J E. Toryila

Department of Human Physiology Faculty of Basic Medical Sciences,

College of Health Sciences, Bingham University, Karu.

G. P Oderinde; W.O hamman, E.U Umana

Department of Human Anatomy, Faculty of Basic Medical Sciences,

College of Health Sciences, Ahmadu Bello University Zaria

All correspondence to: J E. Toryila Department of Human Physiology Faculty of Basic Medical Sciences,

College of Health Sciences, Bingham University, Karu.

ABSTRACT

Background: Antimalaria drugs play important role in treating malaria but some medicinal agents when taken in overdoses or even within therapeutic ranges maybe toxic. Artesunate is one of the successfully used over-the-counter antimalaria drug in treating and preventing malaria but many studies have shown validation of toxicity on several organs. The aim of this study was to investigate the effect of Artesunate on the liver of Plasmodium berhei. infected mice. Methods: 25 mice were divided into 5 groups of 5 animals each, Group 1 received 0.5 ml/kg distilled water, Group 2 received 4 mg/kg/day Artesunate only, Group 3 was inoculated with Plasmodium berghei only, Group 4 and 5 were inoculated with P. berghei and treated with 4 and 8 mg/kg/day Artesunate respectively 72 hours after inoculation. The treatment lasted for a period of 5 days after which the mice were humanly sacrificed. Blood samples were collected by cardiac puncture and stored in 5 ml plain and EDTA bottles for biochemical analysis. The liver tissues were removed and placed in neutral buffered saline. RESULTS: There was no significant difference in the liver function biomarkers (ALT, AST and ALP) examined when compare among the groups. There was also significant increase in SOD activities in Group 5 when compared with Group 1, 2, 3 and 4, also there was a significant decrease in CAT and GSH activities in Group 1 when compared with Group 3, 4 and 5, the MDA activities in Group 5 showed a significant increase when compared with the other Group. There was increase in the liver weight and liver somatic index of Group 3 when compared to Group 1 Histological changes were observed in Group 2, 3, 4 and 5 when compared with Group 1. This research shows that Artesunate and malaria has adverse effect on the liver There is need for caution during the administration of Artesunate in the treatment of malaria. CONCLUSION: The effects of Artesunate on the liver of Plasmodium berghei infected mice were evaluated in this study. The results obtained show that Artesunate induces oxidative stress in mice infected with Plasmodium berghei and causes cytoplasmic vacuolation, traces of periportal mononuclear cells infiltration, and severe and diffused hepatic necrosis with karyolysis in the liver of mice infected with Plasmodium berghei.

INTRODUCTION

Malaria is a preventable and treatable disease. The primary objective of treatments is to ensure complete cure that is the rapid and full elimination of the Plasmodium parasite from the patient’s blood, in order to prevent progression of uncomplicated malaria to severe disease or death and to prevent chronic infection that leads to malaria-related anaemia (WHO 2018).

Artemisinin is currently the most widely used antimalarial drug against drug-resistant malaria. It is a Chinese herb that has been used over thousands of years for the treatment of fever. It is extracted from the leaves of Artemisia annua (sweet wormwood). Artemisinin and its derivatives (artesunate, artemether, arteether and dihydroartemisinin) are highly potent, rapidly eliminated antimalarial drugs

with a broad stage specificity of action. They clear parasitemia more rapidly than all other currently available antimalarial agents. Artesunate is a hemisuccinate derivative of the active metabolite dihydroartemisin. Currently it is the most frequently used of all the artemisinin-type drugs. Its only effect is mediated through a reduction in the gametocyte transmission. It is used in combination therapy and is effective in cases of uncomplicated P. falciparum (Izunya et al., 2010; Ashley et al., 2014).

The liver is a vital organ only found in vertebrates (Abdel-Misih and Bloomston, 2010). The liver is a reddish-brown wedge-shaped organ with four lobes of unequal size and shape. A human liver normally weighs 1.44 – 1.66 kg (3.2 – 3.7 Ib.), and has a width of about 15 cm, it is both the heaviest internal organ and the largest gland in the human body. Located in the right upper quadrant of the abdominal cavity, it rests just below the diaphragm, to the right of the stomach and overlies the gallbladder. The numerous functions of the liver are carried out by the liver cells. The liver is thought to be responsible for up to 500 separate functions usually in combination with other systems and organs. It plays a role in metabolism, regulation of glycogen storage, decomposition of red blood cells and hormone production. It also helps in detoxification of various metabolites, protein synthesis, and the production of biochemical necessary for digestion (Tortora and Derrickson, 2008).

Artesunate is one of the successfully used over-the-counter antimalarial therapies in treating and preventing malaria, M any studies on toxicity of Artesunate have shown validation of toxicity on the stomach, brain stem, and the liver. Administration of Artesunate for the treatment of malaria maybe an additional burden on the liver which is already undergoing insult from malaria parasite invasion of the hepatocytes, this therapy even in normal doses may result in severe hepatic injury to an already damaged organ. Some studies have suggested evidence of toxicity on the brainstem, superior colliculus, stomach, testis and liver in artesunate treated rats (Maude et al., 2009). Moreover, artesunate has been reported to destroy cancer cells and also reduces proliferation, interferes in DNA replication and cell cycle and enhance apoptosis through the intrinsic death pathway by ROS generation. It has been reported that artesunate is toxic at nanomolar concentration to malaria parasites, while micromolar concentration produced toxicity in mammalian cells (Turschner and Efferth, 2009). Evidences showed the neurotoxicity of artesunate at high doses (50 – 100 mg/kg per day oral and IM) in laboratory animals (Brewer et al., 1994) including the cytotoxicity of artesunate on tumor cell lines have been reported (Maudeet al., 2009). Therefore, this research was carried out further help in understanding the adverse effect of Artesunate and malaria on the liver and also provide the need for caution during the administration of Artesunate in the treatment of malaria.

MATERIALS AND METHODS:

Regeants: Randox test kit, Microscope, glass slides and cover slips, Formalin and bottles for fixing tissues, Dissecting set, Beakers and syringes, Anaesthesia (Ketamin), Normal saline, Haematoxylin and eosin. Experimental Desig: The animals were grouped into five groups of five (5) male mice per group. One control group and four treated groups; group III to V were inoculated with malaria on the first (1st) day of the experiment. Group I served as the control group and received 1 ml/kg normal saline intraperitoneally during the first day of the experiment and were given distilled water orally subsequently for a period of 5 days, Group II served as the Artesunate group and received 1 ml/kg normal saline intraperitoneally during the first day of the experiment and was latter administered with 4 mg/kg body weight of Artesunate starting from the 4th (fourth) day of the experiment, Group III served as the Untreated malaria infection groups while Group IV, and V were treated malaria infection group in different doses; the animals were infected with malaria and treated with Artesunate at 4 mg/kg body weight, and 8 mg/kg body weight respectively. Malaria infection was established on the 3rd day before commencement of treatment. The mice were treated with Artesunate for five (5) days before sacrifice. Artesunate was dissolved in distilled water and administered orally with the aid of a stainless metallic feeding cannula. The animals were then humanely sacrificed under anesthesia using Ketamine, through a midline incision on the thoracic wall, the heart was accessed and blood samples were collected by cardiac puncture. The blood samples were stored in 5 ml plain bottle for biochemical evaluation. The liver was quickly excised, rinsed in normal saline, gently blotted between folds of a filter paper and then weighed using an electronic compact digital scale. The liver tissue was cut into two pieces; the first piece was immediately fixed in Neutral Buffered Formalin for subsequent histological and histochemical analysis. The second piece was weighed and homogenized in Phosphate Buffer (pH 7.4) (at a ratio of 1 g to 5 ml respectively) for estimation of Oxidative stress parameters. The liver Enzymes and Oxidative Biomarkers were determined by ELISA technique (Kavishe et al., 2017). The tissues were Processed with Tissue Processing Machine and stained using haematoxylin and eosin (H and technique for general liver histology, Statistical Analysis: Data obtained were expressed as mean ± SEM (Standard Error of Mean). One-way analysis of variance (ANOVA) was used to compare the mean differences followed by LSD post-hoc test, P-value less than 0.05 was considered to be statistically significant. All the results were analyzed using the Statistical Package (SPSS version 20)

RESULTS AND DISCUSSION:

This study revealed that there was no significant difference in the liver weight and liver somatic index when compared between the Groups, although there was increase in the liver weight and liver somatic index of Group 3 when compared to Group 1 (Table 1.0). Group 4 also showed a slight increase in the liver weight and liver somatic index when compared to Group 1, Group 5 showed increase in the liver weight and liver somatic index when compared to Group 1 but it was not significant stastistically (Table 4.2). Rungruang et al (2013) reported that the liver of Plasmodium infected mice showed no significant difference in weight when compared with the control,hepatomegaly was observed on day 5 and 6 post-infection of Swiss Albino mice infected with Plasmodium yoelli nigeriensis (Ahmad and Srivastava, 2007). The reason for increase in liver weight and liver somatic index could be that the liver of infected mice is under very severe condition; part of systemic sequestration and anemia reduce the chance of circulating cells passing into the organs (Thuma et al., 1998).

There were no statistical differences (p<0.05) in all the liver injury biomarkers: Alanine transaminase (ALT), Aspartate aminotransferase (AST), Alanine phosphate (ALP) investigated between the experimental groups and the control groups. However, there was a slight increase in the ALT of Group 3 (128.00±20.06) when compared to other groups, there was also a slight decrease in ALT of Group 2 (106.75±15.34) when compared to the other groups (Table 1.0).

There was also no statistically significant difference (p<0.05) in the AST of the all the experimental groups when compared to the control groups (Table 1.0). There was slight increase in AST of Group 4 when compared to other groups. The AST activities of Group 2 were lower when also compared with the other groups

This investigation also revealed no statistically significant difference (p<0.05) in the ALP of all the groups when compared. Group 5 (155.40±25.52) showed the highest level of ALP when compared with the other Groups. Group 4 (148.40±49.36) showed a slight increase in ALP level when compared with Group 1 (137.25±32.42) and Group 2 (142.00±52.38).

Liver function tests are groups of blood tests that give information about the state of the liver (Lee, 2009). Injury to the liver may affect the integrity of hepatocytes leading to the release of liver enzymes such as alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphate (ALP) since these enzymes are confined to hepatocytes and released into the blood following liver injury. Hence, these enzymes are commonly used as markers of hepatic injuries (Ozer et al., 2008). The liver aspartate transaminase (AST) and alanine transaminase (ALT) are useful biomarkers of liver injury with some degree of intact liver function (Mengel and Schwiebert, 2005). These tests can be used to detect the presence of liver disease, distinguish among different types of liver disorders, gauge the extent of known liver damage and follow the response to treatment. In the present study, there was no significant difference statistically in serum level of AST, ALT and ALP as compared between the groups.

The level of liver dysfunction may be determined by the level of parasitemia in the body (Onyesom and Onyemakonor, 2011). Hamman et al (2011) also reported no statistical significant differences in ALP, AST and ALT levels in Wistar rats that were administered Artesunate at various doses.

The result revealed statistically significant (p<0.05) increase in Superoxide Dismutase (SOD) activities in all the groups; Group 4 (21.24±1.35); Group 5 (24.80±1.64) when compared to Group 1 (17.22±0.84) and Group 2 (16.58±0.45). Group 5 (24.80±1.64) also showed a significant statistical (p<0.05) increased in the SOD activities when compared to Group 1 (17.22±0.84), Group 2 (16.58±0.45), Group 3 (19.52±0.96) and Group 4 (21.24±1.35) There was significant statistical increase in Catalase (CAT) activities in Group 5 (10.14±0.50); Group 4 (10.24±0.39); and Group 3 (9.92±0.36) when compared with Group 1 (7.96±0.43) and Group 2 (8.00±0.33). There was also slight decrease in CAT activities in Group 1 (7.96±0.36) when compared to Group 2 (8.00±0.33) however it was not statistically significant (Table 2.0)

There was significant statistical increased in Glutathione (GSH) activities in Group 5 (38.16±2.97); Group 4 (43.88±1.81); and Group 3 (39.40±2.54) when compared with Group 1 (25.06±2.18) and Group 2 (31.04±1.43). There was also slight decrease in GSH activities in Group 1 when compared to Group 2 but was statistically insignificant (Table 2.0).

There was significant statistical increase in the Malondialdehyde (MDA) activities in Group 5 (37.68±3.15) when compared with other groups. There was also significant statistical increase in the MDA activities in Group 2 (34.44±2.75) when compared to Group 3 (25.58±1.94). This result also showed slight decrease in MDA activities in Group 3 (25.58±1.94) when compared to Group 1 (27.68±1.29) and Group 4 (29.06±2.08), however it was statistically insignificant (p<0.05) (Table 2.0).

The role of oxidative stress during malaria infection is still unclear. Some researchers argued a protective role, whereas other claims a relation to the pathophysiology of the disease (Sohail et al., 2007). However, other studies suggested that the generation of reactive oxygen and nitrogen species (ROS and RNS) associated with oxidative stress, plays a critical role in the development of systemic complications caused by malaria. Malaria infection induces the generation of hydroxyl radicals (OH-) in the liver, which most probably is the main reason for the induction of oxidative stress and apoptosis (Guha et al., 2006). The iron-rich environment within the parasite enables rapid production of specific redox active drug molecules and perhaps ROS that can lead to a rapid reaction and destruction of several specific systems within the parasite such as components of cellular membranes, the redox systems of the parasites, and the mitochondrial electron transport chain, and this may explain the rapid elimination of parasites by Artesunate (Kavishe et al., 2017).

