Geographical distribution and species identification of human filariasis and onchocerciasis in Bioko Island, Equatorial Guinea.

Human filariae are vector-borne parasites and the causative agents of various diseases, including human onchocerciasis and lymphatic filariasis. Onchocerciasis causes a spectrum of cutaneous and ophthalmologic manifestations (including blindness) and has long been a major public health problem in Bioko Island (Equatorial Guinea). Bioko Island has been included in the WHO's Onchocerciasis Control Program since 1987. In Bioko Island, the specificity and sensitivity of clinical Onchocerca volvulus diagnosis is key. The objective of this work was to update onchocerciasis elimination progress in Bioko Island, after 18 years of mass ivermectin intervention, and the general filariasis situation through a rapid and accurate molecular method. A cross-sectional study was conducted in Bioko Island from mid-January to mid-February 2014. A total of 543 subjects were included in the study. Whole blood and one skin snip (from lumbar regions) were analysed with a real time PCR assay. Two other skin biopsies were analysed by an expert microscopist. All positive samples were confirmed by sequencing. Traditional microscopic examination of the skin biopsies failed to detect any microfilariae. However, 11 (2.03%) infections were detected using PCR assay, including one O. volvulus, two Mansonella streptocerca, seven Mansonella perstans and one Loa loa infections. PCR assays in blood detected 52 filariae-positive individuals (9.6%) which harboured M. perstans or L. loa. The low prevalence of O. volvulus confirms the success of the Onchocerciasis Control Programme and suggests that Mass Drug Administration in Bioko Island can be interrupted in the near future. The very high prevalence of M. perstans found in skin snips assays raises doubts about the reliability of microscope-based diagnosis of O. volvulus infections.

Nested multiplex PCR for identification and detection of human Plasmodium species including Plasmodium knowlesi.

OBJECTIVE: To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria. METHODS: In this study the nested multiplex malaria PCR was redesigned, targeting the 18S rRNA gene, to identify the fifth human Plasmodium species, Plasmodium knowlesi, together with the other human Plasmodium (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) by amplified fragment size using only two amplification processes and including an internal reaction control to avoid false negatives. RESULTS: The technique was validated with 91 clinical samples obtained from patients with malaria compatible symptoms. The technique showed high sensitivity (100%) and specificity (96%) when it was compared to the reference method employed for malaria diagnosis in the Instituto de Salud Carlos Ⅲ and a published real-time PCR malaria assay. CONCLUSIONS: The technique designed is an economical, sensitive and specific alternative to current diagnosis methods. Furthermore, the method might be tested in knowlesi-malaria endemic areas with a higher number of samples to confirm the quality of the method.