are also comparable to that obtained by Tarrago et al., 2008; Dankor et al., 2014 but completely differs from that reported by Falade et al., 2009.

CONCLUSION
The highest pneumococcal resistance to penicillin and erythromycin was obtained among patients’ =50 years of age. A prevalence rate of 23.0% pneumococcal infection was established in this study.The study shows that the isolates did not only show resistance to penicillin but also to other alternative antibiotics such as Erythromycin, Azithromycin, Tetracycline, and cefuroxime and should be used carefully in the future. The indiscriminate use of antibiotics at the community level might be probable cause of resistance obtained in this work. The use of penicillin as the best choice for treatment of infections with S. pneumoniae may need reconsideration because of the emerging trends in the antibiotic resistance. It is also pertinent to note that the resistance pattern shown by Penicillin and Erythromycin discourages a common practice in some of our Health care institutions where sensitivity is done on one antibiotic and another is used for treatment due to availability or cost implication. The implication of this study is a high potential for treatment failure with most antibacterial drugs recommended to empirically treat lower respiratory tract infections. Higher prevalence of Streptococcus viridians Group (SVG) recorded in this study than Streptococcus pneumoniae implies that the diagnosis of pneumococci can be problematic and if not chanced occurrence; only the use of specific gene targets may solve the problem of inaccurate identification of pneumococci. Serotype 3 and 13 are the most prevalent serotypes causing disease in this study. Screening for pneumococcal vaccination status should be authorized in patients age 60yrs or older. Events that reduce selective pressures deriving antimicrobial resistance must be encouraged such as: More appropriate use of antimicrobials, Vaccination must be enforced (PCV and PPSV) .

ACKNOWLEDGEMENT
The authors sincerely thank Ahmadu Bello University Teaching Hospital, Zaria and all the staff of department of Medical Microbiology, ABUTH, Zaria for their support.

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8. Calix J J and Nahm M H. (2010).A new pneumococcal serotype 11E has a variable Inactivated wcjE gene. Journal of infectious disease.202:29-38. Centers for Disease control and prevention [CDC, (nd)].PCR deduction of Pneumococcal serotypes. https://www.cdc.gov.

9. David Farrell, Stephen Jenkins, Steven D.Brown,Manish Bruce S.Lavin, and Keith P.Klugman(2005) Emergence and Spread of Streptococcus pneumoniae with erm(B) and mef(A) resistance. Emerging infectious diseases; 11(6):851–858.doi: 10.3201/eid1106.050222.

10. Donkor ES, Newman MJ, Oliver-Commey J, Bannerman E, Dayie NTKD, Badoe EV(2010): Invasive disease and paediatric carriage of Streptococcus pneumoniae in Ghana; Scandinavian Journal of Infectious Diseases 2, 42:254-259.

11. Donkor ES, Adegbola RA, Wren BW, Antonio M (2013) Population Biology of Streptococcus pneumoniae in West Africa: Multilocus Sequence Typing of Serotypes That Exhibit Different
Predisposition to Invasive Disease and Carriage. PLoS ONE 8(1): e53925.doi:10.1371/ journal.pone.0053925.

12. Emele F.E(2000).Etiologic spectrum and pattern of antimicrobial susceptibility in bacterial meningitis in Sokoto, Nigeria. Acta Pediatric; 89:942–6.**

13. Fukushima K.Y, Yanagihara Hirakata, Y.Sugahara K, Morinaga, Y, kohno S, Kamihira S(2008).Rapid identification of penicillin and macrolide resistance genes and simultaneous quantification of Strepto-coccus pneumoniae in purulent sputum samples by use of a novel real-time multiplex PCR assay clinical Microbiology.46 (7):2384-2388.

14. Habib AG, Nwokedi EE, Ihesiulor IU, Mohammed A, Habib ZG (2003). Widespread antibiotic resistance in savannah Nigeria. African Journal of Medical Science; 32:303–5.

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The post Antibiotic resistance and serotypes of invasive streptococcus pneumoniae Isolated from Patients attending Acute care Health facilities in Kaduna state, Nigeria. appeared first on Nigerian Biomedical Science Journal.

FASOGBON, Samuel Ayobami
Public Health In-vitro Diagnostic Control Laboratory, Medical Laboratory Science Council of Nigeria, Yaba-Lagos, Nigeria.
ABDULLAHI, Kabiru
Department of Histopathology, Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria.
OKORIE, Nnaemeka
N.K.S.T. Len Gabrielse’ School of Medical Laboratory Science Mkar, Benue State, Nigeria.
OGUNJIMI, Tolulope Samuel
Population Council, Nigeria, Yaba, Lagos State.
All Correspondences to: Fasogbon, Samuel Ayobami Public Health In-vitro Diagnostic Control Laboratory,
Medical Laboratory Science Council of Nigeria, 8 Harvey Road, Yaba, Lagos State, Nigeria. E-mail: samfash4best@gmail.com

ABSTRACT
Background: The study was to immunohistochemicallydetermine the Correlation of the Expression of Vitamin D Receptor (VDR) and Triple Positive Invasive Ductal Carcinoma (IDC) of Breast tissues. Materials and Method: Fifty six (56) archived female breast Invasive Ductal Carcinoma tissue blocks were experimented Immunohistochemically with ER, PR, HER-2 and VDR.Seven (7) confirmed cases of those that were positive to ER, PR, and HER-2 (Triple Positive) were correlated with VDR expressionand the results were compared. Result: The results show that there was no significant difference (P?0.05) found comparing the immunohistochemical expression of VDR with Triple positive IDC tissues. Conclusion: This study shows that no significant difference was found in the expression of VDR and triple positive IDC tissues; it can therefore be said that VDR can be of therapeutic target and of additional antibody in immunohistochemical diagnosis of breast cancer.

Keywords: Vitamin D receptor (VDR), Triple Positive, Breast cancer, Invasive ductal carcinoma (IDC).

INTRODUCTION

Breast cancer is the predominant malignancy where oncologists use predictive markers clinically to select treatment options, with steroid receptors having been used for many years. Immunohistochemistry has taken over as the major assay method used for assessing markers [1]. The surfacing of molecular technology has brought about new biomarkers along with immunohistochemical and serum biomarkers. Immunohistochemical markers [Estrogen receptor (ER), Progesterone receptor (PR), and Human epidermal growth factor receptor 2 (HER-2)] are often included inguiding treatment decisions, to classify breast cancer into subtypes that are biologically distinct and behave differently, and both as prognostic and predictive factors[1]. Invasive ductal carcinoma (IDC), also known as infiltrating ductal carcinoma, is cancer that began growing in the duct and has invaded the fatty tissue of the breast outside of the duct. The commonest type of breast cancer is IDC, which represent 80 percent of all breast cancer diagnoses [2]. Breast cancer is the most frequent cancer among women, being a heterogeneous disease, with distinct morphologies, metastatic behaviour and therapeutic response [3]. Approximately, 90% of breast cancer deaths are caused by local invasion and distant metastasis of tumor cells [4].According to [5], different types of this neoplasm exhibit variable histopathological and biological features, different clinical outcome and different response to systemic interventions. In fact, global gene-expression analyses have provided an appealing molecular classification for breast carcinomas, which is highly associated with patients’ prognosis [6].In the last decade; a major effort has been made to better inform the choice of the systemic treatment for breast cancer patients.
Vitamin D receptor (VDR) can be called Calcitriol receptor, and is also known as NR1I1 (nuclear receptor subfamily 1, group I, member 1), is a member of the nuclear receptor family of transcription factors [7]. Upon activation by vitamin D, the VDR forms a heterodimer with the retinoid-X receptor and binds to hormone response elements on DNA resulting in expression or transrepression of specific gene products. In addition to VDR regulation of transcriptional responses, it is also involved in microRNA-directed post transcriptional mechanisms [8]. In humans, the vitamin D receptor is encoded by the VDR gene [9].Glucocorticoids are known to decrease expression of VDR, which is expressed in most tissues of the body and regulate intestinal transport of calcium, Iron and other minerals [10].Also, it has recently been identified that VDR as an additional bile acid receptor alongside FXR and may function to protect gut against the toxic and carcinogenic effects some endobiotics[11]. Different researches have shown the evidence of linkage between vitamin D and breast cancer. Women who have breast cancer tend to have low levels of vitamin D in their body. Researchers have found how vitamin D might have a role in breast cancer. On the surface of a cell are also found Vitamin D receptors where they receive chemical signals. These chemical signals direct a cell to do something by attaching themselves to a receptor, for example to act in a certain way, or to divide or die. Vitamin D can bind to vitamin D receptors which are in breast tissue. These can oncogenes to die or stop growing, and can stop the cancer cells from spreading to other parts of the body. It is then said that vitamin D may help in preventing breast cancer, by making cells in the breast smarter. However, the relationship between breast cancer and vitamin D is complex, not fully understood, and is still being studied [12][13][14].A study carried out by [19] discovered VDR expression in patient of large population as inversely related with more aggressive breast cancer, but not with breast cancer survival outcomes. This research therefore, has set out to correlate the immunohistochemical expression of VDR with Triple Positive Breast IDC tissues.

MATERIALS AND METHODS
Area of Study
This study was carried out at Department of Histopathology, National Hospital Abuja, FCT, Nigeria. The Hospital serves most of the states of Nigeria and therefore serving a significant population of the region.

Ethical Consideration
Approval for this research work was obtained and given from the Health Research Ethics Committee (HREC) with assigned number NHA/EC/062/2015 of National Hospital Abuja, FCT, Nigeria.

Sample Size
Fifty six (56) archived female breast Invasive Ductal Carcinoma tissue blocks were experimented. The Seven (7) cases that wereconfirmed triple positive Invasive ductal carcinoma tissues were then obtained for correlation with VDR expression.

Sample Collection/Histopathological Procedures
Paraffin tissue blocks diagnosed of invasive ductal carcinoma of the female breast were experimented and those positive to ER, PR, and HER-2 (Triple positive) of the female breast were selected.
The tissue blocks were sectioned at not more than 2µm each. Seven (7) sections were obtained from each block from which one (1) section was used for Haematoxylin and Eosin staining technique while four(4) sectionswere treated with VDR, ER, PR and HER-2antibodies, while the other two (2) sections were used as negative and positive control.

Haematoxylin and Eosin Staining Technique
The sections were taken to water, stained using Harris Haematoxylin for 5minutes, washed in tap water then differentiated in 1% acid alcohol for few seconds. They were washed in tap water then blued in tap water for 10minutes. The sections were then counterstained in 1%
Eosin for 1minutes. They were then washed in tap water, dehydrated, cleared and mounted using DPX [15].

Immunohistochemical Technique
The method used is the Avidin Biotin Complex (ABC) method and the antibodiesthat are used were Novocastramanufactured. The antibody dilution factor used was 1:100 dilutions for all the antibody markers.
The processed tissues were sectioned at 2µm on the rotary microtome and placed on the hot plate at 700C for at least 1hour. Sections were brought down to water by passing them in 2 changes of Xylene, then 3 changes of descending grades of alcohol and finally to water. Antigen retrieval was performed on the sections by heating them on a Citric Acid solution of pH 6.0 using the Microwave at 1000C for 15minutes. The sections were equilibrated gradually with cool water to displace the hot Citric Acid for at least 5min. Peroxidase blocking was done on the sections by covering them with 3% hydrogen peroxide (H2O2) for 15min. Sections were washed with PBS and protein blocking was performed using avidin for 15min. Sections were washed with PBS and endogenous biotin in tissue was blocked using biotin for 15min. After washing with PBS, sections were then incubated with the respective diluted primary antibody, diluted 1:100 for 60 min. Excess antibodies were washed off with PBS and a secondary antibody (link) was applied on section for 15min. Sections were washed and the (label, in this case which is the Horseradish Peroxidase HRP) was applied on the sections for 15min. A working DAB solution is made up by mixing 1 drop (20µl) of the DAB chromogen to 1ml of the DAB substrate. This working solution was applied on sections after washing off the HRP with PBS for at least 5min. The brown reaction began to appear at this moment especially for a positive target. Excess DAB solution and precipitate were washed with water. Sections were counterstained with Haematoxylin solution for at least 2min and blued briefly. Sections were dehydrated in alcohol, cleared in Xylene and mounted in DPX [16].

Immunohistochemical Analysis
Cells with specific brown colours in the cytoplasm, cell membrane or nuclei depending on the antigenic sites were considered to be positive. The Haematoxylin stained cells without any form of brown colours were scored negative. Non specific binding/brown artifacts on cells and connective tissue were disregarded [16].

Statistical Analysis
Photomicrograph was basically used for correlating the expression and where necessary, Paired T-test statistics method was used to analyze the data generated.

RESULTS
Seven confirmed cases of triple positives tissue blocks already diagnosed as invasive ductal carcinoma of the female breast (Age mean=46.4) were used in the study. The results were presented in tables and figures below:

TABLE 1: CORRELATION OF IMMUNOHIS-TOCHEMICAL EXPRESSION OF VDR WITH TRIPLE POSITIVE (ER, PR AND HER2) IDC

DISCUSSION
There is no significant difference found; comparing VDR expression with Triple positives Breast IDC tissues which indicate that VDR can be used as an additional antibody in the immunohistochemical diagnosis of Breast IDC. This result is supported by earlier related study done; VDR expression was analyzed immunohistochemically in breast cancer patients in whichstrong VDR immunoreactivity was observed in breast cancer specimens, supporting the body of evidence that breast cancer may be a target for therapeutically applied vitamin D analogues[17].
This also support a study carried out that said; there are vitamin D receptors in breast tissue, and vitamin D can bind to these receptors. This can cause oncogenes to die or stop growing, and can stop the cancer cells from spreading to other parts of the body. Therefore, it is thought that vitamin D may help in protecting against breast cancer [12][18].This can also be related to the findings by [19] that VDR expression in patient of large population is inversely related with more aggressive breast cancer.

