Subhasish Kamal Guha

High Prevalence of Asymptomatic Malaria in a Tribal Population in Eastern India

ABSTRACT Asymptomatic infection by Plasmodium falciparum is an important obstacle to eliminating malaria. Asymptomatic carriers do not seek treatment for infection, and therefore they become a reservoir for the parasite. For this reason, these carriers pose a real public health risk. The systematic identification and treatment of asymptomatic infections should reduce the parasite reservoir. A large reduction in this pool will lower the chance of transmission of the disease. In this study, we screened a tribal population of 1,040 individuals in the Purulia district of West Bengal by using a dual-antigen rapid diagnostic kit (RDK), microscopy, and species-specific PCR. All positive individuals were treated with artemisinin-based combination therapy (ACT) (artesunate plus sulfadoxine-pyrimethamine) and followed for 42 days. Polymorphisms in candidate genes were screened by DNA sequencing. A significant proportion (8.4%) of the study population was infected with P. falciparum but showed no clinical manifestations. The PCR method was more sensitive in detecting infection than the RDK or microscopy. The efficacy of the ACT was 97%. In the pfcrt gene, the mutation K76T (the mutated amino acid is indicated by bold type) was found in 100% of the cases. In the pfmdr1 gene, the mutations N86Y and Y184F were noted in 55.5% and 11% of the cases, respectively. Six different haplotypes were identified in the pfdhfr-pfdhps genes. Most importantly, the quintuple mutant A16I51R59N108I164-S436G437E540A581A613 was found in 10% of the isolates, which is potentially important for the development of sulfadoxine-pyrimethamine resistance. A significant proportion of the study population harboring P. falciparum does not seek treatment and therefore serves as a reservoir for the parasite, maintaining the natural cycle. If the National Vector Borne Disease Control Programme (NVBDCP) of India is to eliminate malaria, then this hidden parasite burden needs to be addressed properly. Similar study in other parts of the country could help to determine the magnitude of the problem.

Comparative Efficacies of Artemisinin Combination Therapies in Plasmodium falciparum Malaria and Polymorphism of pfATPase6, pfcrt, pfdhfr, and pfdhps Genes in Tea Gardens of Jalpaiguri District, India

ABSTRACT In India, chloroquine has been replaced by a combination of artesunate and sulfadoxine-pyrimethamine (AS-SP) for uncomplicated P. falciparum malaria. Other available combinations, artemether-lumefantrine (AM-LF) and artesunate-mefloquine (AS-MQ), not included in the national program, are widely used by private practitioners. Little is known about the therapeutic efficacy of these artemisinin combinations and the prevalence of molecular markers associated with antimalarial drug resistance. A total of 157 patients with P. falciparum monoinfection were recruited and randomized into three study groups (AS-SP, AM-LF, and AS-MQ). All patients were followed up for 42 days to study the clinical and parasitological responses according to the WHO protocol (2009). We assessed the polymorphism of the pfATPase6, pfcrt, pfdhfr, and pfdhps genes by the DNA-sequencing method. The PCR-corrected therapeutic efficacies of AS-SP, AM-LF, and AS-MQ were 90.6% (95% confidence interval [CI], 0.793 to 0.969), 95.9% (95% CI, 0.860 to 0.995), and 100% (95% CI, 0.927 to 1.00), respectively. No specific mutational pattern was observed in the pfATPase6 gene. All isolates had a K76T mutation in the pfcrt gene. In the pfdhfr-pfdhps genotype, quadruple mutation was frequent, and quintuple mutation was documented in 6.3% of P. falciparum isolates. The significant failure rate of AS-SP (9.5%), although within the limit (10%) for drug policy change, was due to SP failure because of prevailing mutations in pfdhfr, I51R59N108, with pfdhps, G437 and/or E540. The efficacy of this ACT needs periodic monitoring. Artemether-lumefantrine and artesunate-mefloquine are effective alternatives to the artesunate-sulfadoxine-pyrimethamine combination.

Histoplasmosis in eastern India: The tip of the iceberg?

Systemic histoplasmosis has various clinical presentations and is of especially concern in immunocompromised patients. A high index of suspicion is required for its diagnosis. A total of 38 cases had been reported from India up to 1996. The most frequent occurrence of cases was around Calcutta in eastern India where the previous case was detected 20 years earlier. However, we have diagnosed 5 cases in the past 2 years from eastern India which are reported here. These cases may indicate under-diagnosis and under-reporting of histoplasmosis in India. All 5 patients had disseminated disease with multisystem involvement including 2 with bilateral adrenal enlargement. Two were diabetic and only 1 patient was infected with HIV.

