Santanu K. Kar

Epidemics of severe cholera caused by El Tor Vibrio cholerae O1 Ogawa possessing the ctxB gene of the classical biotype in Orissa, India

BACKGROUND We investigated the epidemic of cholera that occurred in Kashipur and Dasmantpur blocks of Orissa, reported during July-September 2007. METHODS Sixty-two rectal swabs and 28 water samples collected from diarrhea patients at different hospitals and villages were bacteriologically analyzed for the identification, antibiogram, and detection of toxic genes of Vibrio cholerae. RESULTS The cholera outbreaks were caused by V. cholerae O1 Ogawa biotype El Tor in both Kashipur and Dasmantpur blocks. All the V. cholerae isolates from the clinical and environmental samples were sensitive to tetracycline, gentamicin, azithromycin, and chloramphenicol, but were resistant to ampicillin, ciprofloxacin, norfloxacin, co-trimoxazole, nalidixic acid, neomycin, and furazolidone, except the water isolates, which were sensitive to ciprofloxacin and norfloxacin. The multiplex PCR assay revealed that all the clinical and environmental V. cholerae isolates were positive for the ctxA and tcpA genes, showing biotype El Tor. Interestingly, 88% of the clinical and environmental isolates of V. cholerae were El Tor biotype with mutation at the ctxB gene of the classical strain, as confirmed by mismatch amplification of mutation (MAMA)-PCR assay. CONCLUSIONS This is the first report of the El Tor variant of V. cholerae O1 Ogawa having the ctxB gene of the classical strain with altered antibiogram causing epidemics of cholera in Orissa, India.

Molecular investigations of dengue virus during outbreaks in Orissa state, Eastern India from 2010 to 2011

Dengue is one of the most important arboviral diseases in India. Orissa state in Eastern India reported the first dengue outbreak in 2010, followed by extensive outbreaks in 2011, affecting large number of people. Detailed entomological, serological and phylogenetic investigations were performed in mosquitoes and patients serum collected from dengue virus (DENV) affected areas of Orissa. The combination of DENV specific IgM capture-ELISA and reverse-transcription PCR (RT-PCR) detected high DENV positivity in serum samples. DENV was detected in mosquitoes reared from field caught pupae by RT-PCR, which confirmed the vertical transmission of DENV that may have an important role in the recurrence of dengue outbreaks. Phylogenetic analyses revealed the circulation of Indian lineage of DENV-2 (genotype-IV) and DENV-3 (genotype-III) in vectors and patients serum in Orissa from 2010 to 2011, DENV-2 being the prevailing serotype. Selection analyses within the C-prM region showed that the emergence of DENV-2 and DENV-3 in Orissa was constrained by purifying selection which suggested the role of ecological factors like mosquito density and behavior in the recurrent outbreaks. Aedes albopictus was found to be the most abundant vector in the areas surveyed, followed by Aedes aegypti. Indoor breeding spots (earthen pots) were most abundant, with high pupal productivity (38.50) and contributed maximum Aedes species in the affected areas. The DENV infection rate estimated by maximum likelihood estimate (MLE) was high for indoor breeding Aedes (4.87; 95% CI: 1.82, 10.78) in comparison to outdoor breeding Aedes (1.55; 95% CI: 0.09, 7.55). The high MLE in Ae. albopictus (4.72; 95% CI: 1.94, 9.80) in comparison to Ae. aegypti (1.55; 95% CI: 0.09, 7.54) indicated that Ae. albopictus was the main DENV vector responsible for the outbreaks. The results indicated the circulation of two virulent serotypes of DENV in Orissa, mainly by Ae. albopictus with the implication for implementation of intradomecile vector control measures to prevent the spread of dengue.

Molecular investigations of chikungunya virus during outbreaks in Orissa, Eastern India in 2010

