Adebukola Adisa Ekoh-Jolly1

1 Department of Medical Laboratory Science, University of Medical Sciences, Ondo city, Ondo State, Nigeria

Isaiah Nnanna Ibeh2 Erhabor Ikponmwosa2

2 Department of Medical Laboratory Science, University of Benin, Benin City, Nigeria

Samuel Ayobami Fasogbon3*

3Department of Laboratory Accreditation, Medical Laboratory Science Council of Nigeria, Abuja, Nigeria

Ahmed O. Adebayo4

4Department of Medical Laboratory Science, Lagos State College of Health Technology, Yaba-Lagos, Nigeria

Loveth O. Fasogbon5; Monica Osato Solade5

5Department of Medical Laboratory Science, College of Medicine, Ambrose Alli University, Ekpoma, Nigeria

Cyril A. Egbo6

6Department of Community Health, College of Medicine, University of Benin, Benin city, Nigeria

Samuel Ehiremen7

7Department of Anatomy, faculty of Basic medical Sciences, Olabisi Onabanjo University, Ago-iwoye, Nigeria.

Ifeoma G. Onukwue8

8National Quality External Assessment Laboratory, Medical Laboratory Science Council of Nigeria, Zaria, Nigeria.

Ayobami B. Olayera9

9Department of Haematology and Immunology, University of Nigeria Teaching Hospital,

Ituku-Ozalla, Enugu State, Nigeria4

All Corresponding to: Samuel Ayobami Fasogbon,, samfash4best@gmail.com

ABSTRACT

Background: The use of alcohol-based hand sanitizer that contains at least 60% alcohol prevents the spread of infections and thus prevents the spread of antimicrobial resistance. Aim: The aim of this study was to evaluate the antimicrobial efficacy of different brands of hand sanitizers sold in Nigeria. Materials and Methods: Antimicrobial efficacy of eleven popular brands of alcohol-based hand sanitizers sold in Nigeria was evaluated and comparatively analyzed using a combination of Agar well diffusion (Mueller-Hinton Agar for bacteria and Sabouraud Dextrose Agar for fungi), Broth dilution (Nutrient broth) and In vivo Viable Microbial count reduction techniques. A total of 330 clinical isolates comprising 55 each of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris and Candida albicans obtained from culture collection were used as test organisms. Results and Discussions: This study revealed a very high significant difference (P<0.001) on the susceptibility pattern of the test organisms against the various hand sanitizers used. The study showed a very high significant difference between the activity of the various hand sanitizers and the positive control (P<0.001). All hand sanitizers used in this study apart from Purit are less active against Proteus vulgaris but are more active against Staphylococcus epidermidis. The findings in this study showed that isopropyl alcohol based-gel form hand sanitizer had 82.2% efficacy while ethanol-based gel form hand sanitizers had 76.9% efficacy. This study revealed Significant difference (P<0.001) on Minimum Inhibitory Concentration (MIC) of all hand sanitizers used against test organisms except for Evree hand sanitizer which showed no significant difference (P>0.05) on Minimum Inhibitory concentration against test organisms. Of all the hand sanitizers used in this study only Purit has bactericidal activity against Proteus vulgaris not lower than 95% concentration. Conclusion: The results of this study suggest high significant difference in the antimicrobial efficacy of various hand sanitizers sold in Nigeria.

Keywords: Anitimicrobial, Concentration, COVID-19, Efficacy, Sanitizers.

INTRODUCTION

Globally, there are 119,094,168 corona virus cases, 2,640,822 deaths, 94,632,462 recovered and 21,820,884 active cases as at 12th March, 2021 [1][2]. The genome sequence of virus SARS-CoV-2 is similar to SARS Coronavirus (SARS-CoV) [3][4]. The CoVs are members of the same genus Beta coronavirus, sharing similar morphology in the form of enveloped, positive single-stranded RNA viruses [5]. They can be deactivated by certain lipid solvents such as ethanol, ether (75%), chlorine-containing disinfectants, and chloroform, except chlorhexidine [5]. Ethyl alcohol, at concentrations of 60%–80%, is effective viricidal agent inactivating all the lipophilic viruses (e.g., influenza, herpes and vaccinia virus) and many hydrophilic viruses (e.g., adenovirus, enterovirus, rhinovirus, and rotaviruses excluding hepatitis A virus (HAV) or poliovirus) [6].

Regular hand washing with soap and water prevent the spread of infections and decrease the risk of getting sick. When there is non-availability of soap and water, CDC recommends the use an alcohol-based hand sanitizer that contains at least 60% alcohol [6]. Hand hygiene prevents the spread of antimicrobial resistance.

Alcohol-based hand sanitizers generally contain one or more alcohols (ethanol or 1-propanol or 2-propanol) in quantitative ratios that are deferred by brand. The alcohols present in the Alcohol-based hand sanitizers formulations triggers bactericidal action by denaturation of microbial proteins [8]. Some Alcohol-based hand sanitizers may as well contain other chemical constituents that work in synergy with the bactericidal action of the alcohols. Other chemical compounds may confer independent bactericidal activities through unknown mechanisms.

Due to the continuing worldwide COVID-19 pandemic, the demand for hand sanitizers remains at a very high level. In Nigeria, many Pharmaceutical companies, cosmetics industries, even a soft drink bottling company and a University went to the production of hand sanitizers to meet market demand and for profit making. Majority of manufacturers claim 99.9% effectiveness of their hand sanitizers.

Different categories of hand sanitizers (alcohols, quaternary ammonium compounds and triclosan) possess different levels of sensitivities and therefore varying resistance by various infectious agents. For example, some species of Staphylococcus aureus carry a gene that allows resistance to some hand sanitizers [6]. Some hand sanitizers are fungistatic, others are bacteriostatic against gram-positive bacteria and bacteriostatic against some gram-negative bacteria [6]. Like ethanol hand sanitizers, those of the quaternary ammonium compounds are not active against non-enveloped viruses. More so, some are safer, more economical, less messy and more enjoyable to use for kids. This study intends to look into the usage and effectiveness of these different brands of hand sanitizers.

This study examined the antimicrobial efficacy of eleven brands of hand sanitizers sold in Nigeria against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris and Candida albicans, and also to assess and compare their antimicrobial effectiveness.

MATERIALS AND METHODS

Type of Study

This is a cross sectional descriptive study and was conducted to comparatively analyze some hand sanitizers sold in Nigeria.

Study Location

The research was conducted in the Biology Laboratory of the Department of Biological Sciences, Faculty of Science, University of Medical Science, Ondo, Nigeria.

Ethical Considerations

Written/verbal informed consent was received from subjects who participated in the study at the study center. The Ethical committee of the Edo state Ministry of health, Benin City, reviewed and approved the study protocol, HA. 737/83.

Population and Subjects

The population of this study was centered on users of hand sanitizers among health workers, students and civil servants in Benin City and Ondo town of Nigeria.

Test Organisms

A total of Three hundred and thirty clinical isolates comprising fifty-five each of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris, and Candida albicans obtained from Don Bosco Health Centre, Akure, Ondo State, Central Hospital, Benin City and Stella Obasanjo Hospital, Benin City, Edo State were used as test organisms in this study. These micro organisms were chosen because they are commonly implicated in community-acquired infections [8][9].

Test Subjects

A total of Two hundred Subjects comprising of Health Workers, Civil Servants and Students participated in this research after they consented to participate.

Hand Sanitizers

Eleven brands of alcohol-based hand sanitizers (10 waterless gel form – Lifebouy, Purit, Dettol, Carex, Tetmosol, Sanitol, Wind, Seed, Tribute, Evree, and one spray form – 2sure) commonly sold and used in Nigeria were chosen for this study based on their popularity and availability at retail outlets across Nigeria. Each hand sanitizer was stored as recommended by its manufacturer and was used well before the expiration date.

Test Organisms Standardization

Turbidity variability of Bacterial suspensions may affect result, thus the need for standardization of microbial testing; McFarland standards was used as a reference to adjust the turbidity of bacterial suspensions. The McFarland 0.5 turbidity standard was prepared by adding 0.5 ml of 1.175% w/v barium chloride dihydrate (BaCl2·2H2 O) solution to 99.5 ml of 15 w/v sulfuric acid (H2 SO4) [10]. This was mixed uniformly and aliquoted into test tubes identical to the ones used in preparing inoculum suspensions of the test organisms. The accuracy of the density of the standard was verified using a spectrophotometer. The absorbance of the 0.5 McFarland standards at wavelength of 625 nm was 0.08–0.10. The tubes were stored in a well-sealed container in the dark at room temperature until when required [11].

A sterile loop was used to pick a loopful of inoculum from a pure culture of the test organism. This was then transferred and suspended into a tube containing sterile normal saline (NaCl 8.5 g, distilled water 1 L). The tube was compared with the turbidity standard, and the density of the organism was adjusted by adding more bacteria or sterile saline until standardization was attained [12].

Susceptibility of Test Organism to Hand Sanitizers

Disk agar diffusion technique (well-variant) as described by Valgas et al. was used for determining antimicrobial efficacy of hand sanitizers [13]. Sterile Mueller-Hinton agar plates were inoculated with standardized test organisms. A sterile cotton swab was dipped into a test tube containing the inoculum and was rotated properly to allow maximum contact. Excess inoculum was removed by pressing and rotating the swab firmly against the inside wall of the tube above the liquid level. The swab was then streaked over the surface of the medium three times while rotating the plate at 60° angle after each application. The swab was also passed around the edge of the agar surface. The inoculum was left to dry for a few minutes at room temperature with the lid closed.

With the aid of a sterile 4mm cork borer, 3 equally spaced holes were bored in the agar plate. The agar plugs were discarded using a sterile needle. A 100 microliters volume of undiluted hand sanitizer was then introduced into the first well, the second well was filled with an equal volume of sterile water to serve as negative control, while the third well was filled with 90% ethanol as positive control. This was done for all the test organisms and hand sanitizers. The plates were incubated for 24hours at 37°C in an upright position. They were then examined for zones of inhibition which indicated the degree of susceptibility or resistance of the test organism to the antibacterial agent. The point of abrupt diminution growth which corresponds to the complete growth inhibition was taken as the zone edge. The test was carried out five times, and the average of all readings was taken as the zone of inhibition in each case.

Determination of Minimum Inhibitory Concentration (MIC)

Minimum inhibitory concentration is the lowest concentration of an antimicrobial agent that completely inhibits the growth of a test organism as seen by the unaided eye [14]. To determine the MIC, increasing concentrations (5% – 100% with 5% intervals) of each Hand sanitizer were prepared in 9ml tubes of sterile nutrient broth. Exactly 100microlitre of each standardized test organism was then introduced into each tube of Hand Sanitizer. A tube containing only nutrient broth and bacteria without hand sanitizer served as negative control while another tube containing just the sanitizer and broth without bacteria served as positive control. Each tube was incubated for 18hours for bacteria and 48hours for fungi, then examined for visible growth or turbidity. The concentration of the hand sanitizer in the tube in which no visible growth was observed when compared with the controls was taken as the MIC.

Determination of Minimum Cidal Concentration

Minimum bactericidal concentration is the lowest concentration of an antimicrobial that can kill the test organism [11]. To determine the cidal for each Hand sanitizer, samples from the test tubes used in MIC test that showed no visible growth after the period of incubation were inoculated on sterile nutrient agar plates (which had no antimicrobial incorporated) for bacterial and Sabouraud Dextrose Agar for fungi in them using sterile swab sticks. The plates were incubated at 370C for 18 to 24hours and were then observed for growth. The concentration at which absence of growth was observed (bactericidal activity) was then taken as MBC.

In vivo Reduction of Viable Bacteria and Fungi counts on Hands of Subjects

The Hand sanitizers evaluated for their efficacy in reducing baseline bacteria and fungi counts on the hands of subjects. Ten individuals were selected for each products and verbal informed consent was obtained from each subject prior to the conduction of the experiment. Subjects did not apply antimicrobial substance to their hands prior to the experiment. Sterile nutrient agar and Sabouraud Dextrose agar plates were divided into two halves with one half labeled BF (before) and the other labeled AF (After). Subjects were asked to gently make an impression on the surface of the BF side of the agar with the three unwashed fingers. After this, 3ml of the hand sanitizer was then applied to the hands and rubbed thoroughly on the palms, fingers and the back of the hands until the hands became completely dried. Subjects were then asked to repeat the finger impression on the AF part of the plate. This was done by all subjects. The plates were incubated at 370C for 24hours and the numbers of colonies were counted. The percentage of CFU reduction was calculated as follows:

%CFU reduction = CFU/mL on Bf Section – CFU/mL on AF Section x 100 CFU/mL on BF Section [15].

Statistical Analysis

Analysis of Variance (ANOVA), Duncan Multiple Range Test, Unpaired T-Test and Chi-Square were used to Test for Significant Difference on Susceptibility Pattern of Test Organisms to the various Hand Sanitizers.

Paired T-test was used to test the significant difference of Percentage CFU Reduction of Viable Bacteria and Fungi from hands of Subjects by the hand Sanitizers.

Minimum Inhibitory Concentration (MIC), Minimum Cidal Concentration (Bacteriocidal) (MBC) and Fungicidal (MFC) of the Hand Sanitizers against Test Organisms are presented in Charts.

RESULTS

Data Analysis and Presentation

In this study, a total of Three hundred and thirty clinical isolates comprising fifty-five each of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris, and Candida albicans were tested against eleven alcohol-based hand sanitizers commonly sold and used in Nigeria (Table 4.1) for their efficacy.

It was observed that all the hand sanitizers used in this study have varying inhibitory activities against all test organisms and that each of the hand sanitizers used had different level of inhibition against each of the test organisms. A sanitizer very active against a particular test organism is less active against another test organism (Table 4.2).

The study revealed that all the hand sanitizers used in this study have varying Minimum Inhibitory concentration (MIC) against the susceptible test organisms (Table 4.3).

It was observed that not all the hand sanitizers used in this study had cidal acitivity against test organisms and where it did have, the Minimum cidal concentrations were mostly 100% (Table 4.4)

The study revealed varying In-vivo reduction of Viable Bacteria and Fungi from hands of subjects by three hand sanitizers that have cidal activities on test organisms. It was observed that Purit hashigher (82.2%) viable bacterial count reduction than fungi (31.8%). The study also revealed that Evree has equal in-vivo reduction of viable bacteria and fungi (76.9%) while Lifebuoy has highe in-vivo reduction of viable fungi (64.5%) than bacteria (41.6%) (Tables 4.5 and 4.6)

Purit and Lifebouy have inhibitory activity against test organisms not below 60% concentration while Dettol has inhibitory activity not below 70% concentration. Sanitol, Seed and Tribute have inhibitory activity not below 75% concentration. Carex, wind and Evree have inhibitory concentration not below 80% concentration while Tetmosol and 2Sure hand sanitizers have inhibitory activity against test organisms not below 90% concentration

.

DISCUSSION

The use of alcohol-based hand sanitizers has become more popular with the emergence of Coronavirus pandemic. Many brands of alcohol-based hand sanitizers flooded the Nigerian markets with the manufacturers of various hand sanitizers claiming 99.9% effectiveness against infectious organisms. This study became necessary due to increasing reliance on alcohol-based hand sanitizers to prevent community spread of infectious diseases.

This study revealed a very high significant difference (P<0.001) on the susceptibility pattern of the test 0rganisms (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris, and Candida albicans) against the various hand sanitizers used in this study, this is in line with the finding of [16], who also reported significant difference (P<0.001).

The study also show a very high significant difference between the activity of the various hand sanitizers and the positive control (P<0.001). This finding is different from the finding of [17] who reported no significant difference between the activity of the various hand sanitizers and the positive control. Though he did not revealed the brand name of the hand sanitizers used in his study, they were also alcohol-based hand sanitizers, the difference may be due to other ingredients in the hand sanitizers, mores so [17] used a commercial hand sanitizer as his positive control while 90% ethanol was used as positive control in this study.

All hand Sanitizers used in this study apart from Purit are less active against Proteus spp (a Gram-negative bacilli) but are more active against Staphylococcus epidermidis (a Gram-positive cocci), this may be due to differences in their cell wall components.

The findings in this study that isopropyl alcohol based-gel form Hand sanitizer (Purit) has higher efficacy than ethanol-based gel form hand sanitizers is the same with what was observed by [15] that isopropyl alcohol solution has higher efficacy than ethanol solution, but contrary to findings of [18] which was in reverse.

This study also showed Significant Difference (P<0.001) on Minimum Inhibitory Concentration (MIC) of all Hand Sanitizers used against Test Organisms except for Evree Hand Sanitizer which show no significance difference (P>0.05) on Minimum Inhibitory concentration against test organisms.

This study revealed that none of the hand sanitizers sold in Nigeria, used in this study have inhibitory activity (MIC) below 60% concentration which is lower than below 75% concentration that was reported by [17].

2Sure the only Liquid form hand sanitizer used in this study has lower antimicrobial efficacy than other gel form hand sanitizers used, this is the same with the report of [18].

Of all the hand sanitizers used in this study only Purit has bactericidal activity against Proteus spp not lower than 95% concentration. Lifebuoy, Dettol, Sanitol, Seed, Tribute and Evree have bactericidal effects not lower than 100% concentration on other test organisms. Carex, Temosol, 2Sure and Wind hand Sanitizers have no bactericidal effect on test organisms.

This study showed that Purit, Lifebuoy, Tetmosol and Evree have fungicidal effects on Candida albicans at concentration not lower than 100%. Dettol, Carex, Sanitol, 2Sure, Wind, Seed and Tribute hand Sanitizers sold in Nigeria, used in this study did not have fungicidal effect on test organism.

The mean percentage in vivo reduction of viable bacteria and fungi counts observed in this study is higher than what was observed by [17] but lower than 99.9% claim by manufacturers of hand sanitizers.

Conclusion

Based on the findings of this study, the Manufacturers’ claim of 99.9% of antimicrobial efficacy of various hand sanitizers sold in Nigeria, used in this study is in doubt. There is significant difference on the antimicrobial efficacy of various hand sanitizers sold in Nigeria, used in this study and the various hand sanitizers sold in Nigeria are more active against Gram positive bacteria than Gram negative bacteria with more potency against bacteria than fungi. Isopropyl alcohol based-gel form Hand sanitizer (Purit) has higher efficacy than ethanol-based gel form hand sanitizers

Recommendation

The revelation of this study suggests that no hand sanitizer sold in Nigeria, used in this study have 99.9% antimicrobial efficacy, therefore hand washing with water and soap should be practiced more often to physically remove the infectious microbes from the hands then combine with rubbing hand with hand sanitizers.