In the present study there was significant increase in the GSH activities in Group 3, Group 4 and Group 5 when compared to Group 1 and Group 2 (Table 4.4). In Plasmodium parasites, GSH is also involved in the degradation of the toxic ferriprotoporphyrin IX (FP IX), which escapes from hemozoin formation. Additionally, GSH functions as an electron donor for the enzyme ribonucleotide reductase (RNR), and also crucial for DNA synthesis and cellular proliferation (Buchholz et al., 2010). Artesunate may induce DNA double-strand breaks in Plasmodium infection in a physiologically relevant dose and time dependent manner which is accompanied by an increase in the ROS level in the parasites (Gopalakrishnan and Kumar, 2015). Mannitol, a ROS scavenger, reversed the cytotoxic effect of Artesunate and reduced DNA damage and modulation of GSH activities impacted ROS and DNA damage induced by Artesunate (Gopalakrishnan and Kumar, 2015). The present study also showed no statistically significant difference between the group that was inoculated with malaria but not treated and the groups that were treated with Artesunate after malaria inoculation, it was also discovered that at increased dose of Artesunate there was reduce GSH activities when compared with the malaria treated group. This could occur because Plasmodium possesses two redox systems which are the thioredoxin and the glutathione system, glutathione is the most abundant low molecular weight redox active thiol in the parasites existing primarily in its reduced form representing an excellent thiol redox buffer. This allows for an efficient maintenance of the intracellur reducing environment of the parasite cytoplasm and its organelles. During development, malaria parasites are exposed to environmental and metabolic stresses.

H&E stain result of the liver sections from Group 1 showed normal cytoarchitecture of the liver parenchyma; the characteristic appearance of the hepatocytes radiating from the central vein. Fine vascular spaces separate the thin plate of hepatocytes, the sinusoids and Kupffer cells found within the sinusoid lining (Plate I). Liver section from Group 2 showed some abnormal morphological characteristics showing Cytoplasmic Vacuolation (CyV) and Sinusoidal Congestion (SC) (Plate II). Liver section from Group 3 showed Cytoplasmic Vacuolation (CyV) with Kupffer cells Hyperplasia (KH), Central vein congestion (CCV) and Hemozoin (H) (Plate III). Liver section from Group 4 showed Cytoplasmic Vacuolation (CyV), Sinusoidal congestion (SD) and traces of Periportal Mononuclear cells infiltration (PM) (Plate IV). Liver section from Group 5 showed distortion in the cytoarchitecture with severe and difused Hepatic Necrosis with karyolysis (HN), Cytoplasmic vacuolation (CyV), traces of Periportal Mononuclear cells infiltration (PM) and Sinusoidal Congestion (SC) (Plate V).

Table 1 Liver functions biomarkers

Table 2. Oxidative stress markers

Oneway ANOVA test followed by Tukey poct hoc test; Results expressed as mean±SEM, cells carrying same superscripts on each column are significantly different (p<0.05). SOD: Superoxide Dismutase; CAT: Catalase; GSH: Glutathione; MDA: Malondialdehyde.

^CCV CyV

H

KH

Plate III: Transverse section of the Liver from

Group 3 showing Cytoplasmic Vacuolation (CyV) with Kupffer cells Hyperplasia (KH), Central vein Congestion (CCV), Hemozoin (H) (H&E x250)

CyV

SC

CV

HN

PLATE V: Transverse section from the Liver from Group 5 showing distortion in the cytoarchitecture with severe and diffused Hepatic Necrosis with karyolysis (HN), and Sinusoidal Congestion (SC), Cytoplasmic vacuolation (CyV) (H&E x250)

CONCLUSION: This research shows that Artesunate and malaria has adverse effect on the liver There is need for caution during the administration of Artesunate in the treatment of malaria.

REFERENCES

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• Ahmad, R., and Srivastava, K. (2007). Effect of Plasmodium yoelii nigeriensis infection on hepatic and splenic glutathione-S- transferase(s) in Swiss albino and db/+ mice: efficacy of mefloquine and menadione in antimalarial chemotherapy. Parasitology. 134(7):931 – 938
• Ashley, E. A., Dhorda, M., Fairhurst, R. M., Mokuolu, O. A., and Olaosebikan, R. R. (2014). Spread of Artemisinin Resistance in Plasmodium Plate IV: Transverse section of the Liver from Group 4 showing Central Vein, Cytoplasmic Vacuolation (CyV), Sinusoidal Congestion (SD) and traces of Periportal Mononuclear cells infiltration (PM) (H&E x250) falciparum Malaria. New England Journal of Medicine. 371(1)411 – 423

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• Hamman, W. O., Musa, S. A., Muzaffer, F., Sani, S. O., Umana, U. E., Timbuak, J. A., Nok, A. J., and Ojo, S. A. (2011). Acute exposure to Artesunate and its effect on the hematological indices, hepatotoxicity and histology of the liver of adult Wistar rats. Asian Journal of Medical Sciences 3(4):176 – 179
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• Kavishe, R. A., Koenderink, J. B., Alifrangis, M. (2017). Oxidative stress in malaria and artemisinin combination therapy: pros and cons. Federation of European Biochemical Societies. 284(1):2579 – 2591
• Lee, M. (2009). Basic skills in interpreting laboratory data. ASHP. Pp. 259
• Maude, R. J., Plewes, K., Faiz, M.A., Hanson, J. Charunwatthana, P., (2009). Does artesunate prolong the electrocardiograph QT interval in patients with severe malaria? The American Journal of Tropical Medicine and Hygiene. 80(1):126-132.
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The post Evaluation of the Effects of Artesunate on the Liver of Plasmodium Berghei Infected Mice appeared first on Nigerian Biomedical Science Journal.

Abd-Elhafez EA

Department of Anatomy and Histology, Faculty of Veterinary Medicine, Assiut University, 71526, Egypt

All Correspondences to: Fatma, El-Zahraa AM, Department of Anatomy and Histology, Faculty of Veterinary Medicine,

Assiut University, 71526, Egypt e-mail: f.histology@aun.edu.eg

The integrity of uterine circulation is essential for normal pregnancy outcome. The present study describes for the first time the histology, histochemistry, and ultrastructure of the different types of vessels in the uterus at critical early stages of pregnancy. Arteries were congested and showed PAS-positive granules. veins characterized by its wide lumen and thick adventitia rich in collagen and elastic fibers and showed PAS-positive reaction in their wall. Lymphatics showed a positive reaction with PAS-AB combination. Many arteriols observed in the uterine wall and showed PAS-positive granules and venules appeared congested in the endometrium. Spiral artery and arterial sinuses also demonstrated. Anastomosis between arterioles and venules was recorded. Blood vessels of a special structure were demonstrated presenting a vascular wall of variable thickness, unevenly distributed smooth muscle in the wall, smooth muscle fibers arranged in a different direction. Spirally oriented arterioles and venules were also demonstrated. Sclerotic changes observed in the wall of some vessels. Glomus cells observed in the wall of many arteries, arterioles, spirally oriented arterioles, and arteriovenous anastomosis. filopodia-like protrusions and caveolae were the most characteristic feature of the endothelium.

Key Words: Blood vessels; Lymphatics; Glomus cells; Telocyte; Filopodia; Early pregnancy.

The integrity of uterine circulation is essential for normal pregnancy outcome. The clinical relevance of maternal uterine vascular adaptation during pregnancy is underscored by the fact that its aberrance is associated with several common gestational pathologies, including intrauterine growth restriction, gestational diabetes, and preeclampsia [1]. Preeclampsia is a major cause of maternal and neonatal mortality and morbidity

It is known that the vascularization of the uterus is of primary importance in the pregnancy success [3], and vascular disorders may play a role in pathological pregnancies [4]. Furthermore, normal uterine vascular development may have critical effects on the growth and development of the fetus and insufficient uterine vascular establishment is associated with increased risk for cardiovascular morbidity in adult life [5]. The lymphatic circulation plays an important role in regulating content of interstitial fluid and adaptive immunity [6] and lymphatic vessels could also play a role in maternal and fetal immunity [7]. The uterine lymphatics during pregnancy have been scantily studied. Rabbit has a duplex uterus, with the main uteroovarian arteries and veins running parallel to, but well outside of, the uterine wall within the mesometrium. The vessels of the mesometrium are perfused by arterial blood coming from either the uterine or the ovarian end. Secondary vessels analogous to the arcuate arteries in humans may form redundant loops with the main artery, and tertiary radial arteries connect the arcuate loops with the uterine wall. These radial arteries divided into premyometrial arteries.

The premyometrial arteries enter the uterine wall supplies the myometrium [1]. A better understanding of the basic morphological structure of the blood vessels of the uterus of the rabbit may guide the clinicians to the design of strategies to improve their reproductive efficiency and help in solve pregnancy problems which related to the blood vessels. Yet, the available literature lacks any detailed

information concerning the uterine vessels during early stages of pregnancy. The aim of the present investigation was to study the different types of vessels in the uterus at critical early stages of pregnancy. The current study focused on arteries, veins, lymphatics, arterioles, venules, blood vessels of special structure.

MATERIAL AND METHODS Sample collection

The study was approved by the Ethics Committee of Assiut University, Egypt. The material included in this work was originated from the female genitalia of 6 healthy New Zealand rabbits at 0 and 7days of pregnancy. The uterus was taken immediately after slaughtering.

Histological preparation

The collected materials were dissected as soon as possible and immediately fixed in Bouin• fs fluid for 22 hours. The fixed materials were dehydrated in an ascending series of ethanol, cleared in methyl benzoate and then embedded in paraffin wax. Transverse paraffin sections at 1-7 ƒÊm in thickness were cut and stained with Harris haematoxylin and eosin for general histological examination, Crossmon• fs Trichrome for identification of collagenous and muscle fibers and Wigert• fs resorcin fuchsine for identification of elastic fibers. For carbohydrate histochemistery, sections were stained with Periodic Acid Schiff (PAS) technique. For demonstration of neutral mucopolysaccharides, combined alcian blue-PAS technique were used [8].

Semithin sections and transmission electron microscopic preparations

Other small specimens of the uterus were fixed in a mixture of 2.5% paraformaldehyde and 2.5% glutaraldehyde in 0.1M Na-cacodylate buffer, pH 7.3 for 4 hours at 4 Co. They were washed in the same buffer used and then post-

37

fixed in 1% osmic acid in 0.1M Na-cacodylate buffer for further 2 hours at room temperature. The samples were then dehydrated in ethanol and embedded in Araldite-Epon mixture. Semithin sections (1ƒÊm in thickness) were cut and stained with Toluidine blue and examined under a light microscope. Thin sections, obtained by a Reichert ultramicrotome, were stained with uranyl acetate and lead citrate [9] and examined with a Philips EM 400.

Myometrial artery (A) showed tunica media (TM) and vascular tunica adventitia (TA) (Harris haematoxylin and eosin). (G) Longitudinal section in myometrial artery (A) demonstrate PAS-positive granules (blue arrow) (PAS). (H) Myometrial artery (A) showed well developed internal elastic membrane (black arrow) (Wigert’s resorcin fuchsine).

RESULTS

O day of pregnancy

Mesometrial arteries were congested and demonstrated in varies directions cross and longitudinally oriented arteries (Figure 1A and 1B). The wall of these arteries was formed mainly of 3 – 5 layers of smooth muscle fibers tunica media, intermingled by elastic fibers. The tunica intima consisted of a thin layer of flat endothelial cells separated from the coating muscular media by a well-developed highly tortuous internal elastic membrane. The adventitia contained abundant elastic fibers and rich in collagenous fibers (Figure 1A-1D). In addition, there were arteries recorded in the mesometrium with spirally oriented smooth muscle fibers in the tunica media (Figure 1E). Myometrial arteries possessed a wide lumen and surrounded by a 2-4 layer of smooth muscle fiber. In addition, the adventitia showed several blood vessels. These arteries demonstrate PAS-positive granules in their wall and well developed internal elastic membrane (Figure 1F-1H). Mesometrial veins were numerous wide thin-walled vessels of different size. The tunica intima was surrounded by a thin tunica media composing of a 1-2 layer of smooth muscle fibers encircled by a relatively thicker adventitia rich in collagen and elastic fibers (Figure 2A and 2B). At the myometrial region, venae showed PAS-positive reaction in their wall (Figure 2C). Several thin-

Figure 1) Arteries at 0 day pregnancy. (A) Cross section in mesometrial artery (A) showed tunica media (TM) and internal elastic membrane (black arrow) (Combined Alcian blue-PAS). (B) Longitudinal section in the congested mesometrial artery (A) and tunica media smooth muscle fibers (TM) (Harris haematoxylin and eosin). (C) Cross section in mesometrial artery (A) showed tunica media (TM), internal elastic membrane (black arrow), tunica adventitia (TA) and elastic fibers (red arrow) (Wigert’s resorcin fuchsine). (D) Showed tunica media (TM) and tunica adventitia rich in collagenous fibers (TA) (Crossmon’s Trichrome).

1. Showed tunica media formed from spirally oriented smooth muscle fibers (TM) and internal elastic membrane (black arrow) (PAS). (F)

Figure 2) Veins and lymphatics at 0 day pregnancy. (A) Mesometrial vein appeared as wide thin-walled vessels (V) and lymph vessel (L) showed valve (red arrow) (Harris haematoxylin and eosin). (B) Mesometrial vein

1. showed thick tunica adventitia (TA) rich in elastic fibers (blue arrows) (Wigert’s resorcin fuchsine). (C) PAS-positive reaction in the wall of myometrial vein (V) (PAS). (D) Myometrial lymph vessel (L) with a thin valve (black arrow) (Harris haematoxylin and eosin). (E) Myometrial lymph vessel (L) with a valve (black arrow) and showed a positive reaction with PASAB combination (red arrows) (Combined Alcian blue-PAS). (F) Endometrial lymph vessel with a wide irregular lumen (L) (Crossmon’s Trichrome).