CONCLUSION
On the basis of this study and relevant literatures review, VDR has statistically significant correlations when compared with Triple positives Breast IDC tissues; it can therefore be said that VDR can be of therapeutic target and of additional antibody in immunohistochemical diagnosis of breast cancer. Further research is recommended to fully ascertain this fact.

REFERENCES

1. Walker R. A. (2007): Immunohistochemical markers as predictive tools for breast cancer Department of Cancer Studies & Molecular Medicine, University of Leicester, Robert Kilpatrick Building, Leicester Royal Infirmary, Journal of Clinical Pathology; 61:10.1136.

2. Johns Hopkins (2016): Inasive Ductal Carcinoma
(IDC); http://www.hopkinsmedic-ine.org/breast_center/breast_cancers_other_conditions/invasive_ductal_carcinoma.html . Sited November, 2016.

3. Ricardo, S., Vieira.,A.F.,Gerhard, R.,Leitão, D.,Pinto, R., Cameselle-Teijeiro, J, F., Milanezi, F., Schmitt, F., Paredes, J (2011): Breast cancer stem cell markers CD44, CD24 & ALDH: expression distinguishing intrinsic molecular subtype: Journal of clinical pathology; 64 (11): 937- 946.

4. Yifau, W., and Binhua, P.Z (2011): Epithelial-mesenchymal transition in breast cancer progression and metastasis: Chinical journal of cancer; 30 (9): 603-611.

5. Viale, G (2012): The current state of breast cancer classification: Annual Oncology; 23(10):207-210.

6. Sotiriou, C., Neo, S.Y., McShane, L.M (2003): Breast cancer classification and prognosis based on gene expression profiles from a population-based study. Proclaimed National Academic Science; 100:10393–10398.

7. Hosoi, T. (2002): Polymorphisms of vitamin D receptor gene. .Nippon Rinsho. 60Suppl 3: 106–110.

8. Uitterlinden AG, Fang Y, Van Meurs JB, Pols HA, Van Leeuwen JP (2004): “Genetics and biology of vitamin D receptor polymorphisms”. Gene 338 (2): 143–156.

9. Norman A.W. (2007).”Minireview: vitamin D receptor: new assignments for an already busy receptor”.Endocrinology147 (12): 5542–5548.

10. Bollag W.B. (2007): Differentiation of human keratinocytes requires the vitamin d receptor and its coactivators. Journal of Investigative Dermatology127(4): 748–750.

11. Salashor, S., and Woodgett, J.R. (2002): Links between AXIN and carcinogenesis; 58:225-236

12. Rose A.A.N, Elser C, Ennis M. (2013): Blood levels of vitamin D and early stage breast cancer prognosis: a systematic review and meta-analysis. Breast Cancer Resilience Treatment; 141:331-339.

13. Wang, D., Velez de-la-Paz OI, and Zhai JX, (2013): Serum 25-hydroxyvitamin D and breast cancer risk: a meta-analysis of prospective studies. Tumor Biology; 1-9.

14. Welsh J. (2012): Cellular and molecular effects of vitamin D on carcinogenesis. ActaBichemistry and Biophysics; 523b:107-114

15. Avwioro, O. G. (2014): Histochemistry and Tissue Pathology in; Principle and Technique Third edition, page 133-168

16. Marc (2009): Standard Immunohistochemistry
Staining Method; Avidin Biotin Complex (ABC) Method. IHC world life science products and sevices. http://www.ihcworld.com /_protocols/general_IHC/standard_abc_method.htm[cited: 20th October, 2016]

17. Friedrich M, Villena-Heinsen C, Tilgen W, Schmidt W, Reichrat J, Axt-Fliedner R. (2002): Vitamin D receptor (VDR) expression is not a prognostic factor in breast cancer. Anticancer Research May-Jun; 22(3):1919-1924.

18. Fasogbon S.A.; Okechi O.O.; Adisa J. O.; and Madukwe J. U. Immunohistochemical Correlation between the Expression of Vitamin D Receptor (VDR) and Triple Negative Invasive Ductal Carcinoma (IDC) tissues; Am. J. Biomed. Sci. 2017; 9(4), 237-243 doi:10.5099/aj170400237

19. Al-Azhri J, Zhang Y, Bshara W, Zirpoli G, McCann SE, Khoury T, Morrison CD, Edge SB, Ambrosone CB, Yao S(2017): Tumor Expression of Vitamin D Receptor and Breast Cancer Histopathological Characteristics and Prognosis. Clin Cancer Res.23(1):97-103.

The post Expression of Vitamin D Receptor (VDR) in Triple Positive Breast Cancer Tissues appeared first on Nigerian Biomedical Science Journal.

K.N. Elechi-Amadi
Department of Medical Laboratory Science, Rivers State University, Port Harcourt
O.N. Briggs
Department of Medical Laboratory Science, Rivers State University, Port Harcourt
G.A. Obediah
Department of Biochemistry, Rivers State University, Port Harcourt.
All Correspondences to: kemzi89@gmail.com

ABSTRACT

HIV infection is one of the most prevalent infections in Nigeria. The high rate of transmission of HIV in Nigeria is of an immense public health concern. This study evaluated the levels of the antioxidant enzymes, superoxide dismutase and glutathione peroxidase in HIV positive subjects. A total of 173 subjects of age 20 to 70 years were involved in this study; 60 HIV positive patients on HAART (Highly Active Antiretroviral Therapy), 57 HIV patients who are HAART-naïve, and 56 apparently healthy control subjects. Patients who had severe malaria, tuberculosis or diabetes, were excluded from this study. Pregnant female subjects were also excluded. Five millilitres of venous blood was obtained from each subject by standard procedure. CD4 count was done using Fluorescent Activated Cell Sorter (FACSCount) automation, while the antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were determined using enzyme-linked immunosorbent assay (ELISA) technique. It was observed that HIV patients had significantly reduced levels of the SOD than control subjects, with HIV positive subjects on HAART even having lower levels than those who were not on HAART. However, there were no significant differences in the levels of GPx. The results indicate impairment of antioxidant function in HIV positive subjects. Inclusion of antioxidant therapies in the treatment and management of HIV infection may prove a useful treatment modality in ameliorating the negative health implications of a compromised antioxidant system in the subjects.

Keywords: HIV, Superoxide dismutase, Glutathione peroxidase, Antioxidants, HAART, Port Harcourt

INTRODUCTION

The human immunodeficiency virus (HIV) is a retrovirus that affects the human immune system. Retroviruses constitute a large and diverse family of enveloped RNA viruses that use as a replication strategy for the transcription of virion RNA into linear double-stranded DNA with subsequent integration into the host genome (Chatterjea & Shinde, 2008).
Chronic administration of Highly Active Antiretroviral Therapy (HAART) has been reported to induce metabolic disorders including insulin resistance. HIV drugs have also been reported to cause abnormalities in carbohydrate metabolism (Florescu & Kotler, 2007). For example, Protease inhibitors have been reported to cause glucose intolerance and hyperglycaemia (Mulligan et al., 2000, Savès et al., 2002).
The human body possesses a system of antioxidants that co-operate to protect the cells of the body against the dangerous effects of oxidants. Antioxidant system consists of a variety of components, endogenous and exogenous in origin, which functions to neutralize free radicals (Krishnamurthy & Wadhwani, 2012) in the body. The human antioxidant system consists of both enzymes and non-enzymatic systems (Panda, 2012). Essentially, antioxidants are compounds that dispose, scavenge and suppress the formation free radicals or oppose their actions (Oguntibeju et al., 2009). They prevent or delay the oxidation of other molecules by preventing oxidative chain reactions.
Antioxidant enzymes; superoxide dismutase, glutathione peroxide and catalase, are complex proteins that have minerals incorporated into their molecular structure (Kiefer, 2006). Antioxidant enzymes are often the first line antioxidant defence in the human body; they terminate the chain reaction of free-radicals by donating hydrogen or electrons to the free-radicals, thus, converting them to more stable products (Panda, 2012). All the cells in the body possess powerful antioxidant enzymes (Kabel, 2014).
Superoxide dismutase (SOD) belongs to the group of oxido-reductases (Vaskova et al., 2012). It catalyses the dismutation of superoxide (O2-) to oxygen and hydrogen peroxide. The mechanism of dismutation of superoxide by SOD involves successive oxidation and reduction of the particular transition metal in the molecule of the SOD (Krishnamurthy & Wadhwani, 2012). Superoxide dismutase has been reported to be the most powerful natural antioxidant enzyme (Kiefer, 2006).
Glutathione peroxidase (GPx) is responsible for protecting cells from damage due to hydrogen peroxide and lipid peroxides (Krishnamurthy & Wadhwani, 2012). It is an 80kDa enzyme that catalyses the oxidation of peroxide using glutathione substrate. This antioxidant enzyme contains a single seleno-cysteine (Sec) residue in each of the four identical subunits. L-selenocysteine is the most important amino acid at the active site of GPx, and is also responsible for the reduction of hydroperoxides, utilizing a tripeptide substance known as glutathione in the process (Alberto et al., 2010). This study evaluated the levels of superoxide dismutase and glutathione peroxidase in HIV positive subjects.

METHODOLOGY
This work was carried out in one hundred and seventy three subjects. One hundred and seventeen (117) were HIV subjects, sixty (60) of whom were receiving antiretroviral therapy while fifty-seven (57) were not on antiretroviral therapy. Fifty-six (56) HIV negative individuals were used as control subjects. The bio-data and medical history of the subjects were obtained using questionnaire. The subjects that participated in this research gave their informed consent, did not have tuberculosis, diabetes or severe malaria; those with any of these conditions were excluded from the research. Pregnant female subjects were also excluded from the study. Ethical consent was also obtained for this research study.
Five millilitres of blood was collected from each subject; 2ml of this were put in a sample bottle containing ethylene diamine tetraacetic acid ( EDTA), for the assay of CD4 count, while 3ml were put in plain bottles for the assay of superoxide dismutase and glutathione peroxidase. The blood samples were centrifuged at 3,000rpm for 10 minutes and the plasma separated and put in another plain bottle. The plasma samples were then preserved at -20oC in the refrigerator prior to analysis. Analysis of the samples for superoxide dismutase and glutathione peroxidase was done using ELISA technique while Fluorescent Activated Cell Sorter (FACSCount) automation was used for CD4 count. The data generated was analysed using SPSS version 22.

RESULTS

DISCUSSION

The results of this work indicate differences in the levels of antioxidant enzymes in HIV patients compared to the control subjects. SOD levels were significantly reduced in HIV patients than in control subjects. During HIV infection, there is enhanced oxidative stress due to activation of the immune system, which increases the generation of free radicals which results in the depletion in the levels of these antioxidant enzymes. The depletion is probably because they are consumed in the process of providing antioxidant defence in the affected subjects (Suresh et al., 2009). Therefore, HIV infection can cause a significant decrease in SOD levels in HIV-positive subjects.
The levels of the antioxidant enzymes were lower in HIV patients on HAART than in HAART-naive HIV subjects. A similar results have been reported by other researchers (Kostyushov et al.,2009; Brown & Elechi-Amadi, 2016). HAART increases chemically reactive species in humans (Awodele et al., 2012), and has also been associated with increased oxidative stress in human HIV positive subjects (Ngondi et al., 2006). Thus, antiretroviral therapy exerts a negative effect on the activities of antioxidant enzymes.

It was observed that the levels of GPx in HIV subjects did not significantly reduce compared to the control subjects. This finding agrees with the work of Kim et al., 2015. This is may be due to the synthesis of a selenoprotein that has significant homology to mammalian GPx by HIV (Zhao et al., 2000).
Alterations in the levels of antioxidant enzymes have been reported in different disease conditions (Gil et al., 2003). HIV patients suffer constant oxidative stress, and this can lead to malfunctioning of the antioxidant system in these individuals.

CONCLUSION
This study has shown that HIV patients suffer significant oxidative stress. There is impairment of the antioxidant defence mechanism in HIV patients, and this is further affected by drug therapy used in the management of the condition. It may prove useful for treatment of HIV infection to include antioxidant therapy.

REFERENCES

1. Chatterjea, M.N & Shinde, R.(2008). Textbook of Medical Biochemistry. New Delhi. Jaypee Brothers.

2. Mulligan, K., Grungfeld, C., Tai, V.W., Algren, H., Pang, M., Chernoff, D.N., Lo, J.C. & Sehambelam, M. (2000). Hyperlipidaemia as insulin resistance are induced by protease inhibitors independents with HIV infection. Journal of Acquired immune Deficiency Syndrome, 23, 35-43.

3. Savès, M., Raffi, F., Capeau, J., Rozenbaum, W., Ragnaud, J.M., Perronne, C., Basdevant, A., Leport, C. & Chene, G. (2002). Factors related to lipodystrophy and metabolic alterations in patients with HIV infection receiving HAART. Clinical and Infectious Diseases, 34, 1396 ? 1405.

4. Florescu, D. & Kotler, D.P. (2007). Insulin resistance, glucose intolerance and diabetic mellitus in HIV-infected patients. Antiviral Therapy (London), 12, 149-162.

5. Krishnamurhthy, P. & Wadhwani,A. ( 2012). Antioxidant enzymes and human health. In El-Missiry, M.A( Ed.). Antioxidant Enzymes. Croatia. InTech.

6. Oguntibeju, O.O., Esterhuyse, A.J.& Truter, E.J. ( 2009). Possible benefits of micronutrient supplementation in the treatment and management of HIV infection and AIDS. African Journal of Pharmacy and Pharmacology, 3(9), 404-412

7. Panda, S.K. (2012). Assay-guided comparism for enzymatic and non-enzymatic antioxidant activities with special reference to medicinal plants. in El-Missiry, M.A.(Ed.). Antioxidant Enzyme. Croatia. InTech.