Conflict of Interest

Authors declare no conflict of interest

REFERENCES

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2. NCDC, (2021). COVID-19 Coronavirus 2019-n Cov Statistics Update (Live): 4,122,912 Cases and 280,337 Deaths. Available online: https:// virusncov. com/ (accessed on 8 May 2020).

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5. Cascella, M., Rajnik, M., Cuomo, A., Dulebohn, S.C. and Di Napoli, R. (2020). Features Evaluation and Treatment of Coronavirus (COVID-19); StatPearls Publishing: St. Petersburg, FL, USA. pp. 4 – 10.

6. CDC, (2020). COVID-19 Coronavirus 2019-n Cov Statistics Update (Live): 4,122,912 Cases and 280,337 Deaths. Available online: https:// virusncov. com/ (accessed on 8 May 2020).

7. Paulson, D.S., Fendler, E.J., Dolan, M.J. and Williams, R.A. (1999). A close look at alcohol gel as an antimicrobial sanitizing agent. American Journal of Infection Control. 27: 332 – 338.

8. Montravers, P., Alain, L., Dubreuil, L. and Benchimol, D. (2009). Clinical and microbiological profiles of community-acquired and nosocomial intra-abdominal infections: Results of the French prospective, observational EBIIA study. Journal of Antimicrobial Chemotherapy. 63(4): 785 – 794.

9. Rosenthal, K.L., Hendrick, M.J. and Rankin, S.C. (2008). Methicillin-resistant Staphylococcus aureus associated dermatitis in a Congo African grey parrot (Psittacus erithacus erithacus). Medical Journal of Avian Mycology/Bacteriology.12(9): 4 – 10.

10. NCCLS, (1999). Performance standards for antimicrobial susceptibility testing. www.scrip.org.

11. Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries. 2nd edition. New York: Cambridge University Press. pp. 137 – 141.

12. Vandepitte, J., Verhaegen, J., Engbaek, K., Rohner, P., Piot, P. and Heuck, C.C. (2003). Basic Laboratory Procedures in Clinical Bacteriology. 2nd edition. Geneva: WHO; 2003.

13. Valgas, C., De Souza, S.M., Smania, E.F. and Smania, A. (2007). Screening methods to determine antibacterial activity of natural products. Brazil Journal of Microbiology. 38: 369 – 380.

14. CLSI. (2006). Performance standards for antimicrobial disk susceptibility tests. Approved standard. 9th Edition Document M2-A9. Clinical and Laboratory Standards Institute, Wayne, PA.

15. Oke, M.A., Bello, A.B., Odebisi, M.B., El-Imam, A.A. and Kazeem, M.O. (2013). Evaluation of antibacterial efficacy of some alcohol-based hand sanitizers sold in Ilorin (North-Central Nigeria). Ife Journal of Science. 15(1): 111 – 117.

16. Vardhaman, M., Gundabaktha, N., Arun, S. and Vishwakarma, K.G. (2016). Comparative assessment of antimicrobial efficacy of different hand sanitizers: An in vitro study. Dental Research Journal. 13(5): 424 – 431.

17. Imuetinyan, O. (2017). Comparative analysis of the antimicrobial efficacy of some hand sanitizers in use in Benin Metropolis. Journal of Microbiology.23:3-6.

18. Ochwoto, M, Muita, L., Talaam, K., Wanjala, C., Ogeto, F., Wachira, F., Osman, S., Kimotho, J. and Ndegwa, L. (2017). Anti-bacterial efficacy of alcoholic hand rubs in the Kenyan market. Antimicrobial Resistance & Infection Control. 6(1): 17 – 20.

The post Antimicrobial Efficacy of Various Hand Sanitizers Sold in Nigeria appeared first on Nigerian Biomedical Science Journal.

Joseph. Emberga. Toryila, Ayuk Hughes Bisong, Sheba John Ishaku

Department of Physiology, Faculty of Basic Medical Sciences, Bingham University, Karu,

All Corresponding to: Joseph, joetoryila@gmail.com

ABSTRACT

Background: Infertility is a worldwide health concern in the developing world, affecting 8-15% of couples in their reproductive age. Musa paradisiaca (BANANA) is a crop in the genus Musa and all members of the genus are indigenous to tropical and subtropical countries. Banana is wildly consumed by Africans especially by Nigerians. The study was aimed at determining the effect of Musa paradisiaca (BANANA) flour supplement on the reproductive system of male wistar rats. Methodology: Twenty (20) male wistar rats weighing between 85-170g were used for this study and were randomly divided into four (4) groups. Group one served as the control, group 2 and 3 were treated with commercial feed supplemented with banana flour in the ratio 20:80 ratio and 5ml/kg of body weight of lime juice orally for four weeks .Group 4(Positive control) was given 5ml/kg body weight of lime juice orally for four weeks. The animals were sacrificed by cervical dislocation and semen samples were collected. Sperm motility, sperm morphology, sperm count and testosterone levels were investigated. Resuls: The result shows that there was increase in sperm motility in treated groups 2 (65.00±13.23) and 3 (68.33±7.64) compared to group 4 and the control group (60.0±20.0), significant increase (P≤ 0.05) in sperm count in treated groups 2 (2100.00±208.44) and 3 (2700.00±754.44) compared to group 4 and the control group (1933.33±120.43), There was a significant increase (P≤ 0.05) in sperm mortality in treated groups 2 and 3) compared to group 4 and the control group. There was a significant increase (P≤ 0.05) in testosterone level in treated groups 2 (5.73 ± 4.38) and 3 (7.56 ± 5.07) compared to control the group (1.23 ± 0.32). There was a significant increase in sperm abnormalities in group 4 (11.67±7.63) when compared to group 2, 3 and the control (P≤ 0.05). Conclusion: The effect of banana flour supplement on the reproductive system of male wistar rats was investigated and it was shown to increase sperm motility, sperm count, testosterone levels in all treated groups, while decreasing sperm mortality and sperm abnormalities.

INTRODUCTION

Infertility is a couple`s inability to have a child after one year of unprotected sexual intercourse (WHO, 2016). Infertility is a worldwide health concern in the developing world, affecting 8-15% of couples in their reproductive age (Otubu, 2006). The prevalence of infertility in a rural Nigerian community is determined by a systematic random sampling of the population. The overall prevalent rate was 30.3%, giving indices of 9.2% for primary infertility and 21.1% for secondary infertility (Adebayo et al,2021). Infertility leads to stigmatization, marital instability, and enormous psychological stress (Adebayo et al,2021). Infertility is a public health concern in many developing nations of the world due to its high prevalence and especially because of its serious social implications. Infertility has social, economic and personal effects, which go beyond childlessness, and women bear the major brunt of the burden. It is a major cause of marital disharmony and separation, and personal misery in some other population groups (Ola. 2009). The highest prevalence is in low resource countries, particularly in sub-Saharan Africa (Sharma et al 2008). Male infertility can be caused by poor penile erection, abnormal sperm quality and volume, abnormal ejaculation, other causes such as plants like lime, which has been described as a natural spermicide; a contraceptive substance that reduces sperm concentration to prevent pregnancy.

Banana is an elongated, edible fruit botanically a berry (Armstrong et al., 2013) produced by several kinds of large herbaceous flowering plants in the genus Musa (Armstrong et al., 2013). Researches into natural diets like banana (plantain) showed that its consumption by men could enhance some reproductive functions, and also alleviate certain reproductive dysfunctions (Yakubu et al., 2007) (Ojewole et al., 2003). Bananas are considered nutritive with high content of vitamins A, B1 and C (Margard and Briav, 1979). Generally, bananas contain a considerable amount of mineral elements and could therefore serve as a good source of mineral supplement in human/animal diets. It had a medicinal use in people with high blood pressure and stroke and also reported to help to stimulate the production of hemoglobin and also have hypolipidemic effects

Musa paradisiaca promotes healthy digestion, improves affective state, helps in the retention of and serves as good sources of calcium, phosphorus and nitrogen, which build and regenerate tissues in the body, and is also a rich source of iron and vitamins, especially Vitamins C and E. The fruit is also rich in potassium which are essential for production of sex hormones and enhancing the male libido and improve fertility by enhancing semen quality (Hossain et al., 2015). Enzyme bromelain in banana increases sex drive and reverses impotence in men (also found in pineapple). These phytochemicals also have stimulatory effects on sex hormone and semen production (Hossain et al., 2015), as well as sperm motility. Hence the aim of this research was to ascertain the effect of banana flour supplements on the reproductive system of male wistar rats using lime juice to induce sperm mortality.

MATERIALS AND METHODS

Preparation of the banana flour

Green plantain fruits (ripe); were obtained from a farm. The fruits were cut longitudinally into chips of about 5 mm thickness and air dried for 4 days after which they will be grinded and made into flour..Commercial feed supplemented with banana flour in the ratio 20:80 (Manuela et al., 2018).

ANIMAL GROUPING

Twenty (20) male wistar rats weighing between 85-170g were used for this study were divided randomly into four groups:

Group 1: Served as Control group.

Group 2: Commercial feed supplemented with banana flour in the ratio 20:80 ratio and 5ml/kg lime juice orally.

Group 3: Commercial feed supplemented with banana flour in the ratio 20:80

Group 4: Given 5ml/kg body weight of lime juice orally (Ojewole et al., 2009)

The treatment lasted for a period of 14 days.

Twenty four (24) hours after the 14th day of treatment, the rats were euthanized by cervical dislocation and the testes and epididymis excised using a midline abdominal incision. The left caudal epididymis was immediately transferred into sterile bottles containing 2 ml of normal saline for semen analysis. Semen analysis was carried out on sperm motility, sperm morphology and sperm count according to the method of Dolt and Foster, 1972. Blood sample was collected by cardiac puncture and Serum Testosterone level was determined using ELISA technique.

RESULTS

The results indicate significant increase in testosterone level in treated groups 2 and 3 compared to control the group (P≤ 0.05). There was a decrease in testosterone level in lime juice treated group 4 compared to the control group.

Semen Analysis

There was significant increase in sperm motility and sperm count in treated groups 2 and 3 compared to the control group (P≤ 0.05). There was significant decrease in sperm motility and sperm count in lime juice treated group 4 compared to the control group. The result indicates no difference in abnormalities in treated groups 2 and 3 when compared to the control group. However there was an increase sperm abnormality in lime juice treated group 4 when compared to the control group (P≤ 0.05).

Semen Analysis

The results indicate significant increase (P≤ 0.05) in sperm motility in treated groups 2 (65.00±13.23) and 3 (68.33±7.64) compared to the control group (60.0±20.0) due to the banana flour supplement treatment. There was significant decrease in sperm motility in lime juice treated group 4 (60.00±10.00) compared to the control group (60.0±20.0). The results indicate no difference in sperm abnormalities in treated groups 2 (5.00±5.00) and 3 (5.00±5.00) compared to the control group (5.00±5.00). However there was a significant increase (P≤ 0.05) in sperm abnormalities in lime juice treated group 4 (11.67±7.63) when compared to control group (5.00±5.00). There was significant increase (P≤ 0.05) in sperm count in treated groups 2 (2100.00±208.44) and 3 (2700.00±754.44) compared to the control group (1933.33±120.43) due to the banana flour supplement treatment. However was there was a significant decrease (P≤ 0.05) in sperm count in lime juice treated group 4 (316.7±497.17) compared to the control group (1933.33±120.43).

The results indicate significant increase (P≤ 0.05) in testosterone level in treated groups 2 (5.73 ± 4.38) and 3 (7.56 ± 5.07) compared to control the group (1.23 ± 0.32) due to the banana flour supplement treatment. There was a decrease in testosterone level in lime juice treated group 4 (0.77 ± 0.90) compared to the control group (1.23 ± 0.32).

Musa paradisiaca (Banana) contains the enzyme bromelain and other chemicals such as calcium, potassium, alkaloids, flavonoids, vitamins A ,B1 andC . Bromelain, potassium and vitamins B are essential for the production of sex hormone (testosterone), enhancing the male libido and improving fertility by enhancing semen quality (Hossain et al., 2015). They also have stimulatory effects on sex hormone (testosterone) and semen production (Aiddiq et al., 2012), as well as sperm motility.

These results are in line with work done by Yakubu (2007) on the effects of banana fruits in the management of sexual dysfunction in male wistar rats. Although the exact mechanism of action of antioxidants was not stated, the banana fruit was reported to be efficient in the management of sperm concentration (Yakubu et al., 2007). Vitamin A has been studied in combination with other antioxidants including zinc, N-acetylcysteine (NAC), selenium, and vitamins C and E. Scott et al. 1998, reported a 30% increase in motility after a 3-month regimen of vitamin A supplementation combined with vitamins C and E and selenium

For men hoping to increase fertility, the goal is to make sure sperm are healthy. Antioxidant supplements, vitamin A,B1 and C contain in banana can help.

RECOMMENDATION

Infertility is on the increase, eating banana flour as a supplement has proven to affect the male reproductive system positively. Thus medical practitioners should look outside traditional methods for treating infertility in men. Further study should be done on the possible mechanism of action of banana flour supplement on the hypothalamic-pituitary-gonadal axis.

CONCLUSION

The effect of banana flour supplement on the reproductive system of male wistar rats was investigated and it was shown to increase sperm motility, sperm count, testosterone levels in all treated groups. Banana flour supplement should be considered for treatment of infertility in men.

REFERENCES

1. Ketiku AO (1973). Chemical composition of unripe (green) and ripe plantain (Musa paradisiaca). Journal of Science and Food Agriculture 24: 703-707.

2. Brugh V., and Lipshultz L., (2004). Male factor infertility.Journal of Medical Clinics of North America.

3. Caballero B., Finglas P., and Toldra F., (2015). Encyclopedia of Food and Health. Elsevier Science.

4. Corbier, P. Edwards, D.A. Roffi, J. (1992). “The neonatal testosterone surge: a comparative study”. Arch Int Physiol Biochim Biophys. 100 (2): 127–131

5. Hirsh, A. (2003). “Male subfertility”. British Medical Journal. 327 (7416): 669–672.

6. Holstein AF, Schulze W, Davidoff M. Understanding spermato-genesis is a prerequisite for treatment.

7. Reproductive Biology and Endocrinology. 2003; 1:107

Fellers, P.J. Nikdel, S. and Lee, H.S. (August 1990).

8. “Nutrient content and nutrition labeling of several processed Florida citrus juice products”. Journal of America Diet Association.

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10. Chimbatata N., and Malimba C., (2016). Infertility in sub-Saharan Africa. A qualitative review of literature. Journal of Social Science. .

11. Araoye O., (2003). Epidemiology of infertility: social problems of infertile couples. Journal of West African Medicine.

12. Van Balen F., and Bos H., (2009). The social and cultural consequences of being childless in poor-resource countries. Facts, Views and Vision in Obstetrics and Gynaecology. Journal of Medical Gynaecology.

13. Ola T., Aladekomo F., and Oludare B., (2008). Determinants of the choice of treatment outlets for infertility in Southwest Nigeria. Journal of Shawal Medicine.

14. Pantii A., and Sununu Y., (2014). The profile of infertility in a teaching hospital in north West Nigeria. Journal of Shawal Medicine.

15. Scott R et al. The effect of oral selenium supplemen- tation on human sperm motility. British Journal Urology. 1998;82(1):76–80

16. World Health Organization. (2010). “WHO Laboratory manual for the examination and processing of human semen, 5th edition”. Cambridge: Cambridge University Press.

17. Walker, W. H. (2009) “Molecular mechanisms of testosterone action in spermatogenesis”. Steroids 74: 602-607

18. Moench GL, Holt H. (1931) Sperm morphology in relation to fertility. American Journal of Obstetrics and Gynaecology ; 22: 199–210

19. Katz DF, Overstreet JW, Samuals SJ, Niswander PW, Bloom TD et al (1986). Morphometric analysis of spermatozoa in the assessment of human male infertility. Journal of Andrology; 7:203–10

The post Effect of Musa Paradisiaca (BANANA) Flour Supplement on the Reproductive System of Male Wistar Rats appeared first on Nigerian Biomedical Science Journal.

