Moytrey Chatterjee

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.

Prevalence of Polymorphisms in Antifolate Drug Resistance Molecular Marker Genes pvdhfr and pvdhps in Clinical Isolates of Plasmodium vivax from Kolkata, India

ABSTRACT Sulfadoxine-pyrimethamine has never been recommended for the treatment of Plasmodium vivax malaria as the parasite is intrinsically resistant to pyrimethamine. The combination was introduced as a promising agent to treat Plasmodium falciparum malaria in many countries but was withdrawn after a few years due to development and spread of resistant strains. Presently, sulfadoxine-pyrimethamine is used as a partner drug of artemisinin-based combination therapy to treat uncomplicated falciparum malaria, and a combination of artesunate-sulfadoxine-pyrimethamine is currently in use in India. In countries like India, where both P. vivax and P. falciparum are equally prevalent, some proportion of P. vivax bacteria is exposed to sulfadoxine-pyrimethamine due to misdiagnosis and mixed infections. As reports on the in vivo therapeutic efficacy of sulfadoxine-pyrimethamine in P. vivax are rare, the study of mutations in the marker genes P. vivax dhfr (pvdhfr) and pvdhps is important for predicting drug selection pressure and sulfadoxine-pyrimethamine resistance monitoring. We studied the prevalence of point mutations and haplotypes of both the genes in 80 P. vivax isolates collected from urban Kolkata, India, by the DNA sequencing method. Point mutation rates in both the genes were low. The double mutant pvdhfr A15N50R58N117I173 (mutations are in boldface) and the single mutant pvdhps genotype S382G383K512A553V585 were more prevalent, while 35% of the isolates harbored the wild-type genotype. The triple mutant ANRNI-SGKAV was found in 29.9% isolates. No quintuple mutant genotype was recorded. The P. vivax parasites in urban Kolkata may still be susceptible to sulfadoxine-pyrimethamine. Hence, a combination of antimalarial drugs like artesunate-sulfadoxine-pyrimethamine introduced for P. falciparum infection might be effective in P. vivax infection also. Study of the therapeutic efficacy of this combination in P. vivax is thus strongly suggested. (The study protocol was registered in the Clinical Trial Registry-India [CTRI] of the Indian Council of Medical Research under registration number CTRI/2011/09/002031.)

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.

Polymorphisms in voltage-gated sodium channel gene and susceptibility of Aedes albopictus to insecticides in three districts of northern West Bengal, India

Background The control and prevention of dengue largely depends on vector control measures, environmental management, and personal protection. Dengue control programmes are facing great challenges due to development of insecticide resistance among vector mosquitoes. Information on susceptibility status to different insecticides is important for national programmes to formulate vector control strategies. Methods We have studied the larval susceptibility of Aedes albopictus to temephos and adult susceptibility to 4% DDT, 0.05% deltamethrin, and 5% malathion as per WHO protocols in the northern districts of West Bengal. Polymorphisms in the VGSC gene were studied by direct sequencing of PCR products. Results The Ae. albopictus larval population showed sensitive [Resistance Ratio (RR99)<3] to moderate levels of resistance (510) to temephos at different study sites. Adult bioassay results revealed that Ae. albopictus was highly resistant to DDT [Corrected Mortality (CM) < 90%] in all the study sites and susceptible to deltamethrin and malathion (CM > 98%), except in Dhupguri where a low level of resistance to deltamethrin (CM = 96.25%) was recorded. None of the six important kdr mutations (S953P, I975M/V, L978, V980G, F1474C, D1703Y) were found in the VGSC of studied mosquitoes, but we identified 11 synonymous and 1 non-synonymous mutation in the VGSC gene. Conclusion The higher susceptibility level to deltamethrin and malathion, along with the absence of important kdr mutations indicates that these two insecticides are still effective against Ae. albopictus in the study areas. The susceptibility status of temephos should be monitored closely as low to moderate levels of resistance were observed in few sites. A similar study is recommended for monitoring and early detection of insecticide resistance in other parts of the country.

Polymorphisms in Pfcrt and Pfmdr-1 genes after five years withdrawal of chloroquine for the treatment of Plasmodium falciparum malaria in West Bengal, India.