8. Kiefer, D. ( 2006). Superoxide dismutase: boosting the body?s primary antioxidant defence. Life Extension Magazine,6, 73-78.

9. Kabel, A.M. (2014). Free Radicals and antioxidants: role of enzymes and nutrition. World Journal of Nutrition and Health, 2(3), 35-38.

10. Vaskova, J., Vasko, L., & Kron,I. ( 2012). Oxidative processes and antioxidative metaloenzymes. In El-Missiry, M.A(Ed.). Antioxidant Enzymes. Croatia.InTech.

11. Gil,L., Martinez,G., Gonzalez,I., Tarinas,A., Alvarez,A., Giuliani,A., Molina,R., Tapanes,R., Perez,J. & Leon,O.S. (2003). Contribution to characterization of oxidative stress in HIV/AIDS patients. Pharmacological Research, 47, 217-224

12. Suresh, D.R., Annam, V., Pratibha,K. & Prasad, B.V.M. ( 2009). Total antioxidant capacity-a novel early biochemical marker of oxidative stress in HIV infected individuals. Journal of Biomedical Science, 16(61), 186-196

13. Kostyushov, V.V., Bokal, I.I. & Petrov, S.A. (2011). The study of activity of blood antioxidant enzymes in HIV infection. Biochemistry (Moscow). Supplement series B: Biomedical chemistry, 5(2), 193-196.

14. Awodele, O., Olayemi, S.O., Nwite, J.A. & Adeyemo, T.A. (2012). Investigation of the levels of oxidative stress parameters in HIV and HIV-TB co-infected patients. Journal of Infection in Developing Countries, 6(1), 79-85

15. Ngondi, J.L., Oben, J., Forkah, D.M., Etame, L.H. & Mbanya, D. (2006). The effect of different combination therapies on oxidative stress markers in HIV infected patients in Cameroon. AIDS Research and Therapy, 3, 19.

16. Mgbekem, M.A., John, M.C., Umoh,I.B., Eyong, E.U., Ukam,N. & Omotola, B.D. (2011). Plasma antioxidant micronutrients and oxidative stress in people living with HIV. Pakistan Journal of Nutrition, 10(3), 214-219

17. Kim, S., Smith, A.J., Tan, J., Shytle,R.D. & Giunta,B. (2015). MSM ameliorates HIV-1 tat induced neuronal oxidative stress via rebalance of the glutathione cycle. American Journal of Translational Research, 7(2), 328-338

18. Zhao,L., Cox,A.G., Ruzicka,J.A., Zhang,W. & Taylor,E.W. (2000). Molecular modelling and invitro activity of an HIV-1 encoded glutathione peroxidase. Procedures of the National Academy of Science of USA, 97, 6356-6361

19. Brown, H. & Elechi-Amadi, K.N. (2016). Assessment of Antioxidant Enzymes and Cortisol Levels among HIV Patients on HAART. International Journal of Science and Research, 5(4), 633-636

The post Evaluation of Antioxidant Enzyme Levels in HIV Subjects In Port Harcourt, Nigeria appeared first on Nigerian Biomedical Science Journal.

Easter Godwin Nwokah, Samuel Douglas Abbey and Confidence Kinikanwo Wachukwu.
Department of Medical Laboratory Science, Rivers State University, Port Harcourt, Nigeria
All Correspondences to: easternwokah@yahoo.com

ABSTRACT

The problem of Antimicrobial resistance has since become a global health challenge with renewed calls for global action, including surveillance, to contain the menace. Staphylococcus aureus strains are implicated in a wide range of diseases in humans and other animals. The aim of this study was to investigate the antimicrobial susceptibility pattern of Staphylococcus aureus isolates in Port Harcourt, Nigeria. Two hundred and five (205) isolates of Staphylococcus aureus from human sources were randomly collected from three health facilities- University of Port Harcourt Teaching Hospital, Braithwaite Memorial Specialist Hospital and De-Integrated Laboratories- all located in Port Harcourt. Isolates were grouped as Hospital-acquired (n=76) and Community-acquired (n=129) Staphylococcus aureus based on established criteria. The isolates collected were reconfirmed using standard laboratory protocols and thereafter, stored at +4°C. Using the disk diffusion method, the following antimicrobial agents (OXOID, UK) were tested- Cefoxitin(30µg) Vancomycin (30µg), Erythromycin (15µg), Fusidic acid (10µg), Penicillin G (10 Units), Tetracycline, Mupirocin (5 µg), Levofloxacin (30µg), Gentamicin (10 µg), Ceftazidime (30 µg), Cefuroxime (30µg), Clindamycin (2 µg), Amoxicillin/clavulanic acid (30µg), Tigecycline (15µg) and Linezolid (10µg), Quinupristine/dalfopristine (30µg), Ticarcillin/ clavulanic acid (85µg), Sulphamethoxazole/Trimethoprim (25µg), Piperacillin/Toxobactam (110µg). Organisms showed high levels of resistance to Cotrimoxazole (65.4%) and amoxicillin-clavulanic acid (44.9%) while only one isolate was resistant to Tegecycline. All isolates were susceptible to Vancomycin, Linezolid and Quinupristine/dalfopristine. MRSA detection was 12.2% and this, including the PCR methods for mecA status has been previously reported. Study further established the presence of multi-drug resistant (MDR) strains of Staphylococccus aureus (65.4%). Beta-lactamase production was detected in 94.1% of the isolates. There is need for sustained surveillance of antimicrobial resistance of S. aureus in this region to enhance guidance for treatment and also for infection control policies.

KEYWORDS: Antimicrobial Resistance, Staphylococcus aureus, MRSA,

INTRODUCTION

The problem of Antimicrobial resistance has since become a global health challenge with renewed calls for global action, including surveillance, to contain the menace. Staphylococcus aureus strains are implicated in a wide range of diseases in humans and other animals such as boils, deep tissue abscesses, enterocolitis, bacteriuria, osteomyelitis, pneumonia, carditis, meningitis, septicemia and arthritis, with associated morbidities and mortalities and medical costs. Following the introduction of the ß-lactam antibiotic, penicillin in the early 1940s, which improved outcomes, penicillin-resistant strains of S. aureus was reported, and by 1946 it was estimated that 60% of hospital isolates in the UK were resistant to this antibiotic. [1] Since then, successive introduction of new antibiotics- streptomycin, tetracycline, chloramphenicol and erythromycin has, in each case, been rapidly accompanied by the emergence of resistant organisms. [2,3]
It is interesting that many strains acquiring resistance to the latest antimicrobials also usually expressed a ß-lactamase (penicillinase), providing resistance to penicillin while some are resistant to all of the other antibiotics. Introduction of the semisynthetic ß-lactamase-resistant penicillins, such as methicillin and oxacillin, during the early 1960s, led to a general decline in the prevalence of multiple-resistant S. aureus. [3]. However, by the late 1960s to early 1970s, strains resistant to the ß-lactamase-resistant penicillins were isolated with increasing frequency. [4] Till date, there has been an increasing incidence of hospital-associated (nosocomial) and also community-acquired infections caused by multi-drug resistant strains of S. aureus, especially the methicillin-resistant S. aureus (MRSA), which term includes not only resistance to methicillin, but also to many other different antimicrobial compounds, including various biocides. Several genes have been found in strains of MRSA which confer on them high virulence and resistance to several antibiotic classes; these include mecA (that codes for penicillin resistance), lukS-lukF (responsible for widespread skin and soft tissue infections) and tetM (that codes for tetracycline resistance), erm (for macrolide resistance) among others.[5] The acquisition of genes such as mecA that codes for penicillin binding protein (PBP2A) by the strains, confers almost complete resistance to all beta-lactam antibiotics, including the semi-synthetic penicillin. [6-8]
A number of studies have indicated that multi-drug resistant S. aureus is among the most frequently encountered microorganisms in microbiology laboratories in Nigeria.[9-15] It is common knowledge that there is indiscriminate use of antibiotics and poor hygienic practices in our locality, as well as poor infection prevention and control practices in our health care facilities. These are some of the reasons for the success of this pathogen, accounting for is its great variability, occurrence at different periods and places with diverse clonal types and antibiotic resistance patterns within regions and countries.
The ability to characterize S. aureus and monitor antimicrobial susceptibility patterns is important for clinicians selecting empirical antimicrobial therapy, rational formulation of public health care polices, and providing useful information on the global surveillance of this pathogen. However, data on the antimicrobial susceptibility patterns of this pathogen in Rivers State are inadequate, and in most cases, isolates are screened against commonly available first line antibiotics only, most of which have lately become unhelpful. This study was aimed to investigate the antimicrobial susceptibility pattern and Beta-lactamase production in Staphylococcus aureus isolates in Port Harcourt, Nigeria.

MATERIALS AND METHODS
Study Area
Port Harcourt is a cosmopolitan city, located in Rivers State, one of the 36 States of Nigeria. There are two major tertiary healthcare facilities as well as other public and private health facilities. Clinical isolates were collected from the two tertiary health facilities- University of Port Harcourt Teaching Hospital and Braithwaite Memorial Specialist Hospital and De-Integrated Laboratories. Isolates were also grouped as Hospital in-patient (Nosocomial) or Out-patient isolates (Community-acquired) according to the criteria as prescribed by the US Centers for Disease Control and Prevention. [16]

Microbiological/ Identification Tests
Two hundred and five (205) non-duplicate human isolates of Staphylococcus aureus, were used for this study. Reconfirmation of isolates was done following standard microbiological protocols. [17] All confirmed isolates were stored at +4°C and later sub-cultured to carry out phenotypic characterizations.

Antimicrobial Susceptibility Testing
Antimicrobial Sensitivity Discs
The antibiotic sensitivity discs (Oxoid Ltd., Basingstoke, England) and their disc strengths are as follows: Oxacillin(1µg) Cefoxitin(30µg) Vancomycin(30µg), Erythromycin (15µg), Fusidic acid (10µg), Penicillin G (10 Units), Tetracycline (30µg), Levofloxacin (5µg),
Ciprofloxacin (5µg), Gentamicin (10µg), Ceftazidime (30µg), Cefuroxime (30µg), Clindamycin (2µg), Amoxicillin/clavulanic acid (Augmentin) (30µg), Tigecycline (15µg), Linezolid (30µg), Quinupristine/dalfopristine(30µg), Ticarcillin/clavulanic acid(85µg), Sulphamethoxazole/Trimethoprim (Bactrim) (25µg), Piperacillin/Toxobactam (110µg),

Disk Diffusion method
All isolates of Staphylococcus aureus were subjected to in-vitro antimicrobial susceptibility testing on Muller-Hinton agar (MH) as per the method described by Kirby and Bauer (1966) [18]. Briefly, inocula of bacteria were prepared to 0.5 McFarland standards and tested against all the aforementioned antibiotics disks. Following incubation at 37°C for 24 hrs, the zones of inhibition around the discs were measured with ruler and interpreted using the interpretation chart as prescribed by CLSI (2009) [19]. Multi-resistance was defined as resistance to at least three classes of antibiotics.

Test for Decreased Vancomycin Susceptibility
Isolates were screened further for vancomycin resistance using the vancomycin agar screening test whereby isolates were spot-inoculated onto Mueller Hinton agar supplemented with 6 µg/ml of vancomycin from a 0.5 McFarland standard suspension. The plates were incubated at 35°C for 24 h as recommended; and growth of two or more colonies on this agar would be considered as positive. [20]
Detection of Beta-lactamases production:
ß –lactamase test was also carried out on the S. aureus isolates to detect whether the organism would be able to produce the enzyme ß-lactamase- an enzyme that inactivates ß- lactam antibiotics. ß -lactamase production was detected by two different methods: Test tube iodometric technique and filter paper technique using 24 hour old culture and 10,000 units/ml of crystalline penicillin as per the method described by Sykes and Mathew (1979). [21]
Test tube method:
A loopful of heavy inoculum of 24 hours old culture from MH agar was mixed well with 1.0 ml Penicillin solution containing 10000 U per ml. The tubes were left for 60 minutes at room temperature with mixing at 15 minutes interval. Then 2 drops of 1% soluble starch solution was added, followed by one drop of Iodine solution. The tubes were mixed well and the results were recorded as follows- Instant discoloration: ++++ (Strong positive); Discoloration in 1-5 min: +++ (Average positive); Discoloration in 6 to 10 min: ++ (Moderately positive); Discoloration in 10-15 min: + (Weak positive) and No discoloration – (Negative). All test tubes showing discoloration within 10 minutes after adding iodine solution were taken as positive for beta-lactamase production.
Agar Plate method:
Isolates were inoculated on MH agar containing 1% soluble starch and incubated at 37°C for 48 hours. Then the plate was flooded with Penicillin solution containing 10000 U per ml and left at room temperature for 30 minutes. Then the penicillin solution was decanted completely and flooded with 1:5 dilution of iodine follows- More than 10 mm diameter discoloration around the culture: ++++ (Strong positive); 5-10 mm discoloration around culture: +++ (Average positive); 2-4 mm discoloration around culture: ++ (Moderately positive); 1 mm or discoloration below culture: + (Weakly positive) and No discoloration around or below culture: – (Negative).