Animoku Abdulrazaq A.1,5, Mesole Samuel Bolaji2, Okpanachi Omachonu Alfred2,

Yusuf Uthman Ademola3 Shember Pascal Bemseer4

1Department of Anatomy, College of Health Sciences, Kogi State University, Anyigba-Nigeria

2Department of Clinical Medicine, School of Medicine, Eden University-Zambia

3Department of Human Anatomy, School of Medicine, Mulungushi University-Zambia

4Department of Pharmacology and Therapeutics, College of Health Sciences, Benue State University, Makurdi-Nigeria

5Department of Anatomy, Ahmadu Bello University Zaria-Nigeria

All Corresponding to: Animoku Abdulrazaq, animokuaa@gmail.com

ABSTRACT

Background Mercury is a heavy metal contaminant of known toxicity with potential for global mobilization following its give off through air, soil, water and food Aim: The objective of this work was to examine the molecular changes in the DNA caused by a heavy metal pollutant mercuric chloride (HgCl2) in Wistar rats and the role of ascorbic acid administration. Methods: Twenty five adult Wistar rats (average weight 185 g) were randomly divided into five groups of five rats per group; a control group administered normal saline, mercuric chloride (HgCl2; 49.8 mg/kg), HgCl2 with distilled water, HgCl2 with low dose ascorbic acid (595mg/kg) and HgCl2 with high dose ascorbic acid (1,190 mg/kg) groups. All administration was carried out orally for a period of three to six weeks and the animals were humanely sacrificed. Results: The results from molecular studies showed that the level of migration of the genomic DNA were fastest in the mercury treated groups when compared to ascorbic acid and the control groups as characterized on agarose gel electrophoresis. While, significant decrease (p<0.05) in the DNA area and height were observed in mercury treated groups as compared to ascorbic acid and the control groups. Conclusion: The findings from this study showed that ingestion of mercuric chloride has potential deleterious effects on genomic DNA of Wistar rats. However, ascorbic acid administration demonstrate possible ameliorative potential against mercury induced molecular changes

Key words: Mercuric Chloride, Ascorbic acid, DNA, Agarose gel electrophoresis, Wistar rats

INTRODUCTION

Mercury intoxication has been a public health problem for many decades (Burger et al., 2011). Consideration of the role of environmental factors in determining the susceptibility to mercury has recently been renewed by evidence from epidemiological studies (Wang, et al., 2007). Populations are exposed to mercury through the consumption of fishes and sea foods (European Commision, 2005), dental amalgam, mining of silver, gold in industries (WHO, 2007). Mercury readily crosses the blood-brain barrier due to limited lipid solubility and can result in neurological symptoms; seizures, mental retardation, language disorders, vision, hearing and memory loss (WHO, 2003, 2007; Animoku et al., 2018). It is a potential factor in brain damage (Ibegbu et al., 2014), mental impairment, behavioral anomalies (Farina et al., 2011), but little information concerning the potential genotoxicity of mercury compounds (Barregard et al., 1991). However, heavy metals like mercury and lead had been associated with chromosomal aberrations, elongation of X-chromosome, pulverization and birth defects (Valverde et al., 2002), cell death by apoptosis and DNA fragmentation (Hassan et al., 2010). Mercuric salts can be absorbed through the skin of animals (Altmann et al., 2008), Mercury compounds rapidly enter the bloodstream (Berlin et al., 2007), and the level of mercury in the blood is higher after exposure to low dose than a high dose (Berlin et al., 2007). Elimination of mercury is via urine, feaces (WHO 2005), expired air, sweat, and saliva (Booth and Zeller, 2005). In Nigeria, fishes from Lagos Lagoon and the use of “Kohl” a traditional cosmetic had been reported as an agent of mercury toxicity (Onyeike et al., 2002). Presenting symptoms include headache, itching, dizziness, burning, excitability, irritability, restlessness, increased salivation, profuse sweating and frequent urination (Grant and Lipman, 2009; ATDRS, 2011).

Ascorbic acid is an essential nutrient for humans and some other animal species. Vitamin C is an antioxidant that protects our cells against free radicals (Padayatty et al., 2003), pneumonia (Hemila and Louhiala, 2007), prevents scurvy (WHO, 2001) and lowering the incidence of gout (Choi et al., 2009). Ascorbic acid is a well known non-enzymatic antioxidant that can break the chain of lipid peroxidation in cell membranes and scavenges free-radicals such as superoxide ion, hydrogen peroxide, nitric oxide, hydroxyl, alkoxyl and alkyl-peroxyl, collectively called reactive oxygen species (ROS) (Kacuk et al., 2003). Vitamin C is found in high concentration in immune cells and is consumed quickly during infections (Preedy et al., 2010). Ascorbic acid is absorbed in the body by both active transport and simple diffusion (Savini, et al., 2007). Examples of antioxidants are Ascorbic acid (Vitamin C), Vitamin E and Vitamin A (Vasudevan and Sreekumari, 2007). Sources of vitamin C are fruits, vegetables, liver, nutritional supplement, tablets, drinks (Wilson, 2005) and animal products (United Kingdom Food Standard Agency, 2007). Scurvy results from lack of vitamin C, it leads to the formation of brown spot on the skin, spongy gums, and bleeding from all mucous membranes (Stanner et al., 2004).

The aim of the study was to determine the role of ascorbic acid on mercury induced molecular changes and DNA alteration in Wistar rats.

MATERIALS AND METHODS

Twenty five (25) Adult Male Wistar rats of average weight 185g were used for this study. After acclimatization in the Animal House of the Department of Human Anatomy, Ahmadu Bello University, Zaria, the animals were grouped into five groups of five animals each (n = 5). Mercuric chloride (X-N202, May and Bakers, England) was utilized at LD50 of 166 mg/kg body weight as adopted from ATSDR (2011).While; the LD50 of ascorbic acid (S42238, Sam Pharmaceuticals, Nigeria) was adopted from MSDS (2008) as 11,900 mg/kg body weight. The mercury chloride was the approved laboratory grade chemical by Standard Organization of Nigeria, marketed and sold in Nigeria, while the ascorbic acid tablets was approved by National Agency for Food and Drug Administration and Control to be marketed and used in Nigeria. Before the commencement of the study, ethical approval was sort and obtained from the Ahmadu Bello University Zaria Ethical and Animal Use Committee, Faculty of Veterinary Medicine with reference Number ABU/ FVM/ EAUC/ 2015/12. The animals were dosed as follows: control group was administered with normal saline, group II with 30% mercuric chloride (HgCl2, 49.8 mg/kg) only, group III received HgCl2 with distilled water only, group IV received HgCl2 with 5% low dose ascorbic acid (595 mg/kg), while group V received HgCl2 with 10% high dose ascorbic acid (1,190 mg/kg). However, administrations of distilled water and ascorbic acid from weeks 3-6 were done in order to observe for any possible natural recovery and possible ameliorative potentials of ascorbic acid respectively (Table 1). The administration was by oral route daily and lasted for 3-6 weeks, while animal feed and water were allowed ad libitum.

MOLECULAR STUDIES

Brain tissues were collected in eppendorf tubes containing genomic lysis buffer and were taken to Avian Influenza Laboratory, Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria for Molecular analysis. The tissues were homogenized, kept in water bath overnight and were washed twice through centrifugation set at 14,000 rpm with 200µl of DNA pre-wash buffer, 200µl DNA wash buffer and DNA elution buffer respectively so as to extract the DNA. This protocol was according to the manufacturer’s instruction (Zymo research; the epigenetics company USA). 1% agarose was added to 100 µl Tris Acetic Acid (TAE) buffer and was dissolved in micro wave oven for 5 minutes and allowed to cool to about 45 ˚C; this was followed by the addition of 1µl ethidium bromide to the gel and was mixed thoroughly and gently poured into the casting tray containing the combs and allowed to solidify. Running buffer (Tris Acetic Acid EDTA) was poured into the tank while 20 μl of the DNA sample was mixed with 5 μl DNA loading dye and was loaded into the wells. The DNA sample was run for 40 minutes at 75 mV while the Gel was observed in gel documentation system and the DNA was documented.

STATISTICAL ANALYSIS

All the results were analyzed using the Statistical package for Social Scientist (SPSS version 21) and the results were expressed as Mean ± SEM. The Statistical significance between means were analyzed using one-way analysis of variance (ANOVA) followed by post HOC test; Turkey’s multiple comparison test to test for statistically significant difference between control and experimental groups. A p-value < 0.05 was considered statistically significant.

RESULTS:

MOLECULAR STUDIES

The results from molecular studies as observed from the Genomic DNA showed that the level of migration of the genomic DNA on agarose gel electrophoresis of the animals in Groups II and III appeared fastest characterized by lighter DNA bands when compared to Groups I (Control), IV and V as shown in Plate I.

MEASUREMENT OF GENOMIC DNA AREA, WIDTH, HEIGHT, PERIMETER AND LENGTH OF MIGRATION OF DNA FROM WELL

The measurement of Genomic DNA area, width, height, perimeter and length of migration of DNA showed significant decrease (p<0.05) in the measured areas and heights of the genomic DNA in Groups II and III when compared to Group one (Control) and Group V, while, measurement of the length of migration of genomic DNA revealed significant increase (p<0.05) in Group II when compared to Groups I (Control), III, IV and V respectively as shown in Table 2.

DISCUSSION

The present study was on whether or not mercuric chloride induced toxicity was accompanied by changes in the Genomic DNA and the role of ascorbic acid. The study revealed that the level of migration of the Genomic DNA as characterized on agarose gel electrophoresis were fastest in Groups treated with mercury only, suggesting lesser molecular weights of the DNA molecules which could be related to the effect of mercury on the Genomic DNA. The study showed significant increase (p<0.05) in the mean Genomic DNA length measured from the well in Groups exposed to mercury only when compared to ascorbic acid treated groups and animals in the Control Group. This study agrees with the work of Hasan et al., (2010) who reported that liver cell DNA of mercury treated fishes appeared further away from the well when compared to the position of the Control Group and this change in position was related to cell death by apoptosis accompanied by DNA fragmentation. In another study, it was reported that DNA damage in mercury exposed individuals suggests that mercury overload induces an imbalance in the redox cycle (Hasan et al., 2013). The present study revealed significant decrease (p<0.05) in height and area of the Genomic DNA of animals exposed to mercury only when compared to animals treated with ascorbic acid and animals in the Control Group suggesting the toxic effects of mercury and the possible ameliorative effects of ascorbic acid against mercury induced DNA damage. However, further study on Polymerase chain reaction (PCR), DNA sequencing and comet assay (Tice et al., 2000) could further reveal any alteration in the Genomic DNA sequence or if any mutation might have occurred following mercury exposure and if this alteration could possibly be ameliorated by ascorbic acid, since it was suggestive that ascorbic acid can protect against DNA damage (Cail et al., 2001; Diatrich et al., 2003).

CONCLUSION

The findings from the present study justify the ameliorative potential of ascorbic acid against mercury induced molecular changes and DNA alteration; hence the consumption of ascorbic acid should be encouraged to populations exposed to increased risk of mercury poisoning.

Conflict of Interest

None declared.

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3. Animoku AA, Buraimoh AA, Hamman WO, Ibegbu AO, Samuel SB, Uthman AY, Joseph SM, Akpulu, P. (2018). Ameliorative effect of ascorbic acid (vitamin c) on mercury induced temporal lobe damage in rat. Nigerian Journal of Neuroscience. 9(1):9-15.

4. Barregard, L., Hogstedt, B., Schutz, A., Karlsson, A., Sallsten, G. and Thiringer, G. (1991). Effects of occupational exposure to mercury vapor on lymphocyte micronuclei. Scandinavian Journal of Work, Environment and Health, 17(4):263–268

5. Berlin, M., Zalups, R.K. and Fowler, B.A. (2007). “Mercury,” in Handbook on the Toxicology of Metals, G.F. Nordberg, B.A. Fowler, M. Nordberg, and L.T. Friberg, Eds., Chapter 33, Elsevier, New York, NY, USA, 3rd edition.

6. Booth, S. and Zeller, D. (2005). “Mercury, food webs, and marine mammals: implications of diet and climate change for human health,” Environmental Health Perspectives, vol. 113, no pp. 521-526.

7. Burger, J., Jeitne, C. and Gochfeld, M. (2011). Locational differences in mercury and selenium levels in18 species of saltwater fish from New Jersey. Journal of Total Environmental Health;74(13):863- 74.

8. Cail, L., Koropatrick, J. and Cherian, M.G. (2001). Roles of vitamin C in radiation induced DNA damage in the presence and absence of copper. Chem. Biol. Interact., 137: 75-88

9. Choi, M. D., Gao, X., Curhan, G., Xiang Gao, M. D. and Gary Curhan, M. D. (2009). Vitamin C Intake and the Risk of Gout in Men. Archives of Internal Medicine. 169 (5): 502–507.

10. Diatrich, M., Block, G. and Benowitch, N.L. (2003). Vitamin C supplementation decreases oxidative stress biomarker F2-isoprostane in plasma of non-smokers exposed to environmental tobacco smoke. Nutr. Cancer, 45: 176-184.

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12. Farina, M., Rocha, J.B. and Aschner, M. (2011). Mechanisms of methylmercury-induced neurotoxicity: Evidence from experimental studies. Journal of life Sciences 89:555–563

13. Grant, L.D. and Lipman, M. (2009). Human exposure and their effect. Environmental toxicant; 3rded:108-112.

14. Hasan, M.A., Ahmed, M.K., Akhand, A.A., Ahsan, N. and Islam, M.M. (2010). Toxicological effects and molecular changes due to mercury toxicity in fresh water snakehead (Channa punctatus). International Journal of Environmental Research 4(1):91-98.

15. Hasan, F. A., Akram, Umran and Nadhem, H. H. (2013). Oxidative Stress, Antioxidant Status and DNA Damage in Mercury Exposure Workers. British Journal of Pharmacology and Toxicology 4(3): 80-88.

16. Hemila, H. and Louhiala, P. (2007). Vitamin C for preventing and treating pneumonia.Cochrane Database System Rev (1).

18. Ibegbu A.O., Ayuba, M., Animoku, A. A., Brosu, D., Adamu, S. A., Akpulu, P., Hamman, W.O., Umana, U.E. and Musa, S.A. (2014). Effect of Ascorbic Acid on Mercury Induced Changes on the Liver in Adult Wistar Rats. IOSR Journal of Dental and Medical Sciences, Volume 13, Issue 10 Ver. II):10-16.

19. Ibegbu, A.O., Animoku, A.A., Ayuba, M., Brosu, D., Adamu, S.A., Akpulu, P., Hamman, W.O., Umana, U.E. and Musa, S.A. (2014). Effect of Ascorbic Acid on Mercuric Chloride Induced Changes on the Histomorphology of the Cerebellum of Wistar Rats. African Journal of Cellular Pathology, Vol.3:9-15.

20. Kacuk, O., Sahin, N., Sahin, K., Gursu, M. F., Gulcu, F., Ozcelik, M. and Issi, M. (2003).Egg production, egg quality and lipid peroxidation status in laying hens maintained at a low ambient temperature (6%) and fed a vitamin C and vitamin E-supplemented diet. Veterinarni Medicina, 1-2: 33-40.

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The post The Role of Ascorbic Acid on Mercury Induced Molecular Changes and DNA Alteration in Rats. appeared first on Nigerian Biomedical Science Journal.

Olufemi SS, Faborode MB, Mustapha RA, Oyegoke TG

Department of Nutrition and Dietetics, Faculty of Applied Sciences, Rufus Giwa,

Polytechnic, P M B 1019, Owo, Ondo State, Nigeria

Adeniran SM

Department of Nutrition and Dietetics, C.S.T, Kaduna Polytechnic, Kaduna

All Corresponding to: Olufemi SS, olufemisamuel2175@gmail.com

ABSTRACT

Background: Breastfeeding is certainly an important child feeding practice, which promotes the wellbeing and survival of children, especially in the first two years of life. Objective: The objective of the study is to assess the impact of maternal education and health facilities on the breastfeeding practices of mothers in Lagos and Ogun State, Nigeria. Material and Methods Three hundred and two (302) breastfeeding mothers and their children were selected using systematic sampling techniques from (5) government and private hospitals in Lagos and Ogun States. Semi-structured interviewer administered questionnaire was administered to obtain information on socio-demographic status, clinical status, knowledge and attitude of mothers towards breastfeeding practices and impact of health facilities on the breastfeeding mothers. Data obtained were analyzed using descriptive statistics and Pearson correlation method. Results: Results showed that more than 60% (198) of the children were between the ages of 0-8 months of which half were males. Majority (76.8%) of the mothers had post-secondary education. More than half 62.3% earning an income of #30,000-#50,000. Clinical characteristics of the breastfeeding mothers showed that majority of the children were delivered through normal delivery and 2.6% had low birth weight. Mothers’ knowledge of breastfeeding practice revealed that half of the population initiated breastfeeding one hour after delivery and majority (94.4%) of mothers gave colostrum to their children. The result also showed that 48.3% of the mothers preferred private hospital and 33.8% of the mothers preferred government hospital. More than half (55.3%) of the mothers gave birth in the private hospital. Conclusion: The study concluded that prevalence of exclusive breastfeeding is low while a high number of mothers preferred private facilities to public facilities during child birth. Recommendation: There is a need for policies which will promote exclusive breastfeeding, encourage mothers and improve quality of health facilities to promote the practice of breastfeeding in both private and public health facilities in Nigeria.

Keywords: Exclusive breastfeeding, Breastfeeding practices, Health facilities, Children , Mothers

INTRODUCTION

Breastfeeding is the feeding of an infant or young child with breast milk directly from female human breasts (i.e.via lactation) rather than using infant formula from a baby bottle or other container [5]. According to a statement by World Health Organization (WHO), Breastfeeding is the ideal method suited for the physiological and psychological needs of an infant [19]. Breastfeeding is an important public health strategy for improving infant and child morbidity and mortality, improving maternal morbidity, and helping to control health care costs [12]. The Nigerian health care has suffered several down-falls. Despite Nigerian’s strategic position in Africa, the country is greatly underserved in the health care sphere. Health facilities (health centers, personnel, and medical equipments) are inadequate in this country, especially in rural areas [10]. According to the 2009 communiqué of the Nigerian national health conference, health care system remains weak as evidenced by lack of coordination, fragmentation of services, dearth of resources, including drug and supplies, inadequate and decaying infrastructure, inequity in resource distribution, and access to care and very deplorable quality of care [18].

Furthermore, It is difficult to ascertain the size of the private health care sector in Nigeria since a sizeable number of existing health facilities are believed to be operating without appropriate licensure by State Ministries of Health (SMOHs). Furthermore, private facilities seldom forward to the SMOH information reflecting health facility case-loads, available personnel and technologies in any given year [3]. In particular, information on private sector personnel resources is compromised by the fact that a considerable proportion of health workers (especially doctors, pharmacists, radiographers and laboratory technicians) working in the private sector hold full-time appointments in the public sector [10]. Faith-based providers are grouped as part of the private, nonprofit sector, and often integrate themselves into national health systems by agreeing with the government to provide specific services. Yet faith-based providers are often more “public” than “private” in both practice and ethos. They often fill gaps left by others, or are designated district hospitals to which other private or lower-level public facilities report. The most obvious example is in Nigeria, where a coalition of churches and faith-based providers manage and operate more than half the national health system [13]. Therefore, this study aimed at assessing maternal knowledge and impact of health facilities on breastfeeding practices of mothers in Lagos and Ogun State.

MATERIALS AND METHOD

Study Design

A cross-sectional descriptive study was used to assess the impact of health facilities on the breastfeeding practices of mothers in selected health facilities in Lagos and Ogun state.

Study Location

The study was carried out in some selected Local Government Areas (L.G.A) in Lagos and Ogun State

Study Population

The study focused on breastfeeding mothers and their children that are between age 0-24months.