Chikungunya virus (CHIKV), an arthritogenic alphavirus, is transmitted to humans by mosquitoes of genus Aedes, mainly Aedes aegypti and Aedes albopictus. The resurgence of CHIKV in different parts of India is a point of major public health concern. In 2010, chikungunya outbreaks with high epidemic magnitude were recorded in coastal areas of Orissa, Eastern India, affecting more than 15,000 people coupled with severe arthralgia and prolonged morbidites. Detailed entomological, serological and molecular investigation of this unprecendented outbreak was carried out by collecting and studying 1359 mosquito samples belonging to A. albopictus, A. aegypti, A. vittatus, A. edwardsii and Culex species and 220 patients serum from the affected areas. In this study, CHIKV specific IgM capture-ELISA and reverse-transcription PCR (RT-PCR) were done to detect recent infection of CHIKV in serum samples and adult mosquitoes collected from the affected areas. The high maximum likelihood estimate (MLE) (15.2) in A. albopictus mosquitoes indicated that it was the principal vector involved in transmission of CHIKV in Orissa. Phylogenetic analysis revealed that the CHIKV strains involved in the outbreak belonged to the Indian Ocean Lineage (IOL) group within the East, Central and South African (ECSA) genotype. Genetic characterization of envelope glycoprotein (E1 and E2) genes revealed that all the CHIKV isolates from Orissa had the E1-A226V mutation that enhances viral dissemination and transmissibility by A. albopictus mosquitoes along with E2-L210Q and E2-I211T mutations, which play an epistatic role with E1-A226V mutation in adaptation of CHIKV to A. albopictus by increasing its midgut infectivity, thereby favoring its vectorial capacity. Our results showed the involvement of A. albopictus vector in the recent outbreaks in Orissa and circulation of IOL strains of ECSA genotype of CHIKV with E1-A226V, E2-L210Q and E2-I211T mutations in vectors and patients serum.

Human Lymphatic Filariasis: Genetic Polymorphism of Endothelin-1 and Tumor Necrosis Factor Receptor II Correlates With Development of Chronic Disease

BACKGROUND Hydrocele and elephantiasis are 2 clinically very diverse and often mutually exclusive chronic manifestations of human bancroftian filariasis. Plasma levels of endothelin-1 (ET-1), a major angiogenic factor, and tumor necrosis factor receptors (TNFRs) that regulate host inflammation have been associated with development of chronic filariasis, although their genetic basis are not known. METHODS We studied polymorphisms of ET-1 (Ala288Ser) and TNFR-II (Met196Arg) genes by means of the polymerase chain reaction confronting 2 pairs primers method and restriction fragment length polymorphism, respectively. Plasma ET-1 level was measured by enzyme-linked immunosorbent assay. RESULTS Met196Arg genotype frequency of TNFR-II polymorphism was significantly greater in hydrocele patients, compared with elephantiasis patients (OR, 4.34 [95% CI, 2.04-9.20]). Conversely, a significantly high prevalence of the Ala288Ser mutation of ET-1 was observed in elephantiasis patients, compared with hydrocele cases (OR, 2.15 [95% CI, 1.13-4.10]). Decreased plasma ET-1 levels associated significantly with Ala288Ser mutation in the study population. A combined analysis indicated a 23-fold higher risk for developing elephantiasis in individuals with TNFR-II (Met196Met) and ET-1 mutants (Ala288Ser + Ser288Ser). CONCLUSIONS ET-1 (Ala288Ser) and TNFR-II (Met196Arg) polymorphisms are associated with development of one or the other form of chronic disease in bancroftian filariasis.

Analysis of the phylogenetic relationship of Anopheles species, subgenus Cellia (Diptera: Culicidae) and using it to define the relationship of morphologically similar species

Studies on the relationship of various vectors and non-vectors of malaria from the evolutionary point of view are important. Use of molecular methods to define phylogeny helps to understand the interrelationship among the members of the anophelines and elucidate the ambiguity that has arisen from improper classification. It could also help to design molecular markers for species differentiation, particularly in those which pose difficulty when classified, based on morphological features. In the present study, the phylogenetic relationships among the species of the anophelines of subgenus Cellia are inferred from the mitochondrial genes COI and COII, the ribosomal RNA gene, in particular the D3 region, and Internal Transcribed Spacer 2 (ITS2) region. The molecular phylogeny obtained in this work matches with that of the classical morphological taxonomy reasonably well, and was useful in properly defining species positions and resolving the ambiguity that normally arises due to morphological taxonomy. The correct arrangement of the various anopheline taxa as per the traditional morphological character-based classification of anophelines was there when we considered the D3 region of 28S rRNA gene and ITS2 region of rDNA. However, the arrangement of the taxa did not match with that of the morphological classification in some aspects, when we considered the COI and COII region of mitochondrial DNA. It may have been due to the variable degree of the rate of evolution of the different genes within the organism. Thus, a proper selection of those particular genes that evolve at the rate that is reflected at the species differentiation level, could help to construct the correct phylogenetic relationship among the anophelines and could be used to correlate with the grouping pattern done from the morphological perspective.