RESULTS
Two hundred and five (205) non-duplicate isolates of Staphylococcus aureus collected from different clinical specimens were used in this study. Antimicrobial susceptibility pattern revealed varying degree of resistance to various types of antimicrobial agents tested (Table 1). Highest degree of antimicrobial resistance was recorded in cotrimoxazole- 134 (65.4%) out of 205 isolates of S. aureus and followed by amoxicillin-clavulanic acid (44.9%) while only one isolate was resistant to Tegecycline. All isolates were susceptible to vancomycin, teicoplanin, quinupristin/dalfopristin and linezolid. The oxacillin disc susceptibility testing showed that 25 (12.2%) out of 205 isolates of S. aureus were resistant to oxacillin (Table 1).
Table2 shows comparison of antimicrobial
susceptibility between in-patient S.aureus isolates and out-patient S.aureus isolates. Resistance was significantly higher in in-patient S.aureus isolates (p<0.05).
Multi-drug Resistance (resistance to three or more classes of antimicrobial agents) was significantly high (p<0.05) as detected in 134 (65.4%) of the isolates

(Table 3).
MRSA detection was significantly higher in in-patient isolates ((23.7% of 76) than out-patient (5.4% of 129) S. aureus (p = 0.000318) (Table 4).

Table 5 shows distribution of ß-lactamase producing S. aureus isolates. One hundred and ninety-three (94.1%) of the 205 isolates were positive. There was no significant difference (p > 0.05) in ß-lactamase production between MRSA and MSSA. There was also no significant difference (p > 0.05) in ß-lactamase production between in-patient and out-patient isolates.

DISCUSSION
Multi-Drug Resistance among pathogens is a significant challenge in both hospital and community settings that adds to the cost of medical care and the morbidity and mortality of patients. This becomes worrisome in the light of our local economic realities. Continual surveillance is essential to guide therapy and for the establishment of adequate infection control programmes.
This study revealed a multi-drug resistance rate of 65.4% (inclusive of all MRSA isolates) (Tables 3). MRSA detection was 12.2% and this, including the PCR methods for mecA status has been previously reported.[8] The study further confirmed that MRSA are more resistant to various groups of antibiotics compared to MSSA (Table 3), which also agrees with other authors. [22-23] Furthermore, selective pressure on the organisms, occasioned by misuse of antibiotics, as well as the ease of transferability of genetic elements is instructive. Study confirmed that Hospital Acquired-MRSA (nosocomial) are more resistant to antimicrobial agents than the Community-Acquired-MRSA (out-patients) among S. aureus isolates from clinical sources in Port Harcourt.
None of the isolates in this study showed resistance to the glycoprotein- vancomycin (Table 1). This is in consonance with some other investigations in Nigeria. [24-26] However, certain reports have revealed the presence of VRSA in Nigeria. Akambi and Mbe, (2013),[27] reported vancomycin resistance in 4 isolates out of 213 isolates of S. aureus in the University of Abuja Teaching Hospital, although only the disk diffusion method was employed. A VRSA prevalence rate of 57.7% has also been reported in Zaria, Northern Nigeria,[28] 6.3% among MRSA.[29] In another study, in non-clinical isolates, a prevalence rate of 89% was reported.[30] The difference in rates of vancomycin resistance probably reflects differences in levels of over-prescription and abuse in different parts of the country. Vancomycin-resistant strains are a source of concern because until recently, vancomycin was the only uniformly effective treatment for staphylococcal infections, particularly MRSA. Resistance to vancomycin severely limits therapeutic options. It is therefore cheering that there was zero resistance for vancomycin in this study. Also, none of the isolates was resistant to teicoplanin, another glycoprotein.

Nasal mupirocin plays an important role in the eradication of MRSA carriage. [31] Overall, 13 isolates were resistant to mupirocin (Table 1). The 6.3% prevalence of mupirocin resistance in this study is comparable to the 7% reported by Shittu and Lin, (2006) [32] but higher than a study (2%) in South Africa[33] and 1.5% in India.[34] This trend suggests that mupirocin resistance in S. aureus is an emerging feature in this locality, and therefore, underscores the need for routine testing for early detection of resistant isolates and prompt institution of infection control measures.
Five of the isolates in this study showed resistance to fusidic acid (Table 1). Some other investigators have reported full susceptibility of S. aureus to fusidic acid,[32, 35] indicating that fusidic acid is a good and effective agent for the treatment of S. aureus infections. Monotherapy with fusidic acid has been associated with the emergence of resistance and therefore, in order to minimize the emergence of fusidic-acid resistant strains, Howden and Grayson, (2006) [36] advised that monotherapy with fusidic acid should be discouraged and a combination with another anti-staphylococcal agent (ß-lactams, rifampicin or glycopeptides) be recommended.
A number of reports have indicated an increase in the resistance of staphylococci to trimethoprim/ sulphametoxazole (cotrimoxazole) in Nigeria. In one study, resistance rate among MRSA was 92.1%. In the present study, 65.4% of S. aureus isolates was resistant to cotrimoxazole, accounting for the highest level of resistance observed. Twenty-one (84%) of MRSA were also resistance to cotrimoxazole. This antimicrobial has wide clinical application, inexpensive, orally administered and available over-the-counter in Nigeria, where they are sold with or without prescription. This could possibly explain the high level of staphylococcal resistance observed in this study.
Erythromycin is one of the commonest and affordable antimicrobial agents available in Nigeria. An erythromycin-resistance rate of 21.5% in this study is therefore a cause for concern. More worrisome is that 23 out of the 38 erythromycin-resistant S. aureus were inducibly resistant to clindamycin and this has been previously reported.
This study also detected resistance to the fluoroquinolones. 22.9% (30 MSSA and 17 MRSA) of the 205 isolates were resistant to levofloxacin while 21.0% (28 MSSA and 15 MRSA) were also resistant to ciprofloxacin. Fayomi et al., (2011), had reported 12.2% and 9.6% ciproxin resistance among MRSA and MSSA respectively in Ido-Ekiti, Nigeria. These rates, although lower than the findings from one study in Okigwe, Nigeria where resistance to various brands of quinolones ranged from 44% to 88%, constitute a growing concern for a therapeutic option in our setting, especially where the preferred vancomycin are largely unavailable. The reasons for the disparity in rates of quinolone resistance between MSSA and MRSA strains are uncertain but could include antibiotic selective pressure, especially in the hospital setting, resulting in the spread of the more antibiotic-resistant MRSA strains.
In this study, the enzyme, ß–lactamase was detected in 193 (94.1%) of the 205 isolates (Table 5), a rate higher than 64% earlier reported by Terry-Alli et al., (2011) but lower than the 100% previously reported by Olowe et al. (2007). Twenty-five (13.0%) of the 193 ß-lactamase-producing isolates in this study were MRSA. However, there was no significant difference (p>0.05) in ß-lactamase-production between MRSA and MSSA. A previous study reported majority of ß-lactamase producers as methicillin-resistant. The indiscriminate use of antibiotics and the over-the-counter availability of antibiotics without prescription have, to a large extent, contributed to the emergence of resistant strains. ß-lactamase production by S. aureus had been identified to be a risk factor for the prevalence of MRSA in South Western, Nigeria. The finding in this study is instructive for urgent surveillance, control and other intervention programmes.
In Nigeria, systematic reporting of infectious diseases is still rudimentary and most health institutions lack proper infection control programmes. Because there is no national policy or guidelines for screening, reporting and control of MRSA outbreaks, the tendency is either under-reporting or over-reporting of the true prevalence of MRSA and therefore infections/ outbreaks are largely undetected and their contributions to mortality, morbidity and cost of care, associated with hospital-acquired infections are unknown. This problem is expected to be overcome with the recent establishment of a reference laboratory in Nigeria.

CONCLUSION
The degree of Multidrug resistance among S. aureus and especially among MRSA as detected in this study in Port Harcourt, Nigeria is high enough to warrant the need for continuous antimicrobial resistance surveillance as well as molecular epidemiological typing. This will enhance guidance for treatment and also for infection control policies.

ACKNOWLEDGEMENTS
We are grateful for the technical support of all staff of the Medical Microbiology departments in UPTH, BMSH and De-Integrated Laboratories in Port Harcourt, Nigeria.

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8. Nwokah, E. G., Abbey, S.D and Wachukwu C. K. (2016). mecA gene profile of Methicillin- Resistant Staphylococcus aureus isolates from clinical sources in Port Harcourt, Nigeria. American Journal of Biomedical and Life Sciences 4 (3), 41-48.

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14. Orji, I., Nworie, A., Eze, U. A., Agberotimi, I. O., Okereke, E. C. and Azi, S. O. (2012). The prevalence and antimicrobial susceptibility profile of methicillin-resistant Staphylococcus aureus from clinical specimens in tertiary hospital, South-East, Nigeria. Continental Journal of Pharmaceutical Science, 6(1), 23-29.

15. Onelum, O., Odetoyin, B., Onipede, A. and Oyelese, A. (2015). The Role of Methicillin-Resistant Staphylococcus aureus in Clinical infections in Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, South Western Nigeria. Journal of Microbiological Experimentation, 2(2): 00041

16. Garner, J.S., Jarvis, W.R., Emori, T.G., Horan T.C. nosocomial infections. American Journal of Infection Control, 16,128-140.

17. Cheesbrough, M. (2000). District laboratory practice in tropical countries (2), Cambridge University press, UK.

18. Bauer, A. W., Kriby, W. M., Sherris, W. M. and Turk, J. C. (1966). Bauer-Kirby standardized, single disc susceptibility, test for rapid growing pathogens. American Journal of Clinical Pathology, 45, 493-498.

19. Clinical Laboratory Standards Institutes (2009). Performance Standards for Antimicrobials Susceptibility testing; Nineteenth informational supplement. m100-S19, 29(3).

20. National Committee for Clinical Laboratory Standards (2004.) Performance standards for antimicrobial disk susceptibility tests. 12th informational document NCCLS document M100-S14 2004, PA-NCCLS.

21. Skyes, R. B. and Mathews, M. (1979). Detection assay and immunology of beta-lactamases In: Beta Lactamases: Ed: JMT. Hamilton-Miller & JT Smith: Publ: Acad, Press, 1st Ed.UK.

22. Mohanty, S., Kapil, A., Dhawan, B. and Das, B. (2004). Bacteriological and antimicrobial susceptibility profile of soft tissue infections from Northern India. Indian Journal of Medical Science, 58,10-15.

23. Fayomi, O. D., Oyediran, E. I. O., Adeyemo. A. T. and Oyekale, O. T. (2011). Prevalence and Antibiotic Resistance Pattern Of Methicillin-Resistance Staphylococcus Aureus Among In-Patients At A Tertiary Health Facility In Ido-Ekiti, Nigeria. The Internet Journal of Laboratory Medicine, 4 (2), p10

24. Adebayo, S., Johnson, L. and Deboye, K. (2006). Staphylococcus aureus characterization of MRSA in Southwestern Nigeria. Sidebars, 18, 45-51.

25. Terry-Alli, O. A., Ogbolu, D. O., Akorede, E., Onemu, O. M. and Okanlawon, B. M. (2011). Distribution of mec A gene amongst Staphylococcus aureus isolates from Southwestern Nigeria. African Journal of Biomedical Research, 14, 9 -16.

26. Efuntoye, M. O., Omotosho, O.O. and Ashidi, J. S. (2012). Prevalence MRSA and CONS among male students in a private tertiary institution and their enterotoxin -producing potentials. Asian Journal of Pharmaceutical and Health Sciences, 2 (1), 231-234.

27. Akanbi, B. O. and Mbe, J. U. (2013). Occurrence of methicillin and vancomycin resistant staphylococcus aureus in University of Abuja Teaching Hospital, Abuja, Nigeria. African Journal of Clinical and Experimental Microbiology,
14(1), 10-13.

28. Olayinka, B. O., Olayinka, A. T., Onaolapo, J. A. and Olurinola, P. F. (2005). Pattern of resistance to vancomycin and other antimicrobial agents in staphylococcal isolates in a University Teaching Hospital. African Journal of Clinical and Experimental Microbiology, 6, 21?27.

29. Olowe, O. A., Eniola, K. I. T., Olowe, R. A., and Olayemi, A. B. (2007). Antimicrobial Susceptibility and Beta-lactamase detection of MRSA in Osogbo. Southwest Nigeria. Nature and Science, 5(3), 44-48.

30. Onanuga, A., Oyi, A. R., and Onaolapo, J. A. (2005). Prevalence and susceptibility pattern of methicillin resistant Staphylococcus aureus isolates among healthy women in Zaria, Nigeria. African Journal of Biotechnology, 4, 1321-1324.

31. Duckworth, G. J. (1993). Diagnosis and management of methicillin resistant Staphylococcus aureus infection. British Medical Journal, 307, 1049?1052.

32. Shittu, A. O. and Lin, J. (2006). Antimicrobial susceptibility patterns and characterization of clinical isolates of Staphylococcus aureus in KwaZulu-Natal province, South Africa. BioMed Central Journal of Infectious Disease, 6(1), 125- 137.

33. Zinn, C. S., Westh, H. and Rosdahl, V. T. (2004). An international multicenter study of antimicrobial resistance and typing of hospital Staphylococcus aureus isolates from 21 laboratories in 19 countries or states. The SARISA Study Group. Microbial Drug Resistance, 10:160?168.

34. Krishnan, P U, Miles, K and Shetty, N. (2002). Detection of methicillin and mupirocin resistance in Staphylococcus aureus isolates using conventional and molecular methods: a descriptive study from a burns unit with high prevalence of MRSA, Journal of Clinical Pathology, 55, 745?748.

35. Okon, K. O., Basset, P., Uba, A., Lin, J., Shittu, A. O. and Blanc, D. S. (2009). Co-occurrence of predominant Panton-Valentine leukocidin-positive sequence type (ST) 152 and multi-drug resistant ST241 Staphylococcus aureus clones in Nigerian hospitals. Journal of Clinical Microbiology, 47(9), 3000?3003.

36. Howden, B. P. and Grayson, M. L. (2006). Dumb and Dumber ? The potential waste of a useful antistaphylococcal agent: emerging fusidic acid resistance in Staphylococcus aureus. Clinical Infectious Disease, 42, 394-400.