Ethical consideration

A written permission to carry out this research was obtained from each of the hospitals within the study area, verbal consent were also received from the mothers after the purpose and procedure of the study was read to the participants, giving the mothers a fair understanding into the study aims and the chance to freely choose to participate in the study.

Sampling Technique

Five Local Government Areas (LGAs) 5 health facilities were purposively selected for the study (three (3) public hospitals and five (5) private health facilities). One hundred and fifty-two (152) mothers with their children were further selected from government hospitals and One hundred and fifty (150) mothers with their children from private and faith-based hospitals systematic sampling. A total number of Three hundred and two (302) breastfeeding mothers with

Statistical Analysis

Data analysis was carried out with the aid of Statistical Package for the Social Sciences (SPSS) version 21.0. Questionnaires were scrutinized for completeness. Relevant descriptive and inferential statistics was used to analyze data.

RESULTS

Socio-demographic characteristics of the respondent

Table 2 shows that104 (34.4%) of the children were between the age 0-5months, 94 (31.1%) were between the ages of 6-8 months, 43 (14.2%) were between the ages of 9-11 months while 61 (20.2%) were between12-24 months. Result also showed that 153 (50.7%) of the children were males and 146(48.3%) were females. The results also showed that majority (78.6%) had tertiary education and 17.3% obtained secondary education. Estimated family monthly income shows that, 39 (12.9%) earned below N10,000, 61 (20.2%) earned between N10,000- N30,000 while 67 (22.2%) earned

above N50,000.

Clinical Characteristics of the respondents

Table 2 revealed the clinical data of the respondents. It shows that 8 (2.6%) of the children were less than 2.5kg in weight at birth, 248 (82.1%) weighed between 2.5kg-5.0kg at birth while 14(4.6%) weighed above 5.0kg. The results showed 295(97.7%) had no birth defects or malformation as at the time of the study and 3(1.0%) had birth defects. The clinical data also shows that 11 (3.6%) of the children suffered illness and 283 (93.7%) had no illness.

Maternal Knowledge of Mothers on the Practice of Exclusive Breastfeeding

The results in table 3 shows that thirty six (11.9%) of the mothers introduced breastfeeding immediately after delivery, (31.8%) introduced it 30minutes after delivery while (53.6%) of mothers introduced one hour after delivery. In the practice of exclusive breastfeeding, 49.0% of the mothers fed their children with only breast milk, 70 (23.2%) fed their children with breast milk and water. Colostrum was given to the children immediately after birth by 285 (94.4%) of mothers while 12(4.0%) didn’t give colostrum.

Table 4 shows that two hundred and seventy nine (92.4%) revealed that the health facilities were good and encouraged them to adequately breastfeed, 16 (5.3%) indicated average, 2 (0.7%) of the mothers reported that the health facilities were poor. Majority (97.7%) agreed that the health workers are competent and practice good hygiene, 98.3% of the respondents reported that there is no nutritionist in the facilities.

Mothers preference of health facilities as a result of breastfeeding practices

Figure 1 shows that one hundred and forty six (48.3%) preferred the private hospitals for their breastfeeding practices, 102 (33.8%) preferred Public/Government hospital and 48 (15.9%) preferred faith-based hospital.

Figure 1: Mothers preference of health facilities as a result of breastfeeding practices

Health facilities by child delivery

Figure 2 shows that one hundred and sixty-one (53.3%) gave birth to their children in private hospitals, 106 (35.1%) Public/Government hospital hospitals, 21 (7.0%) for faith-based and 6 (2.0%) mothers specify other places.

DISCUSSION

This study assessed breastfeeding practices among nursing mothers attending selected government and faith-based health facilities and the impact of health facilities on breastfeeding practices of mothers in the selected facilities. Proper and Adequate health facility with good nutrition practices during infancy and early childhood is very essential to ensure the optimum growth, healthy lifestyle and development of children to their full potential.

The study revealed that a high number of the children were between the ages of 0-8 years, this is because the study focused on children of age 0-24 months

Majority of the mothers who participated in the study (92.4%) were married and a high number of them were highly educated, with 75.2% having of tertiary education. The impact and support from husband in breastfeeding practices cannot be underestimated. In line with other studies [15], this study showed that educated mothers will have a fair knowledge about the meaning of exclusive breastfeeding and its recommendation as proposed by WHO and UNICEF that infant should be exclusively breastfed for six months. Educated mothers are more likely to expose to health information and make use of such information especially that which concern their children. The study shows that 42.2% of the mothers were trader/business woman; this tends to be higher compared to findings of Onah et al., [17] which reveals that 9.1% of lactating women were traders/business women. The clinical profile revealed that majority of the children (82.1%) had normal birth weight This supports the findings of Akinyemi et al., [1] that 81.7% of live births in urban Nigeria between 2008 and 2018 reported normal birth weight (2,500g and 4,000g. The prevalence of HBW, estimated to be 10.7%, was higher than the LBW estimated at 7.5%. Birth weight is a strong predictor of child’s health and development. A low birth weight (LBW) of less than 2,500g is associated with adverse health outcomes and is a leading risk factor for neonatal morbidity and mortality.

Some of the mothers considered breast milk as the best form of food and nutrition for infants, they agreed to the concept of six months exclusive breastfeeding, however, feeding children with only breast milk, exclusive breastfeeding (EBF) rate of 49% recorded in this studies is far below the expected 90% set by WHO [20] for the EBF to produce a reasonable impact in the population. The introduction of colostrum had increased among the breastfeeding mothers in this study with the rate 94.4%, This is higher than what has been documented by other studies in Nigeria [2].

In this study, timely initiation of breastfeeding (within one hour of birth) found among majority of the respondents in this study is commendable with53.6% of the mothers initiated breastfeeding within the first hour after delivery while 31.8% initiated breastfeeding thirty minutes after delivery. This result is higher compare to the findings of Ọládòyìnbó et al.,[16] which revealed that 28.6% of the infants who initiates breast milk in less than 1 hour. Early breastfeeding initiation has been found to increase mother to child bonding, regulate infant temperature, breathing and enhance breastfeeding longevity and findings on the time of breastfeeding initiation on nutritional status of mother and infant shows that within 24 hours is the best [4].

The results of this survey indicated that 49.0% of the mothers had good knowledge and positive attitude towards exclusive breastfeeding. This study also found out that more than half of the mothers obtained their recommended length of breastfeeding information from health workers, 16.6% from media and 2.0% from family/husband. This is again almost similar to the findings of Bolanle [7], in which 64.5% of the mothers obtained such information from health workers, 9.0% from the media and 7.3% from their husbands. The finding of this study shows that decreased value which was 35.1% of the mothers gave birth at public (government hospitals). This is lower when compared to the reports of Nigeria Demographic and health survey [14], that 43.2% of the mothers in Nigeria give birth in public/government hospitals across the nation. The impacts of health workers cannot be underestimated in breastfeeding, even their knowledge also contribute to the effective practice of breastfeeding [11]. Mothers assessed in this study were satisfied with services of the health workers in terms of competency and good hygiene practices (97.7%). Successful breastfeeding is dependent on an array of factors related to the mother, infant, and supportive environment. Although the majority of the women in the study cited support from the hospital staff and partner or family members. The presence of a support system, whether it is personal or professional, has a positive influence on the duration of exclusive breastfeeding. Research has shown that peers, partners, and families play a major role in helping and supporting mothers to continue EBF for six months, thereby significantly decreasing the risk of early is continuation of EBF before six months [6], [[9].

Conclusion

This study brings to the conclusion that there is awareness on exclusive breastfeeding among women attending public (government owned hospitals) and private hospitals bur the practice of exclusive breastfeeding is still very low in urban areas. In addition, the study gave account of almost no presence of nutritionist could deliver adequate information about diet and nutritional intake of the breastfeeding mothers in all the health facilities

Recommendations

Based on the result findings of the study, the following recommendations were made:

Exclusive breastfeeding counseling should be more elaborative with emphasis on its advantages in both private and government hospitals in the study are. The government should to employ the services of nutrition experts in all the health facilities to improve breastfeeding practices and the consumption of adequate diets that will support growth and development of mothers and their babies.

REFERENCES

1. Akinyemi, J.O., Bamgboye, E.A. & Ayeni, O. (2015) Trends in neonatal mortality in Nigeria and effects of bio-demographic and maternal characteristics. BMC Pediatr.

2. Agbo, H.A., Envuladu, E.A., Adams, H.S., Inalegwu, E., Okoh, E. & Agba, A. (2016) Barriers and facili- tators to the practice of exclusive breast feeding among working class mothers: a study of female resident doctors in tertiary health institutions in Plateau State. J Med Res; 2(1):112-6.

3. Agunbiade, M.O. & Ogunleye, V.O. (2012). Constraints to exclusive breastfeeding practice among breastfeeding mothers in southwest Nigeria: implications for scaling up. International breastfeeding Journal; 7:5.

4. American Academy of Pediatrics. (2005). Policy statement: Prevention of pediatric overweight and obesity. Pediatrics, 112(2), 424-430.

5. Awatef, M., Olfa, G., Imed, H., Kacem, M., Imen, C., Rim, C., Mohamed, B. & Slim, B.A. (2009). Mothers knowledge, attitude, beliefs and practices concerning exclusive breastfeeding in Calabar. African journal of nursing and midwifery. 11(1). 65-75.

6. Bevan, G.& Brown, M. (2014). Interventions in exclusive breastfeeding: A systematic Rates and determinants of exclusive breastfeeding in first 6 months among women in Nova Scotia: A population- based cohort study. CMAJOpen, 1(1), E9-E17.

7. Bolanle, A.J. (2013). Appraisal of Nursing Mothers’ Knowledge and Practice of Exclusive Breastfeeding in Yobe State, Nigeria, Journal of Biology, Agriculture and Healthcare 3(20), available atwww.iiste.org.

8. Brand, E., Kothari, C.& Stark, M. (2011). Factors related to breastfeeding discontinuation between hospital discharge and 2 weeks postpartum. Journal of Perinatal Education, 20(1), 36-44.

9. Demirtas, B. (2012). Strategies to support breastfeeding: A review. International Nursing Education; 18(2):32-39.

10. Lawoyin, T.O., Olawuyi, J.F. & Onadeko, M.O. (2011). Factors Associated with Exclusive Breastfeeding in Ibadan. Nigeria Journal of Human Lactation, 17: 321-325.

11. Inayati, V., Dery, M. & Gaa, P.K. (2012). Knowledge, attitudes and determinants of exclusive breastfeeding practice among Ghanaian rural lactating mothers. Int Breastfeed J;11(1).

12. James, D.C.& Rachelle, L. (2009). “Promoting and supporting breastfeeding.,” J Am Diet Assoc, vol. 109, no. 11, p. 126–142

13. Jones, S.C.,Eval, M.,Telenta, J.&Cert, G., Shorten, A. & Johnson, K. (2011). Midwives and pregnant women talk about alcohol: What advice do we give and what do they receive? Midwifery; 27:489–496.

14. Nigeria Demographic and Health Survey. Abuja: Federal Republic of Nigeria; https:// dhsprogram. com /pubs/pdf/ fr293/fr293.pdf.

15. Oche, M.O., Umar, A.S. & Ahmed, H. (2011). Knowledge and practice of exclusive breastfeeding in Kware, Nigeria. Afr Health Sci 2011, 11(3):518–523.

16. Oladoyinbo, E.N., Antia-Obong, O.N., Udo, J.J. & Edet, E.E. (2016). Maternal exclusive breastfeeding practice in Calabar Nigeria: Some related social characteristics. Nigeria J Clin Pract.;6(2):92–4.

17. Onah, S., Osuarah, D.I.C., Ebenebe, J., Ezechukwu, C., Ekwochi, U. & Ndukwu I. (2014) Infant feeding practices and maternal socio-demographic factors that influence practice of exclusive breastfeeding among mothers in Nnewi south- East Nigeria: A cross- sectional and analytical study. Int Breastfeed J.;9:6

18. Sholeye, O.O. & Abosede, O.A. (2015). Exclusive Breastfeeding and Its Associated Factors among Mothers in Sagamu, Southwest Nigeria. Journal of Health Science; 5(2):25-31.

19. Subbiah, N. (2013). A Study to assess the Knowledge, Attitude, Practice and Problems of Postnatal Mothers regarding Breastfeeding Nursing J India; 94 (8): 177-179

The post Assessment of Maternal Knowledge and Impact of Health Facilities on the Breastfeeding Practices of Mothers in Lagos and Ogun State, Nigeria appeared first on Nigerian Biomedical Science Journal.

Sadeeq AA, Amos A, Bauchi ZM, Tanko M and AI Mukthar

Department of Human Anatomy, Neuroscience Unit, Faculty of Basic Medical Sciences, Ahmadu Bello University, Zaria. Nigeria.

Muhammad Z

Department of Human Anatomy, Faculty of Basic Medical sciences, Damaturu, Yobe state University. Nigeria.

El-ladan IS

Department of Human Anatomy, Faculty of Basic Medical Sciences, Umaru Musa Yar’addua University, Katsina, Nigeria.

El-ladan IS

Department of Forensic Sciences, School of Basic and Applied Sciences, Golgotia University, Uttar Pradesh, India

Rilwan BH

Department of Human Anatomy, Faculty of Basic Medical Sciences, Kaduna State University, Kaduna, Nigeria.

All Correspondences to: SadeeqAA, Neuroscience unit, Department of Human Anatomy, Faculty of Basic Medical Sciences, College of Medical Sciences, Ahmadu Bello University, Zaria. e-mail:aasadeequ@gmail.com

ABSTRACT

The herbicide Paraquat, is a superoxide generator that harm neurons, chunk motor axon conduction,and impair locomotive activities.This study evaluates the ameliorative effects of ethanolic extract of tomato (Solanum esculentum) on Paraquat-induced cerebellar cortex and cervical spinal nerve segment lesion of adult Wistar rats. Twenty (20) apparently healthy rats averagely weighing 144g were divided into five groups of four animals each. Group I (control) received normal saline. GroupsII, III and IV were administered 30mg/kg of Paraquat,were administered 30mg/kg of Paraquat followed by tomato extract (1500mg/kg and750mg/kg respectively). Groups Vwere treated with 1500mg/kg tomato extract only. Treatments was via oral route and lasted for twelve consecutive days. Gripstrength and Footprint test for motor coordination and balance were the neurobehavioral studies carried out to ascertain effects on motor coordination.Cervical spinal nerves segment and cerebellar tissue were accessed by opening the hollow vertebral column and sutures of the skull respectively. Tissues were fixed in Bouins fluid, processed using routine Histological stain H and E.Cyto-morphometric evaluation for cell volume and number using a digimizer v4.0 was carried out. Graphad prism v8.0.1 was used for the analysis. Treatment with Paraquat dichloride impaired motor activities of the forelimbs and balance coordination with significant decrease in time taken to release the grip wire (0.01) with an increase in numbers of foot slip that was significant(0.01).Histopathological changes such as nuclei fragmentation or loss and vacoulation were observed in the cerebellum and cervical spinal segment of the spinal cord. However, administration of tomato extract revealed a neuroprotective potential to the spinal cord by improvement in the footprint distance relative to the control;histopathological lesionswere ameliorated in the treated group whencompared with the control.

Keywords:Paraquat, solanum esculentum, Cerebellum, cervical Spinal segment,foot print, motor coordination and balance.

INTRODUCTION

Paraquat is a wild acting, non-selective bipyridylium herbicide extensively used in numerous countries in the world due to the stumpy cost and efficacy against a range of weeds (Costa et al., 2014). It is a quaternary nitrogen herbicide with chemical name 1,1′- dimethyl 4,4′-bipyridinium (Raghu etal., 2013). Which is extremelylethal to both humans and animals by all routes of exposure, and is labelled with a Danger-Poison signal word.Paraquat has been used for withering of seed crops, weed control in orchard; defoliation and desiccation of cotton; as harvest aid in soya bean, sugar cane and for killing dormant plants (Ecobichon, 1991; Raghu et al., 2013).

Paraquat through blood stream it reaches all the areas of the body, leading to lung damage, liver and kidney damage (Podpresartet al., 2007). Inhalation of this herbicide can lead to sore nose and throat and nose bleeding (Baharuddinet al., 2011). Dermal contact can lead to mild irritation, ulceration, blisters, desquamation (loosing of outer skin layer), necrosis and second degree burns (Marrs and Adjei, 2003). Contact with the eye can lead to eye irritation, inflamed eyelids and decreased visual acuity (Wineket al., 2001).Paraquat was revealed to have cause drop in neurotransmitters (Qamar at al., 2013), dead of both matured and immature cerebellar granule neaurons (Stelmashooket al., 2007), caused cerebral hemmorrage (Saeed et al., 2001). Chronic exposure to paraquat is also a potential etiological factor for development of parkinson’s disease (Abdulwahid and Ahmad 2010). A single large dose administered orally or by injection to animals, can cause excitability and lung cramming, which in some cases leads to convulsions, incoordination, and death by respiratory failure. Many cases of illness and/or death have been recorded.Paraquat is not naturally occurring, it is synthesized. Most poisoning is reported to be human deliberate action for suicidal purpose (Dinis-oliveriaet al., 2008).The herbicide paraquat, is a superoxide generator (Liu et al., 1995). These oxidants destroy neurons, block motor axon conduction, and impair open field locomotion, hind limb function, and inclined plane stability when generated in lumbar spinal cord gray matter (Bao and Liu 2002; Liu 1994;Carolina et al., 2019)

Tomato contains valued phytochemicals and antioxidants including carotenoids such as lycopene and beta-carotene (Canene-Adams et al., 2005). It has health effects which include lowering of cholesterol (Palozza and Catalano 2012), anti-inflammatory and antioxidative effect, causes a decrease in risk of blood clotting (Risoet al., 2006), decreases cancer prevalence (Ford et al., 2011), guard against sun burn and promote vision.

Paraquat is a toxic herbicide widely used in developing countries which causes various neurological disorders to both animals and human beings. People are exposed to the potential risk of herbicide and agricultural pesticide via ingestion of food contaminated by Paraquat or accidental exposure.This study determined the evaluating the ameliorative effect of ethanolic extract of tomato on the cerebellum and spinal cord changes induced by paraquat dichloride.

