RT-PCR assay for the detection of infective (L3) larvae of lymphatic filarial parasite, Wuchereria bancrofti, in vector mosquito Culex quinquefasciatus.

BACKGROUND & OBJECTIVES: Periodic monitoring of vector population for infection and infectivity rates is central to the evaluation of the filariasis elimination strategies in endemic areas to monitor the success of MDA and also to establish endpoints for intervention. The main objective of this study was to develop a RT-PCR assay, based on L3 stage-specific primers to detect the presence of infective stage larvae of filarial parasite, Wuchereria bancrofti in the vector Culex quinquefasciatus. MATERIAL & METHODS: Subtracted probe development technique was employed for the identification of infective stage (L3) specific genes. The subtracted cDNA was labeled by non-radioisotopic method and used for screening cDNA library of L3 stage larvae of W. bancrofti constructed in UniZap XR. Recombinants were probed and identified from the library. The inserts of the recombinant clones were purified and sequenced. Primers were designed based on the sequence information of three recombinant clones for detecting L3 larvae of W. bancrofti in the vector by RT-PCR assay. Preliminary laboratory evaluation was carried out to assess the sensitivity and specificity of WbL31 RT-PCR assay. RESULTS: cDNA library of L3 stage of W. bancrofti constructed in UniZap XR vector, constituted 5 x 10(5) phages with 80-90% recombinant phages and the size of inserts varied from 0.1 to 1.0 kb. When subtracted cDNA was random prime labeled and used for screening cDNA library of L3 stage of W. bancrofti constructed in UniZap XR, 18 clones were identified from the library. Three genes were found up-regulated in the L3 stage, out of which WbL31 (cuticular collagen) was found to be useful in detecting L3 larvae of W. bancrofti in the vector by RT-PCR assay with high specificity and sensitivity (98-100%). CONCLUSION: Present paper marks first report on the development of an infective stage-specific RT-PCR assay (WbL31 RT-PCR assay) to detect L3 stage W. bancrofti in the vector. This assay will have potential application in assessing the transmission of infection and hence in decision-making related to elimination programme.

Laboratory evaluation of Ssp I PCR assay for the detection of Wuchereria bancrofti infection in Culex quinquefasciatus.

BACKGROUND & OBJECTIVES: There is a need to delimit the areas of filariasis transmission in view of the Filariasis Elimination Programme launched in India. Infection rate in vectors is an important parameter in determining transmission and it is conventionally assessed by dissection and microscopy. A PCR assay based on Ssp I repeats of Wuchereria bancrofti has shown potential in the detection of infection in vectors. The aim of the present study was to evaluate the specificity and sensitivity of this assay on W. bancrofti and its vector, Culex quinquefasciatus, prevalent in India. METHODS: The DNA from pools of C. quinquefasciatus to which W. bancrofti microfilariae (mf) were added, was extracted by lysing with 0.1 M NaOH and 0.2 per cent sodium dodecyl sulphate (SDS), followed by silica absorption in the presence of guanidinium thiocyanate. The PCR assay of the DNA samples was carried out using NV-1 and NV-2 primers and the species specific SspI band was visualized on agarose gels stained with ethidium bromide. RESULTS: The Ssp I PCR assay was found to be highly species specific, as it did not detect the DNA of a closely related filarial parasite, Brugia malayi. The assay detected as little as 0.04 pg of W. bancrarofti DNA. Minimum number of parasite detectable in pools of mosquitoes was 1 mf. A pool size of 50 mosquitoes was found to be optimum for the PCR assay. INTERPRETATION & CONCLUSION: The Ssp I PCR assay was found to be highly specific and sensitive in detecting filarial parasite in pools of mosquitoes and therefore has potential application in rapid assessment of transmission of filariasis.

Observations on nocturnal activity and man biting habits of malaria vectors, Anopheles fluviatilis, An. annularis and An. culicifacies in the hill tracts of Koraput District, Orissa, India.

Biting and feeding behavior of malaria vectors were studied in nine villages (5 from Jeypore zone and 4 from Malkangiri zone) of Koraput District. Man biting catches comprised of 16 anopheline species including the incriminated vectors of this area: An. fluviatilis, An. annularis and An. culicifacies. An. fluviatilis was predominant and biting of this species recorded indoors throughout the year in both the zones. The period, during which the biting activity peaked, was different between the two zones and consequently the time of peak transmission was also different between the zones. The biting activity was at its peak between 21.00 and 03.00 hours in both the zones. However, in cold season the biting activity peaked in the first quarter of the night in Jeypore zone. The anthropophilic index (AI) of An. fluviatilis was 26.2% in Jeypore and 83.7% in Malkangiri and of An. culicifacies the AI was 0.4% in Jeypore and 7% in Malkangiri. Analysis of gonotrophic stages of night resting females indicated that in Jeypore zone, the majority of An. fluviatilis female left indoors for outdoor resting before the completion of gonotrophic cycle, but in Malkangiri, the females remained indoors till the end of the gonotrophic cycle. The presence of full-fed females in night resting catches in Jeypore village further suggested that the females do not leave the house immediately after taking blood meal but rest for some time.

Lambdacyhalothrin treated bed nets as an alternative method of malaria control in tribal villages of Koraput District, Orissa State, India.

A village scale trial was carried out to evaluate the efficacy of bed-nets impregnated with lambdacyhalothrin, at the dose of 0.025 g/m2, in reducing malaria transmission in villages of Koraput District of Orissa, India, inhabited by tribals. The nets were distributed before peak transmission season. There was an overall decline in the parasite rate in all the age groups, six months after the supply of impregnated nets while the same increased in control village and in a village where untreated nets were supplied. The vector densities (resting and man landing) were lower in the treated village as compared to untreated and control villages throughout the study period. The reduction in the parasite rate was consistent when the reimpregnation was done at six monthly interval and the same tend to increase when the gap between the two impregnations was increased to one year. Though malaria incidence was reduced, transmission was not completely interrupted during the study period, due to outdoor transmission. The insecticidal effect of bednets was retained upto six months. Washing of bednets by the community did not affect the efficacy. The acceptance and usage was better with impregnated nets as compared to ordinary nets.