37. Ghebremedhin, B., Olugbosi, M. O., Raji, A. M., Layer, F., Bakare, R. A., König, B. and König, W. (2009). Emergence of a community-associated methicillin-resistant Staphylococcus aureus strain with a unique resistance profile in Southwest Nigeria. Journal of Clinical Microbiology, 47(9), 2975?2980.

38. Shittu, A. O., Okon, K., Adesida, S., Oyedara, O., Witte, W., Strommenger, B., Layer, F. and Nübel, U. (2011). Antibiotic resistance and molecular epidemiology of Staphylococcus aureus in Nigeria. BioMed Central Microbiology, 11, 92-99

39. Shittu, A., Oyedara, O., Abegunrin, F. Okon, K. Raji, A. Taiwo, S. Ogunsola, F., Onyedibe, K. and Elisha, G. (2012). Characterization of methicillin-susceptible and -resistant staphylococci in the clinical setting: a multicentre study in Nigeria. Biomed Central Infectious Diseases, 12, 286- 295

40. Nwokah, E. G. and Abbey, S.D. (2016). Inducible-Clindamycin Resistance in Staphylococcus aureus
isolates in Rivers State, Nigeria. American Journal of Clinical and Experimental Medicine, 4 (3), 50-55.

41. Ugbogu, O. C., Nwaugo, V. O., Orji, A. and Ihuoma, N. (2007). Quinolone- Resistant Staphylococcus aureus in Okigwe, Imo State, Nigeria. Journal of Biological Sciences, 7 (4), 697-700.

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The post Antimicrobial Resistance and Beta-lactamase Detection in Staphylococcus aureus isolates from Human Sources in Port Harcourt, Nigeria. appeared first on Nigerian Biomedical Science Journal.

Okorie Nnaemeka
N.K.S.T. Len Gebrielse’ School of Medical Laboratory Science Mkar, Benue State, Nigeria.
Fasogbon Samuel Ayobami
Public Health In-vitro Diagnostic Control Laboratory, Medical Laboratory Science Council of Nigeria, Lagos State, Nigeria
Adeluwoye Adekunle Oluwatosin
Department of Medical Laboratory Science, Lead City University, Ibadan, Oyo State.
Ajileye Ayodeji Blessing
Department of Medical Laboratory Science, College of Medicine and Health Sciences, AfeBabalola University, Ado Ekiti, Ekiti State.

ABSTRACT
The aim of this study is to compare thehistochemicalreaction of unripe orange extract juice-Schiff with that of Periodic acid Schiff reagent on skin and intestinal tissues.Four (4) tissue sections each obtained from a processed skin and intestine of a wistar rat were prepared and used for this study. The unripe orange juice was extracted and the various pH was measured with different timing. The control tissue sections were stained with periodic acid Schiff technique while the test tissue sections were stained with the unripe orange juice extraction obtained replacing the periodic acid solution in the PAS technique.Unripe orange juice (UOJ) which has similar pH with periodic acid Schiff react with the glycogen and mucosubstances on skin and intestinal tissue of the wistar rat used in this study, by staining them purple magenta similar to what is obtainable with periodic acid Schiff (PAS) staining protocol. In conclusion, unripe Orange JuiceSchiff technique has stained the skin and intestine of the Wister rat in similar manner to periodic acid Schiff technique.

Keywords: Histochemical, Periodic acid Schiff, Intestinal tissue, Skin tissue, Mucosubstances, Glycogen.

INTRODUCTION

Periodic Acid Schiff (PAS) is a staining method used to detect polysaccharide such as glycogen, and mucosubstances such as glycoproteins, glycolipid and mucins in tissues. The reaction of periodic acid oxidizes the vicinal diols in these sugars. Usually breaking up the bond between two adjacent carbons not involved in the glycosidic linkage or ring closure in the ring of the monosaccharide unit that are parts of long polysaccharides, and creating a pair of aldehydes at the two free tips of each broken monosaccrharide ring1. The oxidation condition has to be sufficiently regulated so as not tooxidize the aldehydes further. These aldehydes then react with the Schiff reagent to give a purple magenta color. A suitable basic stain is often used as a counter stain2.
The PAS stain is a histochemical reaction in that the periodic acid oxidizes the carbon to carbon bond forming aldehydes which react to the fuchsin-sulfurous acid of the Schiff solution forming the magenta color3.PAS staining is mainly used for staining structures containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans), typically found in e.g. connective tissues, mucus, the glycocalyx, and basal laminae.PAS diastase stain (PAS-D) is PAS stain used in
combination with diastase, an enzyme that breaks down glycogen. Alcian blue/periodic acid–Schiff (AB/PAS or AB-PAS) uses alcian blue before the PAS step4. Presence of glycogen can be confirmed on a section of tissue by using diastase to digest the glycogen from a section, then comparing a diastase digested PAS section with a normal PAS section. The diastase negative slide will show a magenta staining where glycogen is present within a section of tissue. The slide that has been treated with diastase will lack any positive PAS staining in those locations on the slide.
Among several immense use that has been found for Periodic acid Schiff staining in the diagnosis of medical conditions are glycogen storage disease, adenocarcinomas (which often secrete neutral mucins), paget disease of the breast, Alveolar soft part sarcoma, staining macrophages in Whipple’s disease, Pulmonary alveolar proteinosis, and erythroleukemia (leukemia of immature red blood cells). It can be used to diagnose a1-antitrypsin deficiency if periportal liver hepatocytes stain positive, as well as pulmonary interstitial glycogenosis (PIG), a condition where glycogen is identified in lung biopsy specimens of infants.
Orange is one of the largest citrus grown fruit in Nigeria5 and is one of the main sources of vitamin C (ascorbic acid) in Nigerian diets. Other vitamins that are of dietary significance are found in orange and include folic acid and thiamine.Orange juice also contains niacin, riboflavin and pantothenic acid vitamins although in minute quantities of about 2 to 4%6. Orange as one of the citrus fruits is non-climacteric, hence, they do not ripen or improve in quality after harvest. Various methods which can be used for processing of orange juice include the traditional thermal processing method (pasteurization) which kills bacterial and extend the shelf-life of juices, but affects the taste of the product. The pressurized carbon dioxide process, according to7 showed that this process is as effective as heat pasteurization, but does not change the taste and preserves more of the vitamins found in fresh squeezed juice. The periodic acid Schiff reaction is used to basement membrane, glycogen and neutral acidic mucosubstances will appear purple due to positive reaction with both alcianlue and periodic acid Schiff. According to8 periodic acid stain is useful for many things. It stain glycogen, mucin, mucoprotein as well as glycoprotein and fungi. A pre-digestion step with amylase will remove staining for glycogen periodic acid Schiff is useful for outlining tissue structure basement membranes capsules, blood vessels, etc8.

Orange juice have been found to be of medicinal importance in folkloric medicine. Among uses found for orange juice includes the reduction of the presence and effects of “bad” cholesterol, why increasing the amount of “good” cholesterol in the body, boosting of immune system, as well as dissolution of kidney stone.Orange juice neutralizes the pro inflammatory effect of a high-fat, high-carbohydrate meal and prevents endotoxin induced toxicity. Similarly, in cancer prevention, since one of the most important functions of antioxidants is to prevent cancer, orange juice is very rich in vitamin C which works as an antioxidant. Antioxidants keep the DNA of healthy cells from mutating into cancerous cells,hence antioxidants like vitamin C are the first line of defense for cancer and other serious diseases9. Having been observed for it acidity level, this study therefore compare the unripe orange juice with periodic acid as a component solution in the Periodic acid Schiff (PAS) staining technique, which is a staining protocol carried out at an acidic pH of 3.9.This is particularly important as naturally occurring dyes from plants are being viewed as an alternative to synthetic dyes for many developing countries that can no longer afford the ever increasing cost of synthetic dyes.

MATERIALS AND METHOD
Ethical Clearance
Ethical approval for this research study was obtained from the research ethics committee of the N.K.S.T. Len Gabrielse School of Medical Laboratory Science, the experiment was carried out in strict accordance with the guidelines for the care and use of animals for research.

Experimental Animal
Wister rat was used for this scientific research. The animal was handled humanely in conformity with the established ethical guideline for the care and use of laboratory animals.

Extraction of orange juice
For thisstudy, unripe orange fruit where obtained freshly from the orange tree stem at Mkar, Gboko Local Government area of Benue State, Nigeria.The unripe orange fruit were peel using knife and were then sliced into smaller pieces. The sliced unripe orange were parked inside the juice extractor machine, in order to extract the juice separating it from the chaff.The juice was filtered using four layers of gauze, and labeled with date.

Procedure for testing pH of unripe orange juice (UOJ)
The pH meter was inserted in the extracted unripe orange juice (UOJ) to measure the pH of the orange juice solution. The result of freshly prepared orange juice was 3.6. After 24 hours, the pH remains 3.6, only measuring 3.4 after 48hours, an indication of reduction due to fermentation.

Study Design
A total number of eight (8) tissue sections, four (4) each from skin and intestine of a wistar rat were prepared and used for this study. Skin and Intestine tissues were selected for this study as they have been histologically demonstrated to be rich tissue source of localized mucosubstances and glycogen.

Preparation of Slide
Four sections were obtained from a processed intestine tissue of the wistar rat and labeled as follow:
IC – Intestine control at pH 3.9
IT1 – Intestine test 1 at pH 3.6(pH of freshly prepared UOJ)
IT2 – Intestine test 2 at pH 3.6(pH after 24 hours of UOJ)
IT3 – Intestine test 3 at pH 3.4(pH after 48 hours of UOJ)
Also, another four tissue sections were obtained from a processed skin tissue of the wistar rat and labeled as follow:
SC – Skin control at pH 3.9
ST1 – Skin test 1 at pH 3.6(pH of freshly prepared UOJ)
ST2 – Skin test 2 at pH 3.6(pH after 24 hours of UOJ)
ST3 – Skin test 3 at pH 3.4(pH after 48 hours of UOJ)

Staining Procedure Using PAS (Control Slides)
i. Control slide IC and SC were dewaxed and hydrated using descending grades of alcohol (100%, 90%, 70%)
ii. They were taken to water
iii. They were oxidized using 1% Periodic acid at pH 3.9 for 10 minutes
iv. Both slides were rinsed with tapwater
v. They were then stained with Schiff reagent for 20 minutes
vi. Slides IC and SC were again rinsed thoroughly in tapwater
vii. They were counterstained with Cole’s haematoxylin for 10 minutes
viii. They were rinsed with tapwater
ix. Differentiated with 1% acid alcohol
x. Rinsed with water
xi. They were blued in tap water for 10 minutes grades of alcohol (70%, 90%, and 100%).
xiii. Cleared using xylene and mounted using Diphthalenexylene (DPX)

Staining Procedure Using Orange Juice Extract (Test Slides)
i. Sections marked IT1, IT2, IT3 and ST1, ST2, ST3 were dewaxed and hydrated in descending grades of alcohol (100%, 90%, 70%)
ii. Sections were taken to water
iii. Slide IT1, IT2, and IT3 were oxidized with unripe orange juice at pH 3.6 (fresh), 3.6(24hrs), and 3.4 respectively for 10 minutes
iv. Slide ST1, ST2, ST3 were oxidized with unripe orange juice at pH 3.6 (fresh), 3.6 (24hrs), and 3.4 respectively for 10 minutes
v. All the sections were rinsed thoroughly in tap water
vi. They were stained with Schiff solution for 20 minutes
vii. Rinsed thoroughly in tap water
viii. They were counterstained with Cole’s haematoxylin for 10 minutes
ix. They were again rinsed in tap water
x. Differentiated briefly with 1% acid alcohol
xi. They were blued in tap water for 10 minutes
xii. Dehydrated using ascending grades of alcohol (70%, 90%, 100%)
xiii. Cleared using xylene and mounted using Diphthalenexylene (DPX)
All slides were examined under the binocular microscope using 10x, 40x and 100x objectives respectively.

RESULTS
Histology of Intestinal Tissue Sections
Histology of Intestinal Control (IC) pH 3.9 micrograph

Discussion
Cell structures take up specific stains to varying degrees based on their biochemical characteristics. Many stains which are suited for particular purposes and allowing cell structures to be differentiated have been developed by histologists. The unripe orange juice which have the similar pH with that of periodic acid solution in the Periodic Acid Schiff (PAS) stain, stained clearly, the glycogen components of the intestinal tissue at pH3.6 (both fresh and 24hrs), as well as at pH 3.4. However for the intestinal tissue, mucosubstances (glycogen) were clearlydemonstrated at pH 3.4. Goblet cells of the intestine were also stained by the unripe orange juice due to its positive reaction with Schiff solution. For the skin tissue sections, similar to the PAS technique which serves as control,the unripe orange juice stained the glycogen, stratum adiposum, and hair follicles on skin tissues of wistar rat as indicated in figures 6-8 above. It is of note too that neoplastic cells on the skin test sections, (ST1, ST2, and ST3) were also demonstrated (indicated by yellow arrow).
Conclusion
Based on the findings of this study, it is observed that, unripe orange juice which has similar pH with periodic acid Schiff react with the glycogen and mucosubstances on skin and intestinal tissue of the wistar rat, by staining them purple magenta similar to what is obtainable with periodic acid Schiff (PAS) staining protocol.

REFERENCES

1. Thomas J, Lawton G. Breast. Cambridge University Press. 2009 16 November Pp. 55. ISBN 978-0-521-88159-3.

2 Carson, FL, Hladik C, Histotechnology: A Self-Instructional Text (3rdedition.). Hong Kong: American Society for Clinical Pathology Press.2009 Pp. 137–139. ISBN 978-0-89189-581-7.