MATERIALS AND METHODS

Experimental Design

Twenty adult male Wistar rats were obtained from the Faculty of Pharmaceutical Sciences, Ahmadu Bello University, with average weight of 144g. The animals were housed in wooden cages with wire gauze covering to allow free ventilation with sow-dust bedding for comfort. They were allowed to acclimatize for three (3) weeks in the animal house of the Department of Human Anatomy, Ahmadu Bello University, Nigeria. Animals were fed daily with vital feed and clean water was provided ad libithum. Animals were randomly divided into five (5) groups of four (4) animals per group.

Animal Handling and Usage

Animals used for the study was according to Ahmadu Bello University, Nigeria, Committee on Animal Use and Care (ABUCAUC)

Chemical Substance

Paraquat dichloride containing herbicide slasher was manufactured by Sinochem Ningbo Ltd, with a batch number 2017101001. The LD50 was adopted from the manufacturer as 100mg/kg. While tomato ethanoic extract LD50was adopted as 500mg/kg according to Wathita et al., (2013)

Administration of Paraquat

Animals were divided into five groups of four animals per group (Group I,II, III,IV and V). Group I (control) received normal saline. Groups II, III and IV were administered 30mg/kg of Paraquat, were administered 30mg/kg of Paraquat followed by tomato extract (1500mg/kg and750mg/kg respectively). Groups V were treated with 1500mg/kg tomato extract only. Treatments was via oral route and lasted for twelve consecutive days.

NEUROBEHAVIORAL STUDIES

Grip strength test

These tests involve the forepaws of the rats being placed on a horizontally suspended metal wire (measuring 2 mm in diameter and 1 m in length), placed one meter above a landing area filled with soft bedding. The length of time each rat was able to stay suspended before falling off the wire is recorded. A maximum time of 2 minutes is given to each rat after which it will be removed. This test reflects muscular strength and balancecoordination in the animals (Tamashiro et al., 2000).

Footprint test for motor coordination and balance Measurement of motor coordination and balance can be used not only to assess the effects of test compounds or other experimental manipulations on mice and rats, but also to characterize the motor phenotype of transgenic or knockout animals (Carter et al., 2001). To obtain footprints, rat paws were painted with nontoxic paints and the mouse is allowed to walk along a narrow, paper- covered corridor, leaving a track of footprints. According to the method established by (Carter et al., 2001), a wooden open-top runway of 100 cm long, 10 cm wide, with walls 20 cm high with an enclosed goal box at one end was set up in the experimental room.The sheet of paper from the runway was removed and the footprint patterns were allowed to dry in a well-aerated room for more than an hour before storing. Each rat was given three consecutive trials on each of three consecutive days of training. For each step parameter, three values are measured from the middle portion of each runway trial, excluding footprints made at the beginning and end of the trial where the mice initiate and finish movement, respectively and the mean of each set of three values is used in the analysis.

Animal Sacrifice

Animals were humanely sacrificesa day after the last administration. Which was anesthetized using chloroform.The brain was removed by opening through the sagittal suture of the skull and ndfixedin aBouin’s fluid for proper preservation and tissue processing. The cervical spinal cord segment was accessed via the vertebral columnby cutting off the entire vertebrae cervical region which was done after fixingin Bouin’s fluid for proper fixation and easy access to the cervical spinal segment (Sadeeq et al., 2017).

Tissue Processing

Brain tissue (cerebellum) and cervical segment of the spinal cord were processed routinely for histopathological studies using Haematoxylin and Eosin staining procedure. Tissue sections were viewed under light microscope and photomicrographs were taken using digital Amscope (MD-900) microscope camera.

Cytometric Analysis

Cell volume is the amount of space the cell occupies and is found by multiplying the length of the cell by the width and the height of the cell..H&E staining technique was used in order to stain the nucleus of the neural cells purple , cell number was counted when nucleus when it is focused in the optical dissector. Digimizer v 4.0 @ medcalc Software was used for calculating the volume and counting cells.

Statistical Analysis

Data obtained was expressed as Mean Standard Error of

100

?

???

80

60

time(s)

40

20

Mean (SEM)One-way analysis of variance(ANOVA) was used to compared the differences between and within the groups A P- Value less than 0.05 was considered to be statistically significant.

RESULTS

The fore grip strength test in figure 1, indicates a significant (p≤0.001) decrease in time taken to release the grip wire among groups treated with Paraquatalone

(30mgkg) when compared to the control and the extract group (1500mgkg).An increase that was significant (p≤ 0.05) in time taken holding on the wire was observed

among groups treated with Paraquat and 1500mg/kg extract of tomato when compared to the groups that received Paraquat/750mg/kg extract and Paraquat (30mg/kg) alone.

Control

Paraquat + 1500mg/kg extract 30mg/kg (Paraquat)

Paraquat + 750mg/kg extract

1500mg/kg extract

0 Groups

Figure 1: mean time taken for animals to release grip wire during fore grip strength test

Table 1. Distance of the forelimb limb strides and base among experimental animals

One-way ANOVA, **= p ≤ 0.01. RFLS= Right Forelimb Stride; LFLS= Left Forelimb Stride; FB= Forelimb Base,s(time in seconds)

Histological Observation

A section of Wistar rat cerebellum of the control group treated with normal saline. Showing normal histoarchitecture of the cerebellar tissue; Molecular layer (ML), Purkinje layer (PL), Purkinje cell (PC) and Granular layer ,(GL). loss purkinje cells (PCS), Degenerating cells (DC), Clumped cells (CC), Disintegrated nuclei (DN) H and E stain (Mag ×250).

A cross section of the cervical spinal segment of the spinal cord anterior horn cell with normal cytoarchitecture cell body (CB), Nucleolus (NC). Nuclei, and congested nuclei (CN). normal cells (NC),loss of nuclei(LN) , loss of neuronal fibres (LF), disintegration nuclei (DN), compacted cells (CC) nerve fibre (NF) and nuclei intact (NI). H&Ex250.

Mean cell volume

Purkinje cells in the cerebellum indicates a significant decrease(p≤0.05) in the mean cellular volume among group treated with Paraquat alone(30mg/kg) when compared with the control and other treated groups An increase in cell volume was also observed among Groups administered 30mg/kg Paraquat/1500mg/kg extract and 30mg/kg Paraquat/750mg/kg extract but not significant when compared to the control and extract group Though, a significant increase (p≤0.01) of cellular volume was noticed in extract treated group (1500mgkg) when compared with all experimental groups and control. Motor

neuron cells in the cervical spinal segment revealed that there was significant increases in cellular volume among group treated with extract alone (1500mg/kg) when compared to the control group and all experimental groups. While group administered with Paraquat alone (30mg/kg) decrease significantly (p≤0.05)when compared with control and

Paraquat/extract combine groups, shows in figure 2b.

Cell count

Figure 2: Mean cellular volume of the Purkinje cells in the cerebellar cortex and Motor neurons in cervical spinal segment (CSS)

Purkinje cells counted in the cerebellum increases significant (p≤0.01) among group treated with tomato extract alone (1500mg/kg) when compared with the control and other treated groups. While an increase (p≤0.05) in cells number was observed among Groups administered

30mg/kg Paraquat/1500mg/kg extract when compared to the control and 30mg/kg Paraquat/750mg/kg extract group, Though, a significant decrease (p≤0.01) of cellular number

was noticed in group treated 30mg/kg of paraquat when

compared with all experimental groups and control as shown in figure 3 Motor neuron cells in the cervical spinal segment revealed that there was a significant increase in cellular number among group treated with extract alone (1500mg/kg) when compared to the control group and all experimental groups. While group administered with Paraquat alone (30mg/kg) decrease significantly (p≤0.05)

when compared with control and Paraquat/extract combine

groups.

Figure 3: Mean cellular number of the Purkinje cells in the cerebellar cortex and Motor neurons in cervical spinal segment (CSS)

DISCUSSION

An increase in forelimb grip mean time was observed among control groups compared to the seventh day which is possibly due to acclimatization to training, a significant increase was also observed among group 30mg/kg + 1500mg/kg Extract animals who were treated with 30mg/kg Paraquat followed by 1500mg/kg tomato extract in day twelve compared to day seven which is possibly due to the ameliorative potential of tomato against neurotoxins which is similar to the finding of Owoeye and Farombi (2015), who reported that treatment of rats with Tomato extract ameliorated the effect of Mercury chloride on cerebellum and increased forelimb strength and movement. In the footprint test, right forelimb stride, left forelimb stride and forelimb base were used to characterize the level of spinal cord damage in the cervical spinal segment of animal rats model used in this study. A decrease in the distance between the left and right forelimb stride was seen in the group that received paraquat alone this may be due to motor impairment elicited by paraquat as described by Liu et al., (1995). An increase in the distance of the left and right forelimb stride in the group that received the fruit extract, this may be due to the cyto-protection elicited by flavonoids which are present in tomato, this is necessary for the maintenance of proper nerve connections as shown by O’Neill et al., (2001).

It was observed that the group treated with 30mg/kg of paraquat had a loss of purkinje cells which could be as a result of paraquat induced cell degeneration. Jayasinghe and Seneviratne (2016) reported degenerative changes in Purkinje cells and granular cells of the cerebellum as a result of paraquat ingestion. This can in turn lead to loss of motor coordination and balance Songin et al., (2010) reported significantly reduced or disturbed motor activity among rats treated with paraquat. The group treated with 30mg/kg of paraquat followed ethanolic extract of tomato showed some viable cells (purkinje cells) with intact nuclei and a few areas of degenerating cells.. These depicts the protective effect of tomato against induced apoptosis or neurodegeneration this is in line with the work of Kokturk et al., (2013) which reported that tomato extract exerted a protective effect against electromagnetic field-induced apoptosis and neurodegeneration in rat Purkinje neurons and granule cells, during pre-and postnatal periods.

The effect of the tomato extract was seen to be dose dependent as the protective effect was more in the group administered high dose of tomato extract than that lower dose.

Histopathological distortion of the cervical spinal segment of the spinal cord section in the group treated with Paraquat alone manifested as neurodegenerative changes such as degenerating cell, disintegrating nuclei, congested nuclei, loss of neuronal fibres and perineuronal vaculations when compared with the spinal section of the control are indication of paraquat induced spinal cord lesions this is in line with the findings of Ren et al., 2009 who reported that repeated doses of Paraquat (10 mg/kg gavage daily for four months or 10 mg/kg intraperitoneal injection twice weekly for three consecutive weeks) induced damage to the cells in substantia nigra pars compacta (SN) in mid brain sections from mice. Chen et al.(2010a , 2010b) who also demonstrated that after treatment with paraquat which was given orally once daily for 28 consecutive days to mice, cells in the hippocampus were irregular, and cytoplasm was found to be condensed. Number of Nissl bodies found there was reduced and apoptotic or necrotic neurons were observed. Increased response latency was also noted in animals given paraquat. A significant decrease in cerebellar cell volume among animals that received 30mg/kg of Paraquat, and a non significant decrease in cell volume among of animals that received 30mg/kg of Paraquat followed by 1500mg/kg of tomato extract and 1500mg/kg of tomato extract decreased when compared to the control group. A significant decrease in cell number of animals in group II,III and IV and an insignificant increase in the number of cells among animals of group v when compared with the control group was observed.

A decrease in the cell number could be as a result of induced apoptosis by Paraquat. Though a cell could be present with intact nucleus, decrease in cell volume may depict impairment in the normal cellular activity such as Energy metabolism, Enzyme and substrate concentration, protein synthesis, cell division which in turn could affect the normal function of a tissue or an organ. The significant decrease in cell number and volume is possibly due to the action of Paraquat, this finding is similar to that of Lou et al., (2012) who reported a significant decrease of viability and significant increase of apoptosis in dopermegic neurons as a

result of oxidative damage and also the work of Chen et al., (2010a) which showed that after treatment with Paraquat for 28 days, cells in the Hippocampus were irregular and cytoplasm was found to be condensed with number of cells reduced. The increase in cell volume and cell number in some of the groups could be attributed to the ameliorative effect of tomato as a result of Its antioxidative property as also reported by Gonz´ales-Vallinas et al., (2 013) Who reported that Tomato contain carotenoid and its function related to their antioxidant power.It was observed that there was a decrease in cell volume and cell number in the group treated with 30 mg/kg of paraquat when compared to the control which will interrupt proper nerve connections which in turn will affect motor impulse conduction and affect motor action, this is in line with the study of Chen et al., 2010 who observed that number of nissl bodies found there could reduce due apoptotic or necrotic neurons upon administration of paraquat. Increase in cell volume and cell number in the cervical spinal is in concordance with the work of Esposito et al., (2002) who showed that protection of cells in culture against diverse insults (glutamate, AB peptide, and others) by flavonoids has proved to be a useful approach.

CONCLUSION

Paraquat dichloride induces neurotoxicity and damage to the cerebellum and cervical spinal segment of the spinal cord. This is Manifested as neurodegenerative changes such as degenerating cell, disintegrating nuclei, congested nuclei, loss of neuronal fibres and peri-neuronal vaculations which in turn affects motor coordination and balance and histomorphometry of the cerebellum and cervical spinal segment. Tomato has a neuroprotective potential against paraquat induced spinal cord lesion in adult male Wistar rats by maintaining the cytoarchitecture of the cervical spinal segment. This could be consequent on the presence of antioxidant properties and phytochemicals present in tomato.

Conflict Of Interest

The authors hereby declare that, the manuscript has not been submitted for publication anywhere, the authors declare also that there is no conflict of interest in the present study.

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2. Tamashiro KLK, Wakayama T, Blanchard RJ, Blanchard C and Yanagimachi R. (2000). P o s t n a t a l g r o w t h a n d b e h a v i o r a l development of mice cloned from adult cumulus cells. Biological Reproduction, 63: 328–334. Toxicology.Volume 3.
3. Wathita P., Supaporn M., Terdthai T., Jintanaporn W., Wipawee T., Chonlathip T., s Bungorn S. and Panakaporn W., (2013).Acute toxicity study of tomato pomase extract in rodents, Online Journal of Biological Science, 13(1):28-34.
4. Winek, CL, Wahba, WW, Winek, CL Jr., Winek-Balzer, T., (2001).Winek’sdrug and chemical blood level data.

The post Solanum esculentum ameliorates paraquat-induced Cerebellar and Cervical spinal lesions in Wistar Rats. appeared first on Nigerian Biomedical Science Journal.

Francis Ajeneye

Blood Sciences Department, Maidstone and Tunbridge Well Hospitals NHS Trust, TN2 4QJ, U.K All Correspondences to: Francis Ajeneye, f.ajeneye1@nhs.net

INTRODUCTION

Vaccines save millions of lives each year. Vaccines work by teaching the body’s natural defenses, the immune system to recognize and

The risk of vaccination and the untimely release of Covid had led to fewer public health websites than searches without risk as a search term. There is a significant relationship between organization on social media and public doubts of vaccine safety. In addition,

fight off the viruses and bacteria they target. After vaccination, if the body is later exposed to those corresponding viruses, the body immediately recognize to fight the infection. The killer T-cells destroy the infected cells, while the B-cells and helper T-cells support antibody production. The challenge facing government and public health authorities is designing tailored strategies that successfully reach vaccination skeptics.

Designing a vaccine against the new coronavirus is an enormous challenge for researcher world-wide, but if there are things we learnt from history to reduce the hesitance of vaccination uptake during a pandemic, this must be addressed by the policymakers across the world.

Public acceptance of vaccination programs is essential for unnecessary exposures to diseases. However, increasing sectors of the population across the world have expressed hesitancy about participating in such programs, which might lead to the re-emergence of vaccine preventable diseases.

Policymakers need to begin planning for ways to work against the patterns of hesitancy in vaccine uptake by conducting a qualitative research prior to the release of Vaccination and the insights gathered about your audience will help policyholders to develop evidence- informed communication activities prior to introducing any new vaccines. While public outreach and education about the importance of vaccines will likely be the cornerstone of any vaccine deployment. The use of the internet and social media stations spreads, the challenge of vaccination hesitancy presents itself as an increasingly trans-national problem, a characteristic that further complicates policymakers’ tasks of designing effective policies.

There are many studies in this area are guided by an explicit theory vaccination hesitancy, short- or long- term side-effects or are ineffective, attitude factors, religious belief and the distrust of governments and historical abuse of experiments on vulnerable subjects.

there is a substantial relationship between foreign disinformation campaigns and declining vaccination coverage.

Much of the hesitation to get a Covid-19 vaccine stems from the lack of trust in the health care system, the pharmaceutical companies that brought the vaccines to market within a record time, in some vaccination advocates, and in our government that is regulating and promoting it. In addition to strategies to combat misinformation, we must wage an all-out effort to build public trust. To compensate for this, national and state public health authorities and vaccine administration sites could provide real-world safety data to help people of various ethnicities and conditions see how the vaccine is faring in groups that most closely represent them.

According to the Centers for Disease Control and Prevention (CDC): “Researchers have been working with mRNA vaccines for decades. Interest has grown in these vaccines because they can be developed in a laboratory using readily available materials. This means the progression can be standardized and scaled up, making vaccine development faster than traditional methods of making vaccines.”

The COVID-19 RNA vaccine consists of mRNA molecules made in a laboratory that code for parts of the SARS-CoV-2 virus, specifically the virus’ spike protein. Once injected into the body, the mRNA instructs the cells to produce antigens – such as the spike protein which are then detected by immune cells, triggering a response by the body’s lymphocytes. Whoever is exposed to the COVID-19 coronavirus in the future would have an immune system to fight off the infection.

There are good reasons for an immunization programme to conduct its own qualitative formative research before the introduction any new vaccine. The research provides valuable insights into the target audience’s points of view, concerns, and needs, Attendance to the audience’s concerns benefits the overall immunization programme. By identifying the population’s knowledge gaps and misinformation and highlighting programme deficiencies can help immunization staff modify

Reducing Vaccination Hesitancy…

services accordingly and finally Involving key stakeholders and target group members in the research builds the community’s sense of participation in the work of the programme. The public will have a greater sense of ownership of the vaccination programme when they feel they have been heard.

In many of the studies, a large number of the subjects were poor, vulnerable, racial minorities, and/or prisoners. Often, subjects were sick or disabled people, whose physicians told them that they were receiving “medical treatment”. They were used as the subjects of harmful and deadly experiments, without their knowledge or consent. In response to this, interest groups and institutions across the world have worked to design policies and oversight to ensure that future human subject research in the would be ethical and legal.

