Genetic polymorphism in Plasmodium falciparum: differentiation of parasite isolates of high & low virulence by RAPD.

BACKGROUND & OBJECTIVES: The increase in Plasmodium falciparum infections which are associated with severe and complicated malaria and drug resistance has made control of malaria a difficult task. Extensive genetic polymorphism in P. falciparum has been reported from several parts of the world which affects the efficacy of sub-unit vaccines. The knowledge of genotypes of the parasite in a geographical region is therefore, important for effective management and control. The aim of the present study was to investigate the usefulness of random amplified polymorphic DNA (RAPD)-PCR technique for differentiation of P. falciparum isolates from patients presenting with severe (cerebral malaria) and mild malaria. METHODS: Genetic polymorphism in 21 P. falciparum isolates obtained from patients found positive for P. falciparum by light microscopy was studied by RAPD-PCR analysis. Eleven RAPD primers were used for analysis of 21 P. falciparum isolates obtained from cerebral and non-cerebral malaria patients. RESULTS: Of the 11 primers, only three (E-4, E-8, and R-8) produced useful polymorphic patterns. The cluster analysis based on UPGMA demonstrated that isolates causing cerebral malaria cluster separately from those causing uncomplicated malaria. However, the analysis of phylogenic tree showed that P. falciparum isolates causing non-cerebral and cerebral malaria clustered separately but showed relatedness. INTERPRETATION & CONCLUSIONS: The results of the present study showed that the RAPD-PCR was able to differentiate the isolates causing severe and mild malaria. The cluster analysis of the phylogenic tree suggested that the virulent strains evolved from less virulent strains as it clustered separately. RAPD technique may be useful in discriminating between the different isolates of the same species resulting in different clinical profiles.

Anaemia & expression levels of CD35, CD55 & CD59 on red blood cells in Plasmodium falciparum malaria patients from India.

BACKGROUND & OBJECTIVES: Severe anaemia in Plasmodium falciparum (Pf) associated malaria is a leading cause of death despite low levels of parasitaemia. In an effort to understand the pathogenesis of anaemia we studied expression level of RBC complement regulatory proteins, CR1 (CD35), CD55 and CD59 with haemoglobin status in a group of malaria cases from Assam, Goa and Chennai, and in healthy controls. METHODS: Flowcytometry was used to study expression of CR1, CD55 and CD59 in 50 Pf cases and 30 normal healthy volunteers. Giemsa stained thick and thin blood films were used for microscopic detection and identification of malarial parasites and parasite count. RESULTS: No correlation was found between degree of expression of RBC surface receptors CR1, CD55 and CD59 with haemoglobin level. However, expression of CD55 was less in malaria cases than in healthy controls. INTERPRETATION & CONCLUSIONS: The present findings indicate that malaria infection changes the expression profile of complement regulatory protein CD55 irrespective of severity status of anaemia. Further studies are needed to explore the pathophysiology of anaemia in malaria cases in Assam where expression of RBC complement receptors appears to be low even in normal healthy population.

Kinetics of microfilaraemia & antigenaemia status by Og(4)C(3) ELISA in bancroftian filariasis.