3. Crookham J, Dapson R. Hazardous Chemicals in the Histopathology Laboratory, 2nd Edition, Anatech, 1991Pp. 28 – 37.

4. Hauser (2005): https://www.google.com.ng /search?q=Hausar+2005,+glycogen+PAS&dcr

5. Osbeck L. Citrus X sinensis (L) Osbeck (Pro sp) (Maxima x reticulate) sweet orange” plants. USDA. Gov Archived from the original on may 12, 2011. https://www.google.com.ng/search?q=Osbeck+2011,+orange&dcr=0&tbm=isch&t

6. Smoot JM, Nagy S.Effects of storage temperature and duration on total vitamin C content of canned single-strength grapefruit juice. J. Agric. Food Chem., 1980, 28 (2), Pp 417–421.

7. Nicolosi E, Deng ZN, Gentile A, La-Malfa S, Continella G, Tributlato E. citrus phylogeny and genetic origin of important species as investigated by molecular markers” TAG theoretical and Aplied Genetics. Theor Appl Genet 2000 100:1155-1166.

8. Manjunath M. Shenoy S, Teerthanath V K, Karnaker B S, Girisha, Krishna P, Jerome P.Periodic Acid Schiff’s staining. Indian J Dermatol Venereol Leprol 2009 75 (1): 73-74.

9. Lutsenko EA, Cárcamo JM,Golde DW. Vitamin C prevents DNA mutation induced by oxidative stress. J Biol Chem.2002: 277(30):27576-27578.

The post Comparison of Staining Reaction of Periodic Acid Schiff with Unripe Orange Extract-Schiff on Skin and Intestinal Tissues appeared first on Nigerian Biomedical Science Journal.

Bukar Alhaji, Mary Ann Amarachi Umeh, Obi Simon Osita, Waziri Gimba, Medugu Jessy Thomus
Department of Haematology, University of Maiduguri Teaching Hospital, Maiduguri,
Anthony Nwobi, Osakue Eguagie Osareniro
Department of Chemical Pathology, Igbinedion University Teaching Hospital, Okada
Olaniyan Matthew Folaranmi
Department of Medical Laboratory Science, Achievers University, Owo
Jeremiah Zaccheaus Awortu.
Department of Medical Laboratory Science, Niger Delta University
All correspondence to: alhajibukar@gmail.com.

ABSTRACT
Haemoparasite,such as Plasmodium and Microfilaria are animal parasite living in the blood of a vertebrate host. The study was aimed to determine the prevalence of Haemoparasite (Plasmodium and Microfilaria) in blood donors attending University of Maiduguri Teaching Hospital. A total of 230 blood donors were recruited for this study using simple random sampling. A semi-structured questionnaire was used to collect data regarding demographic and social profile of the subjects. Giemsa stained thick blood film was used for the detection of malaria parasite while wet preparation was used for the detection of microfilaria. A total of 78 blood donors had malaria parasites while no filarial parasite was recorded showing a prevalence of 33.9% and 0% respectively. The prevalence of malaria parasites in the blood donors was not significantly associated with usage of insecticide and/or insecticide treated net. The prevalence of malaria parasite was however significantly associated with treatment with antimalarial drugs. It is therefore necessary for the government to improve the sanitary condition of Maiduguri which will in turn reduce the availability of breeding sites for mosquitos.

Keywords: Malaria, microfilaria, blood donors.

INTRODUCTION
Blood transfusion is potentially a lifesaving therapeutic procedure and a common form of tissue transplantation which is aimed to provide patients with blood components which they are deficient.1. Although,blood transfusion is generally believed to save human lives, blood can nonetheless be a route for the transmission of infections generally referred to as transfusion transmissible infections (TTIs). TTI occurs when a patient is infected by the same parasite that was present in the donor’s blood. TTIs are broadly classified into viral, bacterial, amoebal or parasitic.Haemoparasite is an animal parasite such as a haemoflagellate or filarial worm living in the blood of a vertebrate host2. These parasites reside eitherin the blood cells or in the plasma. Malaria parasite and Babesiaare haemoparasites that resides in the red blood cells, while leishmania and filarial wormsresides in the white blood cells and the plasma respectively3.
In Nigeria, malaria and filariasis are more prevalent and over the years varying prevalence has been recorded among Nigerian blood donors4. Haemoparasites constitute a serious threat to human race as they can result in increased morbidity and mortality5. Malaria is sporozoan parasite of the genus Plasmodium, its infection is transmitted naturally through the bite of infected female Anopheles mosquitoes6. In endemic areas, malaria transmission is so intense that a large proportion of the population is infected but not made ill by these parasites7. These carriers harbour low levels of the parasitesand shows no clinical signs of infection as they are immune to parasitic illness but not to the infection and for this reason, blood from such donors contains malaria parasite which can easily be transmitted to recipients by blood transfusion.
A bite from an infected mosquito may cause malaria by introducing as few as 15 parasites while a single parasite identified on a thick film (4ul) is equivalent to approximately 10,000 parasites in 450ml unit thereby causing malaria in transfused patients8. Transfusion-transmitted malaria can however have serious consequences, as infection with P. falciparum may prove rapidly fatal when such blood is transfused especially into children under 5 years, pregnant women, trauma victims with acute blood loss and immuno-suppressed patients9. Malaria destroys red blood cells and converts it to methaemoglobin leading to methemoglobinemia causing illness especially in immune compromised individuals 7, 10.

Filariasis on the other hand is a parasitic disease that is caused by thread-like nematodes (roundworms) belonging to the superfamily Filarioidea. These parasites are transmitted from host to host by blood feeding arthropods, mainly black flies and mosquitoes.
As adults, the worms can survive and reproduce for up to 7 years within which the worms gradually build-up in the vessels of their host. This interfereswith the lymphatic system’s ability to fight infection and causes lymph fluid to accumulate in the arms, legs, breasts and male genitals leading to welling and disfigurement3, 11.
In all species, sexually mature female worms release microfilariae, which are their pre-larval stages into the bloodstream of their infected host.If, the blood from microfilaraemic individuals is transfused into a patient, the transfused microfilariae may persist in the recipient’s circulation for up to 3 years12. Recipient of these blood component usually develop post transfusion allergic reactions due to dying microfilariae13.
In Nigeria, screening forparasitic infections is not routinely done in blood banks, nor stipulated in the current National Blood Guidelines. This is because transmission of parasitic infections such as malaria through blood transfusion is generally not regarded as a serious problem in adult and adolescent whose level of immunity is thought to be sufficiently effective in combating post transfusion malaria in an endemic area like Nigeria6. These parasites are prevalent in Nigeria but the extent to which it currently affects blood donors attending UMTH is unknown, we therefore, considered it necessary to contribute some information on this subject.

MATERIALS AND METHOD
This study was conducted at the University of Maiduguri Teaching hospital (UMTH) from February 2017 to May 2017. A total of 230blood donors which are negative to HIV 1/2, HBsAg, Syphilis and HCVwere recruited for the study. These donors were recruited using simple random sampling. Two millitres of the donor’s venous blood was collected into an EDTA container.ABO and RhD blood groups of the donors were determined using tile method. Malaria parasite was qualitatively determined by making thick blood films in duplicates for each blood samples on a clean grease free glass slide. these was allowed to air-dry after which it was stained with Giemsa stain. Stained films were examined under x100 objective lens of microscope with Immersion oil for any stage of malaria parasite. A slide is defined as negative if no asexual stage of the parasite is found after counting 100 microscopic fields.For, microfilaria parasite, a drop of anticoagulated blood was dispensed on a cleaned grease-free slide and covered with cover slip. It was examined microscopically using x10 and x 40 magnification for motile microfilaria. Result were analysed using, percentage and SPSS 20.0 statistical package. Chi-square was used to determine if prevalence was dependent on certain factors. A P-value of less than or equal to 0.05 (p=0.05) was considered as statistically significant.

RESULTS
A total of two hundred and thirty subjects (230) were recruited for the study. The subjects were within the age group of 18-55 years with the age group 20-29 having the
highest mode (47.6%, 110/230) and age group 50-59 years with the least (3%, 7/230).
Table I shows the prevalence of malaria parasite and filarial worms in the blood donors attending UMTH.Out of the 230 blood donors studied, 78 donors had malaria parasite giving a prevalence rate of 33.9% while none had filarial worm giving a prevalence rate of 0%.Table II shows the prevalence of malaria parasite in blood donors attending UMTH in relation to blood group and donation history. Malaria parasite in respect to ABO blood group, group B donors had the highest prevalence rate of 38.1% (16/42) while blood group AB donors had no malaria parasite in their blood. This difference was not statistical significant (x2 = 1.513, df= 3, p-value = 0.679). Malaria parasite with relation to Rh D blood group, Rh D- donors had a higher prevalence rate of 37.5% (6/16) while Rh D+ donors had a lower prevalence of 33.6% (72/214). This difference was also not statistically significant (x2 =0.99, df =1, p-value = 0.753). Family replacement donors had a higher prevalence of malaria infection 34.2% (77/225) when compared to voluntary donors who had a prevalence rate of 20% (1/4), this difference was also not statistically significant (x2 =0.441, df= 1, p=0.506). There was no commercial blood donor in this study. Repeat donors had a higher prevalence rate of malarial infection 36.1% (49/119) while first-time donors had a lower prevalence rate 35.1% (35/111). This prevalence is not statistically significant (x2 =0.543, df= 1, p=0.461).
Table III shows the prevalence of malaria parasite in relation to some social factors.Female donors had higher prevalence rate of malaria parasite (36.4%, 4/11) compared to male donors who had a prevalence rate of 33.8% (74/219). This difference is not statistically significant (x2=0.31, df= 1, p=0.860). Donors below 20 years had the highest prevalence of malarial infection 40% (4/10) while those within the age range of 40-49 had the least prevalence.It is not statistically significant (x2=3.994, df= 4, p=0.479).The prevalence is higher among the singles, 40.7% (50/123) while no infection was detected among the divorced donor. It is not statistically significant (x2=5.66, df= 2, p=0.059).
Table IV shows the prevalence of malaria parasite in relation to usage of insecticides and/ or insecticide treated net. Blood donors who neither used insecticides nor insecticide treated net had the highest prevalence rate of 50% (18/18) while those who used both insecticides and insecticide treated net had the least prevalence rate of 23.5% (4/17). This difference is not statistically significant.
Table V shows the prevalence of malaria parasite in relation to treatment with antimalarial drugs. Donors who 

said to have never been treated with antimalarial drugs had the highest prevalence of 56.7% (17/30) while donors who self-administered antimalarial drug within the last six months had the least prevalence 14.5% (11/76). This difference is statistically significant. No Filarial parasite is found in any of the donors.
DISCUSSION
Results obtained from this study showed that 78 blood donors had malaria while none had filarial worm showing a prevalence of 33.9% and 0% respectively. The prevalence of malaria parasitaemia in this study was lower than that reported by Abioye et al.15 who recorded a prevalence rate of 56% (140/250) in Abuja and was higher than the report of Garba et al.16 who reported a prevalence of 7.5% (27/360) in Kaduna. These differences in regional prevalence could be attributed to variation in predisposing factors such as present of Anopheles species, environmental conditions, climatic conditions, period of study, the study population and diagnostic test method used. The high prevalence rate may be attributed to current security challenges in Borno which forced people from other villages within other towns of the state to relocate to Maiduguri which in turn increases the population and decreases sanitary condition of the city. The decreased sanitary condition has resulted in increased chocked drainage channels which provide a suitable breeding ground for Anopheles mosquito.However, in relation to filarial worms the result from this study was inconsistent with the report of Bolaji et al.3 who reported a prevalence of 2% with Loa loa, Brugria Malayi and Wuchereria Bancrofti in the following proportion; 4(1.33%), 1(0.33%) and 1(0.33%) respectively.This difference may be attributed to difference in the number of subjects and geographical locations. Although Ochocerca Volvulus is prevalent in some areas in Bornu such as Hawul18,it is not prevalent in Maiduguri possibly due to lacks fast flowing water which is a suitable breeding site for its biological vector (Backfly).

This study further revealed that malaria parasitsitaemia is higher among blood group B donors while no malaria parasite was recorded in AB blood donors. This result does not tally with the report of Agboola et al.5 who reported a higher prevalence among blood group O donors. This difference may be as a result of chance. The difference in malaria among ABO blood groups in this study was however not statistically significant, indicating that susceptibility to malaria parasite is independent of a person’s ABO blood group. Also, a higher prevalence of parasitaemia among Rh D negative blood donors was reported in this study compared to the Rh D+ blood donors. This finding was not similar to a report by Bankole et al.19 who reported a higher prevalence among Rh D+ blood donors. The difference between Rh D blood groups in this study was not statistically significant, indicating that susceptibility to malaria parasite is independent of a person’s Rh D blood group. The study clearly suggests that family replacement donors were the major source of blood for transfusion in UMTH. This is consistent with findings from other researchers19, 20, indicating that family replacement donors were the major source of blood for transfusion in most states in Nigeria.There is higher prevalence of malaria parasitaemia in family replacement donors when compared to voluntary blood donors, this result is in line with the report of Olawumi et al.20, however not statistically significant. The lower prevalence recorded in this study indicates that there is reduced risk of transmission ofmalaria when blood productsare derived from voluntary donors. Result from this study shows a higher prevalence of parasitaemia in repeat donors when compared to first-time donors. This result is consistent with the report of Garba et al.16. This could be as a result of the fact that first-time donors are apprehensive and those having mild symptoms of malaria such as headache are usually excluded to donate blood.