Detection of adult Brugia malayi filariae by ultrasonography in humans in India and Indonesia

In bancroftian filariasis, ultrasonography (USG) is a suitable tool to monitor infection by the detection of adult filariae in addition to antigen detection tests (ICT, Og4C3). However, in brugian filariasis, ultrasound examinations in humans have so far failed to detect adult worms and no antigen test is available to verify infections in patients who are carriers of adult worms but amicrofilaraemic. In this study, we describe the feasibility of detection of adult Brugia malayi filariae by USG. Worm nests were detected in 4 of 32 patients in India and Indonesia, located in the breast, the thigh, the calf and an inguinal lymph node. The study shows that adult filariae of B. malayi in humans can be detected by USG, but the technique is limited by the fact that worm nests seem not to be stable over time in humans, as is the case in bancroftian filariasis.

The development and evaluation of a single step multiplex PCR for simultaneous detection of Anopheles annularis group mosquitoes, human host preference and Plasmodium falciparum sporozoite presence

The Anopheles annularis group mosquitoes, subgenus Cellia Theobald (Diptera: Culicidae), includes five recognized species: An. annularis Van der Wulp, An. nivipes Theobald, An. pallidus Theobald, An. philippinensis Ludlow and An. schueffneri Stanton. From these five, the three most common species found in Orissa were considered for this study because of their remarkable vectorial and behavioral variation and the important role they play in malaria transmission. To identify and understand their role in malaria transmission we developed a single multiplex PCR-based assay. This assay included the detection of human blood feeding habit and Plasmodium falciparum sporozoite presence. Of the 186 An. annularis mosquitoes collected, morphological character-based identification showed that 94 were An. annularis, 54 were An. philippinensis and 38 were An. pallidus. However, the multiplex PCR assay confirmed that 91 were An. annularis, 56 were An. philippinensis and 39 were An. pallidus individuals after adjustments were made for misidentified specimens in the morphological method. Anopheles annularis and An. philippinensis were found positive for human blood, and two samples of An. annularis species were positive for P. falciparum sporozoites. This one-step PCR-based method constitutes a very powerful tool in large surveys of anopheline populations.

Development and evaluation of a single-step multiplex PCR to differentiate the aquatic stages of morphologically similar Aedes (subgenus: Stegomyia) species

Objective  To develop a single‐step multiplex PCR to differentiate the aquatic stages of Aedes aegypti, Aedes albopictus and Aedes vittatus collected from different breeding spots in arbovirus endemic/epidemic areas and to detect the most abundant species by the multiplex PCR.

A unique methodology for detecting the spread of chloroquine-resistant strains of Plasmodium falciparum, in previously unreported areas, by analyzing anophelines of malaria endemic zones of Orissa, India.

Generally, clinical data is referred to study drug-resistance patterns of Plasmodium falciparum in an area. This is only possible after a clear manifestation of drug-resistance parasites inside the human host, and thereafter detection by healthcare persons. The detection of spread of drug-resistant P. falciparum in a population, before any pathological symptoms detected in humans is possible by analyzing the anopheline vectors, transmitting malaria. In the present study we implemented a new strategy to detect the spread of chloroquine-resistant (CQR) strains of P. falciparum by the major malaria vectors prevalent in selected endemic regions of Orissa, India. We screened P. falciparum positive vectors by using polymerase chain reaction (PCR)-based assay and thereafter detected K76T mutation in the Pfcrt gene, the chloroquine-resistance marker, of parasites present within the vectors. This study showed higher transmission rate of chloroquine-resistant P. falciparum parasites by Anopheles culicifacies and Anopheles fluviatilis. This study will help in assigning chloroquine-resistant P. falciparum sporozoite transmission potential of malaria vectors and suggest that by adopting the mentioned methodologies, we can detect the spreading of the drug-resistant P. falciparum in its transmission. This approach of studying the anophelines during regular vector collection and epidemiological analysis will give the knowledge of chloroquine-resistance pattern of P. falciparum of an area and help in devising effective malaria control strategy.

Anopheles culicifacies sibling species in Odisha, eastern India: First appearance of Anopheles culicifacies E and its vectorial role in malaria transmission.

To identify the Anopheles culicifacies sibling species complex and study their vectorial role in malaria endemic regions of Odisha.