BACKGROUND & OBJECTIVE: Bancroftian filariasis caused by Wuchereria bancrofti is endemic in many parts of India. In recent years diagnosis of W. bancrofti infection has been revolutionized with the availability of filarial antigen tests, which is important in monitoring success of chemotherapy. We carried out this study to measure microfilariaemia and antigenemia levels in bancroftian microfilariae (mf) carriers at 1 yr follow up after chemotherapy, in lymphoedema patients and in endemic controls from a filariasis endemic area in Tamil Nadu State using Og(4)C(3) ELISA to identify the best marker to assess success of chemotherapy. METHODS: Serum samples were collected from 30 bancroftian microfilaremic (Mf) carriers pre-treatment and at sequential intervals (7,30,60,90,180 and 365 days) following treatment with diethylcarbamazine (DEC:6mg/kg body weight, single dose), 30 lymphoedema patients (without treatment) at periodic intervals, and 68 control subjects (24 endemic normal subjects in filariasis endemic area in Tamil Nadu State, 24 non-endemic normal subjects residing in Chandigarh, India; 5 brugian filariasis, 5 endemic control subject in brugian filariasis endemic area and 10 other disease controls). The circulating antigen of W. bancrofti was measured quantitatively using Og(4)C(3) ELISA kit. RESULTS: In Mf carriers, there was no significant difference in microfilariae count in pre- and post-treatment (PT) samples till day 30 while significant differences were observed in pre- and sequentially collected post-treatment (PT) samples day 60 to 180 (P<0.001), day 365 (P<0.005). However, there was no significant difference in antigenaemia levels between pre-treatment (day 0) and PT samples collected on day 7 onwards till day 365. Though of the 19 patients who could be followed up till 365 days PT, 4 (21%) were amicrofilaraemic, none became antigen negative. No significant difference was found in antigenaemia levels in sequentially collected samples from lymphoedema patients. Significant differences were observed in antigenaemia levels in samples collected at the start of study in mf carriers as compared to lymphoedema patients and endemic normal subjects (P<0.001). Subjects (non-endemic control) residing in filariasis free area (24), brugian endemic area (5), B.malayi infected patients (5) and patients with other parasitic diseases (10) were found antigen negative. INTERPRETATION & CONCLUSION: Annual single dose of DEC therapy alone may not result in complete clearance of infection and detection of antigenaemia rather than microfilaraemia may be taken into consideration as an indicator of successful chemotherapy. The study supports the earlier view that filarial antigenaemia is relatively common in amicrofilaraemic and asymptomatic subjects in endemic areas and further studies are needed to determine the clinical significance, prognosis and effective management of such infections in endemic areas.

Multidrug resistance in amoebiasis patients.

BACKGROUND & OBJECTIVES: Amoebiasis, caused by Entamoeba sp. a protozoan parasite, is a major public health problem in tropical and subtropical countries. The symptomatic patients are treated by specific chemotherapy. However, there are reports of treatment failure in some cases suggesting the possibility of drug resistance. The present study was therefore planned to assess the presence and expression of mRNA of multidrug resistance (MDR) gene in clinical isolates of Entamoeba histolytica and E. dispar. METHODS: Forty five clinical isolates of Entamoeba sp. [E. histolytica (15) and E. dispar (30)] were maintained in polyxenic followed by monoxenic medium. DNA and total RNA were extracted from clinical isolates of Entamoeba sp. and from sensitive strain of E. histolytica (HM1: IMSS) and subjected to polymerase chain reaction (PCR) and multiplex reverse transcription (RT)-PCR techniques. RESULTS: The 344 bp segment of E. histolytica DNA was seen by PCR using primers specific to EhPgp1 in all clinical isolates and sensitive strain of E. histolytica. Over expression of EhPgp1 was observed only in resistant mutant of E. histolytica; however, transcription of EhPgp1 was not seen in any clinical isolates and sensitive strain of E. histolytica. INTERPRETATION & CONCLUSION: The findings of the present study indicate that, so far, drug resistance in clinical isolates of E. histolytica does not seem to be a major problem in this country. However, susceptibility of clinical isolates of E. histolytica against various antiamoebic drugs needs to be investigated for better management.

Pathophysiology of visceral leishmaniasis - some recent concepts.

Visceral leishmaniasis is characterized by diversity and complexity of clinical manifestations ranging from asymptomatic infection to life threatening illness. Experimental evidence and clinical studies indicate multifaceted role of various factors leading to parasite survival and multiplication. In early stage of infection, generation of reactive oxygen and nitrogen intermediates play significant role in curtailing the parasite multiplication while in later phase on one hand, hepatic resistance is expressed by the dominant role played by nitric oxide synthase (NOS)-2 gene regulation and on the other hand, production of inhibitors of NOS-2 gene expression, interleukin 10 (IL-10) and transforming growth factor beta (TGFbeta) correlate well with reduced parasite killing. The hepatic infection is usually self-limiting due to production of multiple cytokine responses including moderate level of tumour necrosis factor (TNF) while in spleen excess TNF mediates destructive pathology. CD8+ T cells appear to play multiple roles comprising both cytotoxic activity and secretion of cytokines and chemokines. Capacity to produce ThI cytokines is associated with asymptomatic or subclinical self-healing infection. However, in symptomatic patients, Th I cytokine production is not depressed but there appears to be unresponsiveness to the stimuli of these cytokines. Experimental evidences indicate genetic basis for such a phenomenon.

Leishmania donovani: effect of verapamil on in vitro susceptibility of promastigote and amastigote stages of Indian clinical isolates to sodium stibogluconate.