The prevalence of malaria parasite in this study shows high rate among female donors when compared to male donors. The difference in prevalence between the genders may not be conclusive owing to the relatively small number of female donors who participated in the study. Higher prevalence of parasitaemia was found in donors whose age where below twenty and the least prevalence was seen in donors within the age range of 40-49 years of age. This result does not tally with the report of Ekwunife et al.(2011)6 who reported the highest prevalence among donors within the range of 25-29 and the least prevalence among donors within the age range of 50-54 years of age. The difference may be due to chance.There is higher prevalence rate in single (unmarried) blood donors while malaria parasite was not detected in the blood of the divorced donor. The result does not tally with the report of Alli et al.,4 who reported a higher prevalence among married donors which might be probably by chance.
Overall, there is no significant relationship between the prevalence of malaria infection and the usage of personal protection against mosquitos. This indicatesthat the current prevalence of malaria parasite among the blood donors is not dependent on the use of insecticide and/or insecticide treated nets. The results also indicated that there is statistically significant relationship between the prevalence of malaria infection and treatment with anti-malaria drugs. This indicates that treatment with antimalarial drugs significantly reduces the prevalence of malaria among blood donors. This coincides with the report of UNICEF 21, which states that the two major ways to reduce the spread of malaria are the use of insecticide treated mosquito nets and early diagnosis and prompt treatment with antimalarial medications.

CONCLUSION
In conclusion the result from this study shows a progressive increase in the prevalence of malaria parasite among blood of donors attending UMTH when compared with previous results. This increase is alarming as these donors are apparently healthy subjects indicating an increased risk of transmission of malaria through transfusion in Maiduguri. No filarial worm was recorded in this study. No statistically significant relationship was established betweenmalaria infection and the usage of Insecticide and/ or insecticide treated net. However, statistically significant relationship between the prevalence of malaria parasite and treatment with antimalarial drugs is noted.

Recommendations Haemoparasites can be transmitted through transfusion of infected blood derived from asymptomatic donors. This may negatively affect patient’s health and increase the duration of their illness. State government should improve the sanitary condition of Maiduguri and environs which will in turn reduce mosquito breeding sites. Screening donors for parasitic infections should be included in the current nation’s transfusion guidelines. Enlighten donors on better ways of preventing infections with haemoparasites.Encourage prompt and effective treatment of infected prospective donors.In additions incentives such as insecticide treated mosquito net, insect repellent and refreshments should be given to donors as this may encourage voluntary donation and as well reduce the prevalence of haemoparasites in the blood of donors.

REFERENCES

1. Schmaier, A. H., & Petruzzelli, L.M. (2003). Haematology for the medical student. Lippincott Williams & Wikins.

2. Medical Dictionary for the Health Professions and Nursing (MDHPN). (2012). Retrieved May 23rd 2017 from http://medical-dictionary.thefreedic tionary.com/haemoparasite

3. Bolaji, O.S., Uthman-izobo, S.O., Ojurongbe, O., Opaleye, O.O & Adeyeba, O.A. (2014). Filariasis among asymptomatic blood donors in general hospital, Odan Marina-Lagos, Nigeria. International Journal of Research in Applied, Natural and Social Sciences, 2(6), 177-182.

4. Alli, J.A., Okonko, O.I., Abraham, O.A., Kolade,
A.F., Ogunjobi, P.N., Salak, A.O., Ojezele, M.O & Nwanze, J.C. (2010). A serosurvey of blood parasites (plasmodium, microfilaria, HIV, HBsAg, HCV antibodies) in prospective Nigerian blood donors. Research Journal of Medical Science, 4(4), 255-275.

5. Agboola, T.F., Ajayi, M.B., Adeleke, M.A. & Gyang, P.V. (2010). Prevalence of malaria parasite among blood donor in Lagos University teaching hospital, Lagos Nigeria. Scholars Research Library Annals of Biological Research, 1(3), 72-75.

6. Ekwunife, C.A., Ozumba, N.A., Eneanya, C. I. & Nwaorgu, O.C. (2011). Malaria infection among blood donors in Onitsha urban, Southeast Nigeria. Sierra Leone Journal of Biomedical Research 3(1), 21-26.

7. Anthony, C.N., Yee-Ling, L., Jia-Siang, S., Mun-Yik, F., Hany, A., Wai-Linn, Z., Indra, J. & Rohela, M. (2013). Malaysian child infected with plasmodium vivax via blood transfusion: a case report. Malaria Journal 12:308. Retrived on January 10th 2017 from http://www.malariajournal.com/content/12/1/308.

8. Alex. K.O., Christopher, P. & Imelda, B. (2010). Transfusion transmittable malaria in countries where malaria is endemic: A review of the literature from sub- Saharan African. Journal of Clinical Infectious Diseases, 1192-1193.

9. Kitchen, A.D., and Chiodini, P.L. (2006). Malaria and blood transfusion. Vox Sanguiis, 90, 77-84

10. Okeke, C.O., Agbasiere, F.N., Amilo, I.G. & Ifeanyichukwu, O.M. (2017). Methemoglobin levels among malaria parasite-infected blood donors in Nnewi, Southeastern, Nigeria. Tropical Journal of Medical Research, 20(1), 80-83.

11. Ojo-bola, T., Omisakin, C.T., Esan, A.J. & Owoseni, M.F. (2014). Filaria worm among prospective blood donors attending a tertiary health institution in south-west Nigeria. Journal of Dental and Medical Sciences, 13(1), 84-87.

12. Nagwa, M.E. (2015). Recent updates in transfusion transmitted parasitic diseases. Journal of Bacteriology, Virology and Parasitology, 2(1) 1-11

13. Bregant, E.T., Balzarinl, L., Ghiringhelli, C. & Tarsta, P. (2003). Transfusional Mansonella pertans microfiariasis. Parassitologia 45, 71-72

14. Chessbrough, M. (2000). District laboratory practice in tropical countries. Part 1, (2nd ed). Cambridge University Press:UK.

15. Abioye, J.O.K., Abdullahi, D.K., Alalade, O.M., & Olokun, A.L. (2015). Incidence of malaria parasite in blood donors at Kwali General Hospital, FCT Abuja. Journal of Emerging Trends in Engineering and Applied Sciences, 6(3), 212-216.

16. Garba, D.D., Ameh, B.J., Whong, C.M.Z. & Mukhtar, M.A. (2016). Prevalence of malaria parasites among blood donors in Kaduna, Nigeria. International Journal of Research in Medical Sciences, 4(6), 2112-2119.

17. Okoye, I.C., Dakul, D.A. & Wakawa, A.I. (2011). Perception of onchocerciasis by rural Hausa women in northeast Nigeria and the implications for onchocerciasis control. Animal Research International, 8(1), 1309 – 1314.

18. Bankole, H.O., Richard, O., Eguagie, O.O., & Tola,
O.O. (2014) Asymptomatic malaria among blood donors in Benin city Nigeria. Iranian Journal of Parasitology, 9(3), 415–422.

19. Olawumi, H.O., Fadeyi, A., Babatunde, S.K., Akanbi, A.A., Babatunde, A.S., Sani, M.A., & Aderibigbe, A.S. (2015). Malaria parasitaemia among blood donors in Ilorin, Nigeria. African Journal of Infectious Disease, 9(1), 10–13.

20. UNICEF. (2000). The Prescriber: Promoting rational use of drugs and correct case management in basic health services. Retrieved on 5th May 2017, from https://www.unicef.org/ prescriber/eng_p18.pdf

The post Prevalence of Haemoparasites (Plasmodium and Microfilaria) in Blood Donors Attending University Of Maiduguri Teaching Hospital (UMTH) appeared first on Nigerian Biomedical Science Journal.

John B. Kalule, Karen H. Keddy, Anthony Smith, Mark P. Nicol,1 and Lourens Robberts

ABSTRACT

Introduction: Shiga toxin-producing Escherichia coli (STEC) is an emerging infectious pathogen which could lead to haemolytic uremic syndrome. Even though previous studies have compared the performance of CHROMagarTMSTEC to real-time polymerase chain reaction (PCR) in Europe, no study has been done to assess its performance on African isolates. Objectives: This project aimed to validate and test an in-house-developed duplex real-time PCR and use it as a reference standard to determine the performance of CHROMagarTMSTEC on African isolates from diarrhoeic stool samples. Methods: This study evaluated STEC diagnostic technology on African isolates. An in-house-developed duplex real-time PCR assay for detection of stx1 and stx2 was validated and tested on diarrhoeic stool samples and then used as a reference standard to assess the performance of CHROMagarTMSTEC. Real-time PCR was used to screen for stx in tryptic soy broth and the suspected STEC isolates, while conventional PCR was used to detect the other virulence genes possessed by the isolates. Results: The real-time PCR limit of detection was 5.3 target copies/?L of broth. The mean melting temperature on melt-curve analysis for detection of stx1 was 58.2 °C and for stx2 was 65.3 °C. Of 226 specimens screened, real-time PCR detected stx in 14 specimens (6.2%, 95% confidence interval = 3.43% – 10.18%). The sensitivity, specificity, negative predictive value and positive predictive value of the CHROMagarTMSTEC were 33.3%, 77.4%, 95.3% and 11.3%.
Conclusions: The in-house developed real-time PCR assay is a sensitive and specific option for laboratory detection of STEC as compared to CHROMagarTMSTEC in this setting.

INTRODUCTION

Globally, food- and water-borne outbreaks of both O157 and non-O157 Shiga toxin-producing Escherichia coli (STEC) have been successfully detected due to the availability of good baseline data and effective active laboratory-based surveillance systems.1,2,3,4 Early detection of outbreaks is important to minimise morbidity, mortality and associated economic losses.5 There is a lack of good baseline data on STEC in Africa, which can be attributed to a lack of laboratory resources and the surveillance strategy employed. STEC has been implicated in outbreaks of bloody diarrhoea in sub-Saharan countries;6,7,8 however, these have been difficult to track and manage due to laboratory weakness.9,10 Furthermore, typical haemolytic uremic syndrome, which is overwhelmingly caused by STEC, was reported as the leading contributor to acute renal failure in paediatric patients at a South African academic hospital.11 Even though several studies have evaluated the performance of CHROMagarTMSTEC by comparison to molecular and antigen detection methods in developed countries,12,13 no study has so far evaluated its performance in Africa. This is necessary, especially given that there are geographical differences in characteristics of STEC that are dependent on the index of suspicion for the different STEC serotypes and on the availability of suitable laboratory methods to detect them.14
In many South African (and African) laboratories, stool specimens are not routinely tested for STEC, although physicians may request tests specific for E. coli serotype O157:H7, if it is clinically suspected. Testing is based on the non-sorbitol fermenting property using sorbitol MacConkey and only on request by a physician. This practice is of concern, is misleading and underestimates the real magnitude of STEC, since not all serotype O157 strains are non-sorbitol fermenting (O157: NM), and over 470 non-O157 serotypes have been attributed to clinical disease.15 Laboratory capacity for molecular detection is increasingly available in African countries and may, in some cases, be simpler than culture-based detection. This project, therefore, aimed to validate and test diarrhoeic stool samples by using an in-house developed duplex real-time polymerase chain reaction (PCR) and use it as a reference standard to determine the performance of CHROMagarTMSTEC on African isolates. The duplex assay was used to screen tryptic soy broth (TSB) for stx following overnight stool enrichment, and conventional PCR was used to screen for the other diarrhoeagenic E. coli virulence genes. Diarrhoeagenic E. coli were serotyped, and stx-positive isolates were tested for Shiga toxin production using immunochromatography.

Methods
Ethical approval
This study was approved by the ethics and research committee of the Faculty of Health Sciences at the University of Cape Town (HREC REF: 014/2015).

Study design
This study validated an in-house-developed duplex real-time PCR assay for detection of stx1 and stx2. The assay was then tested on diarrhoeic stool samples at a tertiary referral hospital and was used as a reference standard to assess the performance of a commercial chromogenic medium (CHROMagarTMSTEC) for STEC screening (Figure 1).

Summary of methods used in this study.
Target plasmid preparation
The real-time PCR previously described by Grys et al.16 was used to amplify stx1 and stx2 gene targets from a STEC O157:H7 NCTC control strain (C4193-1) with both stx1 (subtype 1a) and stx2 (subtype 2a). PCR amplicon size was confirmed visually by agarose gel detection (~208 bp for stx1 and ~204 bp for stx2) before confirmation by sequencing using the Big Dye® Terminator v3.1 Cycle Sequencing Kit (Life Technologies Corporation, Carlsbad, California, United States). We used primers 1a and 2a (Table 1) for unidirectional Sanger sequencing of the amplicons. Resultant sequences were then trimmed and submitted for BLAST analysis against the NCBI database and confirming stx1 or stx2 target sequences in comparison to O157:H7 EDL933 (NCBI Reference: NC_002655.2).17
Purified amplicons (Mini Elute Gel extraction kit, Qiagen, Madrid, Spain) were cloned using CloneJet PCR cloning kit (Thermofisher Scientific, Austin, Texas, United States) into a pJet 1.2/blunt vector using the sticky end cloning protocol and transfected into the JM109 competent cells by calcium chloride transformation. Plasmids containing stx1 and stx2 were separately extracted using a Genopure plasmid Maxi kit (Roche Life Sciences, Rotkreuz, Switzerland) and quantified by spectrophotometry. To verify successful preparation purified plasmids were subjected to PCR amplification using primers 1a and 1b for stx1 and 2a and 2b for stx2 with amplicon size visually confirmed by agarose gel detection and subsequent sequence analysis. Plasmid quantification was determined spectrophotometrically employing the BioDrop-µLite (Isogen Life Science, B.V, Veldzigt, Netherlands). The A260 was used to calculate the plasmid concentration expressed as the number of molecules/µL.