Figure 3) Arterioles and venules at 0 day pregnancy. (A-C) Mesometrial arterioles (red arrows), venules (black arrows), tunica media (TM), internal elastic membrane (blue arrow) and tunica adventitia which composed mainly of collagen fibers (TA) (figure A stained with Harris haematoxylin and eosin, figure B stained with Wigert’s resorcin fuchsine and figure C stained with Crossmon’s Trichrome). (D and E) Myometrial arterioles (red arrows), tunica media (TM) and internal elastic membrane (blue arrow) (Figure D stained with Harris haematoxylin and eosin and figure E stained with Wigert’s resorcin fuchsine). (F-I) Mesometrial arterioles (red arrows), venule (black arrow), smooth muscle (black arrow head), interrupted internal elastic membrane (green aarrow) and PAS-positive granules (blue arrows) (Figure F stained with Harris haematoxylin and eosin, figure G stained with Crossmon’s Trichrome figure H stained with Wigert’s resorcin fuchsine and figure I stained with PAS).

walled wide-lumened lymphatics were recorded within the mesometrium. Their wall is composed of an intimal tunic surrounded by a thick fibrous layer consisting of collagenous, elastic fibers and connective tissue cells. Some of these vessels demonstrate valves (Figure 2A). Myometrial lymphatics were composed of an intimal tunic surrounded by a fibrous layer of collagenous and elastic fibers. The valves of these vessels were mainly thin and consisted of a single cellular layer. The wall of myometrial lymph vessels showed a positive reaction with PAS-AB combination (Figure 2D and 2E). The lymphatic vessel in the endometrium appeared as wide irregular thin walled vessels (Figure 2F). Several arterioles were demonstrated within the mesometrium. These blood vessels were composed of 1-2 smooth muscle fiber-thick tunica media encircling a thin tunica intima with interrupted internal elastic membrane and surrounded by tunica adventitia which composed mainly of collagen fibers (Figure 3A-3C). In addition, arterioles of the myometrium were composed of 1-2 smooth muscle fiber-thick tunica media surrounded a thin intimal tunic with interrupted internal elastic membrane and was surrounded by fibrous adventitial tunic which mainly consisted of collagen fibers (Figure 3D and 3E). Many arterioles were observed within the endometrium with 1-2 smooth muscle fiber-thick tunica media encircling a thin tunica intima with interrupted internal elastic membrane and surrounded by tunica adventitia which contained mainly elastic and collagenous fibers. In addition, few PASpositive granules observed on the wall (Figure 3F-3I). Venules wall consists of an intimal tunic surrounded by one cell-thick tunica media of circularly arranged smooth muscle fibers coated by thick adventitial tunic consisting of collagenous fibers and connective tissue cellular elements.

However, venules of endometrium appeared as dilated thin wall vessels engorged with blood (Figure 3A and 3G). Spiral artery also demonstrated and characterized by the wide lumen and spiral course. Its wall consists of tunica intima of endothelial cells surrounded by a thin layer of smooth muscle fibers and thin adventitia (Figure 4A). In addition, arterial sinuses demonstrated and characterized by wide irregular lumen, one layer of smooth muscle tunica

media and unevenly disreputed elastic fibers (Figure 4B-4D). Anastomosis between arterioles and venules was recorded in mesometrium and showed an abrupt change from the arteriole to a venule. Another arteriovenous anastomosis in the mesentery with several glomus cells and its lumen

is closed (Figure 4E and 4F). Anastomosis between arterioles and venules was also recorded in the myometrium and (Figure 4G). Blood vessels of a special structure were demonstrated within the mesometrium presenting a vascular wall of variable thickness. The thicker portions of the wall showed a tunica media of 2-3 layers of smooth muscle fibers. However, the thin portions of the wall showed tunica media consisting of an interrupted layer of smooth muscle fibers (Figure 5A). In addition, another vessel showed 2-3 layers of longitudinally-arranged smooth muscle fibers at one side only (Figure 5B). Various special structures arteries observed within the mesometrium.

Characterized by the wide lumen and 2-3 layer of glomus cells (Figure 5C). Another special type of artery characterized by smooth muscle fibers arranged at various directions and fibrinoid deposition (Figure 5D). Various special structures veins observed within the mesometrium and characterized by a wide irregular lumen. The flattened endothelial cell lining was surrounded by 2-3 cell thick muscular media composing of circularly arranged smooth muscle fibers. These muscle cells were oriented into several protrusions, within the consecutively irregular lumen, bearing as muscular pads. The relatively thick

Figure 4) Spiral artery, arterial sinuses, and Anastomosis between arterioles and venules at 0 day pregnancy. (A) Spiral artery (SA) with the wide lumen and spiral course (Harris haematoxylin and eosin). (B-D) arterial sinuses (AS), arteriole (red arrow) and elastic fibers (black arrows) (figure B stained with Harris haematoxylin and eosin, figure C stained with Crossmon’s Trichrome and figure D stained with Wigert’s resorcin fuchsine). (E and F) Anastomosis between arterioles and venules in the mesometrium and (G) Anastomosis between arterioles and venules in the myometrium, arterioles (blue arrows), venules (red arrows) and glomus cells (black arrows) (figure E, F and G stained with Harris haematoxylin and eosin).

Figure 5) Blood vessels of a special structure in the mesometrium at 0 day pregnancy. (A) Blood vessel showed vascular wall of variable thickness (blue arrows) (Harris haematoxylin and eosin). (B) Special vessel showed longitudinally-arranged smooth 3 muscle fibers at one side only (black arrow) (Harris haematoxylin and eosin). (C) Special structures arteries characterized by the wide lumen and glomus cells (red arrows) (PAS). (D) Special type of artery characterized by smooth muscle fibers arranged at various directions (black arrows) and fibrinoid deposition (black arrowhead) (PAS). (E and F) Special structure vein characterized by smooth muscle cells oriented into several protrusions within the consecutively irregular lumen an showed PAS-positive granules (black arrows) (E stained with Harris haematoxylin and eosin and F stained with PAS). (G) Small arterioles of a special structure with few glomus cells (red arrows) (Harris haematoxylin and eosin). (H) Arteriole with an occlusive lumen and one layer of glomus cells (red arrows) (Harris haematoxylin and eosin).

39

Figure 6) Blood vessels of the special structure in the myometrium at 0 day pregnancy. (A) Artery with triangular shape lumen and surrounded by tunica media of spirally oriented smooth muscle fibers (black arrow) (Harris haematoxylin and eosin). (B) Artery with patches of longitudinally smooth muscle (blue arrow) in between circularly arranged smooth muscle (black arrow) (Harris haematoxylin and eosin).

1. Spirally oriented artery (A) with a clear internal elastic membrane (green arrow) (Wigert’s resorcin fuchsine). (D) Spirally oriented arterioles with glomus cells in the tunica media (red arrows) (Wigert’s resorcin fuchsine). (E) Artery of a special structure with the narrow lumen (red arrow) and unevenly distributed elastic fibers in the wall (green arrows) (Wigert’s resorcin fuchsine).

Figure 7) Blood vessels of the special structure in the myometrium at 0 day pregnancy. (A) Arteries characterized by the wide irregular lumen (A) and unevenly distributed elastic fibers in the wall (green arrows) (Wigert’s resorcin fuchsine). (B) Veins of special structure (V) with irregular lumen and thick adventitia (TA) (Harris 4 haematoxylin and eosin). (C) Vein of special structure (V) with an irregular lumen and thick adventitia rich in elastic fibers (green arrows) (Wigert’s resorcin fuchsine). (D) Small arterioles surrounded by glomus cells (red arrows) (Harris haematoxylin and eosin).

adventitia mainly consists of collagenous and elastic elements. In addition, several islets of longitudinally-arranged smooth muscle fibers surrounded these vessels and characterized by PAS-positive granules (Figure 5E and 5F).

Small arterioles of a special structure were also observed and lined with 2-3 endothelial cells and surrounded by smooth muscle fibers of tunica media with few glomus

Figure 8) Digital colored TEM micrographs of uterine blood vessels at 0 day pregnancy. (A) Endometrial arteriole endothelial cells (EC), red blood cell (green arrow), smooth muscle cells (black arrows). cytoplasmic processes between endothelial cells and smooth muscle cells (red arrows) and filopodia-like protrusions of the endothelial cell toward the lumen (blue arrows). (B) Capillary with many caveolae (blue arrows), vesicles (red arrows), free ribosomes (black arrowhead), mitochondria (black arrow) and myaline figure (green arrow). (C) Spirally oriented capillary

(C). (D) Endothelial cells contained abundant ribosomes (black arrows) and small caveolae (red arrows) and large caveolae (green arrow).

cells (Figure 5G). Another arteriole with an occlusive lumen and one layer of glomus cells also demonstrated (Figure 5H). Blood vessels of the special structure were demonstrated also within the myometrium. Our observation revealed that there were arteries with triangular shape lumen and surrounded by tunica media of spirally oriented smooth muscle fibers (Figure 6A). In addition, some arteries demonstrate 2-4 layer of circularly arranged smooth muscle fibers and patches of longitudinally smooth muscle fibers in between (Figure 6B). Spirally oriented arteries were present within the myometrium with a wide lumen, clear internal elastic membrane and elastic fibers distributed at a various layer of the vessels wall (Figure 6C). Another spirally oriented arterioles observed and characterized by clear glomus cells in the tunica media (Figure 6D). In addition, there was another special type of arteries which characterized by the narrow lumen, thick wall and unevenly distributed elastic fibers in the wall (Figure 6E). Although, there was another type of arteries characterized by the wide irregular lumen and unevenly distributed elastic fibers in the wall (Figure 7A). Veins of special structure could also be observed within myometrium bearing an irregular lumen and relatively thick fibrillar adventitia mainly consists of collagenous and elastic elements (Figure 7B and 7C). Small arterioles of a special structure were lined with 2-3 endothelial cells and surrounded by glomus cells (Figure 7D).

Transmission electron microscopic observations of uterine blood vessels at 0day pregnancy showed that there were endometrial arterioles with endothelial cells surrounded by two smooth muscle layers. Endothelial cells and the inner smooth muscle cells gave cytoplasmic processes to each other and numerous filopodia-like protrusions of the endothelial cell toward the lumen (Figure 8A). Capillary with overlapping endothelial cell ends also observed and characterized by many caveolae of different size distributed singly or in small groups, numerous small vesicles, many free ribosomes, some mitochondria, and myaline figure (Figure 8B). In addition, spirally oriented capillary demonstrated and characterized by abundant ribosomes and rich in caveolae which small in size and arranged in rows or large in size and distributed singly (Figure 8C and 8D)

7 days of pregnancy

The most prominent characteristic features of this stage of pregnancy were the observation of numerous vessels of special morphological character. Some arteries in the myometrium contained batches of longitudinal smooth muscle in between the circular smooth muscle of the tunica media (Figure 9A). In some arteria, smooth muscle of the tunica media appeared in different directions (Figure9B). Other arterial tunica media showed different thickening of the smooth muscle from 1 to 3 layer (Figure 9C). In addition, there was arteria showed inner circular and outer longitudinal epitheloid cells in tunica media and thick adventitia (Figure 9D).

Our observation also revealed that Internal elastic lamina of some arteries appeared less developed but the external elastic lamina clearly well developed (Figure 9E) and in other arteries the elastic fibers distributed in one side of the wall only (Figure 9F). PAS-positive granules and PAS-positive material demonstrated in the wall of the blood vessels in the large amount (Figure 10A and 10B). Moreover, our observation also revealed that some arterioles surrounded by numerous venules (Figure 10C). Other arterioles of different size characterized by a very narrow lumen and some of them with dumbbelllike appearance (Figure 10D and 10E).

At this stage of pregnancy Arteriovenous anastomosis also observed with thick fibrous adventitia. The arterial side of the anastomosis may show narrow lumen and thick media (Figure 10F). However, another artery tunica media formed from epitheloid cells (Figure 10G).

Endometrial arteries showing special character one of them showed extensive venous or arterial profiles at the tunica adventitia of many arteries (Figure 11A). Spirally oriented arterioles and venules also observed at the endometrium (Figure 11B and 11C). In addition, arteriovenous anastomosis observe at the endometrium (Figure 11D). Extensive capillary network observed in lamina propria and take strong PAS positive reaction (Figure 11E).

At this stage of pregnancy transmission electron microscopic observations of uterine blood vessels showed that endothelial cells of the arteriole appeared swell to a plump with abundant mitochondria, numerous cytoplasmic filopodic extensions toward the lumen and few microvacuoles. The nuclei of these endothelial cells change to large nuclei (Figure 12A). However, the endothelium of the venule characterized by the presence of abundant free ribosomes, many mitochondria, some caveolae, few rough endoplasmic reticulum, scant microvacuoles and several filopodic extensions toward the lumen (Figure 12B-12D). Capillary also observed and encircled by telocyte (Figure 13A). Endothelium of the capillary contained caveolae and

Figure 9) Arteries at 7 days pregnancy. (A) Myometrial artery showed batches of longitudinal smooth muscle (blue arrows) in between the circular smooth muscle of the tunica media (TM) (Harris haematoxylin and eosin). (B) Myometrial artery with smooth muscle of the tunica media appeared in different directions (blue arrows) (Harris haematoxylin and eosin). (C) Myometrial artery tunica media showed different thickening of the smooth muscle (blue arrows) (Harris haematoxylin and eosin). (D) Myometrial artery showed inner circular epitheloid cells (blue arrow) and outer longitudinal epitheloid cells (red arrow) in tunica media and thick adventitia (TA) (Harris haematoxylin and eosin). (E) Myometrial artery showed less developed internal elastic lamina (red arrow) and well developed external elastic lamina (green arrows) (Wigert’s resorcin fuchsine). (F) Myometrial artery with elastic fibers distributed in one side of the wall only (green arrows) (Wigert’s resorcin fuchsine).

Figure 10) Myometrial vessels. (A and B) PAS-positive granules (black arrow) and a large amount of PAS-positive material demonstrated in the wall of the blood vessels (blue arrows) (figure A and B stained with PAS).

1. Arteriole (blue arrow) surrounded by numerous venules (black arrows) (Harris haematoxylin and eosin). (D and E) Semithin sections of arterioles of different size characterized by the very narrow lumen (black arrows) and some of them with dumbbell-like appearance (red arrows). (F and G) Arteriovenous anastomosis showed the arterial side of the anastomosis (blue arrow), venous side of the anastomosis (black arrow) and epitheloid cells (red arrows) (figure F and G stained with Harris haematoxylin and eosin).