Although pentavalent antimonials are the first-line drug for treatment of visceral leishmaniasis all over the world, yet, in India, increasing number of patients are being reported to be unresponsive to sodium stibogluconate. Verapamil, a calcium channel blocker, affects drug uptake by preventing its efflux and thereby accumulation within the cell. In the present study, effect of verapamil on in vitro susceptibility of both promastigote and amastigote stages of 15 clinical isolates and standard strain of Leishmania donovani to sodium stibogluconate was evaluated by detection of acid phosphatase. Amastigotes were found more susceptible to sodium stibogluconate than the promastigotes (p<0.05) and in the presence of verapamil, IC(50) value of sodium stibogluconate was reduced only for those isolates, which had a higher IC(50). Verapamil alone did not have any effect on the parasites. The results indicate that amastigotes are more susceptible to sodium stibogluconate than promastigotes and verapamil can reverse the in vitro drug resistance of L. donovani clinical isolates to sodium stibogluconate.

Drug resistance in amoebiasis.

Amoebiasis caused by Entamoeba histolytica, is a major public health problem in developing countries. Morphologically similar E. dispar is non pathogenic. Because of the redefinition of E. histolytica and E. dispar, and the limited number of antiamoebic drugs available, a new approach to treat such individuals is necessary. The cost of treating asymptomatic individuals is highly exorbitant and not justifiable. The indiscriminate use of antiamoebic drugs can result in increased minimum inhibitory concentration (MIC) values against Entamoeba species, and treatment failure may emerge as an important public health problem. Development of new antiamoebic drugs is still in infancy and vaccine development appears to be distant dream. In future, the development of drug resistance may seriously affect the control of disease. This review discusses the factors involved in drug resistance mechanisms developed by the parasite.

Plasmodium falciparum: polymorphism in the MSP-1 gene in Indian isolates and predominance of certain alleles in cerebral malaria.

Polymorphism in the block-2 region of merozoite surface protein-1 gene in 69 North Indian Plasmodium falciparum isolates was studied by PCR and RFLP using Dra-1 endonuclease. On the basis of molecular weight of the PCR products, considerable size polymorphism in target gene was seen and 69 isolates were classified into five allelic types. On RFLP, the isolates in three allelic types were further divided into two sub-allelic types each and thus eight genetic types could be identified. Interestingly, all five allelic types were identified in 47 isolates from uncomplicated (non-cerebral) malaria patients while only two allelic types (Type 2 and 3) were seen amongst 22 isolates from cerebral malaria patients. Furthermore, on RFLP, one subtype (2A) was predominantly seen in cerebral malaria patients and one subtype (3A) was exclusively found in cerebral malaria patients. These observations suggest that a few, comparatively more virulent isolates prevalent in an area may cause severe disease (cerebral malaria) which can be identified by molecular techniques like PCR-RFLP.

Genetic polymorphism in Plasmodium falciparum vaccine candidate antigens.

Malaria is still a major public health problem in many tropical and subtropical countries. Malaria vaccine is highly desirable as an adjunct to existing malaria control measures. The polymorphisms in malaria vaccine candidates antigens might be a hurdle in developing an effective vaccine. The present article reviews the genetic polymorphism in several antigens expressed on the parasite surface, which are targets for immunological responses of the host and are good candidates for vaccine development against P. falciparum. Variable regions of most genes are generally dimorphic probably as a result of intragenic recombinations. Each allele in turn shows polymorphism resulting from point mutations, or other mechanisms. Several antigens like merozoite surface protein-1 and 2 (MSP-1 and MSP-2) and S antigen show high polymorphism while in others like circumsporozoite protein (CSP), apical membrane antigen-1 (AMA-1) and erythrocyte binding antigen-175 (EBA-175) functional constraints limit the degree of polymorphism. Polymorphism reported in these genes is discussed.

Cytokine mRNA expressions in symptomatic vs. asymptomatic amoebiasis patients.

Infection with Entamoeba histolytica results in high mortality worldwide. Studies on the cytokine response in symptomatic and asymptomatic amoebiasis (caused by E. histolytica, the pathogenic species and E. dispar, the non-pathogenic species) subjects and their correlation with symptomatology are lacking. The present study reports the cytokine response (TNF-alpha, IFN-gamma, IL-4, IL-10 and TGF-beta) in such subjects as measured by RT-PCR. The results showed significantly (< 0.05) higher expressions of IL-10 and TGF-beta in the symptomatic group as compared to the asymptomatic and healthy controls. The cytokine profile indicated the role of suppressive immune response in symptomatic amoebiasis patients.