In Vivo Therapeutic Efficacy of Chloroquine Alone or in Combination with Primaquine against Vivax Malaria in Kolkata, West Bengal, India, and Polymorphism in pvmdr1 and pvcrt-o Genes

ABSTRACT Plasmodium vivax malaria, though benign, has now become a matter of concern due to recent reports of life-threatening severity and development of parasite resistance to different antimalarial drugs. The magnitude of the problem is still undetermined. The present study was undertaken to determine the in vivo efficacy of chloroquine (CQ) and chloroquine plus primaquine in P. vivax malaria in Kolkata and polymorphisms in the pvmdr1 and pvcrt-o genes. A total of 250 patients with P. vivax monoinfection were recruited and randomized into two groups, A and B; treated with chloroquine and chloroquine plus primaquine, respectively; and followed up for 42 days according to the WHO protocol of 2009. Data were analyzed using per-protocol analyses. We assessed polymorphisms of the pvmdr1 and pvcrt-o genes by a DNA-sequencing method. Out of the 250 patients recruited, 204 completed a 42-day follow-up period, 101 in group A and 103 in group B. In group A, the non-PCR-corrected efficacy of CQ was 99% (95% confidence interval [CI], 0.944 to 1.00), and in group B, all cases were classified as adequate clinical and parasitological response (ACPR). Day 3 positivity was observed in 11 (5.3%) cases. No specific mutation pattern was recorded in the pvcrt-o gene. Eight nonsynonymous mutations were found in the pvmdr1 gene, three of which were new. The Y976F mutation was not detected in any isolate. Chloroquine, either alone or in combination with primaquine, is still effective against P. vivax malaria in the study area. (The study protocol was registered in CTRI [Clinical Trial Registry-India] of the Indian council of Medical Research under registration no. CTRI/2011/09/002031.)

Predictors of mortality among HIV-infected patients initiating anti retroviral therapy at a tertiary care hospital in eastern India.

OBJECTIVE To assess early mortality and identify its predictors among the ART naive HIV-infected patients initiating anti retroviral therapy (ART) available free of cost at the ART Centres. METHODS A retrospective cohort analysis of routinely collected programme data was done for assessing mortality of all ART naive adult patients who received first-line ART at a government tertiary care hospital in eastern India during 1st March 2009 and 28th February 2010. Bivariate and multiple regression analyses of the baseline demographic, clinical and laboratory records using SPSS 15.0 were done to identify independent predictors of mortality. RESULTS The mortality rate at one year was estimated to be 7.66 (95%CI 5.84-9.83) deaths/100 patient-years and more than 50% of the deaths occurred during first three months of ART initiation with a median time interval of 73 days. Tuberculosis was the major cause of death. ART naive patients with baseline serum albumin <3.5 mg/dL were eight (OR 7.9; 95%CI: 3.8-16.5) at risk of death than those with higher serum albumin levels and patients with CD4 count <100 cells/μ L were two times (OR 2.2; 95%CI1.1-4.4) at risk of death compared to higher CD4 counts. CONCLUSIONS Risk of mortality is increased when ART is initiated at advanced stages of immunosuppression denoted by low serum albumin levels and CD4 cell counts. This highlights the importance of early detection of HIV infection, early management of opportunistic infections including tuberculosis and timely initiation of the antiretroviral drugs in the resource-limited countries, now available free in the Indian national ART programme.

Laryngeal involvement during post kala-azar dermal leishmaniasis in India

Summary Post kala‐azar dermal leishmaniasis (PKDL) involving the mucus membranes is relatively rare on the Indian sub‐continent. We describe 3 cases of PKDL presenting with hoarseness of voice. In one case the skin, nasal, oral, oropharyngeal and laryngeal mucosa had nodular and nodulo‐ulcerative lesions; in the 2 other cases, genitalia and anorectal mucosa were also affected. Laryngoscopic examination revealed nodular lesions on the vocal cords. Biopsy smear and culture confirmed their leishmanial origin.

Recurrence of kala-azar after PKDL: role of co-factors.

Recurrence of kala‐azar after post kala‐azar dermal leishmaniasis (PKDL) has remained uncommon. We report here two patients with recurrence of kala‐azar (KA) after development of PKDL. In one case the second attack of KA was preceded by repeated attacks of malaria and tuberculosis, and in the other the recurrence of KA followed an attack of measles. While measles has earlier been suggested as co‐factor in inducing transformation from sub‐clinical to clinical kala‐azar, malaria was demonstrated to enhance the virulence and invasiveness of Leishmania in an experimental model as well as under natural condition. We propose that in our cases, measles and repeated attacks of malaria or tuberculosis led to immunosuppression and recurrence of visceral leishmaniasis (VL).