Figure 11) Endometrial vessels. (A) Artery with extensive venous and arterial profiles at the tunica adventitia (black arrows) (Harris haematoxylin and eosin). (B and C) Semithin sections showed spirally oriented arterioles (black arrows) and venules (red arrows). (D) Arteriovenous anastomosis showed the arterial side of the anastomosis (blue arrow), venous side of the anastomosis (black arrow). (E) Extensive capillary network observed in lamina propria showed strong PAS positive reaction (black arrows) (PAS).

Figure 12) Digital colored TEM micrographs of uterine blood vessels at 7 days pregnancy. (A) Arteriole endothelium (E), nucleus (N), mitochondria (blue arrows), cytoplasmic filopodic extensions (black arrows) and microvacuoles (red arrow). (B) Venule endothelium (E), nucleus (N) and filopodic extensions (black arrows). (C and D) Venule endothelium (E), nucleus (N) and filopodic extensions (black arrows), free ribosomes (black arrowheads), mitochondria (red arrows), caveolae (green arrows), rough endoplasmic reticulum (blue arrows), scant microvacuoles (red arrowhead).

Figure 13) Digital colored TEM micrographs of uterine blood vessels at 7 days pregnancy. (A) Capillary (C), telocyte (T), telopodes (black arrows) and microvacuoles 6 (red arrow). (B) Capillary (C), telocyte (T), nucleus (N), caveolae (green arrows) and free ribosomes (blue arrow), microvacuoles (red arrows) and rough endoplasmic reticulum (black arrows).

free ribosomes and large microvacuoles resemble the microvacuoles which present in the nearby telocyte (Figure 13B).

DISCUSSION

Rabbit is considered one of the widely used laboratory animals. Rabbits have been used extensively for basic research in drug and bacteria, toxicology, healing, tissue and organ culture, mycology, skin sensitivity, immunology, ophthalmology, oncology and reproductive biology [10]. The rabbit is believed to be a helpful model for comparative biology in humans, concerning sperm capacitation and the general reactivity of the female genital tract during the reproductive cycle [11].

In this study, we established for the first time a detailed investigation of the arterial, venous and lymphatic mapping of the uterus at early stages of pregnancy in the rabbit. The viability of this vessels were very important for the female reproduction and fetus maintenance. It was recognized in the present study that arteries were congested and demonstrated in varies directions in the mesometrial region.

Rabbit has uteroovarian arteries running within the mesometrium and giving secondary and tertiary vessels (radial arteries). These radial arteries give the premyometrial radial arteries enter the uterine wall supplies the myometrium. The uterus is drained by a venous system that parallels the arterial tree, with closely apposed arteries and veins [1]. The present study demonstrated that arteries of mesometrial and myometrial regions were characterized by well-developed elastic lamina which helps the vessel to bear changes in blood pressure during pregnancy.

Large increases in uteroplacental blood flow during gestation are essential for normal foetal growth and survival and occur in every mammalian species studied, including human beings [12]. During pregnancy, the passive (fully dilated) diameter of the human uterine artery is approximately doubled [13], with similar changes reported in rodents, sheep, pigs and guinea pigs [14,15]. The myometrial arterial supply that course between the outer and middle thirds of the myometrium and this zone is referred to as the ‘vascular zone’.

Because of their semicircular course, these arteries are referred to as ‘arcuate’ arteries [16]. Our observation revealed that myometrial arteries characterized by its wide lumen, thin muscular coat, and vascularized adventitia. Arteries and veins are arranged in close apposition in a number of species [17]. Our observation revealed that mesometrial and myometrial veins characterized by its thin wall encircled by a thick adventitia rich in collagen and elastic fibers. The uterus is an immunologically unique organ in that it must be able to protect itself effectively from invading pathogens, but at the same time is faced with periodic exposures to foreign cells and tissues, namely allogeneic spermatozoa and the fetal-placental unit. Even though the uterus seems to be an effective site of immunization for some antigens [18]. In rats, mice, and similar small mammals, there is only one main lymphatic plexus lying between the circular and longitudinal muscles. In addition, Endometrium possessed few or no lymphatics [19].

Our observation revealed that several thin- walled wide-lumened lymphatics were recorded within the mesometrium. Their wall is composed of an intimal tunic surrounded by a thick fibrous layer consisting of collagenous, elastic fibers and connective tissue cells. Some of these vessels demonstrate valves. The rat showed similar observation [20].

Myometrial lymphatics were composed of an intimal tunic surrounded by a fibrous layer of collagenous and elastic fibers. The valves of these vessels were mainly thin and consisted of a single cellular layer. The wall of myometrial

lymph vessels showed a positive reaction with PAS-AB combination. The lymphatic vessel in the endometrium appeared as wide irregular thin walled vessels similar observation obtained in the rat [21]. Rabbit arterial sinuses as arterial device affecting the pulse pressure and linear velocity of maternal blood flow and regulate optimal condition for nutritional exchange [22]. The present study revealed that Several arterioles, venules observed at the different layer of the uterus. in addition, spiral artery also observed with a wide lumen and spiral course. As well as, arterial sinuses characterized by wide irregular lumen, a single layer of smooth muscle and unevenly distributed elastic fibers.

Arteriovenous anastomosis demonstrated at a various layer of the uterus. Some of this vessels showed several glomus cells. Arteriovenous anastomoses are one of the key adaptations for thermoregulation [23]. The glomus cell observed at this study in the wall of arteries, arterioles, arteriovenous anastomosis, and spirally oriented arterioles. the glomus cells can be considered as a highly reactive special type of cell, which cause a reduction in the diameter of the lumen by their ability to swell or even complete obliteration of the vessel lumen [24-26].

The histological and histochemical investigation of the uterine vasculature in the rabbit revealed several blood vessels of special regulatory devices which indicate a unique function. These vessels were observed at different uterine parts.

Our observation revealed that there was fibrinoid deposition at some arteries similar observation reported in women [27,28]. In addition, Fibrinoid deposition and elastic fibers contents of vessels will increase at 7 days pregnancy of pregnancy. gestation sclerosis discused as a type of sclerosis developed due to circulatory demands increased [29]. However, gestational sclerosis considered as physiological processes as they are not uniform in nature, intensity and distribution [26].

The present study demonstrated some spirally oriented arterioles with extreme thickening of their wall by glomus cells in addition to arterioles with a narrow lumen. This constriction of arterioles increases

peripheral resistance to blood flow giving an important role in the regulation of systemic blood pressure. Similar observations were recorded in camel ovaries [26] and camel skin [30].

In our study, Various vessels of special structure detected at different parts of the uterus. The vessels of the special structure are supposed to possess an important regulatory function for the peripheral circulation. They exert an active function on the blood flow and pressure regulation. This function is attained either through the contraction of the smooth muscle fibers or by the presence of glomus cells which cause a reduction in the diameter of the lumen by their ability to swell [25]. Ultrastructure study revealed that numerous filopodia-like protrusions at the arteriole endothelium toward the lumen. The formation of filopodial structures described under inflammatory conditions and in response to the inflammatory mediators [31]. Mild but significant inflammatory activity is involved in the development of normal pregnancy, which might have important physiological roles [32,33]. The maternal inflammatory response is supposed to be modulated to allow the establishment and maintenance of a viable pregnancy.

Abundant caveolae of different sizes and different arrangement were observed in the endothelial cells of the capillary. The permeability of endothelial cells is usually described in electron microscope studies as related to the presence of caveolae [34]. With the advancement of pregnancy, endothelial cells of some vessels appeared swell to a plump and Similar observation obtained at early stages of human pregnancy [35].

Our data revealed that telocyte encircled the capillary with similar vacuoles in both. The telocyte discussed as play a role in juxta and paracrine signaling [36]. In addition, telocyte expressed estrogen and progesterone receptors and

act as a hormonal sensor [37,38]. However, it always needs to be noted the exceptional cases out of such rules, and cutaneous horns at the gross level should prompt consideration of biopsy for the definite diagnosis. Our case

#10 also seems to support such speculation.

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The post A Histological, Histochemical and Ultrastructural Characterization of Uterine Vessels at Early Stages of Pregnancy appeared first on Nigerian Biomedical Science Journal.

,¹ Jim Pruckler,¹ William Bibb,¹ Nguyen Thi My Thanh,² Tran My Trinh,²

Nguyen Thi Minh,³ Sumathi Sivapalasingam,¹ Amita Gupta,¹ Phan Thu Phuong,⁴ Nguyen Tran Chinh,⁵ Nguyen Vinh Chau,⁵ Phung Dac Cam,⁴ and Eric D. Mintz1*

Foodborne and Diarrheal Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia,¹ and Pasteur Institute² and Hospital for Tropical Diseases,⁵ Ho Chi Minh City, Cai Lay Medical Center, Cai Lay,3 and National Institute of Hygiene and Epidemiology, Hanoi,⁴ Vietnam

Corresponding author. Sonja J. Olsen Mailing address: Foodborne and DiarrhealDisease Branch, Centers for Disease Control and Prevention.

ABSTRACT

Laboratory diagnosis of typhoid fever requires isolation and identification of Salmonella enterica serotype Typhi. In many areas where this disease is endemic, laboratory capability is limited. Recent advances in molecular immunology have led to the identification of sensitive and specific markers for typhoid fever and technology to manufacture practical and inexpensive kits for their rapid detection. We evaluated three commercial kits for serologic diagnosis of typhoid fever. Patients presenting with > 4 days of fever were enrolled at two hospitals in Southern Vietnam. Cases were patients with serotype Typhi isolated from blood samples, and controls were patients with other laboratory-confirmed illnesses. Serotype Typhi isolates were confirmed and tested for antimicrobial susceptibility at the Pasteur Institute in Ho Chi Minh City. The Widal test was run at the hospitals and the Pasteur Institute. Sera were shipped frozen to the Centers for Disease Control and Prevention and tested by using Multi-Test Dip-S-Ticks, TyphiDot, and TUBEX to detect immunoglobulin G (IgG), IgG and IgM, and IgM, respectively. Package insert protocol instructions were followed. We enrolled 59 patients and 21 controls. The sensitivity and specificity findings were as follows: 89 and 53% for Multi-Test Dip-S-Ticks, 79 and 89% for TyphiDot, 78 and 89% for TUBEX, and 64 and 76% for Widal testing in hospitals and 61% and 100% for Widal testing at the Pasteur Institute. For all assays, the sensitivity was highest in the second week of illness. The Widal test was insensitive and displayed interoperator variability. Two rapid kits, TyphiDot and TUBEX, demonstrated promising results.

Typhoid fever, caused by Salmonella enterica serotype Typhi, is a major cause of morbidity and mortality worldwide, causing an estimated 16.6 million new infections and 600,000 deaths each year (14). In Vietnam, typhoid fever is highly endemic, with the southern provinces most heavily affected. In a study conducted in Dong Thap Province in 1995 and 1996, the incidence of confirmed serotype Typhi infection was 198 per 100,000 for all ages (11).Isolation of serotype Typhi from blood, urine, or stool is the most reliable means of confirming an infection. However, this requires laboratory equipment and technical training that are beyond the means of most primary health care facilities in the developing world. Most serotype Typhi infections are diagnosed purely on clinical grounds and treated presumptively. As a result, the diagnosis may be delayed or missed while other febrile illnesses are considered, and patients without typhoid fever may receive unnecessary and inappropriate antimicrobial therapy. Emerging drug resistance among circulating serotype Typhi strains in Vietnam (6, 15) and elsewhere (16) has complicated the treatment of typhoid fever and heightened the need for rapid accurate diagnosis and the appropriate and selective use of antimicrobial agents to which the organism has thus far remained susceptible.

Serodiagnosis of typhoid fever has been attempted since the late 19th century when Widal and Sicard showed that the serum of patients with typhoid fever agglutinated typhoid bacilli (20). Unfortunately, neither the Widal test, which remains in widespread use in the developing world, nor any of the serodiagnostic tests that have since been developed has proven sufficiently sensitive, specific, and practical to be of value in areas where this disease is endemic (9). Recent advances in molecular immunology have led to the identification of potentially more sensitive and specific markers in the blood and urine of patients with typhoid fever and have enabled the manufacture of practical and inexpensive kits for their detection.

Here we report the results of an evaluation of three commercial serodiagnostic assays for diagnosis of acute serotype Typhi infection with specimens collected in southern Vietnam.

MATERIALS AND METHODS

Specimen collection. Specimens were collected from patients at two hospitals in Southern Vietnam: Cai Lay District Hospital (180 beds) in Tien Giang Province and the Hospital for Tropical Diseases (Cho Quan Hospital) (500 beds) in Ho Chi Minh City. Patients 3 years old who presented with 4 days of fever between October 2000 and April 2002 were eligible for enrollment. Patients who met the criteria were asked to give informed consent and answer a brief questionnaire about clinical signs and symptoms, antimicrobial treatment, and history of typhoid fever and vaccination. Participants gave 5 ml of blood (3 ml from children 3 to 5 years old) upon routine venipuncture for blood culture. Only patients with a laboratory-confirmed etiology of their fever were included in the analysis.

Blood samples were centrifuged, and the serum was divided into aliquots and stored at 20°C. In order to minimize the degradation of the antibodies in the serum, the specimens were frozen immediately and remained frozen until the time of testing. At routine intervals, personnel from the Pasteur Institute retrieved the isolates and serum specimens from the hospitals; serum was stored at 70°C. All isolates were confirmed at the Pasteur Institute, and serum was reevaluated by using the Widal test. Serum specimens from all patients with a laboratory-confirmed illness were batched and shipped on ice every

few months to the Centers for Disease Control and Prevention (CDC) in Atlanta, Ga., for further testing with the commercial assays. Patients with serotype Typhi isolated from blood were compared to patients with other laboratory-diagnosed pathogens by three commercial kits for rapid diagnosis of acute typhoid fever.

