Diagnosis of Chikungunya dominated co-infection with dengue during an outbreak in south India (2010 and 2012).

Following a report of dengue outbreak from January 2010 to 2012 in the Tirunelveli, Theni, Dharmapuri and Thiruvallur districts of Tamil Nadu state, India, an investigation was carried out. The study was to demonstrate the probable presence of Chikungunya viral antibodies in patients clinically suspected of dengue fever. Out of 331 samples analysed, dengue viral antibodies were observed in 14.8% (n = 49) of patients, while 16.6% (n = 55) were positive for Chikungunya viral specific IgM antibodies. In the four districts surveyed, patients found positive for Chikungunya were found to be higher than dengue. The clinician should consider Chikungunya in the differential diagnosis of dengue-like infection appearing in the community.

Dengue NS1 and prM antibodies increase the sensitivity of acute dengue diagnosis test and differentiate from Japanese encephalitis infection.

Accurate and early diagnosis of dengue infection is essential for dengue case management. In outbreak conditions, it is essential to include two different tests to diagnose dengue and the choice depends on the number of days after the onset of illness in which the sample is collected. During the laboratory diagnosis of dengue in late acute and convalescent phase by MAC-ELISA, it is necessary to rule out possible cross reactions of closely related flavivirus, such as Japanese encephalitis virus which is commonly co-circulating. In the present investigation, the usefulness of dengue virus NS1 and prM antibodies in diagnosing and differentiating dengue from Japanese encephalitis infection was assessed using samples collected during out-breaks. It was shown here that, detection of antibodies against dengue NS1 and prM proteins increases the sensitivity of dengue diagnosis until 15days. Moreover, detection of antibodies against both proteins was able to differentiate dengue from Japanese encephalitis infection.

Description of a new species, Toxorhynchites (Toxorhynchites) tyagii (Diptera: Culicidae), from Nilgiri hills, Western Ghats, southern India.

Specimens of Toxorhynchites (Toxorhynchites) tyagii, sp. n., were collected from the fringe areas of Gudaloor town, Ooty in the Nilgiri hills at an altitude of 1000 m above sea level in Western Ghats ranges in southern India during October 2011 and from Darjeeling ranges in the northern hilly region of Raymatang TG in Jalpaiguri district, West Bengal, in eastern India during February 2012. The adults, pupa and fourth-instar larva of this species are described and illustrated to distinguish it from Toxorhynchites (Tox.) splendens and Toxorhynchites (Tox.) edwardsi, which are the closest allies of Tx. (Tox.) tyagii. Besides possessing remarkable distinguishing male genital characteristics, Tx. tyagii differs from Tx. splendens also by the presence of conspicuous yellow scale-patches over the wing root that extend to the scutellum, and differs from Tx. edwardsi by having midtarsomeres 3-5 all dark whereas in Tx. edwardsi tarsomeres 3 and 4 and a larger part of 5 are white.

Evaluation and use of NS1 IgM antibody detection for acute dengue virus diagnosis: report from an outbreak investigation.

The usefulness of detecting circulating non-structural protein 1 (NS1) IgM antibodies for diagnosing acute dengue virus infection was evaluated during an outbreak investigation along with other routinely used laboratory diagnostic methods. For the first time, the samples were also tested for NS1 antigen detection. NS1 IgM antibody detects all the serum samples that were positive for NS1 antigen detection within first 5 days of infection. The sensitivity of the NS1 IgM ELISA was higher when compared with RT-PCR and therefore, it could be used for early diagnosis.

Detection of Wolbachia endobacteria in Culex quinquefasciatus by Gimenez staining and confirmation by PCR.

BACKGROUND & OBJECTIVES: Wolbachia are common intracellular bacteria that are found in arthropods and nematodes. These endosymbionts are transmitted vertically through host eggs and alter host biology in diverse ways, including the induction of reproductive manipulations, such as feminization, parthenogenesis, male killing and sperm-egg incompatibility. Since they can also move horizontally across species boundaries, Wolbachia is gaining importance in recent days as it could be used as a biological control agent to control vector mosquitoes or for paratransgenic approaches. However, the study of Wolbachia requires sophisticated techniques such as PCR and cell culture facilities which cannot be affordable for many laboratories where the diseases transmitted by arthropod vectors are common. Hence, it would be beneficial to develop a simple method to detect the presence of Wolbachia in arthropods. METHOD: In this study, we described a method of staining Wolbachia endobacteria, present in the reproductive tissues of mosquitoes. The reliability of this method was compared with Gram staining and PCR based detection. RESULTS: The microscopic observation of the Gimenez stained smear prepared from the teased ovary of wild caught and Wolbachia (+) Cx. quinquefasciatus revealed the presence of pink coloured pleomorphic cells of Wolbachia ranging from cocci, comma shaped cells to bacillus and chain forms. The ovaries of Wolbachia (-) cured mosquito did not show any cell. Although Gram's staining is a reliable differential staining for the other bacteria, the bacterial cells in the smears from the ovaries of wild caught mosquitoes did not take the stain properly and the cells were not clearly visible. The PCR amplified product from the pooled remains of wild caught and Wolbachia (+) Cx. quinquefasciatus showed clear banding, whereas, no banding was observed for the negative control (distilled water) and Wolbachia (-) Cx. quinquefasciatus. INTERPRETATION & CONCLUSION: The Gimenez staining technique applied, could be used to detect the members of the endobacteria Wolbachia easily, even in a simple laboratory without any special facilities or even in the field condition and for handling large number of samples in a shorter duration.