Drug resistance in malaria.

Ever since the discovery of the first case of chloroquine resistance along the Thai-Combodian border in the late 1950s, Southeast Asia has played an important role as a focus for the development of drug resistance in Plasmodium falciparum. Although the first case of quinine resistance had been reported much earlier from South America, the onset of chloroquine resistance marked the beginning of a new chapter in the history of malaria in Southeast Asia and by 1973 chloroquine finally had to be replaced by the combination of sulphadoxine and pyrimethamine (SP) as first line drug for the treatment of uncomplicated malaria in Thailand and more than 10 African countries have also switched their first line drug to SP. In 1985, eventually SP was replaced by mefloquine. The rapid development of resistance to this new drug leads to the introduction of artemisinin as a combination drug in the mid-1990s. It is mandatory to mention here that therapeutic regimens for prevention and treatment of chloroquine-resistant P. falciparum are associated with higher costs and side-effects compared to chloroquine. Additionally, some of these alternative treatments are associated with more side-effects, take longer time for cure and are more difficult to comply with than chloroquine. Urgent efforts are needed to identify effective, affordable, alternative antimalarial regimens. Molecular markers for antimalarial resistance have been identified, including pfmdr-1 and pfcrt polymorphisms associated with chloroquine resistance and dhfr and dhps polymorphisms associated with SP resistance. Polymorphisms in pfmdr-1 may also be associated with resistance to chloroquine, mefloquine and artemisinin. Use of such genetic information for the early detection of resistance foci and future monitoring of drug resistant malaria is a potentially useful epidemiological tool, in conjunction with the conventional in vitro and in vivo drug sensitivity assessments. The purpose of this review is to describe the state of knowledge regarding drug resistant malaria and to outline the changing patterns of drug resistance including its determinants, current status in diverse geographical areas, molecular markers and their implications to limit the advent, spread and intensification of drug resistant malaria.

Decreased level of 2,3-diphosphoglycerate and alteration of structural integrity in erythrocytes infected with Plasmodium falciparum in vitro.

2,3-Diphosphoglycerate (2,3-DPG), an intracellular metabolite of glycolytic pathway is known to affect the oxygen binding capacity of haemoglobin and mechanical properties of the red blood cells. 2,3-DPG levels have been reported to be elevated during anaemic conditions including visceral leishmaniasis. 2,3-DPG activity in P. falciparum infected red blood cells, particularly in cells infected with different stages of the parasite and its relationship with structural integrity of the cells is not known. Chloroquine sensitive and resistant strains of P. falciparum were cultured in vitro and synchronized cultures of ring, trophozoite and schizont stage rich cells along with the uninfected control erythrocytes were assayed for 2,3-DPG activity and osmotic fragility. It was observed that in both the strains, in infected erythrocytes the 2,3-DPG activity gradually decreased and osmotic fragility gradually increased as the parasite matured from ring to schizont stage. The decrease in 2,3-DPG may probably be due to increased pyruvate kinase activity of parasite origin, which has been shown in erythrocytes infected with several species of Plasmodium. The absence of compensatory increase in 2,3-DPG in P. falciparum infected erythrocytes may aggravate hypoxia due to anaemia in malaria and probably may contribute to hypoxia in cerebral malaria. As 2,3-DPG was not found to be increased in erythrocytes parasitized with P. falciparum, the increased osmotic fragility observed in these cells is not due to increased 2,3-DPG as has been suggested in visceral leishmaniasis.

Transfusion associated infections: a threat to life.

Blood transfusion is indispensable in the management of many haematological diseases and has become the mainstay in major surgical procedures. Transfusion-transmitted infections have been a major threat to life since the dawn of transfusion therapy. The authors have highlighted the different viral, parasitic and bacterial infections associated with transfusion and have focussed on the precautionary measures that can be implemented for prevention of the infections along with a brief review of the literature.

A serological study of human toxocariasis in north India.