Laboratory analysis. (i) Blood culture. At Cai Lay Hospital, 5 ml of patient blood was added to blood culture medium (biphasic tryptic soy agar and brain heart infusion broth with SPS [0.6 mg/ml]) supplied by the Pasteur Institute. The blood culture bottle was then incubated at 37°C for 24 h before being tilted so that the liquid flowed over the solid medium. The broth was subcultured on blood agar after 1, 2, 3, and 7 days, and the solid medium was subcultured any time there was a colony visible on the slant. Isolates were Gram stained and identified by standard biochemical methods. Serotyping was performed by using agglutination with Salmonella O, H, and Vi antisera. If there was no growth after 10 days, the culture was considered negative. The Hospital for Tropical Diseases used the BACTEC system and surveyed the results after 5 days. If there was any growth, colonies were subcultured to blood agar and identified as described above.

1. Confirmation and antimicrobial susceptibility testing of isolates at the Pasteur Institute. The identification of suspect serotype Typhi isolates was confirmed at the Pasteur Institute by standard biochemical tests and Salmonella serotyping. Antimicrobial susceptibility testing was done by using the Kirby-Bauer disk diffusion method. The following antimicrobial agents (zone size for resistance) were used: ampicillin ( 17 mm), tetracycline (19 mm), chloramphenicol ( 18 mm), ceftriaxone ( 21 mm), ciprofloxacin ( 21mm), ofloxacin (16 mm), norfloxacin ( 17 mm), nalidixic acid ( 19 mm), and gentamicin (15 mm).
2. Laboratory confirmation of other pathogens. Confirmation of other pathogens was done as follows: blood smear for malaria, acid-fast bacilli (AFB) sputum smear for tuberculosis, blood or urine cultures for other bacterial pathogens, or serum immunoglobulin M (IgM) detection by antibody-capture enzyme immunosorbent assay (MAC EIA) for dengue. AFB smears and blood and urine cultures were done in the hospitals; sera were sent from Cai Lay Hospital to the Center for Preventive Medicine in Tien Giang province for dengue testing by using a MAC EIA kit produced by the Pasteur Institute (validated by comparison to an Omega, UK, commercial kit). The Hospital for Tropical Diseases did not test or refer specimens for dengue serology.
3. Widal test. Widal testing was done by using the Sanofi qualitative agglutination test kits (Bio-Rad) by two different methods. In both methods, serum was serially diluted, starting at 1/10, in physiological saline and then further diluted 1/10 in suspensions containing serotype Typhi O and H antigens, separately. Cai Lay Hospital used the rapid centrifugation technique in which the tubes were centrifuged at 3,000 rpm for 5 min. The precipitate was resuspended by tapping the bottom of the tube; if agglutination was visible, the results were considered positive. The Hospital for Tropical Diseases and the Pasteur Institute used the classical technique with incubation in which the tubes were incubated in a 37°C water bath for 2 h for H suspensions and at room temperature overnight for O suspensions; if agglutination was visible, the results were considered positive.
4. Rapid tests. Serum was evaluated by using the following three commercially available rapid diagnostic kits: Multi-Test Dip-S-Ticks (PANBIO INDX, Inc., Baltimore, Md.), TUBEX (IDL Biotech, Sollentuna, Sweden), and Typhi-Dot (Malaysian Biodiagnostic Research SDN BHD, Singapore, Malaysia).

Briefly, the Multi-Test Dip-S-Ticks tests for five pathogens, including Salmonella serotype Typhi. The test is in a dipstick format that detects anti-O, anti-H, anti-Vi, IgM, or IgG antibodies in patient serum, plasma, or heparinized whole blood. We evaluated the IgG kit only. The TyphiDot is a DOT enzyme immunoassay that detects either IgM or IgG antibodies against a specific antigen on the outer membrane protein of serotype Typhi. For specimens that are indeterminate (IgM negative and IgG positive), a confirmatory test, TyphiDot-M, is recommended by the manufacturer. Due to manufacturing problems with the TyphiDot-M, only the TyphiDot was used in this evaluation. These first two tests, the Multi-Test Dip-S-Ticks and the TyphiDot, are qualitative. The third test was the TUBEX, a semiquantitative test that uses polystyrene particle agglutination to detect IgM antibodies to the O9 antigen. Specimens were run according to the protocol listed on the packet inserts.

Ethical review. The study protocol was approved by the institutional review boards of the CDC and the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam.

Statistical analysis. Analyses were performed by using SPSS version 11.0.1 (SPSS, Inc., Chicago, Ill.). Medians were compared by using the median test for nonparametric data that calculates a chi-square statistic. For each assay, we calculated the sensitivity, specificity, and positive and negative predictive values.

Fleiss quadratic 95% confidence intervals were calculated by using Epi Info 6 (CDC, Atlanta, Ga.). The patient age was calculated by using a mid-year birth date and date of interview.

RESULTS

We enrolled 59 serotype Typhi cases and 20 controls with other laboratory-confirmed febrile illnesses. The control diagnoses were as follows: 7 subjects with dengue fever, 4 subjects with Escherichia coli cultured from blood, 1 subject with E. coli cultured from urine, 2 subjects with malaria (Plasmodium falciparum), 2 subjects with tuberculosis, 2 subjects with Klebsiella pneumoniae cultured from blood, and 2 subjects with S. enterica serotype Paratyphi A cultured from blood. Serum was missing from one case and one control (serotype Paratyphi A).

The demographic characteristics of the serotype Typhi patients and controls are listed in Table 1. In the Hospital for Tropical Diseases there was a slightly longer, but nonsignificant, time between fever onset and enrollment compared to Cai Lay Hospital (median number of days, 11 versus 8 [P 0.07]). Twenty-five patients recalled taking antibiotics after fever onset (10 of 15 cases versus 6 of 10 controls), however, 74 of 75 (99%) reported taking any medicine in the same week.

Most persons (54 of 79 [68%]) did not know if the medicine they took was an antibiotic or not. The dates of fever onset in patients with serotype Typhi ranged from January 2001 to March 2002, peaking in April through October (Fig. 1). None of the participants reported vaccination for typhoid fever; one patient and two controls reported having a history of typhoid fever.

A comparison of the three assays in presented in Table 2. A refrigerator is needed for storage of all of the kits but very little additional equipment is needed. The Multi-Test Dip-S-Ticks method requires a water bath, and the TyphiDot requires a calibrated pipette. At approximately $10 per test, the Multi-Test Dip-S-Ticks is the most expensive, followed by TUBEX at approximately $4 per test and the TyphiDot at approximately $1 per test. The sensitivity, specificity, and predictive values are shown in Table 3. Although the sensitivity of the Multi-Test Dip-S-Ticks was quite high (89%), it had low specificity (50%). The Typhi-Dot and the TUBEX both had high sensitivities (79 and 78%, respectively) and specificities (89 and 94%, respectively). The Widal test was the least sensitive of the assays, and the results varied by place performed (64% sensitive and 76% specific in the hospitals and 61% sensitive and 100% specific at the Pasteur Institute).

We examined the sensitivity of the assays by week after fever onset (Fig. 2). These were not tests performed on specimens from the same patient at weeks 1, 2, and 3 but rather the merged results of single samples collected at a single time point from each patient. The sensitivity of all tests was highest for serum specimens obtained during the second week of illness. The numbers were too small to do a meaningful evaluation of the specificity of the assays by the week after fever onset (data not shown).

There were no discernible differences between the 10 controls with a false-positive result on one of the three commercial assays and the 9 other controls, although the numbers were small. The controls with a false-positive result were slightly younger (mean age, 31 versus 37 years [P 0.6]) and less likely to be women (50% versus 55%, P 1.0); these differences are not statistically significant. Nine of the ten controls were positive on the Multi-Test Dip-S-Ticks, and four of these had dengue fever. One control with

FIG. 1. Time of fever onset in patients with Salmonella Typhi by hospital. a false-positive result reported having a history of typhoid fever.

Antibiotic susceptibility. A total of 58 of the 59 serotype Typhi isolates were available for testing. Of the 58 isolates tested, 14 (24%) were pansensitive. All of the remaining 44 isolates were resistant to nalidixic acid; 33 were also resistant to chloramphenicol and tetracycline, and 29 of these were also resistant to ampicillin. Only two isolates were also resistant to cefotaxime, one of which was also resistant to norfloxacin. Among the 57 cases with serologic results, there was no statistically significant difference in the typhoid assay results by sensitivity as defined by pansensitivity or resistance to at least one antimicrobial agent.

DISCUSSION

We evaluated three commercial rapid diagnostic kits for serotype Typhi with sera collected from patients with acute febrile illness of 4 days’ duration at two hospitals in Vietnam. Overall, the TyphiDot and TUBEX, both of which detect IgM antibodies, demonstrated the most promising results. However, the performance of the TyphiDot assay may not have been optimized since we were unable to run our 15 indeterminate specimens (seven cases and eight controls) on the TyphiDot-M assay for confirmation. The Multi-Test Dip-S-Ticks, which only detects IgG antibodies, had poor specificity. The Multi-Test Dip-S-Ticks to detect IgM was not evaluated. The Widal test had low sensitivity and was highly operator dependent. Since the other three assays were performed at only one laboratory, we could not assess their operator variability.

The hospitals participating in this evaluation were quite different and had the potential to enroll patients at different stages of illness. Cai Lay is a small, rural hospital with minimal laboratory capability, whereas the Hospital for Tropical Diseases is a large, urban referral hospital with good laboratory capability. Despite this, there were few differences in the patients enrolled. Patients at the Hospital for Tropical Diseases were slightly older, more likely to be

women, and were seen a median of 2 days later in their illness.

In our evaluation, the sensitivity of the TyphiDot was high beginning in the first week of illness onset. Presumably, this is because the TyphiDot relies more heavily on IgM results that occur earlier in the course of the illness, whereas IgG rises later; however, we did not see this effect with the TUBEX, which also detects IgM antibodies. In the Widal test, the O and H agglutinins usually appear around day 8 and days 10 to 12, respectively. The Multi-Test Dip-S-Ticks was the most costly assay, presumably because the dipstick measures antibodies to five different pathogens. Although all three assays were relatively easy to use, the TUBEX was the simplest. A limitation of the TUBEX test, which uses a colorimetric reaction, is the potential for difficulty in interpreting the results of hemolyzed samples. Another concern is that the TUBEX may produce a false positive in persons with recent S. enterica serotype Enteritidis infection and result in inappropriate antibiotic treatment (13). Ideally, the Widal test should be run on both acute- and convalescent-phase sera to detect an increase in the agglutination titer. However, to inform treatment decisions before convalescent samples can be obtained, it is common for a single acute-phase serum sample to be run. The results from a single sample are difficult to interpret because high background rates of circulating antibodies to serotype Typhi or other Salmonella serotypes may produce a false-positive result. In Vietnam, an area of high endemicity, a single Widal test can lead to many false-positive and false-negative results (17). Operator variability also contributes to unreliable results, as evidenced in the present study. Each of these three commercial kits has been previously evaluated but, to our knowledge, this is the first time they have all been included together in the same evaluation. In a recent evaluation of the Multi-Test Dip-S-Ticks in Singapore, the sensitivity varied greatly depending upon the case definition (5). Among clinical and blood culture-positive cases it was 51%, and among blood culture-positive patients only it was 78%. The specificity among control patients who had a clinical diagnosis of typhoid but were culture negative, had other laboratory diagnoses, or had pyrexia of unknown origin was 81%. The TyphiDot was evaluated in Pakistan and Singapore by using a variety of case definitions (2, 5). The sensitivity ranged from 59 to 93% for the TyphiDot and 73 to 84% with the addition of the TyphiDot-M. Specificity was consistently higher when TyphiDot-M was used; 89% compared to 77% or lower with only the Typhi-Dot. An evaluation of the TyphiDot in India was 100% sensitive and 80% specific compared to a blood culture “gold standard” (8). In an early evaluation, the TUBEX demonstrated 100% sensitivity and specificity (10). However, this was before the kit was commercially available. In Vietnam, it was 87% sensitive among blood culture positive patients and 76% sensitive among hospitalized patients with fever (7).

One limitation in the previous and current evaluations is that blood culture-confirmed cases were used as the gold standard. Since blood culture is less sensitive than bone marrow culture for diagnosing typhoid fever (4), the results should be interpreted with caution. It is possible that the rapid diagnostic tests are more sensitive than blood culture. If so, a result that appears to be a false-positive test compared to a blood culture may in fact be a true positive. This hypothesis requires further evaluation. Alternatively, a false-positive may be the result of past infection with serotype Typhi or another nontyphoidal Salmonella serotype that shares common antigens. Researchers continue to search for the ideal rapid test to diagnose acute typhoid fever. Several urine assays have been developed (1, 3, 12, 18), although none has proved optimal. In the field, there is a definite advantage to collecting urine instead of blood; urine collection is simple, less invasive than venipuncture, and requires less training and equipment. In addition, some antigens may be excreted in higher concentration in the urine. With the recent sequencing of the entire serotype Typhi genome, it may now be possible to identify other antigens, such as fimbrial antigens, that may produce an antibody response specific to serotype Typhi (19). More sophisticated molecular techniques for diagnosis, such as PCR, are also being explored. However, their use in developing countries will most likely be limited.

FIG. 2. Sensitivity by week after fever onset.

ACKNOWLEDGMENTS

We thank Tran Thi My Trinh and Bui Thu Hien of the Pasteur Institute in Ho Chi Minh for their assistance with laboratory testing.

REFERENCES

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The post Evaluation of Rapid Diagnostic Tests for Typhoid Fever appeared first on Nigerian Biomedical Science Journal.

Tunji Akande

Department Of Medical Laboratory Sciences Bingham University Karu, Nigeri a

All Correspondences to:Tunji Akande Department Of Medical Laboratory Sciences Bingham University Karu, Nigeri a

ABSTRACT

Background: Heart disease is a common and delibilitating condition that affects millions of patients globally. Measurement of troponin assays has been a tremendous boon to clinical diagnosis. Objective: To evaluate the clinical utility and the biochemical characteristics of serum troponin as an early and accurate cardiac marker for diagnosis of acute myocardial infarction. Method: a review of serum troponin as choice biomarker for diagnosis of Acute Myocardial Infarction (AMI). Result: Troponin T allows for early and late diagnosis of AMI. Troponin is only found in the myocardium in adults making it extremely specific for cardiac disease. Troponin I or T is replacing total CK and CK-MB detection as the marker of choice for cardiac dysfunction as seen in AMI. Conclusion: There is value in detection myocardial damage early after its onset. Clinical laboratories should move rapidly to implement the new cardiac troponin standard for the early and accurate diagnosis of AMI.

