Malaria vector sibling species distribution in different endemic districts of Punjab, India.

BACKGROUND & OBJECTIVES: Malaria transmission in Punjab, India is mainly seasonal with variation in its endemicity that may be due to varying vector behaviour in different areas of the state, primarily attributed to the existence of sibling species complexes among the vector species. So far there is no report regarding the existence of malaria vectors sibling species in the state of Punjab, therefore, the present study was planned to investigate the status of sibling species of two main vectors of malaria viz. Anopheles culcifacies and Anopheles fluviatilis in different districts of Punjab. METHODS: Mosquito collections were made through hand catch in the morning hours. Malaria vector species An. culicifacies and An. fluviatilis were morphologically identified and man hour density was calculated. Both the vector species were subjected to molecular assays for sibling species identification through amplification of D3 domain of 28S ribosomal DNA by allele-specific PCR. RESULTS: Four sibling species of An. culicifacies, were identified viz. A, B, C and E. Species A was identified from Bhatinda district, species B, C and E from. S.A.S. Nagar and species C from Hoshiarpur. Two sibling species S and T of An. fluviatilis were identified from districts S.A.S. Nagar and Rupnagar. INTERPRETATION & CONCLUSION: Presence of four sibling species of An. culicifacies and two sibling species of An. fluviatilis in Punjab necessitates planning of longitudinal studies to ascertain their role in disease transmission so that appropriate interventions may be applied to achieve malaria elimination.

Prevalence of submicroscopic malaria in low transmission state of Punjab: A potential threat to malaria elimination.

BACKGROUND & OBJECTIVES: Submicroscopic malaria infections with low parasite density serve as a silent reservoir for maintaining residual transmission in the population. These infections should be identified and targeted to be eliminated for sustained malaria control. The conventional methods of diagnosis such as light microscopy and rapid diagnostic kits often fail to detect low density infections. Therefore, the more sensitive molecular techniques should be employed to detect low density infections. The objectives of the study was to explore the prevalence of sub-microscopic infections in low transmission areas of Punjab using highly sensitive molecular tool. METHODS: A total of 1114 finger prick blood samples were collected through active surveillance and tested for malaria diagnosis using light microscopy, RDT and PCR. Nested PCR amplification was performed using a pair of Plasmodium genus-specific primers from the 18S rRNA small subunit gene (18S rRNA). The amplified PCR products were analysed using a 2% agarose gel, stained with ethidium bromide and observed under transilluminator. RESULTS: Test positive rate (TPR) by microscopy, RDT and PCR was 4.4, 3.95 and 5.75%, respectively. Microscopy and RDT failed to detect mixed infections whereas 0.26% cases were found to be mixed infection in PCR. Compared to LM and RDT, PCR has detected 1.3% additional positive cases. However, of the total positive cases detected by PCR, 23.4% infections were found to be submicroscopic, which could not be detected by conventional methods of diagnosis. INTERPRETATION & CONCLUSIONS: The molecular study revealed the existence of submicroscopic malaria cases in the study population which would have remained undetected by conventional methods of diagnosis. This is particularly important because Punjab state is in malaria elimination phase and targeted to achieve elimination in 2021. However, such undetected parasite positive cases may pose bigger problem any time due to continued transmission. Therefore, application of more sensitive diagnostic tools like PCR and LAMP with conventional methods may be much more useful in case detection particularly in low transmission settings for malaria elimination.