Study of Hepatic Dysfunction Associated with Dengue Epidemiology in a Tertiary Care Hospital, Kolkata.

Dengue is a common arthropod-borne life-threatening febrile illness. This disease affects liver functions with an imbalance of liver enzymes followed by other clinical manifestations. The dengue serotypes can cause asymptomatic infection to more severe versions of hemorrhagic fever and dengue shock syndrome in West Bengal and around the globe. The main aim of this study is to establish how different liver enzymes act in identifying markers for dengue prognosis for the early detection of severe dengue fever (DF). The diagnosis of dengue patients was confirmed by enzyme-linked immunosorbent assay, and associated clinical parameters [aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase, total bilirubin, total albumin, total protein, packed cell volume, and platelet count] were analyzed. Furthermore, the viral load estimation was also carried out by RT PCR analysis. The majority of these patients had elevated AST and ALT levels; ALT levels were higher than AST levels, which were partially observed in all non-structural protein 1 antigen- and dengue immunoglobulin M antibody-reactive patients. Almost 25% of patients had very low platelet count or thrombocytopenia. Furthermore, the viral load shows a significant association with all the clinical parameters with a p-value of <0.0001. All these liver enzymes are significantly correlated with an increased level of T.BIL, ALT, and AST. This study depicts that the intensity of hepatic involvement may play a critical role in the morbidity and mortality of DF patients. As a result, all of these liver parameters can be useful early markers for determining the severity of the disease, allowing for early detection of high-risk cases.

Contrasting Distribution of SARS-CoV-2 Lineages across Multiple Rounds of Pandemic Waves in West Bengal, the Gateway of East and North-East States of India.

The evolution of viral variants and their impact on viral transmission have been an area of considerable importance in this pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We analyzed the viral variants in different phases of the pandemic in West Bengal, a state in India that is important geographically, and compared the variants with other states like Delhi, Maharashtra, and Karnataka, located in other regions of the country. We have identified 57 pango-lineages in 3,198 SARS-CoV-2 genomes, alteration in their distribution, as well as contrasting profiles of amino acid mutational dynamics across different waves in different states. The evolving characteristics of Delta (B.1.617.2) sublineages and alterations in hydrophobicity profiles of the viral proteins caused by these mutations were also studied. Additionally, implications of predictive host miRNA binding/unbinding to emerging spike or nucleocapsid mutations were highlighted. Our results throw considerable light on interesting aspects of the viral genomic variation and provide valuable information for improved understanding of wave-defining mutations in unfolding the pandemic. IMPORTANCE Multiple waves of infection were observed in many states in India during the coronavirus disease 2019 (COVID19) pandemic. Fine-scale evolution of major SARS-CoV-2 lineages and sublineages during four wave-window categories: Pre-Wave 1, Wave 1, Pre-Wave 2, and Wave 2 in four major states of India: Delhi (North), Maharashtra (West), Karnataka (South), and West Bengal (East) was studied using large-scale virus genome sequencing data. Our comprehensive analysis reveals contrasting molecular profiles of the wave-defining mutations and their implications in host miRNA binding/unbinding of the lineages in the major states of India.

The Status of Susceptibility 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.

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 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.

Genetic diversity and multiplicity of infection of Plasmodium falciparum isolates from Kolkata, West Bengal, India.

The study of genetic diversity of Plasmodium falciparum is necessary to understand the distribution and dynamics of parasite populations. The genetic diversity of P. falciparum merozoite surface protein-1 and 2 has been extensively studied from different parts of world. However, limited data are available from India. This study was aimed to determine the genetic diversity and multiplicity of infection (MOI) of P. falciparum population in Kolkata, West Bengal, India. A total of 80day-zero blood samples from Kolkata were collected during a therapeutic efficacy study in 2008-2009. DNA was extracted; allelic frequency and diversity were investigated by PCR-genotyping method for msp1 and msp2 gene and fragment sizing was done by Bio-Rad Gel-Doc system using Image Lab (version 4.1) software. P. falciparum msp1 and msp2 markers were highly polymorphic with low allele frequencies. In Kolkata, 27 msp1 different genotypes (including 11of K1, 6 of MAD20 and 10 of Ro33 allelic families) and 30 different msp2 genotypes (of which 17 and 13 belonged to the FC27 and 3D7 allelic families, respectively) were recorded. The majority of these genotypes occurred at a frequency below 10%. The mean MOI for msp1 and msp2 gene were 2.05 and 3.72, respectively. The P. falciparum population of Kolkata was genetically diverse. As the frequencies of most of the msp1 and msp2 alleles were low, the probability of new infection with genotype identical to that in pretreatment infection was very rare. This information will serve as baseline data for evaluation of malaria control interventions as well as for monitoring the parasite population structure.

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.

Impact of Artemisinin-Based Combination Therapy on Falciparum Malaria in Urban Kolkata: A Clinic-Based Report.

In India, artemisinin-based combination therapy (ACT; specifically artesunate + sulfadoxine-pyrimethamine) has been implemented for uncomplicated falciparum malaria since 2010. But for vivax malaria drug policy remained unchanged i.e., chloroqine and primaquine. We observed the impact of this intervention in urban Kolkata by analyzing data from the Malaria Clinic from 2001 to 2013. In Kolkata, we observed that Plasmodium vivax was perennial, whereas P. falciparum infection was seasonal. Before ACT implementation, the proportion of P. falciparum was as high as 50% and it steadily decreased during 4 successive years post intervention. No change was observed in the number of P. vivax cases. ACT may be an effective measure in reducing falciparum malaria cases. Artemisinin-derivative combination therapies should be explored in vivax malaria to reduce the overall burden of malaria.

Prevalence of polymorphisms in antifolate drug resistance molecular marker genes pvdhfr and pvdhps in clinical isolates of Plasmodium vivax from Kolkata, India.

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.).

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.