Key Words: AMI, Troponin, Cardiac Marker, Early diagnosis.

INTRODUCTION

Cardiovascular diseases are the leading cause of death globally. Cardiovascular disease includes coronary artery disease (CAD) such as angina and myocardial infarction commonly known as a heart attack The World Health Organization has established three criteria for the diagnosis of acute myocardial infarction (AMI) including a history of chest pain, evolutionary changes on the ECG, and elevations of serial cardiac markers [2] however it was rare for a diagnosis of AMI to be made in the absence of biochemical evidence of myocardial injury. [3] .Numerous biomarkers have been monitored to assess myocardial injury. Most are myocardial proteins and differ in their location within the myocyte, release kinetics after damage and clearance from the circulation [4] . The European Society of Cardiology/American College of Cardiology (ESC/ACC) has published consensus guidelines for the diagnosis of AMI [5,6 7]. A Corner stone of the consensus document is predicated on cardiac biomarker especially cTnI or CTnT [8,9]. Because of its dire consequence, great efforts have been made to determine the best tools for the early and accurate diagnosis of AMI. Therefore this article aimed at assessing some cardiac markers that could provide an early and accurate diagnosis for acute myocardial infarction

Myocardial infarction (MI)

Acute myocardial infarction is one of the largest killers globally. Its diagnosis is usually made on the clinical presentation and electrocardiagraphic (ECG) findings and confirmed by the characteristic changes in plasma enzyme activities or troponin levels Symptoms and ECG abnormalities however may be absent or non specific. Thus, the diagnosis of an acute MI has increasingly depended upon evaluation of Cardiac biomarkers particularly troponins [10,11]. Acute myocardial infarction is defined as an imbalance between myocardial oxygen supply and demand resulting in injury and the eventual death of myocyte. When the blood supply to the heart is interrupted, gross necrosis of myocardium results. Such extensive necrosis is most often associated with a thrombotic occlusion superimposed on coronary atherosclerosis. Myocardial infarction is seen as a spectrum of disease ranging from angina pectoris through to acute myocardial infarction; this stratification is based upon cardiac markers reflecting ischemic damage. [12,13]. Myocardial ischemia and infarction are usually segmental diseases. The American College of Cardiology and European Society of cardiology have defined acute myocardial infarction as a typical rise and fall of biochemical marker, for example plasma creatine kinase isoenzyme MB (CK-MB) or troponin, with at least one of the following: Ischemic symptoms, new pathological Q waves, on electro cardiogram (ECG), ischemic ECG changes (ST.depression or elevation) and coronary artery intervention [14]

The patients previously classified as having unstable angina or minor myocardial injury are now reclassified as having non-ST segment elevation myocardial infarction (NSTEMI), therefore myocardial infarction is now regarded as a spectrum of disease ranging from angina pectoris through to acute myocardial infarction. Acute myocardial infarction and NSTEMI have a common pathophysiological pathway (15)

Cardiac markers

Cardiac markers are biomarkers measured to evaluate heart function. They are clinical laboratory tests useful for detecting AMI or minor myocardial injury. They are most useful when patients have non-diagnostic ECG tracings.

Most efforts to date have been placed on the development of an ideal cardiac marker for the early and accurate diagnosis of AMI. Many factors must be considered in the selection of the most clinically diagnostic effective, cost effective and cost efficient laboratory tests for patients with chest pain which include

1. the time that has elapsed after onset of chest pain;
2. Any concomitant diseases;
3. The possibility of skeletal muscle injury
4. The ease of measurement and turn around time for results.
5. Assay specificity, sensitivity and interferences. (17, 18)

Serum enzymes such as aspartate aminotransferase (AST), Creatine Kinase (CK), Lactate dehydrogenase (LD) and their iso enzymes have all been used as biomarkers of AMI. Decades ago they were thought to be sensitive indicators of myocardial necrosis and could be used to correlate with other signs and symptoms such as abnormalities in ECG pattern [19,20] All three of these enzymes, however are found in other tissues as well, making them less specific to myocardial damage. AST for example is also found in skeletal muscle, liver parenchyma cells, and erythrocytes, while CK is found in skeletal muscles, brain tissue and embryonic and malignant tissue. LD is the least specific of these three enzymes in that it is found in virtually all tissues and is associated with damage to liver, skeletal muscle, cardiac muscle, erythrocytes renal cells and many other tissues as well as ovarian and testicular tumors. [21] Historically myocardial infarction was detected by looking for the CK Iso enzyme CK-MB. This marker is released into circulation from necrotic heart muscle. As the heart muscle becomes damaged this CK Isoenzyme is released into the blood stream and may be detected.

Cardiac proteins

Several proteins may be monitored in suspected cases of AMI to give significant diagnostic information myoglobin an oxygen-binding protein is rapidly released from striated muscles (both skeletal and cardiac muscle0 when damaged. However because of its small size, myoglobin is rapidly cleared by the kidney, making it an unreliable long-term marker of cardiac damage. [22] Myoglobin is significantly more sensitive them CK and CK-MB activities during the first hours after chest pain onset. It starts to rise within 1-4hrs and is detectable in essentially all AMI patients between 6 and 9 hours from chest pain onset returning to baseline levels within 18-24 hours. If myoglobin concentration remains within the reference range 8 hours after onset of chest pain, AMI can essentially be ruled out [23] Myoglobin is released into circulation with any damage to muscle tissue, including myocardial necrosis. Since skeletal muscle contains myoglobin, this measurement is quite non-specific for myocardial infarctions. The benefit lays in the fact that a detectable increase is seen only 30 minutes after injury occurs, unlike

troponin and creatine Kinase which can take 3-4 hours [24,25,26] Troponin I and troponin T are normal proteins important in the contractile apparatus of the cardiac myocyte. They are released into the circulation about 3-4 hours after myocardial infarction and are still detectable for 10 days afterwards. The long half-life allows for the late diagnosis of myocardial infarction [27,28]. All of the initial cardiac markers were enzymes, so the earliest techniques measured the catalytic activity of the marker. Immunoassay techniques measure the mass of a marker and they are the predominant methodology used in clinical laboratory practice today. Analytically, they offer lower limits of detection, improved precision, and faster assay time on both highly automated central laboratory Platforms and Point of Care Testing (POCT). [29,30] Over the past 15 years numerous manufacturers have described the development of monoclonal anthody –based diagnostic immunoassays for the measurement of troponins (CTnI and CTnT) in serum. Troponin assays has been a tremendous boon to clinical diagnosis. Troponins released from heart muscle remains in the blood stream for up to 10-14 days after onset of AMI, making them preferred marker for detection of an AMI [31]. Troponins as cardiac markers appear to have many advantages primarily due to their quick release following heart muscle damage.

Recommendations

The National Academy of clinical Biochemistry recommends that two biochemical markers be used for routine diagnosis of AMI: an early marker that is reliably increased within 6 hours after onset of symptoms and a definitive marker that remains increased after 6-9 hours but has high sensitivity and specificity for myocardial injury and remains abnormal for several days. [33]. To assist in differentiating patients with AMI from non – AMI, the European Society of cardiology/American College of cardiology (ESC/ACC) has published consensus guidelines for redefinition of AMI [34]. A cornerstone of this is predicated on cardiac biomarkers, especially CTnI or CTnT. The cardiology recommendations imply that for clinical laboratories that cannot move as rapidly as others to implement the new cardiac troponin standard, CK-MB (Preferable mass should be used. Although it is suggested that CK-MB be used together with cardiac troponin for assisting in timing the onset of myocardial injury, infarct sizing or determination of reinfarction at present there is no strong evidence to support dual testing for cTn and CK-MB.

Conclusion

The diagnosis of acute myocardial infarction can be difficult, but it is important to make prompt diagnosis as thrombolysis needs to be given early. The diagnosis of an acute myocardial infarction has increasingly depended upon evaluation of cardiac biomarker particularly troponins. Troponins are more specific cardiac marker than the CK. Also, plasma troponin stays elevated for longer than CK after an infarct. Troponins are therefore useful for the late diagnosis of myocardial infarction. Clinical Laboratories should move rapidly to implement the new cardiac troponin standard for the early and accurate diagnosis of myocardial infarction.

Table I. The time sequence of changes in plasma cardiac markers after myocardial infarction

CK= Creatine kinase; AST= aspartate transaminase

LDH= lactate dehydrogenase; HBD= Hydroxybutyrate dehydrogenase

REFERENCES

• Mendis; pekka puska; Bo Norring; World Health Organization. Global Atlas on cardiovascular Disease Prevention and Control. World Health Organization in Collaboration with the World Heart Federation and the World Health Organization 2011. Pp 3-18
• WUAHB, Apple FS, Gilber WB. National Academy of Clinical Biochemistry standards of Laboratory practice: recommendation for the use of cardiac markers in coronary artery disease clin chem. 1999;45 (7): 1104-1121

• Alpert JS, Thygesen K, Antman E, Barssand JP Myocardial infarction redefined-a consensus document of the Joint European Society or cardiology /American College of cardiology committee for redefining of myocardial infarction. JAm coll cardio 2000; :36 959-9
• Cardiac Marker Diagnostic Testing Markets Trimark Publications, LLC November 2011
• Cortina MAQ, Pituley AJ, Shin H, Lin J, Kadjevic L, Bilandzic LM Comparison of spectral’s eenhanced point- of – care test for markers troponin I and myoglobin with clinical analyzers clinchem 2003; 49 suppl 6: 471
• Cowie MR, Jourdam P, Maisel A, Dahestrom U, follath F, Isnard R. clinical applications of B-type natriuretic Peptide (BNP) testing. E Hear J 2003; 24 1710-18.
• Kinngawa T, Kato M, Oyino K, Osaki S Igawa O, Hisatome I Plasma endothelin-I levels and Clinical correlates in patients with chronic heart failure. J card fail 2003; 9:318-24.
• Jaffe As, Ravkilde J, Robert R, Naslund U, Apple FS. Galvani.M. It’s time for a change to a troponin standard. Circulation 2000; 102: 1216-20
• LIPPi G, Mattuzzi C, Comelli I, Cervellin G. Glycogen Phosphorylase isoenzyme in the diagnosis of acute myocardial infarction. Biochem med 2013, 23, (1); 78-82
• Yeh RW, Go AS. Rethinking the epidemiology of acute myocardial infarction: challenges and opportunities. Arch interm Med 2010; 170: 759-64
• Thygesen K, Alpert JS, Jaffe AS, Third universal definition of myocardial infarction. J.AM coll cardiol 2012; 60:1581-98.
• Lindahl B. Acute coronary syndrome. The present and future role of biomarkers. Clinchem Lab Med 2013; 1699-706
• Arbustini E, Namla N, Kodama T. Clinical Imagin of AC with ruptured or intact fibrous caps. JACC Cardiovascular imagin 2015; 8:576-8
• Sala F, Komukai K, Capodanno Eroded versus Ruptured Plaques at the culprit site of STEMI: Invivo Path physiological features and Response to primary PCI JACC Cardiovascular imaging 2015; 8: 566-75.
• Iannaccone M, Quadri G. Tahas prevalence and predictors of culprit plaque rupture at OCT in patients with coronary artery disease: a meta analysis Eur Heart J cardiovascular imaging 2015 oct 27
• SwaPCJ, Nagurney JT Value and limitations of chest pain history in the evaluation of patients with suspected acute coronary syndromes JAMA. 2005; 2623-2629
• Naghavi M, Libby P, Falk E, Casscells. From vulnerable plaque to vulnerable patient; a call for new definitions and risk assessment strategies circulation. 2003; 108:1664-1672
• Biomarkers definition Working Group. Biomarkers and surrogate end points; preferred definitions and conceptual frame work. Clin pharmacol ther 2001; 69: 89-95
• Morrow DA, de lemos JA, Sabatine MS, Antman EM. The search for biomarker of cardiac ischemia clinchem. 2003; 49: 537-539
• How industry is approaching the search for new diagnostic markers and biomarkers. Mol cell proteomics 2004; 3: 345-354
• Labner J. So you want to look for biomarkers. J proteome Res 2005; 4 1053-1059
• Reddy GC Cardiac troponin-T and CK-MB (mass) levels in cardiac and non cardiac disease Ind J. Clin Biochem 2004; 19(2): 91-94
• Diserio F: Analytical evaluation of automated immunoassay for cardiac troponin I: the Vidas toponin I Assay. Clin chem. 2003: 10: 1363-1368

• Collinson Po, Boa FG, Gaze DC. Measurement of cardiac troponins. Ann clin Biochem 2001, 38: 423-49
• Apple FS, Wu AHB Jaffe AS. European society of cardiology and American College of cardiology guidelines for redefinition of myocardial infarction: how to use existing assays clinically and for clinical trials. Am heart J 2002; 144: 981-6
• Panteghini M. standardization of cardiac troponin I measurements: the way forwar? Clin chem. 2005; 51: 1594-1597
• Heidenreich PA, Alloggiamento T, Melsop K, The prognostic value of troponin in patient with non-ST elevation acute coronary syndrome: a meta analysis. J.Am coll cardiol. 2001; 38: 478-485
• Thygesen K, Mair J. Katins H. recommendations for the use of cardiac troponin measurement in acute cardiac Care Eur Heart J 2010; 31: 2197
• Wallance TW, AbdullahSM. Drazner MH. Prevalence and determinants of troponin T elevation in the general population circulation 2006; 113: 1958
• De lemos JA, Drazner MH, Omland T, Association of troponin T detected with a highly sensitive assay and cardiac structure and mortality risk in the general population JAMA 2010; 304;2503
• Higgins JP, Higins JA. Elevetion of cardiac troponin I indicates more than myocardial ischemia. Clin invest med 2003; 26 :133
• Jaffe AS, Wright RS. High- sensitivity cardia troponin and primary prevention: An important New Role J. Amcoll cardial 2016; 68: 2729
• Thygesen K, Alpert JS, Jaffe As. Third universal definition of myocardial infarction circulation 2012; 126: 2020
• Thygesen K, Alpert JS. White HD. Joint ESC/ACCF/AHA/WHF Task force for the redefinition of myocardial infarction. Universal definition of myocardial infarction. Eur Heart J 2007; 28; 252

The post Early Diagnosis of Acute Myocardial Infarction (Heart Attack): Immunoassay for Serum Troponin Marker appeared first on Nigerian Biomedical Science Journal.

