Improved limit of detection for zoonotic Plasmodium knowlesi and P. cynomolgi surveillance using reverse transcription for total nucleic acid preserved samples or dried blood spots.

Background: Zoonotic P. knowlesi and P. cynomolgi symptomatic and asymptomatic infections occur across endemic areas of Southeast Asia. Most infections are low-parasitemia, with an unknown proportion below routine microscopy detection thresholds. Molecular surveillance tools optimizing the limit of detection (LOD) would allow more accurate estimates of zoonotic malaria prevalence. Methods: An established ultra-sensitive Plasmodium genus quantitative-PCR (qPCR) assay targeting the 18S rRNA gene underwent LOD evaluation with and without reverse transcription (RT) for P. knowlesi, P. cynomolgi and P. vivax using total nucleic acid preserved (DNA/RNA Shield™) isolates and archived dried blood spots (DBS). LODs for selected P. knowlesi-specific assays, and reference P. vivax- and P. cynomolgi-specific assays were determined with RT. Assay specificities were assessed using clinical malaria samples and malaria-negative controls. Results: The use of reverse transcription improved Plasmodium species detection by up to 10,000-fold (Plasmodium genus), 2759-fold (P. knowlesi), 1000-fold (P. vivax) and 10-fold (P. cynomolgi). The median LOD with RT for the Kamau et al. Plasmodium genus RT-qPCR assay was ≤0.0002 parasites/µL for P. knowlesi and 0.002 parasites/µL for both P. cynomolgi and P. vivax. The LODs with RT for P. knowlesi-specific PCRs were: Imwong et al. 18S rRNA (0.0007 parasites/µL); Divis et al. real-time 18S rRNA (0.0002 parasites/µL); Lubis et al. hemi-nested SICAvar (1.1 parasites/µL) and Lee et al. nested 18S rRNA (11 parasites/µL). The LOD for P. vivax- and P. cynomolgi-specific assays with RT were 0.02 and 0.20 parasites/µL respectively. For DBS P. knowlesi samples the median LOD for the Plasmodium genus qPCR with RT was 0.08, and without RT was 19.89 parasites/uL (249-fold change); no LOD improvement was demonstrated in DBS archived beyond 6 years. The Plasmodium genus and P. knowlesi-assays were 100% specific for Plasmodium species and P. knowlesi detection, respectively, from 190 clinical infections and 48 healthy controls. Reference P. vivax-specific primers demonstrated known cross-reactivity with P. cynomolgi. Conclusion: Our findings support the use of an 18S rRNA Plasmodium genus qPCR and species-specific nested PCR protocol with RT for highly-sensitive surveillance of zoonotic and human Plasmodium species infections.

A protocol and training guidelines for mosquito sampling in remote areas with limited power supply.

Mosquito-borne diseases pose a significant threat in many Southeast Asian countries, particularly through the sylvatic cycle, which has a wildlife reservoir in forests and rural areas. Studying the composition and diversity of vectors and pathogen transmission is especially challenging in forests and rural areas due to their remoteness, limited accessibility, lack of power, and underdeveloped infrastructure. This study is based on the WHO mosquito sampling protocol, modifies technical details to support mosquito collection in difficult-to-access and resource-limited areas. Specifically, we describe the procedure for using rechargeable lithium batteries and solar panels to power the mosquito traps, demonstrate a workflow for processing and storing the mosquitoes in a -20 °C freezer, data management tools including microclimate data, and quality assurance processes to ensure the validity and reliability of the results. A pre- and post-test was utilized to measure participant knowledge levels. Additional research is needed to validate this protocol for monitoring vector-borne diseases in hard-to-reach areas within other countries and settings.

Communicable disease control in Afghanistan.

Among public health challenges in Afghanistan, communicable diseases still predominate because the epidemiologic transition to chronic disease has not yet occurred. Afghanistan's 10-year journey to improve its response to communicable disease is reflected in varying degrees of progress and innovation, all while long-standing conflict and geographic inaccessibility limit outreach and effective service delivery to vulnerable populations. Although Afghanistan is close to achieving polio elimination, other reportable communicable diseases are only slowly achieving their goals and objectives through targeted, sustained programmatic efforts. The introduction of disease early warning systems has allowed for identification and investigation of outbreaks within 48 hours. Tuberculosis case detection has risen over the last 10 years, and treatment success rates have been sustained at World Health Organization targets over the last 5 years at 85%. These successes are in large part due to increased government commitment, Global Fund support, training of community health workers and improved laboratory capabilities. Malaria cases dropped between 2002 and 2010. HIV/AIDS has been kept at low levels except in only certain sub-sectors of the population. In order to build on these achievements, Afghanistan will need a comprehensive strategy for all communicable diseases, with better human and infrastructure development, better multi-sectoral development and international collaboration.