Polymorphisms in pfdhfr and pfdhps genes after five years of artemisinin combination therapy (ACT) implementation from urban Kolkata, India.

BACKGROUND: In India, sulphadoxine-pyrimethamine (SP) is now in use as a partner drug of ACT (AS+SP) to treat uncomplicated falciparum malaria since 2010. Declined trend of AS+SP efficacy has been reported from north-eastern states of the country. It is not possible to determine the efficacy of SP alone from any study with ACT. So, this work was designed to study the pattern of polymorphisms in pfdhfr and pfdhps genes to predict the SP resistance status among parasite population of urban Kolkata after five years of ACT implementation. METHODS: A total of 125 P. falciparum positive patients were enrolled in the study during December 2014 to July 2016 and treated with AS+SP. Parasitic DNA was isolated and subjected to sequencing of pfdhfr and pfdhps genes directly from purified PCR products. RESULTS: Genotyping of both the genes was successfully done in 113 isolates. In pfdhfr, 94.69% (107/113) isolates showed mutations at codon 59 and 108. A double mutant genotype ANRNI was mostly prevalent (107/113, 94.69%), while wild-type genotype ANCSI was found only in 5.3% (6/113) isolates. In pfdhps, mutations were recorded at codon 436 and 437 in 65.49% (74/113) and 23.01% (26/113) isolates, respectively. In combined pfdhfr-pfdhps genes, triple mutant ANRNI-FAKAA was most prevalent (45/113, 39.82%) followed by double mutant ANRNI-SAKAA (37/113, 32.74%) and quadruple mutant ANRNI-FGKAA (24/113, 21.24%). CONCLUSION: SP resistance hallmark mutations i.e., quadruple (AIRNI-SAEAA) or quintuple (AIRNI-SGEAA) genotype in pfdhfr and pfdhps was absent which indicates that SP components of used ACT is still effective in the study area. It is also evident by the clinical response of AS+SP. Monitoring the efficacy of this combination (both by therapeutic and molecular marker study) at a regular interval is highly suggested to record any development of SP resistance in near future.

Asymptomatic leishmaniasis in kala-azar endemic areas of Malda district, West Bengal, India.

Asymptomatic leishmaniasis may drive the epidemic and an important challenge to reach the goal of joint Visceral Leishmaniasis (VL) elimination initiative taken by three Asian countries. The role of these asymptomatic carriers in disease transmission, prognosis at individual level and rate of transformation to symptomatic VL/Post Kala-azar Dermal Leishmaniasis (PKDL) needs to be evaluated. Asymptomatic cases were diagnosed by active mass survey in eight tribal villages by detecting antileishmanial antibody using rK39 based rapid diagnostic kits and followed up for three years to observe the pattern of sero-conversion and disease transformation. Out of 2890 total population, 2603 were screened. Antileishmanial antibody was detected in 185 individuals of them 96 had a history of VL/PKDL and 89 without such history. Seventy nine such individuals were classified as asymptomatic leishmaniasis and ten as active VL with a ratio of 7.9:1. Out of 79 asymptomatic cases 2 were lost to follow up as they moved to other places. Amongst asymptomatically infected persons, disease transformation in 8/77 (10.39%) and sero-conversion in 62/77 (80.52%) cases were noted. Seven (9.09%) remained sero-positive even after three years. Progression to clinical disease among asymptomatic individuals was taking place at any time up to three years after the baseline survey. If there are no VL /PKDL cases for two or more years, it does not mean that the area is free from leishmaniasis as symptomatic VL or PKDL may appear even after three years, if there are such asymptomatic cases. So, asymptomatic infected individuals need much attention for VL elimination programme that has been initiated by three adjoining endemic countries.

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.

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.

Therapeutic efficacy of artemisinin combination therapies and prevalence of S769N mutation in PfATPase6 gene of Plasmodium falciparum in Kolkata, India.

OBJECTIVE: To study the in vivo efficacy of these two ACTs in the treatment of Plasmodium falciparum (P. falciparum malaria) in Kolkata and to determine the prevalence of mutant S769N codon of the PfATPase6 gene among field isolates of P. falciparum collected from the study area. METHODS: A total of 207P. falciparum positive cases were enrolled randomly in two study arms and followed up for 42 days as per WHO (2009) protocol. A portion of PfATPase6 gene spanning codon S769N was amplified and sequenced by direct sequencing method. RESULTS: It was observed that the efficacy of both the ACT regimens were highly effective in the study area and no mutant S769N was detected from any isolate. CONCLUSIONS: The used, combination AS+SP is effective and the other combination AM+LF might be an alternative, if needed.

