Mini-TAS as a confirmatory mapping tool for remapping areas with uncertain filarial endemicity to exclude/ include for mass drug administration: A report from field validation in India.

India has targeted elimination of lymphatic filariasis (LF) through mass drug administration (MDA) by 2027. Mapping of LF endemic areas is a priority for implementation of MDA. Current national LF remapping tool for unsurveyed/uncertain districts, have many limitations. The WHO has recommended a sensitive and rapid remapping protocol (Mini-TAS), that needs validation in Indian setting. Hence, in the present study a comparative assessment of these two protocols (national protocol vs Mini-TAS) was undertaken in two non-MDA districts of Odisha, with unknown filarial endemicity but reporting chronic cases. Purposive sampling was done in five top sites based on filarial case count as per the national protocol. Random 30 cluster survey was done by conducting school based Mini-TAS, Microfilariae (Mf) survey among adults (>10 years) in villages/wards with schools and Molecular Xenomonitoring (MX) of infection in vectors. Costing by activity and items of the surveys was acomplished using itemized cost menu. In Kalahandi, one of the five purposive sampling sites showed Mf prevalence above threshold (> 1%). But except Mini-TAS neither MX nor house-hold Mf survey among adults could detect the infection above the threshold. While in Balangir, Mf prevalence in all purposive sampling sites,Mini-TAS, Mf prevalence among adult and MX were above the respective thresholds confirming endemicity of LF in the district. The per sample cost of purposive sampling for Mf was the lowest INR 41, followed by adult Mf sampling INR 93. Mini-TAS and MX were expensive with INR 659 and 812 respectively. The study demonstrates that though all the sampling methods could detect filarial infection above the threshold in high-risk areas, Mini-TAS could only detect infection in low-risk areas. Therefore, in the national programme Mini-TAS can be used as a decision-making tool to determine whether to exclude/ include a district having uncertain endemicity for MDA.

Prevalence and transmission potential of Wolbachia in Aedes albopictus population circulating in endemic coastal districts of Odisha, India.

Wolbachia, known for its reproductive manipulation capabilities in insects, are being implemented to control dengue and chikungunya. To understand Wolbachia biology and its utility as a bio-control for vector mosquito's populations, we investigated its dissemination pattern in field in collected Ae. albopictus along with its maternal transmission efficacy over generations in regions of endemic dengue (DENV) transmission. Field collected Ae. albopictus were subjected to PCR for Wolbachia screening. Overall mean Wolbachia infection frequency in Ae. albopictus was found out to be 87.3% wherein a trend was observed in the pattern of maternal transmission across generations. χ2 for trend revealed a significant variation between Wolbachia infections and non-infections in Ae. albopictus generations. Linear regression analysis revealed the involvement of a strong negative correlation, implying that overall Wolbachia infection tends to decrease in places with high dengue cases.The reduction in Wolbachia infection frequency may be attributed to several environmental factors with the probability of being the cause for endemicity of dengue in the studied areas.This study reports on the transmission efficacy of naturally occurring Wolbachia in successive generations of Ae. albopictus and its correlation with dengue cases in clusters of Odisha, India. Studying the transmission trend of Wolbachia along with transovarial transmission of DENV might be indicative towards the interplay of Wolbachia infection in presence/absence of DENV.

Implementation of molecular method in routine malaria diagnosis and entomological studies.

BACKGROUND & OBJECTIVES: Molecular methods for malaria vector species and parasite identification have received great attention in recent years. Accurate and precise identification of the target species has direct medical and practical implications, such as in malaria diagnosis and vector dynamics study. Translation of molecular techniques will help in evaluation of epidemiological and entomological profile of malaria even in highly inaccessible areas where there is lack of an expert microscopist or entomologist. METHODS: In the present study, we have developed a simple yet accurate molecular tool for malaria diagnosis as well as for malaria vector studies. We have standardized, simplified and improvised the DNA isolation (using Chelex; a cationic exchanger), its storage and multiplex PCR for parasite detection from dried blood spot (DBS) filter paper as well as malaria vector identification and infection status study. RESULTS: The chelex-PCR based molecular method was highly sensitive (sensitivity >90%) and specific (specificity >80%) for parasite detection as well as vector species identification. This method has proven readily adaptable for use in the clinical diagnostic/research laboratory for epidemiological investigation and vector dynamics study that can challenge the conventional gold standard approach such as microscopy/ morphological methods not only in response to accuracy but also in relation to cost, time and technical expertise. INTERPRETATION & CONCLUSION: Transfer of this molecular technology from laboratory to field condition is highly essential for its availability to the common public rather than being restricted to only academic research. This can be achieved by implementation of the technology in terms of conducting mass training and awareness programs in various resource-limited endemic zones for the purpose of malaria elimination.

Comparison between different methods of DNA isolation from dried blood spots for determination of malaria to determine specificity and cost effectiveness.

DNA extraction from filter paper by using different methods was compiled through a thorough review of many research articles published in various journals. When performing malaria epidemiological surveys in remote area, it is difficult to collect blood samples and transport it. In field particularly in remote area where facilities for storing and processing of samples does not exist, there surveillance and diagnosis of malaria is very difficult. In this review we are focused upon four simple methods of DNA isolation from the field collected blood and mosquito abdomen blood meal spotted on Whatman No. 1 or No. 3 filter paper. The main DNA isolation methods are Chelex-100, Tris-EDTA (TE) buffer; Methanol based DNA extraction and Phosphate buffer saline (PBS) using Lysis buffer and Phenol-Chloroform method. Efforts have been taken to identify the methods which are cost-effective and take less time to extract DNA from dried blood spots (DBS) and whole mosquitoes. The purpose of this paper is to update the knowledge and find a method to extract DNA from DBS which will be specific, rapid, cost-effective, less time consuming and feasible for epidemiological survey in remote area.

Comparative analysis of three methods from dried blood spots for expeditious DNA extraction from mosquitoes; suitable for PCR based techniques.

The objective of this work was to compare the quality, purity and quantity of DNA isolated from dried blood spots (DBS) by three methods (Chelex-100, QIAamp DNA mini kit, and TE (Tris EDTA)-Buffer). Sample collection was performed in six districts in Odisha, India and screened for cases of clinical malaria and dengue and vector density. Mosquito abdomens were spotted on Whatman 3MM (MERCK) Filter paper and dried for 10 min at room temperature. DNA was isolated from DBS using three methods (Chelex-100, QIAamp DNA mini kit, and TE-Buffer), and PCR was used to determine the feeding behaviours of vector mosquitoes. DNA was quantified using a UV-spectrophotometer, and q-PCR was used to determine the target gene copy number to compare the methods. The QIAamp DNA mini kit method was used as the reference method. The yield and purity of DNA extracted with Chelex-100 and TE were 14-72 ng/µl and 1.51-1.85 and 9-50 ng/µl and 1.68-2.1, respectively. DNA extracted using the Chelex-100 method was stored for over 1 month at - 20 °C and was suitable for later use. The Chelex-100 method had a sensitivity of 99.5% and specificity of 78%. A Bland-Altman plot suggested that the Chelex-100 method was similar to the QIAamp DNA mini kit method for determining the feeding behaviours of vector mosquitoes. The Chelex-100 method is simple, cost-effective, and safe and requires minimal time for DNA extraction from dried blood spots. In malaria and dengue research, detecting the feeding behaviours from mosquito DNA from dried blood spots on filter paper by PCR is an easy, minimally invasive and inexpensive molecular technique that can be performed in remote areas.