BACKGROUND The emergence of resistant power against different antimalarial agents particularly by Plasmodium falciparum is a challenge to combat malaria. Regular monitoring is essential not only to determine the efficacy and development of resistance by the parasite but also to detect early sign of regaining sensitivity to any anti-malarial agent that has been withdrawn for a long period. Studies on molecular markers associated with antimalarial drug resistance of prevailing Plasmodium population play an important role in this aspect. The present protocol was designed to study the polymorphisms in pfcrt and pfmdr-1 gene to determine any sign of regaining sensitivity to chloroquine among P. falciparum after five years of artemisinin combination therapy (ACT) implementation. METHODS Clinical isolates were collected from P. falciparum positive patients attending the malaria clinic of Calcutta School of Tropical Medicine during December 2014 to December 2015. Genomic parasitic DNA was extracted and subjected to sequencing of pfcrt and pfmdr-1 gene directly from purified PCR products. RESULTS A total of 89 isolates were sequenced for pfcrt and 73 isolates for pfmdr-1 genes. In pfcrt gene mutant K76T was detected in all isolates and all were SVMNT haplotype. Out of three important polymorphisms in pfmdr-1 gene mutant Y184F was detected among all isolates. One synonymous G182G and one non-synonymous S232F/Y, mutation were detected in 99% isolates. CONCLUSION All isolates carrying mutant K76T in pfcrt gene, considered as hall mark for CQ resistance, indicate that there is no sign of regaining CQ sensitivity among the prevailing P. falciparum population of the study area after five years of ACT implementation.

Insecticide susceptibility status of Phlebotomus argentipes and polymorphisms in voltage-gated sodium channel (vgsc) gene in Kala-azar endemic areas of West Bengal, India

Rational use of insecticides, as advocated by World Health Organisation, plays a crucial role for vector control in eliminating visceral leishmaniasis from endemic countries. Emergence and spread of resistance among vector sand flies is of increasing concern for achieving these goals. Information on insecticide susceptibility status of sand fly populations and potential association between the former and polymorphisms in the insecticide target genes is important for formulating proper vector control measures. The present study was designed to evaluate the susceptibility status of vector sand fly species (Phlebotomus argentipes) against deltamethrin (type II pyrethroid), DDT (organochlorine) and malathion (organophosphate) and to detect polymorphisms in voltage gated sodium channel (vgsc) gene and investigating their association with type II pyrethroid and DDT susceptibility in three Kala-azar endemic districts of West Bengal, India. Adult sand flies were collected from human dwelling and cattle sheds of the study areas and subjected to insecticide bioassay using insecticide impregnated papers as per WHO protocol. Polymorphisms in domain II segment 6 of vgsc gene of pyrethroid and DDT susceptible and tolerant P. argentipes were detected by DNA sequencing. P. argentipes population of the study area was found to be susceptible to deltamethrin and malathion with corrected mortality rate between 98.02% to 98.80% and 98.81% to 100% respectively, but resistant to DDT (corrected mortality rate = 65.62%-76.33%). Two non-synonymous mutations L1014S and L1014F were detected of which L1014F was found to be associated with deltamethrin/DDT resistance. The replacement of DDT by synthetic pyrethroid is aptly done by national vector borne disease control programme (NVBDCP). The prevalence of L1014F mutation in vgsc gene and its association with type II pyrethroid tolerability is an indication of emergence of resistance against it. Malathion may be used as an alternative in the study areas if needed in future. Similar studies at a regular interval are highly suggested for monitoring susceptibility of used insecticide and to detect early signs of emergence of resistance against them.

Susceptibility status of Japanese Encephalitis vectors to insecticides in endemic areas of northern districts of West Bengal, India

Emergence and spread of resistance among vectors toward different insecticides is a serious problem for the Japanese encephalitis (JE) control program. Regularly monitoring the status of susceptibility of vector species to insecticides is important for formulating proper vector control measures. In this study, we studied the susceptibility status of major JE vectors from northern West Bengal, toward 4% DDT, 0.05% deltamethrin, and 5% malathion. Two- to three-day-old unfed female mosquitoes were subjected to a susceptibility bioassay using a World Health Organization kit. Corrected mortality (CM) and knockdown times were estimated. Culex tritaeniorhynchus, Cx. vishnui, Cx. pseudovishnui, and Cx. gelidus were the major JE vectors present in the study areas. All 4 vector species were highly tolerant to DDT with CM ＜ 90%. Cx. tritaeniorhynchus, Cx. vishnui, Cx. pseudovishnui, and Cx. gelidus were tolerant to deltamethrin with CM ＜ 90%, except for Cx. gelidus of Darjeeling and Malbazar. At most of the study sites, malathion was effective against Cx. vishnui, Cx. pseudovishnui, and Cx. gelidus with CM ≥ 98%. In contrast, Cx. tritaeniorhynchus was tolerant to malathion in all study areas. Predominant JE vector populations were highly tolerant to all 3 analyzed insecticides, except deltamethrin for Cx. gelidus and malathion for Cx. vishnui, Cx. pseudovishnui, and Cx. gelidus. The results of this study may be useful for better planning and implementing a JE control strategy.

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.