High prevalence of asymptomatic malaria in a tribal population in eastern India.

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 A(16)I(51)R(59)N(108)I(164)-S(436)G(437)E(540)A(581)A(613) 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.

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.

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.).

Efficacy of oral miltefosine in visceral leishmaniasis in rural West Bengal, India.

CONTEXT: Visceral leishmaniasis (VL), also known as Kala-azar (KA) is a public health problem of tropical and subtropical countries, which infects about 12 million people annually, out of which about 1.5 million are new cases. India contributes a major share of the global burden of VL. For many years leishmaniasis has been treated with pentavalent antimonials. Antimony resistance is a problem in India and in other different geographic areas of the world. Amphotericin B deoxycholate and pentamidine isethionate are effective by parenteral administration and associated with toxicities. The quest for an effective, orally administered, non-toxic and less expensive alternative resulted in the identification of miltefosine (hexadecylphosphocholine). In India, therapeutic efficacy of miltefosine in VL was assessed by many groups of scientists, mainly from Bihar and Uttar Pradesh. No such data is available from West Bengal. AIMS: The present study was designed to observe the efficacy of miltefosine in VL in rural West Bengal. MATERIALS AND METHODS: A total of 71 parasitologically proven VL patients participated in the study who received miltefosine in accordance with the National Vector Born Disease Control Programme (NVBDCP) of India and were followed up for the following one year. RESULTS: The overall efficacy of the drug was 93% and no significant adverse side effects were observed during the study period. CONCLUSIONS: The study concludes that miltefosine is effective, well tolerated, and easily administrable drug in the treatment of visceral leishmaniasis at the field levels.

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.

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, I(51)R(59)N(108), with pfdhps, G(437) and/or E(540). The efficacy of this ACT needs periodic monitoring. Artemether-lumefantrine and artesunate-mefloquine are effective alternatives to the artesunate-sulfadoxine-pyrimethamine combination.

Symmetrical peripheral gangrene: a rare complication of dengue fever.

Symmetric peripheral gangrene is associated with a variety of infective and non-infective etiologies. SPG is always presented with disseminated intravascular coagulation (DIC) and carries a higher mortality. Herein, we describe a 42-year-old female with dengue fever and rash developed bilateral symmetric dry gangrene of 2 nd and 3 rd toes. There was no history of taking B-blockers, ergot etc. All the peripheral pulses of the affected limbs were palpable. Color Doppler of lower limb vessels was done, which indicated normal flow. Blood was positive for Fibrin degradation products and D dimers. Patient was managed with IV fluids, LMWH, FFP etc. Her general condition improved within 72 hours with no further progression of gangrene.

Efficacy of chloroquine and sulphadoxine-pyrimethamine either alone or in combination before introduction of ACT as first-line therapy in uncomplicated Plasmodium falciparum malaria in Jalpaiguri District, West Bengal, India.

OBJECTIVE: In India, till recently, Chloroquine was used as first-line therapy in areas with Chloroquine sensitive Plasmodium falciparum malaria cases. The National Vector Borne Disease Control Programme (NVBDCP) has introduced artemisinin combination therapy (ACT) as first-line option to treat all P. falciparum cases in the country. This study was carried out to ascertain the efficacy of Chloroquine and Sulphadoxine-Pyrimethamine, either alone or in combination, before the launch of ACT by NVBDCP. METHODS: A total of 300 P. falciparum malaria cases were enrolled randomly in three study arms, Chloroquine (CQ), Sulphadoxine-Pyrimethamine (SP) and Chloroquine plus Sulphadoxine-Pyrimethamine (CQ + SP). All patients were followed up for 28 days as per WHO (Assessment and Monitoring of Antimalarial Drug Efficacy for the Treatment of Uncomplicated Falciparum Malaria, Geneva, 2003) Protocol. Paired blood samples of treatment failure cases were collected and subjected to MSP 1, MSP 2 and GLURP genotyping for differentiation between re-infection and recrudescence. The data were analysed by Kaplan-Meier survival curve according to WHO standard procedures. RESULTS: The overall failure rate including both early treatment failure (ETF) and late treatment failure (LTF) of CQ, SP and CQ + SP were 61%, 14% and 8%, respectively, in the study area. Of 60 recurrent malaria cases, genotyping was successful in 49 cases, revealing that most of the (46/49; 94%) cases of recurrent malaria were due to recrudescence. CONCLUSION: In Jalpaiguri District the overall failure rate of CQ was 61% and of SP 14%, which was well above the WHO recommended cut-off threshold level (10%) for change of drug policy.

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.