BACKGROUND: Human toxocariasis owing to lodgement of the larvae of Toxocara canis in different organs can result in serious clinical syndromes such as visceral larva migrans or ocular larva migrans. Detection of an antibody response to Toxocara canis excretory-secretory (TES) antigen in serum samples is sensitive and specific for diagnosis and epidemiological surveys. To assess the extent of this problem in northern India, we tested the antibody response to the TES antigen by ELISA technique in subjects residing in a rural area near Chandigarh and in patients attending Nehru hospital, Chandigarh and clinically suspected to have toxocariasis. METHODS: Serum samples were collected from 94 randomly selected subjects, residents of Kheri village, Ambala district, Haryana; 30 patients clinically suspected to have toxocariasis attending Nehru hospital, Chandigarh; 25 control patients and 15 normal healthy individuals. These were subjected to ELISA technique for detection of an antibody response to TES antigen usinga commercial kit (LMD Laboratories Inc. Ca. USA). All the samples were tested in duplicate and positive samples were tested by a different kit (Melotec Biotechnology, Spain). RESULTS: Of the 94 subjects residing in Kheri village and 30 clinically suspected toxocariasis patients, 6 (6.4%) and 7 (23.3%), respectively, were seropositive for anti-Toxocara antibody response. A history of pica and/or contact with puppies could not be obtained from all the subjects/patients, hence the exact mode of transmission could not be ascertained. However, 3 (3.2%), 2 (2.13%) and 1 (1.06%) seropositive subjects in Kheri village were in the age groups of 1-10, 11-20 and 21-30 years, respectively, while 4 (13.33%) and 3 (10%) seropositive patients who attended Nehru hospital, Chandigarh were in the age groups of 1-10 and 21-30 years, respectively. None of the control patients/healthy individuals were seropositive. CONCLUSION: A positive antibody response to TES antigen in 6.4% subjects residing in a rural area near Chandigarh and in 23.3% of patients clinically suspected to have toxocariasis indicates that human toxocariasis may be endemic in certain regions of northern India. A detailed epidemiological study is needed to determine the extent of this problem.

Specific IgA response, T-cell subtype and cytokine profile in experimental intravaginal trichomoniasis.

Trichomoniasis caused by Trichomonas vaginalis may lead to either a complete absence of symptoms or to severe inflammatory manifestations in infected women. Studies of the role of immune responses in the pathogenesis and varied symptomatology of this disease are lacking. Mice may prove useful as an experimental model for intravaginal trichomoniasis in developing an understanding of the role of local immune responses in the pathogenesis and varied symptomatology of this disease. The present study reports the levels of anti-Trichomonas IgA antibodies in serum and vaginal washes, and T-cell subtype and cytokine profile in vaginal cervical tissues of mice infected intravaginally with T. vaginalis isolates from 15 symptomatic and 15 asymptomatic women. It also correlates the responses with symptomatology of the patients. Successful intravaginal infection was established by inoculating T. vaginalis in BALB/c mice preinoculated with Lactobacillus acidophilus and pretreated with oestradiol. A significant increase in specific IgA antibody levels was detected with enzyme-linked immunosorbent assay in vaginal secretions and serum samples collected on the 7th post-infection day from animals infected with isolates from asymptomatic women when compared with mice infected with isolates from symptomatic women. T-cell subset analysis showed significant differences, with increased CD4+ T-cell count in animals infected with isolates from asymptomatic women compared with animals infected using isolates from symptomatic women. No difference in CD8+ T cells was observed between the two groups. Cytokine profile revealed significantly higher (P < 0.001) production of gamma-IFN and IL-2 in mice infected with asymptomatic isolates compared with animals infected with symptomatic isolates, using T. vaginalis crude antigen extract and nonspecific mitogen (ConA) as stimulants for vaginal cervical lymphocytes. However, no difference in IL-4 levels was observed in the two groups of animals. In contrast, significant increase in tumour necrosis factor (TNF-alpha) levels was observed in animals infected with asymptomatic isolates compared with those infected with isolates from symptomatic women and controls, thereby indicating that TNF-alpha may play an important role in the inflammatory response to trichomoniasis. The study further suggests that specific IgA antibodies might help to protect asymptomatic individuals from severe infection and T-lymphocytes may play an important function in the eradication of the parasite. The cytokine profile indicated the involvement of Th-1 like responses in mice infected with asymptomatic isolates, compared with those infected with symptomatic isolates.

Detection of antigen B of Cysticercus cellulosae in cerebrospinal fluid for the diagnosis of human neurocysticercosis.