To assess the potential for PCR cross-reactivity and assess the analytical specificity of the hybridisation probe-based real-time PCR described by Grys et al.,16 the primer and probe sequences were subjected to BLAST analysis on the NCBI database. The PCR reaction was optimised for use on

the LightCycler®480 Instrument II (Roche Life Sciences, Rotkreuz, Switzerland) employing the LightCycler® 480 Probes Master mastermix (Roche Life Sciences, Rotkreuz, Switzerland) with modification to the thermal cycling conditions for amplification consisting of denaturation at 95 °C for 10 min followed by 45 cycles of 95 °C for 5 s, 56 °C for 5 s and 72 °C for 15 s. A positive amplification signal was defined as an increase in fluorescence signal that crossed the threshold before 30 cycles. Amplicon identity was determined using the melt-curve analysis program of 95 °C for 30 s, 40 °C for 60 s and 85 °C for 5 s with continuous fluorescence acquisition. The Multi-color HybProbe detection format was used for analysis, combining the Red 610, Red 640 and FAM filter pairs (LightCycler®480 Instrument II Manual, Roche Life Sciences, Rotkreuz, Switzerland). The resulting amplicon size was visualised using agarose gel electrophoresis and subjected to DNA sequencing and BLAST alignment to reference stx1a and stx2a sequences (NC_002655.2). To mimic the sample matrix for sensitivity determination, TSB was inoculated with a pea-size amount of stool (from a single donor) shown to be stx-negative by PCR. To this inoculated broth 1 mL of plasmid stock (5.3*106 copies/µL) containing both stx1 and stx2 was added and serially diluted eight times in 9 mL of TSB, to a lowest dilution of 1:108 (53 plasmid copies/ml). Nucleic acid extraction was performed on 200 µL broth employing the MagNApure LC instrument (Roche Diagnostics, Rotkreuz, Switzerland) to yield 100 µL of extract. Initially, real-time PCR was performed in triplicate using a template from each of the eight dilutions to estimate a limit of detection (LOD). Subsequently, real-time PCR was performed in eight replicates on the dilution with the estimated LOD, as well as one dilution above and one dilution below the estimate. The LOD was defined as the lowest plasmid concentration spiked into TSB, before nucleic acid extraction, yielding a positive signal, as described above in all eight replicates. Nucleic acid extractions from STEC subtypes 1d (Reference strain MH1813, GenBank accession No. AY170851), 2b (Reference strain EH250, GenBank accession No. AF043627), 2c (Reference strain 031, GenBank accession No. L11079), 2d (Reference strain C165-02, GenBank accession No. DQ059012), 2e (Reference strain S1191, GenBank accession No. M21534), 2f (Reference strain T4/97, GenBank accession No. AJ010730) and 2g (Reference strain 7V, GenBank accession No. AY286000) were also subjected to PCR amplification to assess impact of strain variation on detection. The reproducibility of melting temperature assessment for stx1 and stx2 differentiation was determined by testing 24 replicates of TSB spiked with cloned stx1 and stx2 plasmids. To further assess the reproducibility of melting temperature across the subtypes, three stx1 subtypes and seven stx2 subtypes were tested similarly.

Clinical specimen testing
Between September 2014 and May 2015, we collected same day residual stool after routine testing from 226 consecutive stool specimens (the stool samples were transported in a temperature regulated box and processed within 12 h of collection) from the National Health
Laboratory Services located at the Groote Schuur Hospital in Cape Town, South Africa, a tertiary care academic teaching laboratory affiliated with the University of Cape Town. This tertiary academic hospital serves the greater Cape Town area.

A pea-sized stool sample was inoculated in 90 mL of TSB and vortexed before incubation at 37 °C for 18 h. Two hundred microlitres of broth were subsequently extracted employing the MagNA Pure LC Total Nucleic Acid isolation kit (Roche Diagnostics, Rotkreuz, Switzerland) using the total variable elution volume protocol and following the manufacturer’s manual (version 14) to yield 100 µL of nucleic acid extract.

In addition, CHROMagarTMSTEC (CHROMagar Microbiology, Paris, France) was inoculated with a loop full of overnight inoculated broth and incubated at 37 °C for 18 h. Bright mauve colonies (up to five mauve colonies were picked per sample, depending on the number of mauve colonies formed) were sub-cultured onto MacConkey agar with crystal violet, sorbitol MacConkey agar, and 2% blood agar (Green point Media, National Health Laboratory Service, Albertynshof, South Africa). E. coli was presumptively identified as lactose-positive, oxidase-negative, spot indole-positive and pyrrolidonyl arylamidase (PYR)-negative with confirmatory identification using VITEK 2 (bioMerieux, Inc., Durham, North Carolina, United States).

Isolate characterisation
Isolates yielding mauve colonies on CHROMagarTMSTEC and presumptively identified as E. coli were subjected to stx characterisation employing the real-time PCR assay characterised herein. Other diarrhoeic E. coli virulence genes, including the fimbrial adhesion gene for diffusely adherent E. coli, the anti-aggregation protein transporter gene for enteroaggregative E. coli, heat-stable and heat-labile enterotoxin genes of enterotoxigenic E. coli, the intimin coding gene eae for enteropathogenic E. coli (EPEC) and the bundle-forming pili gene for the typical EPEC, were determined using standard gel-based PCR as previously described using primers as shown in Table 2.18

ST, heat-stable; LT, heat-labile.
To confirm Shiga toxin production among stx-positive isolates, the Immunocard STAT!® EHEC (Meridian Biosciences, Inc., Cincinnati, Ohio, United States) was used to detect Shiga toxin 1 and 2 (by employing immunochromatography with toxin-directed monoclonal antibodies labelled with red-coloured gold particles). All mauve isolates found to carry virulence genes were serotyped at the Centre for Enteric Diseases, National Institute of Communicable Disease, Johannesburg, by employing antisera (Statens Serum Institut, Copenhagen, Denmark) and the detection of somatic O-antigens as previously described.19,20 H-antigen serotyping was not undertaken.
Statistical analysis
Data on the possession of virulence genes, cultural characteristics on different media, serotypes and Shiga toxin production was entered in Microsoft Office Excel 2010 (Microsoft Corp.,Redmond, Washington, United for Disease Control and Prevention, Atlanta, Georgia, United States) for analysis. Using the LightCycler 480 II software, efficiency of the in-house real-time PCR assay was determined. The amplification curves and the melting peaks were used to differentiate between stx1 and stx2.

Results
Real-time polymerase chain reaction validation
The BLAST analysis of the primers and probe sequence specificity yielded no significant homology to non-stx targets (data not shown). Real-time PCR amplicons generated were confirmed as 208 bp for stx1 and 204 bp for stx2 (Figure 2). Sequencing and BLAST analysis confirmed the identity of both stx1 and stx2 amplicons. The serially diluted plasmid-stool-TSB was successfully amplified in

8/8 replicates in the sixth dilution, whereas the seventh dilution yielded an amplification signal in 3/8 replicates, yielding a LOD of 5.3 target copies/µL of broth. All other stx subtypes investigated (stx1a, stx1b, stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g) were successfully amplified by this assay (data not shown). stx1 and stx2 were successfully distinguished by a melting temperature of 58.2 °C (SD = 0.033) and 65.3 °C (SD = 0.037) (Figures 3–6). The Tm for stx2 subtypes 2a, 2b, 2c, 2d, 2e, 2f and 2g were the same at 65.3 °C (SD = 0.037, 0.041, 0.035, 0.039, 0.034, 0.033 and 0.032, respectively), whereas that of 1d was 44.7 °C (SD = 0.042). The efficiency of the assay was 1.99 as calculated from the amplification curves generated using the Light Cycler® 480 software. The duplex assay detected both targets in the same run, and these could be differentiated by the melt curve with two distinct peaks at 58.2 °C for stx1 and 65.3 °C for stx2 (Figure 7).

Performance of CHROMagarTMSTEC
Of the 53 mauve isolates, 48 were negative for stx genes using the validated real-time PCR assay. Of the 14 broths that were positive on PCR, nine did not yield any mauve colonies on CHROMagarTMSTEC culture.

Isolate characteristics
Forty-four (83%) of the 53 mauve colonies fermented lactose on MacConkey agar with crystal violet. Eleven (25%) of the 44 lactose fermenters were non-sorbitol-fermenting. Real-time PCR on the 44 E. coli confirmed the presence of stx genes in five (11%), whereas 39 were negative for the stx gene. Real-time PCR was not done on the nine non-lactose fermenting isolates as these were found not to be E. coli on biochemical testing. Four of the five stx positive E. coli colonies were also positive in the real-time PCR broth assay. Of the 39 stx-negative E. coli, only four (12.5%) carried eae genes, whereas four possessed aat genes. Of the four eae positive isolates, two also had the bfp genes and were typical EPEC. The other two eae positive isolates did not possess the bfp genes and were classified as atypical EPEC. The four enteroaggregative stx-negative isolates all belonged to E 

coli serotype O104. All the typical EPEC belonged to serotype O55, whereas one of the two atypical EPEC belonged to serotype O101. The atypical EPEC serotype O101 was from stx2 positive broth. The other atypical EPEC isolate was untypeable. No diffusely adherent E. coli, enteroinvasive E. coli or enterotoxigenic E. coli were detected. None of the 53 E. coli isolates that were screened by immunochromatography was positive for Shiga toxins. For the CHROMagarTMSTEC, sensitivity was 33.3%, specificity was 77.4%, negative predictive value was 95% and positive predictive value was 9.4% (Table 3).

DISCUSSION
We validated the use of a previously described duplex real-time PCR assay with modification able to detect and differentiate stx1 (melting temperature = 58.2 °C) and stx2 (melting temperature = 65.3 °C) from overnight broth enrichment with a LOD of 5.3 target copies/µL broth. This assay was able to detect both stx1 and stx2 in the same run, thus potentially reducing process turn-around time in a busy laboratory setting. Timely reporting of STEC infections is important, because use of certain antibiotics is contraindicated in STEC infections.
Compared to the validated duplex real-time PCR, CHROMagarTMSTEC showed a sensitivity of 33%, specificity of 77.4%, negative predictive value of 9.4% and positive predictive value of 95% for detection of STEC in stool following TSB enrichment. Of the 53 mauve isolates, 48 were negative for stx genes on use of the validated real-time PCR, whereas nine of the 14 PCR-positive broths did not yield any mauve colonies when cultured on CHROMagarTMSTEC. Reasons for the poor performance of this medium in relation to the in-house developed duplex real-time PCR include the following: (1) delays in reporting of diarrhoea cases to a tertiary hospital (where samples were collected) may have led to loss of stx genes; STEC numbers are sharply reduced in stool after one week of illness, and the Shiga toxin genes might be lost by the bacteria.21 (2) CHROMagarTMSTEC selects for tellurite resistant strains but misses the tellurite susceptible STEC whose prevalence in this setting is not known.
For a chromogenic medium to be considered for routine screening purposes, it must have high specificity so as not to waste scarce laboratory resources on false positives. The false positivity rate in this setting (48/53 [90.6%]) is higher than has previously been reported in Europe (16.3% reported by Gouali et al., 2013 and 18.3% by Wylie et al., 2013).12,22
Similar studies to evaluate this medium were done in Canada, Finland, and Germany, all of which reported high sensitivities for STEC serotypes O26, O111, O121, O145, O118, and O157.13,22,23 The specificity of 77.4% noted in this study was low compared to values (between 95.8% and 98.9%) reported in similar studies done in Europe. The difference in sensitivity and specificity could be explained by the differences in the patient characteristics (whether they present with haemolytic uremic syndrome and or bloody diarrhoea or not). Unlike the studies in Europe, this study did not focus on only patients with haemolytic uremic syndrome or bloody diarrhoea. Additionally, the distribution of tellurite resistant STEC (which is targeted by CHROMagarTMSTEC) in this setting is not known. The prevalence of stx genes in stool samples (6.2%) was lower than the 9% previously reported by Kullin et al., 2015. Among the 53 isolates that formed mauve colonies on CHROMagarTMSTEC, five were STEC (two serotype O26 and others non-typeable), four were enteroaggregative E. coli (serotype O104), two were atypical EPEC (serotype O101 and non-typeable) and two were typical EPEC (serotype O55). Serotypes O26 and O104 are among the top six STEC serotypes globally.25,26 CHROMagarTMSTEC is intended for STEC culture; however, we detected other diarrhoeic E. coli pathotypes using this medium. The other pathotypes detected might have been hybrid strains that lost the stx genes or hybrid strains whose stx genes could not be detected using the primers employed in this study. Notably, bacteriophages carrying the stx genes are very quickly lost both in vivo and in vitro,27 and not all stx primers can detect all the stx gene variants.28

Limitations
Not all STEC are tellurite resistant and may have been missed on CHROMagarTMSTEC culture. This study only focused on the strains that formed mauve colonies. The stool samples in this study were collected between September 2014 and May 2015, and therefore the results of this study as regards prevalence of STEC may not reflect the entire year or areas of South Africa other than Cape Town.

Conclusions
The in-house developed real-time PCR assay is a sensitive and specific alternative to the currently used diagnostic strategy. Due to the high false positivity rate, CHROMagarTMSTEC can only be used as an adjunt to a more sensitive and specific assay such as real-time PCR.

Reliability
We confirm that the experiments conducted in this study will yield the same results during repeated trials using the same reagents and detection platforms.

Validity
To the best of our knowledge, the findings of this study, as obtained using the methods we employed, are valid for the study area and season. The in-house developed real-time PCR may, however, be adopted in other laboratories in developing countries.

Acknowledgements
We acknowledge the Center for Enteric Disease Research Unit, National Institute of Communicable Disease (Sandringham, Johannesburg, South Africa) for serotyping of the E. coli isolates. We also acknowledge the National Research Foundation, South Africa, the ACP-RISE scholarship fund and the ADB-HEST fund.

REFERENCES

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