Eventually, for certain groups, policymakers may need to employ accessible ways for people to get adapted health information about the vaccine. This is especially important for marginalized populations who have less access to doctors. Possible approaches range from a simple text messaging system, influential community leaders, religious leaders, charity organizations to encourage immunization. Availability in multiple languages will be crucial.

Often the absolute complexity of the health care system prevents people from getting the right care. Once there is interest in getting the vaccine, people need to know when and where to get it. Because the vaccine is being rolled out in waves to different populations,

We should set up vaccination sites to offer easy access, minimize waiting times, and provide more time and attention to those who need it. When it comes to access, convenience is key. Besides, we can leverage the innovation and infrastructure built for Covid-19 testing. For example, drive-through testing sites that allow people to stay in their car could add a vaccination service . Vaccination sites could designate certain time slots for anyone who wants or needs a higher-touch experience, such as children, the elderly, those with physical challenges, and those who fear needles.

The hardest part of the vaccination process is actually the day one to day three after someone has received the shot when a significant proportion of people experience side effects, which range from pain at the injection site to headaches to low-grade fevers. Managing expectations about these side effects is important. If people expect no side effects but feel terrible, they will have a bad experience; conversely, if they feel less discomfort than anticipated, they will have a better experience.

Unfortunately, getting the shot doesn’t mean people can immediately go back to their pre-Immunity to the vaccine takes days to build, a second dose of the vaccine is vital, and we still need to wear masks after getting vaccinated.

Social media can add a function that makes it easy for people to notify the members of their networks that they have scheduled or received the first shot and then the second.

CONCLUSION:

Policymakers need to begin planning now for ways to work against the patterns found in many studies about vaccination hesitancy. While public outreach and education about the importance of vaccines will likely be the cornerstone of any vaccine deployment. More research on the influence of different sources of information is needed to determine the best way to disseminate information to public.

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2. Harmsen, I.A., Mollema, L., Ruiter, R.A. et al. Why parents refuse childhood vaccination: a qualitative study using online focus groups. BMC Public H e a l t h 1 3 , 1 1 8 3 ( 2 0 1 3 ) . https://doi.org/10.1186/1471-2458-13-1183.
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7. Bond L, Nolan T.(2011). Making sense of perceptions of risk of diseases and vaccinations: a qualitative study combining models of health beliefs, decision-making and risk perception. BMC Public Health. 2011 Dec 20;11:943. doi: 10.1186/1471-2458-11-943. PMID: 22182354; PMCID: PMC3260331
8. K.F. Brown, J.S. Kroll, M.J. Hudson, M. Ramsay, J. Green, S.J. Long, et al. (2010). Factors underlying parental decisions about combination childhood vaccinations including MMR: a systematic review Vaccine, 28; pp. 4235-4248.
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Ibiere Allwell Pepple, Catherine Omo Osin and Serekara Gideon Christian*

Department of Medical Laboratory Science, Rivers State University, Nkpolu-Oroworukwo, Port Harcourt, Nigeria All Correspondences to: serekara.christian1@ust.edu.ng

ABSTRACT

This study was aimed at evaluating levels of vitamin B12 and the presence of hypersegmented neutrophils in pregnant women attending Rivers State University Teaching Hospital. It is a comparative and case control study designed to evaluate the levels of vitamin B12 and presence of hypersegmented neutrophils in pregnant women. The study comprises of apparently healthy women in three groups of twenty-five females each (pregnant women who have not had miscarriage, pregnant women with previous miscarriage, and control subjects who have never been pregnant), aged between 30 to 35 years. The study was carried out from July through August 2019. Vitamin B12 levels were determined using ELISA method. Thin films were made and stained using Leishman stain to identify presence of hypersegmented neutrophils. Data was analyzed using Graph Pad prism 8.0.2 statistical package; p<0.05 was considered statistically significant. The results showed significant increase of vitamin B12 in women with miscarriages (193.78±110.63μg/day), when compared to that of women without miscarriages (174.80±53.14 μg/day), and that of non- pregnant women (98.03±9.50 μg/day). Hypersegmented neutrophils were found to be high among women with miscarriages (16%) when compared to those without miscarriage (4%), and control (0%). The miscarriages recorded among the women could be as a result of the presence of hypersegmented neutrophil which is indicative of the tendencies towards miscarriages. However, the high significant level of vitamin B12 in pregnant women with miscarriages may be due to their sea food diet despite the presence of hypersegmented neu trophils; which requ ires fu rtherinvestig a tion.

Keywords: Vitamin B12, Hypersegmented Neutrophils, Pregnancy, Miscarriage

INTRODUCTION

Pregnancy is the period from conception to birth. Pregnancy begins with the fertilization of an ovum (egg) and its implantation. The egg develops into the placenta and the embryo grows to form the foetus. Most eggs implant into the uterus upon fertilization by sperm cell. A normal pregnancy last around 40 weeks from the first day of the woman’s last menstrual period. Normal pregnancy consists of three trimesters of 3 months each (1). Miscarriage (biochemical pregnancy loss) is the pregnancy loss, which occurs after a positive urinary or serum human chorionic gonadotropin (hCG), but before ultrasound or histological detection of pregnancy (<6 weeks) (2). It can also be said to be the loss of the fetus before the 24th week of pregnancy or viability (the ability of the fetus to survive outside the uterus without artificial support). Majority of miscarriages occurs in the first trimester and may be mistaken sometimes for a late menstrual flow (1).

Vitamin B12 (cobalamin or cyanocobalamin) is a water- soluble vitamin that plays a vital role in the activities of several enzymes in the body. It is important in the production of red blood cells in the bone marrow and in the utilization of folic acid and carbohydrate in the diet and the functioning of the nervous system (1). Vitamin B12 sources for humans is food of animal origin. The highest amounts are found in liver and kidney (up to 100μg per 100 g), but it is also present in shellfish, organ and muscle meats, fish, chicken and dairy products (eggs, cheese and milk) in small amounts (6μg/L). Vegetables, fruits and all other foods of non-animal origin are free from cobalamin unless they are contaminated by bacteria. Cooking does not usually destroy cobalamin(3). Vitamin B12 maintains normal folate metabolism which is essential for cell multiplication during pregnancy.

Neutrophil hypersegmentation can be defined as the presence of neutrophils whose nuclei have six or more lobes or the presence of more than 3% of neutrophils with at least five nuclear lobes( 5 ). The presence of hypersegmented neutrophils is suggestive of cobalamin or folate deficiency( 6 ) . So pregnant women with hypersegmented neutrophils correlates with the low amount of vitamin B12 which is detrimental to red blood cell formation and a likely predisposition to neural tube defect and also pregnancy loss (miscarriage). The presence of hypersegmented neutrophils in pregnant women is also an indication of megaloblastic anaemia (5), which puts the pregnancy at risk of a miscarriage. According to Tavasoli et al., the presence of hypersegmented neutrophils indicates low levels of vitamin B12 which results in pregnancy loss due hypercoagulable states and bleeding (7).

In Sub-Saharan Africa, Nigeria inclusive, iron and folate deficiencies were reported to be the most common causes of anaemia in pregnant women (8) Lack and/or insufficient levels of iron supplementation was also reported to be among the most significant risk factors for anaemia to occur during pregnancy(9,10). An anaemic pregnant woman by implication of her condition lacks enough red blood cells for normal metabolic activities and that of the foetus. The resultant effect is that the foetus is deprived of adequate supply of the necessary nutrients required for foetal development.

There is paucity of scientific research information on the levels of vitamin B12 and presence of hypersegmented neutrophils in pregnant women in Rivers State, Nigeria, even though so much effort has been made to improve on the amount of vitamin B12 taken by pregnant women during their routine ante-natal clinic visits. This study was therefore undertaken to ascertain if levels of vitamin B12 and hypersegmented neutrophils could cause pregnancy loss or miscarriage in pregnant women. Hence the need to give this claim a scientific backing.

The aim of the study was to estimate the levels of vitamin B12 and the presence of hypersegmented neutrophils in pregnant women attending River State University Teaching Hospital. The specific objectives of the study are: To estimate the levels of vitamin B12 among pregnant women attending River State University Teaching Hospital; To estimate the number of hypersegmented neutrophils in women attending River State University Teaching Hospital; To ascertain the relationship in values of vitamin B12 estimation with the presence of hypersegmented neutrophils in pregnant women and non- pregnant women.

MATERIALS AND METHODS

Study Design

This is a comparative and case control study which is designed to assess and evaluate the levels of vitamin B12 and the presence of hypersegmented neutrophils in pregnant women attending River State University Teaching Hospital. This study was carried out from July through August 2019.

Study Area

This study was carried out in Rivers State University Teaching Hospital. The hospital is located in Port Harcourt, Rivers State, Nigeria. Port Harcourt is located on GPS coordinates of 4° 49′ 27.0012” N and 7° 2′ 0.9996” E. Rivers State University Teaching Hospital formally Braithwaite Memorial Specialist Hospital (BMSH) is a government-owned hospital, which was named after Eldred Curwen Braithwaite, a British doctor and a pioneer of surgery. It is located in Old GRA, Rivers State. It was established in March 1925.

Study Population

The subjects in this study comprised of apparently healthy pregnant women. Blood samples were drawn from seventy-five (75) women into ethylene diamine tetra acetic acid (EDTA) containers. Their age bracket was 30 to 35 years. They comprised of three (3) groups of 25 subjects each: pregnant women who have not had miscarriage(s), pregnant women that have had previous miscarriage(s) and control subjects who are the non – pregnant women. Women who were less than 30 years or more than 35years were excluded from the study. Convenient sampling method was adopted in recruiting participants.

Informed Consent/Ethical Approval

Informed consent was obtained from the pregnant women before their samples were collected upon clearance from the Department of Medical Laboratory Science, Rivers State University.

Eligibility Criteria

Only apparently healthy pregnant women attending Rivers State University Teaching Hospital and apparently healthy non pregnant women were recruited for this study.

Sample Collection and Storage

A total of 3ml of venous blood was collected by venipuncture with the use of vacutainer needle from each subject and added into individualized vacutainer tube containing 0.5ml of 1.2mg/ml dipotassium ethylene tetra- acetic acid (EDTA). The blood samples in EDTA containers were centrifuged to obtain plasma. The plasma obtained was used to analyse for vitamin B12 using an ELISA reader capable of reading absorbance at 450nm. Thin films were also made immediately before the plasma was separated.

Sample Analysis

The parameters that were analysed were vitamin B12 and presence of hypersegmented neutrophils in thin films.

• • 1. Determination of Vitamin B12 Using Human Vitamin B12 ELISA Kit, CALBIOTECH, Inc., El Cajon,

U.S.A. Lot No VBE5774; Expiry Date: 2020/05

Principle: It makes use of solid phase ELISA methodology based on the principle of delayed competitive binding. Streptavidin coated wells are incubated with extracted vitamin B12 standards, controls, samples and intrinsic Factor-Biotin conjugate at room temperature for 45 minutes. During the incubation, the biotin-labelled intrinsic factor binds to vitamin B12 in the sample, standard or quality control plasma, after the 45-minutes incubation, vitamin B12 enzyme conjugate is added which competes with the vitamin B12 in the sample, standard, or quality control plasma for the remaining sites on the intrinsic factor for an additional 30 minutes. All unbounded conjugates are then removed and the wells are washed, Next, a solution of tetramethylbenzidine (TMB) reagent is added and incubated at room temperature for 15 minutes, resulting in the development of blue colour. The colour development is stopped with the addition of stop solution, and the absorbance is measured spectrophotometrically at 450 nm. The colour intensity is inversely proportional to the amount of vitamin B12 in the sample. The total procedure run time is 1.5 hours.

Procedure: The EDTA blood samples were centrifuged to obtain plasma. All reagents and specimens were allowed to come to room temperature before use. Desired number of coated strips was placed into the holder. 50µl of extracted Vitamin B12 standards, controls and samples was dispensed into appropriate wells. 50 µl of biotinylated intrinsic factor reagent was dispensed into each well. The microplate was shaken gently for 30 seconds to mix. It was incubated for 45 minutes, at room temperature (250oC). 50 µl of enzyme conjugate was added into all the wells. The microplate was gently shaken for 30 seconds to mix. It was incubated for 30 minutes at room temperature (250oC). The contents were briskly shaken out of the wells. The wells were rinsed 3 times with wash buffer. The wells were stroked sharply on absorbent paper to remove residual water droplets. 100 µl of tetramethylbenzidine (TMB) substrate was dispensed into each well which resulted in the development of a blue coloured solution. The absorbance was read spectrophotometrically at 450nm.

Procedure for Thin Blood Film Preparation

One micro litre of blood was dropped near the end of a slide. The edge of the spreader was placed in front of the blood at an angle of 45o. the spreader was drawn back until it touched the drop of blood and the drop spread along the line of contact between the spreader and the slide on which the film was made. The spreader was moved along the slide with a smooth movement. The film was allowed to air dry. The subject identification was written directly on the frosted end using a lead pencil.

Film Staining using Leishman Staining Technique

Principle: Leishman stain is a mixture of eosin and methylene blue. The acidic dye, eosin variably stains the basic components of the cell which is the cytoplasm and the

basic stain, methylene blue stains the acidic components, especially the nucleus.

Procedure

The slide was placed on the staining rack. The film was flooded with Leishman stain and the stain was allowed to stain for 2 minutes. The stain was later diluted with equal volume of buffered water (pH 6.8) and was allowed to stand for 8 minutes. After 8 minutes, the stain and buffered water was washed off and slide allowed to drain. The back of the slide was then cleaned with cotton wool soaked with 70% alcohol. The film was dried on a rack in a vertical position. The stained f i lmed was examined microscopically using oil immersion objective (100x), ( Olympus microscope) for the presence of hypersegmented neutrophils.

Statistical Analysis

The data generated from this study was analysed and calculated to determine the mean, standard deviation, p- value, f-value using analysis of variance; Tukey’s multiple comparison test was done to check for significance in between groups. Graph pad prism 8.0.2 statistical package was used for the analysis.

RESULTS

• 1. Demographic Details of Participants

A total of seventy-five (75) females were recruited for this study. Fifty (50) of the females were pregnant women and were grouped into two (25 were pregnant women that had previous miscarriage(s) and the other 25 were pregnant women that have not had miscarriage(s). Twenty-five (25) women served as control subjects and were non-pregnant. Their age range was between 30-35 years and they were all residents of Port Harcourt, Rivers State. Details are shown in Table 1.

Table 1: Demographic Details of Participants

Age (Years)

No. of Pregnant Women with previous Miscarriage

No. of Pregnant Women without Previous Miscarriage

Control Subjects (Non-pregnant without history of Miscarriage)

30-35 25 25 25

Analysis of Variance of Vitamin B12 in Study Population

Table 2 showed the comparison of vitamin B12 level in pregnant women with previous miscarriage(s), pregnant women without previous miscarriage(s) and non-pregnant women (control)-without history of miscarriage.

Comparatively, the analysis showed that there was statistically significant difference (p<0.05) in the values of vitamin B12 of women with miscarriages (193.78±110.63 μg/day), women without miscarriages (174.80±53.14 μg/day) and the control group (98.03±9.50 μg/day).

Table 2: Comparison of vitamin B12 level using analysis of variance in the study population

Parameter PW+M (A) PW-M (B) Control (C) p-value F-value Inference Tukey’s Multiple Mean±SD Mean±SD Mean±SD Comparison Test

Vitamin 193.78±110.63 174.80±53.14 98.03±9.50 <0.0001 11.95 HS A vs B0.4231 B12 A vs C 0.0001

(µmol/day) B vs C 0.0001

Key: PW+M = Pregnant women with previous miscarriage(s); PW-M = Pregnant women without previous miscarriage(s); HS = Highly significant; SD = Standard deviation. (Applicable to all Tables).

• 1. Percentage Distribution of Hypersegmented Neutrophils in Percentage Rate in the Study Population Table 3. shows the percentage distribution of hyper- segmented neutrophils in women with miscarriages, without miscarriages and non-pregnant women. The result showed that the percentage rate of hyper-segmented

neutrophils in women with miscarriages was 4(16%) while the percentage rate of hyper-segmented neutrophils in women without miscarriages was 1(4%). No hyper- segmented neutrophils were found in non-pregnant women.

Table 3: Percentage distribution of hyper-segmented neutrophils in women with miscarriages, without miscarriages and Non-pregnant women

Parameter No. of Women with hyper-segmented Neutrophils Percentage (%)

4.0 DISCUSSION

From this study, it was observed that there was a significant increase (p<0.05) in the values of vitamin B12 in women with previous miscarriages than those without a history of miscarriage and control participants. Non-pregnant (control) in this study recorded low level of vitamin B12 as a result of them not being on drug supplements and also not having huge appetite for food when compared to pregnant women. The level of vitamin B12 in our study subjects were within the range as reported by VanderJagt et al., (11).

Though it has been reported that maternal vitamin B12 deficiency and increased presence of hypersegmented neutrophils have been associated with increased risk of common pregnancy complications, including spontaneous abortion (miscarriage), low birth weight, intrauterine

among the women could be as a result of the presence of hypersegmented neutrophil which is indicative of the tendencies towards miscarriages. However, the high significant level of vitamin B12 in pregnant women with miscarriages may be due to their sea food diet despite the presence of hypersegmented neutrophils; which requires further investigation.

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findings of the study indicated that pregnant women with history of miscarriages and those without history of miscarriage had high level of vitamin B12; and so, our findings are at variance with the above reports, probably as a result of the management of the conditions that aforetime may have caused the miscarriages.

The presence of hypersegmented neutrophils is an important diagnostic feature of megaloblastic anaemia; and deficiency of vitamin B12 have been associated with megaloblastic anaemia. In regards to the percentage rate of hypersegmented neutrophils in women with history of miscarriage(s), without history of miscarriage and non- pregnant women, the result showed 4(16%) 1(4%) and 0% in the same order. The hypersegmented neutrophils were found to be high amongst women with history of miscarriage(s) (16%), and this indicates that hypersegmented neutrophils could be associated with miscarriages in some cases in women. The presence of hypersegmented neutrophils in pregnant women with history of miscarriage(s) may probably be only a reflection of their previous miscarriage(s) and tendency towards a miscarriage.