Wolbachia endobacteria in a natural population of Culex quinquefasciatus from filariasis endemic villages of south India and its phylogenetic implication.

Understanding Wolbachia mosquito interactions have been recognized as an important concept to develop novel vector control strategies. The prevalence of Wolbachia endobacteria in a natural population of the filariasis vector Culex quinquefasciatus was determined by the polymerase chain reaction method. Earlier workers had estimated the infection rates of Wolbachia with only one or very few individuals per species. In our study large number of specimens were assayed, and a total of 750 adult Culex quinquefasciatus mosquitoes were collected from three south Indian villages of Tirukoilur and Mugaiyur blocks, monthly for a period of five months (December 2006 to April 2007) and screened for the presence of Wolbachia. The percentage prevalence in adult males ranged from 88% to 96%; while in females from 84% to 100%. An overall prevalence of 91.2% was observed. There was no significant difference observed in the proportion of mosquitoes positive for Wolbachia between males and females, and also between different months of the survey; except during the month of February '07. The wsp gene sequence of the Wolbachia strain of Cx. quinquefasciatus detected was BLAST analysed and showed 99% sequence similarity with Wolbachia sp. of Culex pipiens isolated from different geographical regions. Phylogenetic analysis based on wsp gene fragments showed that the present Wolbachia isolate was closely related with Wolbachia from Culex pipens pipiens, Niphotettix virescens (Order: Hemiptera) and Cnaphalocrosis medinalis (Order: Lepidoptera).

Wolbachia endobacteria in a natural population of Culex quinquefasciatus from filariasis endemic villages of south India and its phylogenetic implication

Understanding Wolbachia mosquito interactions have been recognized as an important concept to develop novel vector control strategies. The prevalence of Wolbachia endobacteria in a natural population of the filariasis vector Culex quinquefasciatus was determined by the polymerase chain reaction method. Earlier workers had estimated the infection rates of Wolbachia with only one or very few individuals per species. In our study large number of specimens were assayed, and a total of 750 adult Culex quinquefasciatus mosquitoes were collected from three south Indian villages of Tirukoilur and Mugaiyur blocks, monthly for a period of five months (December 2006 to April 2007) and screened for the presence of Wolbachia. The percentage prevalence in adult males ranged from 88% to 96%; while in females from 84% to 100%. An overall prevalence of 91.2% was observed. There was no significant difference observed in the proportion of mosquitoes positive for Wolbachia between males and females, and also between different months of the survey; except during the month of February ‘07. The wsp gene sequence of the Wolbachia strain of Cx. quinquefasciatus detected was BLAST analysed and showed 99% sequence similarity with Wolbachia sp. of Culex pipiens isolated from different geographical regions. Phylogenetic analysis based on wsp gene fragments showed that the present Wolbachia isolate was closely related with Wolbachia from Culex pipens pipiens, Niphotettix virescens (Order: Hemiptera) and Cnaphalocrosis medinalis (Order: Lepidoptera).

Molecular identification of mosquito vectors using genomic DNA isolated from eggshells, larval and pupal exuvium.

Correct and precise identification of mosquito vectors is important in many respects including development of vector control strategies. Conventional identification methods have limitations for sibling and closely related species of mosquitoes, stage and quality of the specimen used and this could be overcome by DNA-based identification methods using molecular markers such as nuclear ribosomal internal transcribed spacer (ITS) which do not demand intact or undamaged specimen. Genomic DNA is usually isolated from whole mosquito, legs, wings etc. Alternate sources for genomic DNA isolation such as eggshells, larval and pupal exuviae were explored in this study by amplifying the ITS markers. Standardization of genomic DNA extraction and ITS amplification were carried out with laboratory specimens. The same was applied to specimens collected from the field. The results show that PCR amenable genomic DNA could be isolated from fresh exuviae collected in the laboratory and not from older and/or field specimens. But exuviae of larvae and/or pupae collected in the field reared to adulthood in the laboratory yielded PCR amenable genomic DNA. The results also revealed that the ITS2 marker could very well differentiate Aedes aegypti and Aedes albopictus by producing amplicons of ~330 bp and ~520 bp, respectively. The genomic DNA from these alternate sources also supported the species-specific PCR to distinguish the Culex vishnui subgroup mosquitoes.

In-silico homology modeling of three isoforms of insect defensins from the dengue vector mosquito, Aedes aegypti (Linn., 1762).

Dengue is a serious public health problem in tropical and subtropical countries. It is caused by any of the four serologically distinct dengue viruses, namely DENV1-4. The viruses are transmitted by Aedes mosquitoes. Understanding various defence mechanisms of insects has become a prime area of research worldwide. In insects, the first line of defence against invading pathogens includes cellular mechanisms and a battery of antimicrobial peptides such as defensins, cecropins etc. Defensins--cationic, cysteine-rich peptides consisting of approximately 40 amino acids with broad-spectrum activity against Gram-positive bacteria--have been reported from a wide range of organisms. In the dengue vector mosquito, Aedes aegypti, three isoforms of defensins are reported to be expressed in a spatial and temporal fashion. This report presents the three-dimensional structures of the three isoforms of Ae. aegypti defensins predicted by comparative modeling. Prediction was done with Modeller 9v1 and the structures validated through a series of tests. The best results of the prediction study are presented, and may help lead to the discovery of new synthetic peptides or derivatives of defensins that could be useful in the control of vector-borne diseases.