Neurocysticercosis (NCC) is a major cause of morbidity and mortality in developed and developing countries. The diagnosis of this disease remains a problem. We report the detection of specific antigenic fraction (antigen B) of Cysticercus cellulosae by enzyme-linked immunosorbent assay (ELISA) in various fractions of cerebrospinal fluid (CSF) obtained by high performance liquid chromatographic (HPLC) separation, for the diagnosis of human NCC. Forty patients attending or admitted to Nehru Hospital, Chandigarh were included in the study: 10 with suspected NCC, 20 with other neurological diseases and 10 undergoing surgery under spinal anaesthesia for non-neurological conditions, who served as controls. CSF samples collected from all patients and controls were subjected to chromatographic separation on an HPLC system. Antigen B (AgB) was detected in separated fractions by an ELISA test and compared with the detection of antibody response in CSF samples by indirect haemagglutination (IHA) technique. Antigen B was detected in 9 out of 10 patients with suspected NCC based on clinical symptoms and radioimaging reports, but in none of the control subjects. However, antigen B was also detected in 9 out of 20 patients with other neurological disorders, mostly tubercular meningitis. Antibody response by IHA was found positive in only 2 of 10 cases clinically suspected of NCC. In conclusion, antigen B detection in CSF samples may be a useful adjunct to clinical suspicion and radiological reports for the diagnosis of NCC as there is no gold standard criteria to confirm this disease. However, the test needs to be evaluated on more patients in countries where tuberculosis and cysticercosis are endemic due to the high cross reactivity with samples from tubercular meningitis patients.

Role of viral serology in the diagnosis of acute retinal necrosis syndrome.

Due to the devastating nature of acute retinal necrosis syndrome (ARNS), early diagnosis is essential. 5 cases of clinically diagnosed ARNA were investigated for CMC, herpes simplex and varicella zoster virus (VZV) infections. Of the three VZV IgM positive cases, two were positive in acute blood samples and one in vitreous fluid. Thus VZU can be incriminated as the causative agent of ARNS cases in North India.

Ca2+ signaling in the transformation of promastigotes to axenic amastigotes of Leishmania donovani.

The present study is an attempt to look into the role of Ca2+ in signaling the transformation of promastigotes to axenic amastigotes. An estimation of intracellular free calcium concentration at 6 h intervals during the conversion of promastigotes to axenic amastigotes (72 h) revealed a 10 fold increase in [Ca2+]i at the initial 6-12 h during the conversion. This was followed by declining levels till 60 h and the concentration thereafter remained constant. Axenic amastigotes (72 h) had a 5 fold higher [Ca+]i as compared to the promastigotes. A 30-40% decrease in [Ca2+]i after pretreatment of cells with dentrolene and a gradual rise of intracellular Ca2+ in [Ca2+] free medium indicates the role of intracellular calcium pools in the elevation of [Ca2+]i. A sudden increase in [Ca2+]i on addition of NH4Cl (20 mM) in the cells grown in Ca2+ free medium indicates the presence of acidocalcisomes, as intracellular Ca2+ storing pool, in L. donovani. To study the role of Ca2+ influx from the external medium in the morphogenetic transformation and in the elevation of [Ca2+]i a 45Ca2+ uptake study was performed. Maximum uptake of 45Ca2+ was observed in the initial 24 h of transformation and maximum Ca2+ ATPase activity was also observed between 24-42 h. So the presence of low Ca2+ in the cytosol, existence of intracellular Ca2+ pools and presence of mechanisms to maintain the Ca2+ homeostasis in the cells suggests that Ca2+ can be an appropriate candidate for a second messenger during the morphogenetic transformation of L. donovani.

Prevalence of hepatitis B virus infection in Chandigarh over a six year period.

A total of 6418 samples were received between January 1993 to December 1998 from patients with hepatitis. Blood samples were also collected from 946 apparently healthy subjects. All these samples were tested for Hepatitis B surface antigen (HBsAg) by third generation micro ELISA. The overall HBsAg prevalence rate was 12.8%. The highest prevalence was noted in renal transplant patients (21.7%) followed by patients with acute hepatic disease (15.3%), pregnancy with jaundice (9.4%), chronic renal failure (8.8%), nephrotic syndrome (3.1%), whereas the prevalence rate in control group was 2.4%. The prevalence rate of HBsAg was higher in subjects between 21 to 30 years of age with a male preponderance (Male:Female = 2.8:1).