Kevin E. Aghatise

Department of Medical Laboratory Science, Igbinedion University Okada

Ivie Osula and Anslem O. Ajugwo

Department of Medical Laboratory Science, Madonna University Nigeria

All Correspondences to: Kevin E. Aghatise, Department of Medical Laboratory Science, Igbinedion University Okada

ABSTRACT

Anaemia constitutes a serious health problem in many tropical countries including Nigeria because of the prevalence of malaria and other parasitic infections which possibly leads to decrease of hemoglobin. Telfairia occidentalis (fluted pumpkin), Vernonia amygdalina (bitter leaf) and Gongrone malatifolium (utazi leaf) have been reported to be very good in building the constituents of the blood and also replacing them. This study was designed to investigate the effect of fluted pumpkin, bitter leaf and utazi leaf extracts on haematolgical parameters of anaemia induced albino wistar rats. Fifteen Wistar albino rats with weight range from (122-190g) obtained from the animal house of Madonna University, Elele Nigeria were separated into three groups of five rats each for the study. Rats in group B and C were injected 400mg/kg 2,4 dinitrophenylhydrazine for five days to induce anaemia, while rats in group A were fed with grower’s mash and water throughout the experiment. All analysis were done using the standard methods. The result of this study show that, there was a significant (p<0.05) difference when the PCV was compared among the Groups, the control groups had 41.0+2.65% anemia induced group had 25.3+6.8% and the treatment group recorded 33.6+4.9%. The anemia induce group recorded a reduced PCV but was raised in the treatment group and this was statistically significant (p<0.05). There was also a significant difference when the Haemoglobin concentration was compared among the groups, the control group had 14.1+0.7g/dl, anaemia-induced group has 8.3+1.9g/dl and the treatment group had 10.+2.15g/dl. There was no significant difference (p>0.05) in all other parameters estimated. The result of this research indicate that aqueous leaf extract of fluted pumpkin, bitter leaf and utazi leaf could elevate the packed cell volume, red blood cells and hemoglobin concentration in anaemia-induced rats. It can be concluded that aqueous leaf extract of the combination of fluted pumpkin, bitter leaf and utazi leaf are a potential blood booster.

Keywords: anaemia-induced, blood booster, haematological, dinitrophenylhydrazine.

INTRODUCTION

The use of herbal products for medicinal benefits has an important role in nearly every culture on earth. Herbal medicine was practiced by ancient people of Africa, Asia, Europe and the Americas (Abosi and Raseroka, 2003). Over 50% of all modern clinical drugs are of natural product origin and natural products play an important role in drug development programs of the pharmaceutical industry. The consumption of a variety of local herbs and vegetables by man is believed to contribute significantly to the improvement of human health, in terms of prevention, or cure of disease because plants have long served as a useful and rational source of therapeutic agents (babalolaet al., 2003). Regular consumption of plant foods are associated with numerous health benefits rooted in their various physiological effects as a result of their physiochemical and nutritional constituent (Hunter and Fletcher, 2002).

Green leafy vegetables are particularly important in

promoting health because of their rich sources of nutrients (Gupta and Prakash, 2009). Telfairia occidentalis (fluted pumpkin) is a common tropical green leafy vegetables native to many African countries especially Eastern Nigeria (Burkett, 1968). It thrives in humid climate and well drained soils and is usually cultivated in garden and family farms around homes. Telfairia occidentalis is traditionally used by an estimated 30 to 35 million people indigenous people in Nigeria, including the Efik, Ibibio, and Urhobo (Akoroda, 1990). However, it is predominantly used by the Igbo tribe, who continue to cultivate it for food sources and traditional medicines (Okoli and Mgbeogu, 1983). A reoccurring subject in the Igbo’s folklore, the fluted gourd is noted to have healing properties and was used as a blood tonic, to be administered to the weak or ill (Akoroda, 1990). Bitter leaf is a medicinal plant of the family Asterceae. It is a small perennial shrub that grows in tropical Africa. Vernonia amygdalina is commonly called bitter leaf because of its bitter taste and it can be propagated either by cutting or seedling. Today, the plant is widely known throughout the continent and nearly 85% of Nigerians cultivates the plant due to its nutritional and medicinal values. It is locally called ‘ewuro; in Yoruba land (Abosi and Raseroka, 2003). The leaves of the plant may be consumed either as a vegetable (macerated leaves in soup) or aqueous extract as tonic for the treatment of various illnesses (Abosi and Reseroka, 2003).

Gongrone malatifolium also called utazi belongs to class of medicinal plants that are beneficial in preventing and treating certain disease and ailments that are detrimental to human health. Gongrone malatifolium leaf, which can be chewed infused or used for cooking is mainly used in the western part of America for nutritional and medicinal reason. Pharmacological studies suggest that utazi has both analgesic, antimicrobial, antibacterial, anti ulcer and antioxidant properties (Eguyoni et al., 2009). Utazi plant is highly medicinal in nature, which suggest why its health benefits cannot be overemphasized. Researchers agree that the fundamental ingredients used for medical purposes are stored in the various parts of the utazi plants such as leaves, fruits, roots etc (Eguyoni et al., 2009). The vital medicinal ingredients stored in the various parts of the utazi leaf can be extracted either through blending the fresh leaves, chewing the utazi seed, leaves or fruits, infusing either the dry or fresh utazi leaves and decoction. However these various methods of preparing and using utazi leaves for medical purpose mainly depends on the part of utazi plants where the active medicinal ingredient are present (Eguyoni et al., 2009).

Anaemia is decrease in the amount of red blood cells (RBCs) or hemoglobin in the blood. It can also be defined as a lowered ability of the blood to carry oxygen (Rodak and Bernadtte, 2007). When anaemia sets in slowly, the symptoms are often vague and may include: feeling tired, weakness, shortness of breath or a poor ability to exercise. Anaemia that comes on quickly often has greater symptoms, which may include; confusion, feeling like one is going to pass out, loss of consciousness, or increased thirst. Anaemia must be significant before a person becomes noticeably pale (Rodak and Bernadette, 2007).Use of plants has long been known to be used as local remedy for treatment of anaemia especially pumpkin leaf which is used in villages to treat anaemia especially when quality drugs cannot be accessed. This work was carried out to determine the effects of combination of herbal plants in anaemia-induced condition using haematological parameters.

Materials and Methods

Animal Model/Experimental design

Fifteen (15) Wistar strain albino rats with weight range of

(122 – 190g) obtained from the animal house, Madonna University Elele, Nigeria were used for the study. The rats were housed in wire meshed cage under standard conditions (temperature 25 – 29°C, 12 hours light and 12 hours darkness cycles) and fed with standard rat pelleted diet and water. They were housed in 3 meshed cages containing five

1. rats in each cage. The rats were made to acclimatize for 1 week before the experiment began. They were allowed to feed on standard feed and water freely throughout the period the experiment lasted.

Ethical Approval

The research was approved by the ethical committee of the institution. The standard, rules and regulations of use of animal for research purposes was strictly adhered to as approved by the committee.

Order of Placement

Group1: The rats in this group served as control and were

administered with feed and water only. Group2: The rats in this group served as test and were administered with feed, water and 1 ml of phenyl hydrazine for five days. Group3: The rats in this group served as treatment group and were administered with feed, water, 1 ml of phenyl hydrazine for five days and subsequently treated with 0.5 ml of a blend of the local plants for another five days. Preparation and Administration of the plant blend

Large quantities of fresh leaves of Telfairia occidentalis (fluted pumpkin), Vernonia amygdalina (bitter leaf) and Gongrone malatifolium (utazi leaf) were purchased from the Central Market Elele. Identification and authentication of the plants was done by Mr Eze of the Faculty of Science. The leaves were washed and dried air-dried for eight days until there was no sign of moisture and grinded into powder. The powder was dissolved in 100 ml of deionized water and administered to the treatment group 0.5 ml in the morning and evening for 5 days.

Sample Collection

2ml of blood sample was collected by ocular puncture from each of the animal model using capillary tube and was dispensed into commercially prepared concentrations of ethylene diamine tetra acetic acid containers.

Sample Analysis

Blood samples collected were analyzed within six hours of collection for Hb, PCV, RBC count, Total WBC and differential analysis using standard manual methods (Cheesebrough, 2000)

RESULTS

Mean±S.D of hematological parameters of the control group (A), test group (B) and treatment group ©

20

DISCUSSION

The consumption of a variety of local herbs and vegetables by man is believed to contribute significantly to the improvement of human health, in terms of prevention, and or cure of diseases because plants have long served as a useful and rational sources of therapeutic agents (Babalola et al., 2003). Regular consumption of plant foods are associated with numerous health benefits rooted in their various physiological effects as a result of their physiological and nutritional constituents (Hunter and Fletcher, 2002).

From the research work there was a significant (p<0.05) difference when the PCV was compared among the groups, the control group has 41.0±2.65%, anaemia induced group has 25.3±6.8% and the treatment group recorded 33.6±4.9%. The anaemia induced group recorded a reduced PCV but was raised in the treatment group and this was statistically significant (p<0.05). This definitely is attributable to the local blend of plants. Previous work have shown that pumpkin is capable of improving haematological parameters (Alada,2000; Adias et al., 2013).

There was also a significant difference when the Hb was compared among the group, the control group had 14.1±0.7g/dl, anaemic induced group had 8.3±1.9g/dl and the treatment group had 10.5±2.15g/dl, the anaemic group recorded a reduction 8.3+1.9/dl but was elevated in the group given the extract. The reason for the significant increase in these haematological parameters could be related to the chemical composition of the leaves used in this research work. The chemical composition of Telfairia occidentals includes protein, fat, carbohydrates, calcium, iron, vitamin A, thiamine and riboflavin(Tindal, 1968; Adisa et al., 2014). Most of these constituents have positive impact on blood production (Akube, 1980). For instance, iron is a well established haemopoetic factor and deficiency of if produces anaemia (Ganong, 1997). There was no significant (p>0.05) difference in the RBC, the control group had 6.0±0.56×1012/1, the anaemic group had 4.4±062×1012/1, while the treatment group had 5.2±1.0×1012/l. The extract increased the RBC count of the anaemia-induced group from 4.4+062×1012/1 to 5.2+1.0×1012/1 but this was not significant. For TWBC there was no significant (p>0.05) difference when the test groups B (13.7±5.4×109/1) was compared to the control group A (9.3±1×109/1) although the TWBC was increase in the anaemic group (13.7±5.4109/1) and reduced in the treatment group (11.7±2.11×109/1).

There was also no significant (p>0.05) difference in platelets count when the groups B (261±39.5×109/1) and C (302±39.2×109/1) was compared with control group

(272±24.0×109/1) although the platelets count decreased in the anaemic group and increased in the treatment group but this was not significant (p>0.05). No significant difference was seen in all the differential count parameters. In conclusion, the combination of the extract of Telfairia occidentalis (fluted pumpkin), Vernonia amygdalina (bitter leaf) and Gongrone malatifolium (utazi leaf) have been shown to improve blood parameters (PCV and haemoglobin concentration). This extract can be used in management of anaemia especially when the conventional blood builders are not readily available.

Conflict of interest

The Authors declare that we have no conflict of interest.

REFERENCES

• Abosi, A. O. and Raserok, B. H. ( 2003) “In vivo antimalarial activity of Vernonia amygdalina” British Journal of Biomedical Science. 8 (60) 89 – 91.
• Adias, T. C., Ajugwo, A. O., Erhabor, T. and Nyenke, C. U. (2013). “Effect of Pumpkin Extract (Telfairia occidentalis) on Routine Haematological Parameters in Acetone-Induced Oxidative Stress Albino Rats.” American Journal of Food Science and Technology 1 (4): 67 – 69. doi: 10.12691/ajfst-1-4-1.
• Adisa, W. A., Okhiai, O., Bankole, J. K., Iyamu, O. A. and Aigbe, O. (2014). Testicular damage in Telfairia occidentalis extract treated Wistar rats” American Journal of Medical and Biological Research. 2 (2): 37 – 45.
• Akoroda, M. O. (1990) “Ethnobotany of Telfairia occidentalis among Igbos of Nigeria” Economic Botany. 1 (230): 29 – 39.
• Akube, A. (1980). Chemical composition of Telfairia occidentalis. Plantamedia 38 33 – 43.
• Alada, A. R. (2000). The haematological effect of t diet preparation” African Journal of Biomedical Research. 3 (3): 185 – 186.
• Babalola, O. O., Anetor, J. I., and Adeniyi, F. A. (2001) “Amelioration of carbon tetrachloride induced hepatoxicity by terpenoid extract from leaves of Vernonia amygdalina” African Journal Medicine Science. 2 (30): 91 – 93.
• Burkett, H. M. (1968). The results of plants of West Africa. Vol 1. Royal Botanic Garden Kews, England. 603 – 604.
• Cheesebrough, M. (2000). Haematological tests. In: District laboratory practice in tropical countries. Part 2 Cambridge University Press U.K. 297.
• Eguyoni, A., Moody, J. O. and Eletu, O. M. (2009). “Anti-sickling activities of two ethnomedicinal plant recipes used for the management of sickle cell anaemia in Ibadan, Nigeria” African Journal of Biotechnology. 8 (1): 20 – 25.
• Ganong, W. F. (1997). A review of medical physiology. Appleton and Large. 496
• Gupta, S. and Prakash, J. (2009).”Studies on Indian green leafy vegetables for their antioxidant activities” Plant Foods Human Nutrition. 64 (1): 39 – 45.
• Hunter, K. J. and Fletcher, J. M. (2002) “The antioxidant activity and composition of fresh, jarred, frozen and canned vegetables” Innovative Food Science and Emergency Technology. 3 (90): 34 – 37.