5.0 CONCLUSION

The study revealed that women with miscarriages recorded

2 0 0 5 ; 2 0 ( 1 1 ) : 3 0 0 8 – 1 1 . d o i : 1 0 . 1 0 9 3 /

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The post Evaluation of vitamin B12 levels and Hypersegmented Neutrophils in pregnant women attending Rivers State University Teaching Hospital, Port Harcourt appeared first on Nigerian Biomedical Science Journal.

Akpulu S.P and Hamman W.O

Department of Human Anatomy, Faculty of Basic Med. Scin, Ahmadu Bello University Zaria.

Oladele S.B

Department of Vet.Anatomy, Ahmadu Bello University, Zaria.

Ahmed S.A

Department of Pathology, Faculty of Medicine, Ahmadu Bello University, Zaria, Nigeria All Correspondences to: Akpulu S.P E-mail: petosw2000@yahoo.com

ABSTRACT

Since time immemorial, xylene has been one of the most commonly used tissue clearing and dewaxing agent in histology /histopathology laboratories. However, off late, xylene has been reported to have many toxic effects. Numerous solutions have been suggested as possible alternatives to xylene during tissue processing and staining. Most of these suggested alternatives are at best act like xylene. This study compared the efficacy of Citrodora oil, as a possible alternative to xylene in tissue processing and staining procedures. The study was carried out in the department of Human Anatomy ABU Zaria. The Citrodora oil used was extracted in NARICT Zaria. Two pairs of neutral buffered formalin-fixed brain and liver tissues were histologically processed and stained. One pair was cleared and dewaxed in xylene during the tissue processing and staining, while the other pair was treated similarly in Citrodora oil under the same condition. The paraffin sections were stained with H and E, Gordon and Sweet and Golgi, employing the method of Bancroft and Steven 2008. The section and staining quality was evaluated by direct microscopic observation and graded as described by Kunhua, 2012. Data generated were analyzed using SPSS version 20.0. Results from this study showed a similarity in the efficacy of Citrodora oil and xylene as clearing and dewaxing agent and no significant difference (p≥0.05) in section and staining quality when compared. In conclusion, Citrodora oil can be an effective, eco-friendly, and safer alternative to xylene as a clearing and dewaxing agent in the histology /Histopathological laboratories.

Keywords: Citrodora oil, clearing, staining, xylene and alternative.

INTRODUCTION

Biological tissues have to undergo series of ’tissue clearing agents such as: xylene, toluene, chloroform, acetone, kerosene, diaxane, benzene, petrol, methyl salicylate and cedar wood oil. Most clearing agents are processing’ procedures before they are ready to be examined under the microscope. The various steps of tissue processing include fixing, dehydration, clearing and infiltration. Clearing refers to the process of replacing the dehydrant with a substance that is miscible with the embedding medium. It is one of the most critical steps of tissue processing and largely affects the clarity of the final section and hence the precision of diagnosis.

Clearing agents are used to remove alcohols from the tissue before the tissue can be infiltrated with paraffin wax. Clearing agents are sometimes called “de-alcoholization agents” or ante medium. They act as intermediary between the dehydrating and infiltrating solutions. They are miscible with both solutions and have refractive indices similar to proteins with different levels of toxicity (Kieranan, 2010). Most Histology and Histopathology Laboratories use either aromatic solvents, such as xylene, toluene or aliphatic petroleum distillates for the purpose of clearing and de-waxing in the paraffin histological technique (Ankle and Joshi, 2011). There are many

derivatives of aromatic hydrocarbons such as benzene, while others are derived from natural essential oils such as cedar wood oil and olive oil (Hans et al., 1995).

Xylene has probably been the most commonly used chemical in the histology laboratory despite its hazards. Xylene is an aromatic hydrocarbon consisting of a benzene ring with two methyl substituent (C6H4 (CH 3)2 ). It is expensive, but work well for short time clearing of small tissue blocks. Its high solvency factor allows maximum displacement of alcohol and enhancing paraffin infiltration. (Tardif and Brodeur1992, Carson and Hladik, 2009). Xylene does tend to harden tissues a little, but this does not usually interfere with sectioning qualities.(Kieranan, 2010). Long term immersion of tissue in xylene results in tissue distortions (Visfeldt et al., 1982). Xylene has been reported to affect skin, eyes, nervous system, blood, liver and kidneys of animals exposed to it and it can potentially contaminate the working environment (Ankle and Joshi, 2001).

The need to reduce laboratory hazard has been a challenge.

During tissue processing and staining, most of the clearing agents used are among the most noxious and hazardous chemicals with different levels of toxicity (Dapson and Richard, 2005. Several toxicities believed to be caused by intermediate products of xylene metabolism, such as metylbenzaldehyde have been reported by Indu et al., 2014). These include central nervous system disorders, respiratory depression, abdominal pain, dryness and redness of skin, dermatitis, liver diseases, nephrotoxicity, conjunctivitis, and teratogenic and fetotoxic effects. These are in addition to environmental pollution from unsafe disposal of xylene (Ankle et al., 2011) and tissue distortions as a result of long-term immersion of tissue in xylene (Hans et al., 1995).

There have been several attempts to substitute xylene as clearing agent. Recently, xylene alternatives as clearing agents was developed by mixing vegetable oils such as groundnut oil, palm kernel oil and coconut oil either alone as mixture with other clearing agents (Adeneyi et al., 2016). Orange based oil as clearing agents has also been reported by Rene (2000). Some essential oil such as olive, Clove, Coconut oil and Cedal wood oil has been reported (Hans et al., 1995). However, most of these commercially available xylene alternatives are less effective, more expensive, and are not as readily available as xylene (Gosselin et al., 1984; Amdur et al., 1991; Luna, 1992). To the best of our knowledge, there is little or no report of work on the use of Citrodora oil as xylene substitute in tissue processing. Most of these commercially available xylene substitutes are less effective, more expensive, not readily available and are constitute health hazard as or more than xylene itself (Udonkang et al.,2014).

Citrodora oil is extracted from the Eucalyptus plant which belongs to the; Kingdom: Plantae.Order: Myrtales. Family: Genus: Backhousia. Species: Backhousia citriodora. Citrodora oil is a concentrated hydrophobic liquid containing volatile aroma compounds (Pino et al., 2006). It is extracted from the leaves of Eucalyptus plant and also known as eucalyptus oil. It is an essential oil with a clear, sharp, fresh and very distinctive smell, is pale yellow in color and watery in viscosity (Julia, 995). It has molecular weight of 154.25 and the structural formula of C10H180. The main chemical components of citrodora oil are a- pinene, b-pinene, a-phellandrene, 1,8-cineole, limonene, terpinen-4-ol, aromadendrene, epiglobulol, piperitone and globulol. It has been reported to be nontoxic, nonhazardous, nonflammable, biodegradable and used in aromatherapy (Jean-francois, 2011).

MATERIAL AND METHODS

The essential oil of eucalyptus plants was extracted in National Institute for Chemical Research Technology (NARICT), Zaria by hydro distillation method.

500g of the fresh leaves of eucalyptus and citrus peel was separately weighed and packed into a distillation flask fitted with condensers. Heat was supplied to the flask through a steam generator at constant flow. The essential oil which vaporizes with the steam was condensed into a collecting funnel. The oil was then separated by gravity, dried over anhydrous sodium sulphate, measured, labeled and stored in a brown bottle.

Experimental Protocol

The tissues were taken in pairs. One pair is labelled as Citrodora Tissue and the other as Xylene tissue. The two pairs of brain and liver tissues, 5mmx5mm x3mm thick neutral buffered formalin fixed, were histologically processed simultaneously by dehydration, clearing. Infiltration and embedding. All the tissues were subjected to the same treatment except for the clearing. The Citrodora pair tissue was cleared and dewaxed in Citrodora oil while the Xylene pair tissue was cleared and dewaxed in xylene

Figure I: Eucalyptus plant. Source: Biological Technology Co.Ltd

during the processing and staining respectively. Two paraffin sections of 4 and 8-micron thickness were cut from each of the paired tissue blocks using a rotary microtome (Leica RM2 125 RTS) made in England. Tissues sections sets of 4 microns were stained using Hematoxylin and eosin (H and E) to demonstrate the general tissue structures, Gordon and Sweet for reticular fibers while the brain tissues were stained with Golgi silver stain for nerve fibers. The tissues were dewaxed and cleared with their respective clearing agents during the staining and before cover slipping. The section and staining quality was evaluated by direct microscopic observation and graded as described by Kunhua, 2012. Data generated from the study were expressed as mean plus or minus (±) standard deviation (SD). Student t test was used to compare the efficacy of the Citrodora oil with that of the xylene. Data was analyzed by SPSS 20.0. P value less than or equal to (P ≤ 0.05) were

considered statistically significant.

RESULTS

Table 1.1: Clearing and dewaxing effect of Citrodora oil and xylene on sections and staining quality of liver

Section Quality P value

Citrodora Oil 3.500±1.049

Xylene 4.167±0.753

Staining quality (H and E)

Citrodora Oil 3.333±0.816

Xylene 3.333±0.516

This result indicated there were no statistically significant (P ≥ 0.05) between the Citrodora oil and xylene. At the end of clearing and staining, xylene shows no significant

difference in section quality (4.167±0.753) of liver when compared to Citrodora oil (3.500±1.049).

Table 1.2: Clearing and dewaxing effect of Citrodora oil and xylene on sections and staining quality of Brian

Section Quality P value

Citrodora Oil 3.333±0.816

Xylene 3.500±1.225

Staining quality (H and E)

Citrodora Oil 3.333±1.033

Xylene 3.000±0.894

This result indicated there were no statistically significant (P ≥ 0.05) between the Citrodora oil and xylene. The Brain section and staining quality showed no statistical

significant difference both in section and staining quality.

Photomicrographs of Stained Brain and Liver Tissue Sections

Plate 1: Shows transverse sections of H and E stained liver (L1) and brain (B1) cleared and dewaxed in Citrodora oil and L2 and B2 were cleared and dewaxed in xylene. L3 and L4 are reticular fibers (black arrow) of liver tissue cleared and dewaxed in Citrodora oil and xylene, while B3 and B4 are brain sections demonstrating neurons (blue arrow), were cleared and dewaxed in Citrodora oil and xylene respectively. There was no statistically significant difference across the groups both in the section and staining quality. (H and E X 250).

DISCUSSION

The results of photomicrographs from the present study showed no significant difference in section and staining quality. The present study agrees with the work of Rasmussen et al (1992) on the use of vegetable oils mainly olive and coconut oils instead of xylene in tissue processing, where it was stated that the xylene processed tissues and the vegetable oils’ processed tissues showed only minor or insignificant difference in staining and section quality. According to Kinast (2003), most vegetable oils have higher viscosity between 2 and 5.7 cp at 28% which is reduced by transesterification processes. This may be the reasons for most of the clearing effect on the section and staining qualities observed with some vegetable oil when used as tissue clearing agents. Other physical and phytochemical properties may be responsible for the clearing ability of the Citrodora oil in the present study.

CONCLUSION:

This present study concludes that Citrodora oil can clear and dewax Wistar rat tissues during tissue processing and staining as xylene. Also cytoplasmic and nuclear structures as well as reticular and neural fibers and be demonstrated in Wistar rat tissues cleared in Citrodora oil. Therefore, Citrodora oil can be an effective, eco-friendly and safer alternative to xylene as a clearing and dewaxing agent in the histology /Histopathological laboratory.

Recommendations:

Studies using advanced techniques such as immunohistochemistry, Molecular and enzymatic study, Fluorescence, and electron microscopy techniques of the effects of these essential oils in tissue clearing.

Acknowledgment

The authors sincerely thanked the National Institute for Chemical Research Technology (NARICT), Zaria and their staff for the extraction of the oil, and also the department of human anatomy, Ahmadu Bello University, Zaria for the enabling environment to carry out this work.

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The post CITRODORA OIL: A biosafer alternative to xylene as tissue clearing and dewaxing agent appeared first on Nigerian Biomedical Science Journal.

Francis Ajeneye*

Pathology Department, Blood Transfusion Maidstone and Tunbridge Well Hospitals NHS Trust, TN2 4QJ, U.K

Oladimeji Olofin

Pathology Department, Haematology Maidstone and Tunbridge Well Hospitals NHS Trust, TN2 4QJ, U.K

Christy Chinyere Fredrick

College of Health sciences, Department of Pathology, University of Abuja, Nigeria All correspondence to: Francis Ajeneye f.ajeneye1@nhs.net

ABSTRACT

The link between ABO/Rh and various diseases have generated much interest over the last decades and have shown some inconsistency. Some diseases are definitely associated with ABO. Do blood groups have biological roles? Many literatures had documented an

association between blood groups and diseases, particularly some neoplasm and hematologic disorders. The loss of blood group A and B antigen expression had been widely documented, which is beyond the scope of this communique. There are also awareness in defining bacterial and parasitic receptors which are closely associated to some known blood groups. Finally, recent studies had observed statistical relationship of blood group with SARS-CoV-2, the association with blood group and SARS-CoV-2 remains insubstantial and evidence must be approached with a sound research design.

Key words: Blood group SARS-CoV-2 Malaria Carcinoma

Over the past few years ABO association with diseases had been widely discussed with emerging research on ABO association and diet, personality and socio-economic statues which is beyond the scope of this short communication.(1,2) Few literatures had proving evidence of increase of group A compared to group with some types of cancer. (3) There is convincing study relating to cancer of the stomach, the association more common in blood group A compared to blood group O. More research across the world agreed with the incidence. ABO association with carcinoma of the stomach seem indisputable(3). Similar association had been also be found in cancer of the colon, and the salivary gland(5). The risk of chorioncarcinoma is critical related to the ABO Blood group of the woman and the partner, women of blood group A with a partner of group O seem to have a highest risk whereas women of blood group A with a partner of blood group A has the lowest risk. There have been some proven relationship of early abortion due to anti-P, Anti-P1 and anti-Pk. The anti-P had been shown to be present in the placenta and anti-PP1PK had been shown to be cytotoxic. (4)

Aird and Bentall (5, 6) showed that group O were 20% more likely to develop peptic ulcer than blood group A. There appears to be a true association between ulceration and absence of secretion. It is not surprising that large quantity of ABH substance are found in the gastrointestinal mucosa,

expression well demonstrated in some human malignancies, the loss of A and B antigen expression was found in 21 of 25 oral carcinoma tumour and correlate with tumour lacking A and B gene expression(7)

There appears to be an association with blood group A, thrombosis, high cholesterol and myocardial infarction. Mourant and colleagues.(8) analysed data from the world literature that patient with thromboembolic diseases include a raised proportion of group A . This applied to women taking oral contraceptive, to pregnancy and puerperal women. There is more evidence of raised thromboembolic episodes in A women than O women. Five year studies found out that A1,B and A1B had a high incidence of myocardial Infarction than O blood group.(9) It was suggested that the etiological pathway of cardiovascular diseases may be differ in patients of different ABO groups owing to difference in their rheology and plasma protein activities.(10,11) These studies consisted of Western Europeans ethnic background, the association did not hold for Asian, African-American or children. It has been shown that cholesterol association may be differ by race.

Mourant(5) made a challenging point earlier that blood

group A individual have a higher level of Factor VIII than group O individual. Group O tend to haemorhage rather than thrombose into the arterial walls thus sustaining more tissue damage. There have also been association with other

failure to secrete ABH substance may lead to peptic ulcer.

coagulation factors such vWF, FV and FIX.(12) Many

There is substantial evidence of decrease activity of glycosyltransferases activities with loss of A and B gene

bacteria such as gram negative organism like Escherichia Coli, have known to have chemical moieties on their

surface that mimics blood group antigens. Springer tested vitro Rosette had been described by to be stronger in

bacteria and found that some bacteria strains showed A, B Group A individuals.(17) The main antigenic ligands

and H (O) specificity.(13) Most blood group scientists responsible for both cytoadherence and antigenic

believe that a similar mechanism must operate in human for the production of naturally occurring antibodies. Small pox virus possess an antigen similar to A antigen, humoral resistance may be more effective in patient with blood group B and O who possess anti-A in their plasma.

The Asian and African distribution of A gene supports the theory of a selective disadvantage among individual infected with smallpox virus. In area like China, India, parts of Russia has a relative increase of the B gene. Resistance to several bacterial and viral infection has been associated with blood groups. Patient with Cholera were likely to be of group O and a one-ninth as likely to be a group AB, several other groups have reported similar associations. (14)

The association of malaria and Duffy blood group system was described by Miller(15), red cell lacking Fya and Fyb were shown to be resistant to the invasion of malaria. It has been shown that black people are resistant to infection by P.Vivax. P.Vivax infection do not occur in individual with Fy(a-b-). The ligand for P. Falciparum is different from the P.Vivax. P.Falciparum invades Fy(a-b-) equally to Fy(a+b+). The parasites exploits adhesion ligands on the endothelial (CD36 and ICAM1), red cell rosette (Blood group A and B), CR1/CD35 and platelets rosettes – platelets glycophorin IV (Cd36) (16) CD36 negative group O appears to be common in malaria endemic areas, this might be a co-incidence or a product of malaria selection, there is also a reduced cyto-adhesion in group O individuals. In-

variation are the members of the P. Falciparum Erythrocyte Membrane Protein-1 (PfEMP1). The encoded PfEMP1var2 carries a two-cysteine-signature associated with rosetting and antibodies to the protein avidly stain the pRBC suggesting that FCR3S1.2 var 2 is the dominant var gene expressed in this parasite.(18)

The parasite ligand-host receptor interactions that mediate cytoadherence is therefore critical to improving our understanding of malaria pathogenesis and developing a vaccine to alleviate disease severity. The second mechanism is to evade specific immune responses through antigenic variation. This involves switching the clonal expression of PfEMP1 antigens, by enabling iRBCs to navigate clear of immunoglobulin (IgG) responses that prevent their cyto-adherence and opsonize them for phagocytosis. PfEMP1 variants thus play a critical role in parasite survival and are prime targets for naturally acquired immunity in African children. Repeating this immunity through PfEMP1 vaccination has been difficult to co-ordinate, especially as thousands of diverse.

