Ardhendu Kumar Maji

Genetic diversity of Plasmodium vivax in Kolkata, India

BackgroundPlasmodium vivax malaria accounts for approximately 60% of malaria cases in Kolkata, India. There has been limited information on the genotypic polymorphism of P. vivax in this malaria endemic area. Three highly polymorphic and single copy genes were selected for a study of genetic diversity in Kolkata strains.MethodsBlood from 151 patients with P. vivax infection diagnosed in Kolkata between April 2003 and September 2004 was genotyped at three polymorphic loci: the P. vivax circumsporozoite protein (pvcs), the merozoite surface protein 1 (pvmsp 1) and the merozoite surface protein 3-alpha (pvmsp 3-alpha).ResultsAnalysis of these three genetic markers revealed that P. vivax populations in Kolkata are highly diverse. A large number of distinguishable alleles were found from three genetic markers: 11 for pvcs, 35 for pvmsp 1 and 37 for pvmsp 3-alpha. These were, in general, randomly distributed amongst the isolates. Among the 151 isolates, 142 unique genotypes were detected the commonest genotype at a frequency of less than 2% (3/151). The overall rate of mixed genotype infections was 10.6%.ConclusionThese results indicate that the P. vivax parasite population is highly diverse in Kolkata, despite the low level of transmission. The genotyping protocols used in this study may be useful for differentiating re-infection from relapse and recrudescence in studies assessing of malarial drug efficacy in vivax malaria.

Genotyping of Plasmodium vivax Reveals Both Short and Long Latency Relapse Patterns in Kolkata

Background The Plasmodium vivax that was once prevalent in temperate climatic zones typically had an interval between primary infection and first relapse of 7–10 months, whereas in tropical areas P.vivax infections relapse frequently at intervals of 3–6 weeks. Defining the epidemiology of these two phenotypes from temporal patterns of illness in endemic areas is difficult or impossible, particularly if they overlap. Methods A prospective open label comparison of chloroquine (CQ) alone versus CQ plus unobserved primaquine for either 5 days or 14 days was conducted in patients presenting with acute vivax malaria in Kolkata. Patients were followed for 15 months and primary and recurrent infections were genotyped using three polymorphic antigen and up to 8 microsatellite markers. Results 151 patients were enrolled of whom 47 (31%) had subsequent recurrent infections. Recurrence proportions were similar in the three treatment groups. Parasite genotyping revealed discrete temporal patterns of recurrence allowing differentiation of probable relapse from newly acquired infections. This suggested that 32 of the 47 recurrences were probable relapses of which 22 (69%) were genetically homologous. The majority (81%) of probable relapses occurred within three months (16 homologous, 10 heterologous) and six genetically homologous relapses (19%) were of the long latency (8–10 month interval) phenotype. Conclusions With long follow-up to assess temporal patterns of vivax malaria recurrence, genotyping of P.vivax can be used to assess relapse rates. A 14 day unobserved course of primaquine did not prevent relapse. Genotyping indicates that long latency P.vivax is prevalent in West Bengal, and that the first relapses after long latent periods are genetically homologous. Trial Registration Controlled-Trials.com ISRCTN14027467

Genetic association of Toll-like-receptor 4 and tumor necrosis factor-α polymorphisms with Plasmodium falciparum blood infection levels