• Okoli, B. E. and Mgbeogu, C. M. (1983).”Fluted pumpkin: West African Vegetable crop” School of Biological sciences, University of Port Harcourt. 37 (2): 145 – 149.
• Rodak, A.and Bernadette, F. (2007). “Hematology: Clinical Principles and applications (3rd edition). Philadelphia
• Tindal, H. D. (1968). Commercial vegetable growing. Oxford Press, London. 69

The post Local Blend of Plant Improves Blood Parameters In Anaemia-induced Albino Wistar Rats. appeared first on Nigerian Biomedical Science Journal.

Assessment of Antibiotic Susceptibility Pattern on Staphylococcus aureus isolated from Suya (Roasted Meat) and Nunu (Milk) Sold in Jos Metropolis

Ejinaka, OR; ; Lote-Nwaru IE; Agbalaka P.I; Ajik, H.

Federal School of Medical Laboratory Science, Jos-Nigeria

Jwanse, R.I

Health and Development Support Programme (Hands), Jos, Nigeria

All correspondence to: Obeta M.U. E-mail: uchejesoobeta@gmail.com;

ABSTRACT

The assessment of antibiotic susceptibility pattern on Staphylococcus aureus isolated from Suya (Roasted Meat) and Nunu (Milk) sold in Jos was carried out in Jos Metropolis. This study was aimed at the occurrence of staphylococcus aureus in suya and nunu and its sensitivity to antibiotics. A total of 15 Suya and 15 Nunu sample were collected, the sample were cultured and identified by routine bacteriological method. Prevalence of S.aureus in Nunu was 40% and Suya was 33.3%. The susceptibility profile of staphylococcus aureus isolates from Suya (roasted meat) on an antimicrobial agents revealed a high susceptibility to Gentamycin (80%), Levofloxacin (80%), Rifampin (80%) Norfloxacin (80%) and had high resistance to Chloramphericol (80%), Ciprofloxacin (80%), Amoxil (80%), Ampiclox (80%) and Erythromycin (60%). The susceptibility profile of staphylococcus aureus isolates from Nunu (milk) on an antimicrobial agents revealed a high susceptibility to Gentamycin (80%), Levofloxacin (83.3%), Norfloxacin (83.3%), Erythromycin and Ciprofloxacin (66.7%) respectively, Rifampin and Levofloxacin 50% respectively and had high resistance to Chloramphericol, Amoxil and Ampiclox (66.7%) respectively; Rifampin and Levofloxacin 50% respectively. The S. aureus was present in Suya (Roasted Meat) and Nunu

(Milk) sold in Jos and resistant to some commonly used antibiotics in Jos.

Keywords: Staphylococcus aureus, Antibiotic Susceptibility, Suya, Nunu, Jos.

INTRODUCTION
Staphylococcus aureus is one of the major food borne abdominal cramping, with or without diarrhea. The disease is usually self-limiting and typically resolves within 24 – 48h after onset. Occasionally it can be severe enough to pathogens, frequently causing disease globally as a warrant hospitalization, particularly when infants, elderly result of food ingestion contaminated with or debilitated people are concerned (Argud´ın et al., 2012;
staphylococcal endotoxin (Mendoza, et al., 2010). S.
Mendoza et al, 2013).
S. aureus can cause wide spectrum of infections, ranging from superficial skin infections to severe, and potentially fatal, Jhalka et al., (2014) suggests the contributing factors for invasive disease due to combination of toxin-mediated the low incidence of SFD to include misdiagnosis, virulence, invasiveness, and antibiotic resistance. An improper sample collection in laboratory during important impediment to the control of the Staphylococcus examination, lack of seeking medical attention by the aureus infection is the tendency to gain resistance to almost affected persons, and lack of routine surveillance of all classes of antimicrobial agent (Jhalka et al., 2014).
clinical for S. aureus or its enterotoxins and unavailability
Staphylococcus aureus does not compete well with
of implicated foods for confirmation of laboratory testing
indigenous microbial in raw foods, contamination is
at the time of outbreak.
mainly associated with improper handling of cooked or
processed foods. However, S. aureus is also present in food,
Food handlers such as Suya meat and Nunu vendors are
animals, and dairy cattle, sheep and goats (Larkin et al.,
carrying enterotoxin-producing S. aureus in their noses or
2009). The S. aureus bacteria were then termed methicillin-
their hands which are regarded as the main source of food
resistant S. aureus (MRSA) usually show resistant to many
contamination, via manual contact or through respiratory
antibiotic (Ray et al., 2013). Staphylococcal food
secretions thereby contaminating raw foods, as a result of
poisoning (SFP) is an intoxication that results from the
improper handling of cooked or processed foods, followed
consumption of foods containing sufficient amounts of one
by storage under condition which allows growth of S.
(or more) preformed enterotoxin symptoms of SFP have a
aureus in food and animals. Food that have been frequently
rapid onset (2-8h), and include nausea, violet vomiting,
incriminated in staphylococcus intoxication include meat

and meat products, poultry and egg products, milk and dairy products, salads, bakery products, particularly cream-filled pastries and cakes, and sandwich filling. Salted food products (SFP) such as ham, have also been implicated according to the capacity of S. aureus to grow at relatively low water activity.

S. aureus have been found to survive in materials, clothes, hand nails, ATMs, mobile phones aside skins and nasal areas of the body (Oviasogie et al., 2014; Ekrakene & Igeleke, 2007; Adetona & Olabisio, 2010). It is noteworthy that Suya and Nunu hawkers come in contact with clothes, phones, ATMs and other materials while doing the merchandise without any consideration of washing before handling the food.

SFP is common disease whose real incidence is probably underestimated for a number of reasons, which include misdiagnosis, unreported minor outbreaks, improper sample collection and improper laboratory examination. (Argud´ın et al., 2010)

Nowhere has this issue been of great concern than with the gram-positive bacteria pneumococci, enterococci and staphylococci where multidrug resistance has become a norm among these pathogens. S. aureus is perhaps the pathogen of great concern because of its intrinsic virulence its ability to cause a diverse array of life-threatening infection, and its capacity to adapt to different environment conditions (Argud´ın et al., 2012, Hennekinne et al., 2012). The mortality of S. aureus bacteremia remains approximately 20-40% despite the availability of effective antimicrobial agents (Hassen et al., 2006). S. aureus is now the leading overall cause of nosocomial infections and, as more patients are treated outside the hospital setting is an increasing concern in the community as it is popular practice in Jos where drug vendors and Chemists are patronized without diagnosis or medical advice and such could also lead to increasing resistance to a great number of antimicrobial agents. Inevitably this has left fewer effective bactericidal antibiotics to treat these often life-threatening infections. As rapidly as new antibiotics are introduced, staphylococci have developed efficient mechanism to neutralize them.

In Jos metropolis, most infection are been treated with available drugs in the clinic or just by visiting drug vendors otherwise called Chemists where they are given antibiotics without recourse to medical laboratory diagnosis to identify the disease causing organism and possible susceptible drugs for the disease. Such common antibiotics have been found to be resistant to S. aureus as reported by Wang et al., (2017).

This study is aimed to determine the presence and antibiotics susceptibility profile of pathogenic staphylococcus aureus in Nunu and and Suya meat sold in Jos metropolis.

Materials and Methods

Study Area

The study was carried out in Jos Metropolis, Jos North Local Government Area in locations: Hamaz, Gangare, Railway, Yan Taya and Plateau riders Park. Sample Size / Collection

A total of 15 roasted meats (Suya) and 15 raw milk (Nunu) were collected from the select areas in and around Jos metropolis which includes: Hamaz, Gangare, Railway, Yan Taya and Plateau Riders Park. The Suya (roasted meat) and Nunu samples were obtained 3 each from every point of sales in Hamaz, Gangare, Railway, Yan Taya and Plateau riders Park into a sterilized universal bottle, packed into a polythene bags, and transported into the medical laboratory for analysis. While the Nunu sample were also collected in sterile polythene bags and transported in an icebox and transported to the Federal School Research Laboratory, Jos for analysis within 3 hours.

Isolation of Staphylococcus Aureus in Nunu and Suya 1mL of each sample was transferred to flask containing 9ml of peptone water for enrichment. According to ISO 6888, the plates were incubated under aerobic conditions at 370C for 24hr and those that yielded growth were subjected to another 24hr incubation for susceptibility assessment.

The total counts were carried out using nutrient agar. In serial dilution preparation, 1.0g of sample was aseptically, transferred into 9.0ml of diluted water and homogenized by vortex. Subsequent serial dilutions up to 10-5 were made in line with Kalalou et al. (2004). The enumeration of microorganisms in the samples was by the pour plate technique. At the end of the incubation, resultant microbial colonies (staphylococcus aureus) were counted.

Identification of Staphylococcus Aureus from Nunu and Suya

Pure isolates of microorganism grown on the different chocolate agar plates were further identified biochemically using Cowan and Steel (1974) technique using oxidase, coagulase, maltose, glucose and gram’s staining. The staphilococcus aureus were the only organisms isolated from suya meat and nunu samples obtained from the food vendors in Jos metropolis as identified from the samples.

Antibiotic Susceptibility Test of the Isolates of Staphilococcus Aureus

All S. aureus isolates were screened for multi-drug-resistance using disc diffusion in line with Bauer et al., (1966). This was performed on nutrient agar plates. Furthermore, the antibiotic susceptibility pattern of S. aureus strains was determined by disc diffusion method for Ciprofloxacin (10mcg), Gentamycin (10mcg), Norfloxacin (10mcg), Erythromycin (30mcg), Streptomycin (30mcg), Levofloxacin (20mcg), Chloramphenicol (30mcg), Amoxil, Ampiclox and Rifampin. Chloramphenical, Erythromycin and Norfloxacin susceptibility was determined by the e-test according to the manufacturers’ guidelines (Biometriux, France).

The diameter of the zone of inhibition produced each antibiotic disc was measured, recorded and the isolates were classified as “resistant”, “intermediate” and “sensitive” based on the standard interpretative chart updated according to the CLSI (2015). Multi-resistance was defined by resistance of the strain to at least three antibiotic agents.

15

RESULTS

Prevalence of Staphylococcus aureus from suya (roasted meat) sample

Key: X = Number Positive, Y = Number of sample examined, A = Hamas, B = Gangare, C = Rail Way, D = Yantaya, E = Plateau riders

Prevalence of Staphylococcus aureus from Nunu in Jos Metropolis

Key: X = Number Positive, Y = Number of sample examined, A = Hamas, B = Gangare, C = Rail Way, D = Yantaya, E = Plateau riders

Antibiotic Susceptibility Profile for Suya (Roasted Meat)

Key: S = Sensitive, R = Resistance

16

Assessment of Antibiotic Susceptibility Pattern on Staphylococcus…

Antibiotic Susceptibility Profile for Suya (Roasted Meat)

Key: S = Sensitive, R = Resistance

Discussion

Staphylococcus aureus is an important health care and community acquired infection in every region of the world (Wang et al., 2017). This studies shows that Staphylococcus aureus is prevalent in Suya (33.3%) and Nunu (40%) and could possess a potential health hazard (Sofos, 2008) to consumers in Jos Metropolis. Studies on isolation and antibiotic susceptibility frequency of isolation of S. aureus isolates from meat and milk may be due to the possible susceptibility of S. aureus isolates from Suya and Nunu to Levofloxacin, Erythromycin, Norfloxacin, Gentamycin and Rifampin as observed in this study. However, this report was not different from other studies reported by Waters et al. (2011). The high susceptibility of S. aureus to these antibiotics may be due to high costly or injectable forms and the possibility of abuse of such antibiotics may be low. The low susceptibility of chloramphenicol, amoxil, ampiclox observed in this study was not different from the other studies reported by Le et al., (2003). The low susceptibility of S. aureus to antibiotics mentioned may be due to inappropriate use of antibiotics without doctor’s prescription and inclusion of antibiotics in animal feeds as growth promoter (Le et al., 2003). From this study, it was however observed that the frequency of isolation of S. aureus observed in this study was high and isolates were more susceptible to flouroquinolones and aminoglycosides. In view of this, such drugs may be useful for treatment of staphylococcal infections, but there is a need to conduct appropriate medical laboratory diagnosis to find out the causative organism S. aureus for example before treatment especially in Jos Metropolis. The indiscriminate use of antibiotics/antimicrobials agents for prophylactic as well as other therapeutic purpose could be the reasons for increased antimicrobial resistance of S. aureus in animals and humans.

Conclusion

Staphylococcal food poisoning (SFP) is a major concern in public health programs worldwide. S. aureus may be present in cow milk as a result of milk collection from the animal suffering from disease condition and excreting S. aureus in milk or due to unhygienic conditions during production, processing, storage and handling of milk products, which are the main causes of food borne

diseases. The result of this study clearly indicated that some Nunu and Suya available in the Jos metropolis were contaminated with S. aureus, which could pose a risk of food poisoning to Jos metropolis. Thus, more hygienic preventive measures are required to reduce the bacterial contamination, so as to increase the wholesomeness of these Nunu and Suya. The study also revealed common antibiotics used in Jos which are resistant to S. aureus. This study highlights the need for continuous surveillance of antibiotic sensitivity pattern of staphylococcus aureus with a view to selecting appropriate therapy. This study also recommends that:

1. All Suya meat and Nunu vendors should imbibe adequate hygiene measures while handling the food products
2. The Suya and Nunu vendors should use nose masks while processing handling their products
3. The Suya and Nunu vendors in Jos metropolis should avoid using dirty rags to clean their hands while processing and dispensing their products to the public

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