There have been other reports associated blood groups with Covid-19, the association of Covid-19 infection and blood groups remains intangible and must be approach with vigilance. The research designs and methodologies identified in recent literatures are not robust and

Cite as: Francis Ajeneye*, Oladimeji Olofin & Christy Chinyere Fredrick (2021). Association of ABO Blood groups and some diseases – Do Blood groups play a biological role?

convincing. A detailed research design that identifies dependent and independent variables and adjust for confounding factors could be a way forward. However, Covid-19 infection could be the results of complex interactions of factors that could vary from genetics, behavioural, metabolic, psychological, social status and environmental risk factors, ABO blood type distribution worldwide varies considerably in different races and should be addressed in studies. In addition to inflammatory response associated with Covid-19 infection,(19) we cannot alterations of the blood group ABO gene in oral overlook mechanism purported by earlier studies that suggested ABO blood group profound influence on the haemostasis, a major determinant of plasma levels of Von

squamous cell carcinoma. Int J Cancer. 2004 Mar 20; 109(2):230-7. doi: 10.1002/ijc.11592. PMID:

14750174.

Willebrand Factor (VWF).(20,21) Literatures also defined

blood group O as a risk factor for increased severe bleeding while blood group non-O is a risk factor for thromboembolic events. The risk of VTE is probably related to the level of VWF and factor VIII in non-group O subjects. A, B, and H blood group antigens are expressed on N-glycans of vWF and influence the half-life of the protein (10 hours for group O and 25 hours for non-O subjects).

CONCLUSION

The relationship between ABO blood groups and overall cancer risk still remains unclear but several meta-analyses conducted over decades suggested there is a strong link. The geographic distribution of ABO antigens worldwide is consistent with a survival advantage in malaria among group O individuals; clinical studies had provided supporting evidence of the effect of ABO group on malaria severity; and that recent discoveries suggest biologic mechanisms linking disease pathogenesis to ABO antigen expression. The mechanisms underlying the associations between ABO blood group and Cardiovascular diseases risk remain unclear, however, several studies of evidence support its potential cardiovascular effects. More research is required to explore and understand the association of Blood groups and SARS-CoV-2.

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Arnela Salkić, Nermina Durmić

Department of Information Technologies, International Burch University,

Faculty of Engineering and Sciences, Sarajevo, Bosnia and Herzegovina

All Correspondences to: Arnela Salkić Department of Information Technologies, International Burch University,

Faculty of Engineering and Sciences, Sarajevo, Bosnia and Herzegovina

ABSTRACT

Aim of this paper is to give insight in Covid 19 data and to try to predict whether individual person will recover from this virus. Furthermore, this paper aims to give some answers how information like the country, the age, and the gender of the patient, the number of cases in their country and whether they’re from or have visited Wuhan can be used to make that prediction.

Study uses Novel Corona Virus (COVID-19) epidemiological dataset. Logistic regression model and Random Forest algorithm are used in order to make prediction, and the Chi-Square test of independence is used to determine if there is a significant relationship between two nominal (categorical) variables. Paper reveals that recovery/survival is supposed to depend on the age of the patient, gender and country from which patient come. Information, whether the patient is from Wuhan or has visited Wuhan, does not affect recovery/survival of patient.

Keywords: Covid 19; Pandemic; Logistic Regression; Random Forest; Chi-Square Test.

INTRODUCTION

Coronaviruses are frequent RNA viruses, from the Coronaviridae family, which are responsible for digestive and respiratory infections in humans and animals. The virus owes its name to the appearance of its viral particles, bearing growths which evoke a crown.[1] COVID-19 is the infectious disease caused by the last coronavirus that was discovered. This new virus –and disease- was unknown before the outbreak occurred in Wuhan, China in December 2019. COVID-19 is now pandemic and affects many countries around the world. The most common symptoms of COVID-19 are fever, dry cough, and fatigue. Other less common symptoms may also appear in some people, such as body aches and pains, nasal congestion, headache, conjunctivitis, sore throat, diarrhea, loss of taste or smell, rash, or discoloration of the fingers on the hand or foot. These symptoms are generally mild and appear gradually. Some people, although infected, have only very mild symptoms.[2]

Most patients (approximately 80%) recover without the need for hospitalization. About one in five people with the disease have severe symptoms, including difficulty breathing. Older people and those with other health conditions (high blood pressure, heart or lung problems, diabetes or cancer) are more likely to have serious symptoms. However, anyone can get COVID-19 and become seriously ill. People of any age who have a fever and or cough associated with difficulty breathing / shortness of breath, chest pain / pressure, or loss of speech or difficulty getting around should see a doctor immediately. It is recommended, if possible, to call the care provider or health facility in advance, so that the patient is referred to the appropriate service.[2]

Data science will play a main role in the global response to the Covid-19 pandemic by analyzing data which is collected daily and by searching patterns in those datasets which would help humanity to fight pandemic.

Aim of the study is to give insight into Covid-19 pandemic by bringing answers like whether information like the country, the age, and the gender of the patient, the number of cases in their country and whether they’re from or have visited Wuhan can be used to predict whether a random patient whose data we have will recover from this virus. The purpose of this study consists in providing prediction model for future recovery trend which can be used to specify additional measures which would help fighting Covid 19 pandemic.

This paper is organized as follows: section 2. Where several studies about Covid 19 topic have been reviewed and where research question of this paper have been introduced. In section 3. Used datasets and details have been presented as well as methods which were used to get answers about Covid 19 which later lead to the final conclusion about research question. Results are presented in section 4. Using visualization for better understanding, while last two sections 5. And 6. Are discussion and conclusion about conducted study.

LITERATURE REVIEW AND RESEARCH QUESTION

Previous studies and data exploration have been made concerning this topic, however the goal of each study differs. For some data scientists on Kaggle, the model to develop was a regression model to predict the number of cases each day in each country. These studies findings suggested that China and Italy, who were the first to contract the virus, didn’t record many cases during the month of January and then skyrocketed to the top of the coronavirus world meter list, which conveys that the virus spreads at a very high speed. The most affected countries were also in the northern hemisphere, hence the hope that the virus would disappear little by little as the weather around the globe gets calmer. The findings also included China’s astonishing results after lockdown which encouraged many countries to also undergo a confinement mode. Models used comprised of linear regressors and learning algorithms such as XgBoost. [3][4]

Nadia AL-Rousan and Hazem AL Najjar in their work [5] are analyzing Covid-19 pandemic in South Korea based on recovered and death cases. Their study is based on statistically analysis the effect of factors such as region, sex, birth year, infection reasons and released or diseased date on the reported number of recovered and deceases cases. The X2 test is used to find the impact of the previous attributes on the number of recovered and deceases cases. Result of their work shows that confirmed date and infection reasons do not affect deceased cases, while on the other side confirmed date and region variables are significant with recovered cases. Besides, the results found that multinomial logistic regression could give initial indicator about the possibility to survive or die based on the collected data. It is found that the results of multinomial logistic are in line with the results of the X2 test.

Dr. Anis Kouba in his work [6] aims to answer several questions about Covid-19 pandemic: How does COVID-19 spread around the world? What is its impact in terms of confirmed and death cases at the continent, region and country levels? How does its severity compare with other epidemic outbreaks, including Ebola 2014, MERS2012, and SARS 2003? Is there a correlation between the number of confirmed cases and death cases? Data analysis is based on Novel Coronavirus COVID-19 Data Repository provided by Johns Hopkins University. Tableau Professional software was used to analyze the collected data and to develop visualization dashboards about the Coronavirus disease. Methodology consists in creating descriptive models of the Coronavirus outbreak using statistical charts to understand the nature of the spread and its impact. Analysis was developed at three levels, namely, at the country-level, at region-level and continent-level. Each level provides different granularities towards understanding the distribution of the disease around the world. The descriptive model provides different types of statistical charts, including bar charts, geographic maps, heatmaps, box plot, and packed bubbles, to represent different features of the COVID-19 outbreak. Dr.Kouba also develop some predictive models using linear and polynomial regressions to predict the evolution of the outbreak, given the historical data.[6]

In [7], the authors investigated the impact of preventive measures, such as social distancing, lockdown in the containment of the virus outbreak. They developed prediction models that forecast how these measures can reduce the mortality impact of aged people. Mathematical model is performed on the spread of the Novel dataset coronavirus that considers both, the age and social contact structure. Authors conclusion is that the three-week lockdown will be insufficient in India. Their model suggests that sustained periods of lockdown with periodic relaxation will reduce the number of cases to levels where individualized social contact tracing and quarantine may become feasible.

The authors of [8] addressed the question about how the virus has spread from the epicenter of Wuhan city to the whole world. They have also analyzed the impact of preventive measures such as quarantine and city closure in mitigating the adverse impact of the spread. The authors have demonstrated visual graphs and developed a mathematical model of the disease transmission pattern.

Research question of this paper aims at studying the chances that a patient diagnosed with Covid-19 must recover from this illness. This research aimed to identify which information (variables) about patient are dependent with patient recovery. Questions which are stated at the beginning of this study are: whether patient age is important in patient recovery/survival; whether patient gender is important in patient recovery/survival; whether information is patient from Wuhan or has visited Wuhan is important in patient recovery/survival; whether patient country from which comes from is important in patient recovery/survival. It is a classification problem (target variable is a response “Survived” 0 or 1) rather than a regression problem.

METHODOLOGY

A. Data Collection

With more than 10 million cases worldwide among with only half have recovered while the other half remains active, 500 000 deaths, the Coronavirus has become a pressing issue in every part of the world.

Because no country knows precisely the number of infected people among its citizens, the number of cases relies on the tests that the country performs and provides for the people.

In the data-oriented world that we live in today, all countries have been keeping track of all tests that they’ve done daily along with the results. Which fortunately enabled us to do this study.

Both datasets that have been worked with have been retrieved from Kaggle. The data set is the “Novel Corona Virus (COVID-19) epidemiological dataset”. The data is compiled by the Johns Hopkins University Center for Systems Science and Engineering (JHU CCSE) from various sources including the World Health Organization (WHO), DXY.cn, BNO News, National Health Commission of the People’s Republic of China (NHC), China CDC (CCDC), Hong Kong Department of Health, Macau Government, Taiwan CDC, US CDC, Government of Canada, Australia Government Department of Health, European Centre for Disease Prevention and Control (ECDC), Ministry of Health Singapore (MOH), and others. JHU CCSE maintains the data on the 2019 Novel Coronavirus COVID-19 (2019-nCoV) Data Repository on Github.[9]

This dataset contains 2 files, the first file “time_series_covid_19_recovered.csv” which provides us with daily information on the affected people in about 200 countries from the 22nd of January 2020 to the 16th of April 2020, and the second data set “COVID19 linelist data.csv” that contains observations of patients around the world where authorities have kept track of confirmed cases, recoveries and deaths.

B. Data Analysis Method

This study consists of exploring the two datasets thoroughly, understanding the variables and seeing their distributions, visualizing the evolution of numerical variables (time series of the numbers of cases in each country) and relationships between categorical ones (statistical testing). Afterwards, a simple logistic regression was performed to predict whether a patient recovers from Covid19 or not and some clustering techniques have been used to try to understand better recovery rates and how it is linked to the age of the patient. Logistic regression model and Random Forest algorithm are used, and the Chi-Square test of independence is used to determine if there is a significant relationship between two nominal (categorical) variables.

Author of the paper would have opted for Text analysis of the columns “Summary” and “Symptoms” but were unable to do so due to lack of data (portion of Nas in the columns). However, other analysis methods have been used such as descriptive data analysis for the time serial evolution of the number of cases and inferential data analysis when performing statistical tests to reveal the dependence between some variables.

To conclude with predictive analysis with “Survived” as a target variable based on a percentage that conveys how much a patient is likely to be treated and survive the virus.

C. Data Cleaning and Preprocessing

Some countries are divided into provinces/district in both datasets. At first, it has been thought that each district has to be treated individually in order to join the two datasets. But different regions exist in the two data frames, most importantly some regions in the patients list do not exist in the time series data frame and would therefore stay empty if this data frame was filled with the number of cases by specific regions of a country. Which is why was decided to eliminate regions and work with total numbers of cases in a country.

Taking care of Nas is a very important part of every data exploration process. Using the ggpubr library in R, the number of Nas was visualized in each variable of the dataset and decide to get rid of all variables that contain more than 250 Nas (a little less than the 1/4th of the data). Following variables were selected to continue this study: age, gender, number of cases in country, from Wuhan, visiting Wuhan, death, recovery and country.

D. Visualization

* Plotting time series(number of cases by country)

In the initial time series data frame, each day is a variable and that is not a proper structure for visualizing time series. A series of steps is required to obtain a data frame that can be worked with:

• The data frame was transformed to have a column where each line is a day
• The header was removed and some variables that will not be used for this visualization
• All variables were converted to numeric type
• The created column containing dates was set as date-time so that it is recognizable as such by R when dates on the x-axis were plotted.

The number of cases per country were chosen to be visualized continent by continent. Those graphs will enable to follow a country’s covid-19 outbreak and perceive at what moment the exponential spread has started. * Plotting survival against other categorical data

Using the ggplot2 library in R, data set has been grouped by category of the qualitative variable against which Survival using the “group_by” method was plotted.

The result is a histogram illustrating the counts of Survival values (0 or 1) in each category.

E. Chi-Square Test of Independence

This test is used to assess the existence or not of a relationship between two characteristics within a population, when these characteristics are qualitative or when one characteristic is quantitative and the other qualitative, or even when the two characteristics are quantitative but that the values have been combined. Note that this test makes it possible to check the existence of a dependence but in no case the direction of this dependence. Like in any statistical test, there is a null hypothesis H0 that has to be tested – determine whether there is enough evidence to “reject” this hypothesis. For this study test there is:

H0 : the two variables are independent

H1 : the two variables are not independent

á = 0.05 : tolerated risk

The mathematical steps were hardcoded into R to perform this test. The statistic that need to be calculated to perform this test is the following:

Where O is the observed score and E is the expected score. [10]

The observed scores are in a table of the values that already exist in our dataset and are already in our disposition. The expected scores table is one of the same dimensions as the observed scored. It is obtained by multiplying each value of the observed values with the sum of its row and dividing by the total number of observations.

Once these two tables are set, it can be proceeded to calculating the chi-squared statistic. The pvalue is calculated then. This is done by calculating the chi statistic for multiple random samples of our data, the pvalue is the proportion of the X2 statistic that are higher than the real X2 statistic of the sample.

If pvalue < 0.05 the null hypothesis is rejected, meaning that the two qualitative variables are independent.

If pvalue . 0.05 the null hypothesis is accepted, meaning that the two qualitative variables are dependent.

RESULTS

After cleaning and preprocessing the two datasets here are the resulting data frames that have been used for this study. There are 85 countries in the first data frame and the number of cases that appear each day in that said country.

Table 1

After cleaning the observations file and removing the variables that contained too many NAs, the following variables were decided to be kept: age, number of cases in country, gender, from Wuhan, visited Wuhan and the country of the patient. The two datasets were also joined by summing the number of cases during each month that was available in the first time series dataset for each country.

Table 2

By joining two datasets, plotting the evolution of cases in each country during these 4 months was also possible. Figure1 shows the results for the most popular countries during this pandemic (those that have been hit the most and most spoken of in the news).

Fig 1:- Countries affected the most with Covid-19

As it can be noticed, China, the epicenter of this pandemic, was the first to witness an exponential rise in the number of cases early February. The other countries followed suite during the month of March.

To extract some insights from the data concerning the relationship between the survival of the patient and their gender and age was also possible. As it might have been expected before, gender is independent of the survival of the patient, but age does play a role in whether a patient will recover or not.

Fig 2:- Recovery by gender

Fig 3:- Recovery by age

Another thing that might have been suspected is that a person would be less likely to recover if they’re coming from

Wuhan or if they’ve visited Wuhan. Here are the illustrations from the data:

Fig 4:- Recovery dependency if patient visited Wuhan or not

To confirm these hypotheses, and because these are categorical variables, it has been decided to do a Chi-square test of independence to see whether survival was dependent on any of those variables. Results are summarized in the following table:

Table 3:- Categorical variables dependency

The final step of the study was creating a classification model with the data that have been prepared and try to predict whether a person would survive the virus or not. The logistic regression model gave an accuracy of 84% and random forest one of 91%. Random forest gave a better result than logistic regression because the model weighs certain features as more important than others (feature selection), the absence of assumption of a linear relationship like regression models do, and because random forest takes random samples from the data set, forms many decision trees, and then averages out the leaf nodes to get a clearer model (ensemble learning).

DISCUSSION

The analysis confirms that it is indeed possible to predict the recovery -or no- of a covid19 patient given some information about his situation such as age, gender, and the country it comes from. It is to author grand surprise that the statistical tests indicate that the Survived variable is actually dependent on the gender of the patient. Figure2 shows that males are more likely to be infected than females, based on data in our dataset. On the other hand, whether the patient is from Wuhan or has visited Wuhan does not affect in any way the outcome of the observation – author expectations were otherwise.

In line with this research paper question testing, Survival is supposed to depend on the age of the patient as all of us have witnessed during the previous months. The death rates were particularly high for the elderly while adults, teenagers and kids seemed to recover way more successfully from the illness. The country is also supposed to affect the outcome of the treatment since the latter depends on the country’s means and dispositions to treat the patient. This study can contribute to predict future pandemic recovery trend by giving global picture of how many patients could be recovered from this virus and based on that information define further measures which can be overtaken. Future studies should take into account some variables that were in the “COVID19_line_list_data.csv” dataset but that couldn’t have been used because of the number of missing data in those columns, these columns include : “cases_in_country”, “reporting_date”, “symptoms_on_set”, “host_visit_date”, “exposure_start” and “exposure_end”. This information would certainly have been valuable to predict the survival of the patient.

CONCLUSION

This research aimed to identify which variables -pieces of information- about a patient is important to predict whether a person diagnosed with covid19 would survive the virus or not. Results conveyed that age, gender and country are the variables on which the target variable Survived depends.

Further research is needed to determine the reliability of these results since the dataset that have been worked with contains data until April 2020 only, while the pandemic is persisting and will still persist in the future.

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