Characterization of Treatment-Naive HIV/HBV Co-Infected Patients Attending ART Clinic of a Tertiary Healthcare Centre in Eastern India

Objective The study was designed to assess the hepatitis B virus (HBV) and hepatitis C virus (HCV) co-infection scenario among the human immunodeficiency virus (HIV) infected patients attending a tertiary healthcare unit in eastern India. Additionally, clinical and virological characterization of these viruses, prior to antiretroviral therapy (ART) initiation was also done for better understanding of the disease profile. Methods Pool of ART-naive HIV/HBV co-infected and HIV mono-infected patients, participating in two different studies, were included in this study. HBV DNA was detected by nested-PCR amplification followed by HBV genotype determination and HBV reverse transcriptase (RT) region amplification and direct sequencing for detecting drug resistance. Results The prevalence of HBsAg (11.3%) was higher compared to anti-HCV (1.9%) among the HIV infected ART-naive patients. Moreover, majority of the HBeAg positive HIV/HBV co-infected patients (87.7%) had HBV DNA ≥20,000 IU/ml with median HBV DNA significantly higher than that of HBeAg negative subjects (5.7 log10 IU/ml vs. 4.2 log10 IU/ml; p<0.0001). Multivariate analysis also showed that HBeAg-positive status was independently associated with higher HBV DNA level (p = <0.001). Notably, 60.9% of the HBeAg negative co-infected subjects had HBV DNA ≥2,000 IU/ml of which 37.0% had HBV DNA ≥20,000 IU/ml. Genotype HBV/D (68.2%) was the predominant genotype followed by HBV/A (24.3%) and HBV/C (7.5%). Anti-HBV drug resistant mutations were detected in two (3.8%) of the ART-naive patients. Conclusion The prevalence of HIV/HBV co-infection was relatively higher in our study subjects. HBeAg testing might provide clue for early treatment initiation. Furthermore, HBeAg negative patients are also associated with high HBV DNA levels and therefore require appropriate medical attention. Pre-treatment screening for anti-HBV drug resistant mutations is not necessary before ART initiation.

Genetic characterization of HIV-1 from semen and blood from clade C-infected subjects from India and effect of therapy in these body compartments

Biologic and genetic differences between HIV-1 clade C in India and clade B in US suggest that the effect of anti-viral therapy in various body compartments may differ between these two clades. We examined the effect of therapy on viral loads in semen and blood of HIV-1-clade C infected subjects from India and evaluated whether HIV-1 in the semen is different from that in blood in these subjects. HIV-1 RNA was detected in semen and blood at all stages of the disease. Viral loads in semen and blood were strongly correlated with each other, but not with the CD4+ T cell count. Anti-viral treatment reduced viral load drastically in blood and semen within one month of post therapy. Genetic characterization of HIV-1 in the semen and blood demonstrated that they were highly compartmentalized. These data have important implications of sexual transmission of HIV-1 in clade C HIV-1 infected subjects.

Molecular Characterization of HBV Strains Circulating among the Treatment-Naive HIV/HBV Co-Infected Patients of Eastern India

Previously we reported that the exposure to hepatitis B virus (HBV) infection serves as a major threat among the treatment naive HIV infected population of eastern India. Hence, molecular characterization of these strains is of utmost importance in order to identify clinically significant HBV mutations. A total of 85 treatment naive HIV/HBV co-infected participants were included of whom the complete basal core promoter/precore region, the core and the whole envelope gene could be successfully sequenced for 59, 57 and 39 isolates respectively. Following phylogenetic analysis, it was found that HBV/D was the predominant genotype with HBV/D2 (38.5%) being the most prevalent subgenotype followed by HBV/A1. The major mutations affecting HBeAg expression includes the A1762T/G1764A (13.6%), G1896A (22%) and G1862T mutation (33.9%) which was predominantly associated with HBV/A1. Moreover, the prevalence of G1896A was considerably high among the HBeAg negative HIV/HBV co-infected subjects compared to HBV mono-infection. The main amino acid substitutions within the MHC class II restricted T-cell epitope of HBcAg includes the T12S (15.8%) and T67N (12.3%) mutation and the V27I (10.5%) mutation in the MHC class I restricted T-cell epitope. PreS1/S2 deletion was detected in 3 isolates with all harboring the BCP double mutation. Furthermore, the frequently occurring mutations in the major hydrophilic loop of the S gene include the T125M, A128V and M133I/L. Therefore, this study is the first from India to report useful information on the molecular heterogeneity of the HBV strains circulating among the treatment naive HIV/HBV co-infected population and is thus clinically relevant.