Dysregulated innate immune responses due to inappropriate signaling by Toll-like receptors (TLRs) and aberrant production of pro-inflammatory cytokines are implicated in the immunopathology and disease outcome in Plasmodium falciparum malaria. This study investigates the relationship between polymorphic variability of candidate genes including TLR-2, -4, -9, tumor necrosis factor-alpha and lymphotoxin-alpha and blood infection level in Indian mild malaria patients. Genotyping was carried out by PCR-RFLP and sequencing. Association of parasite load with genotypes was examined using model based and model free approaches. Allele and haplotype based risk assessment for disease severity was performed by stratifying the patients into high and low parasitemic groups on the basis of a threshold value derived by employing a two-component mixture model and expectation-maximization algorithm. The mean parasitemia was significantly increased for variant homozygous genotype (C/C) at TNF-alpha promoter -1031 and major homozygous genotypes encoding Asp/Asp and Thr/Thr at codons 299 and 399, respectively, on TLR4 polypeptide. Individuals harboring combined genotype C/C-Asp/Asp-Thr/Thr on TNF-alpha and TLR4 presented the highest parasite load. The frequencies of variant allele C in TNF-1031 (OR=1.91 with 95% CI=1.24-2.94) and TNF-alpha promoter haplotypes C-C-G-G (OR=1.99 with 95% CI=1.21-3.27) and C-C-G-A (OR=2.96 with 95% CI=1.19-7.37) pertaining to loci TNF-1031/-857/-308/-238 were significantly elevated in the high parasitemic group. On the contrary, the frequencies of variant allele encoding Ile at 399 (OR=0.55 with 95% CI=0.32-0.94) and haplotype corresponding to Gly-Ile (299-399) (OR=0.51 with 95% CI=0.28-0.9) in TLR4 were higher in low parasitemic group. In silico analysis indicate differential binding of transcription factors to TNF-alpha promoter haplotypes and alteration in the surface charge distribution of the TLR4 variant proteins. Our results support a genetic role of TLR4 and TNF-alpha in controlling the blood infection level in mild malaria.

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.

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.)

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).

PKDL—A Silent Parasite Pool for Transmission of Leishmaniasis in Kala-azar Endemic Areas of Malda District, West Bengal, India

Post Kala-azar Dermal Leishmaniasis (PKDL) is a chronic but not life-threatening disease; patients generally do not demand treatment, deserve much more attention because PKDL is highly relevant in the context of Visceral Leishmaniasis (VL) elimination. There is no standard guideline for diagnosis and treatment for PKDL. A species-specific PCR on slit skin smear demonstrated a sensitivity of 93.8%, but it has not been applied for routine diagnostic purpose. The study was conducted to determine the actual disease burden in an endemic area of Malda district, West Bengal, comparison of the three diagnostic tools for PKDL case detection and pattern of lesion regression after treatment. The prevalence of PKDL was determined by active surveillance and confirmed by PCR based diagnosis. Patients were treated with either sodium stibogluconate (SSG) or oral miltefosine and followed up for two years to observe lesion regression period. Twenty six PKDL cases were detected with a prevalence rate of 27.5% among the antileishmanial antibody positive cases. Among three diagnostic methods used, PCR is highly sensitive (88.46%) for case confirmation. In majority of the cases skin lesions persisted after treatment completion which gradually disappeared during 6–12 months post treatment period. Reappearance of lesions noted in two cases after 1.5 years of miltefosine treatment. A significant number of PKDL patients would remain undiagnosed without active mass surveys. Such surveys are required in other endemic areas to attain the ultimate goal of eliminating Kala-azar. PCR-based method is helpful in confirming diagnosis of PKDL, referral laboratory at district or state level can achieve it. So a well-designed study with higher number of samples is essential to establish when/whether PKDL patients are free from parasite after treatment and to determine which PKDL patients need treatment for longer period.

No Polymorphism in Plasmodium falciparum K13 Propeller Gene in Clinical Isolates from Kolkata, India

Molecular markers associated with artemisinin resistance in Plasmodium falciparum are yet to be well defined. Recent studies showed that polymorphisms in K13 gene are associated with artemisinin resistance. The present study was designed to know the pattern of polymorphisms in propeller region of K13 gene among the clinical isolates collected from urban Kolkata after five years of ACT implementation. We collected 59 clinical isolates from urban Kolkata and sequenced propeller region of K13 gene in 51 isolates successfully. We did not find any mutation in any isolate. All patients responded to the ACT, a combination of artesunate + sulphadoxine-pyrimethamine. The drug regimen is still effective in the study area and there is no sign of emergence of resistance against artemisinin as evidenced by wild genotype of K13 gene in all isolates studied.