Evaluation of LAMP for the diagnosis of Loa loa infection in dried blood spots compared to PCR-based assays and microscopy

Background: Loa loa is a filarial species found exclusively in West and Central Africa. Microscopy is the traditional diagnosis method for human loiasis. Several molecular methods have developed as an alternative approach for identification of L. loa filarial parasites. Objectives: The aim of this study was to evaluate a Loa-Loop-mediated isothermal amplification (LAMP) assay to diagnose loiasis disease on dried blood spots (DBS) samples, compared to microscopy, filaria-real time-polymerase chain reaction (PCR) and nested-Loa PCR. Methods: A total of 100 DBS samples and 100 blood smears were used for this study. DNA was extracted using saponin/Chelex method. DNA isolated was assayed by a Loa-LAMP assay in parallel to microscopy, filaria-real time PCR and nested-Loa PCR. The sensitivities and specificities of Loa-LAMP assay was computed comparing to each one of the reference methods. Findings: Loa-LAMP's sensitivity was more than 90% and specificity was nearly 100% when compared to molecular methods. On the other hand, sensitivity was decreased a bit when Loa-LAMP faced microscopy, but keeping the other statistical values high. Main conclusions: Loa-LAMP is an appropriate method for loiasis diagnosis in endemic areas. Though, it has disadvantages like the reagents' high price at the moment and not to be able to detect more filarial species at once.Financial support: Fundación Estatal, Salud, Infancia y Bienestar Social (Institute of Health Carlos III) and Cooperative Research Network on Tropical Diseases. T-HT-T is hired at the Malaria and Neglected Tropical Diseases Laboratory of National Centre of Tropical Medicine with a contract named ISCIII-SARA BORRELL CD17CIII/00018, financed by the Institute of Health Carlos III.S

Colorimetric and Real-Time Loop-Mediated Isothermal Amplification (LAMP) for Detection of Loa loa DNA in Human Blood Samples

Loiasis, caused by the filarial nematode Loa loa, is endemic in Central and West Africa. Loa loa has been associated with severe adverse reactions in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. Diagnosis of loiasis still depends on microscopy in blood samples, but this is not effective for large-scale surveys. New diagnostics methods for loiasis are urgently needed. Previously, we developed a colorimetric high-sensitive and species-specific LAMP for Loa loa DNA detection. Here, we evaluate it in a set of 100 field-collected clinical samples stored as dried blood spots. In addition, Loa loa-LAMP was also evaluated in real-time testing and compared with microscopy and a specific PCR/nested PCR. A simple saponin/Chelex-based method was used to extract DNA. Colorimetric and real-time LAMP assays detected more samples with microscopy-confirmed Loa loa and Loa loa/Mansonella perstans mixed infections than PCR/nested-PCR. Samples with the highest Loa loa microfilariae counts were amplified faster in real-time LAMP assays. Our Loa loa-LAMP could be a promising molecular tool for the easy, rapid and accurate screening of patients for loiasis in endemic areas with low-resource settings. The real-time testing (feasible in a handheld device) could be very useful to rule out high-microfilariae loads in infected patients.This research was funded by the Institute of Health Carlos III, ISCIII, Spain (www.isciii.es), grants: RICET RD16/0027/0018 (A.M.), RD16/0027/0000 (A.B.), FCSAI-ISCIII (P.N.) and PI19/01727 (P.F.-S.), European Union co-financing by FEDER (Fondo Europeo de Desarrollo Regional) ‘Una manera de hacer Europa’. We also acknowledge support by the Predoctoral Fellowship Program of Junta de Castilla y León co-financing by Fondo Social Europeo (BDNS (Identif.): 422058 and BDNS (Identif.): 487971), by the ISCIII-Sara Borrell contract CD17CIII/00018 financed by the Institute of Health Carlos III and Predoctoral Fellowship Program of University of Salamanca, and co-financing by Santander Bank.S

Evidence for Suppression of Onchocerciasis Transmission in Bioko Island, Equatorial Guinea

<div><p>Onchocerciasis or "river blindness" is a chronic parasitic neglected tropical disease which is endemic both in mainland and insular Equatorial Guinea. We aim to estimate the current epidemiological situation of onchocerciasis in Bioko Island after vector elimination in 2005 and more than sixteen years of Community Directed Treatment with Ivermectin (CDTI) by using molecular and serological approaches for onchocerciasis diagnosis. A community-based cross-sectional study was carried out in Bioko Island from mid-January to mid-February 2014. A total of 544 study participants were recruited. A complete dermatological examination was performed and three skin snips were performed in every participant for parasitological and molecular assessments. Blood spots were also taken for determination of Ov16 IgG4 antibodies trough an “in-house” ELISA assay. Overall, we found 15 out of 522 individuals suffering any onchocerciasis specific cutaneous lesions and 16 out of 528 (3.0%) with onchocercal nodules in the skin. Nodules were significantly associated with age, being more common in subjects older than 10 years than in younger people (3.9% vs. 0%, p = 0.029). Regarding the onchocerciasis laboratory assessment, no positive parasitological test for microfilaria detection was found in the skin snips. The calculated seroprevalence through IgG4 serology was 7.9%. No children less than 10 years old were found to be positive for this test. Only one case was positive for <i>Onchocerca volvulus</i> (<i>O</i>. <i>volvulus</i>) after skin PCR. The present study points out that the on-going mass ivermectin treatment has been effective in reducing the prevalence of onchocerciasis and corroborates the interruption of transmission in Bioko Island. To our knowledge, this is the first time that accurate information through molecular and serological techniques is generated to estimate the onchocerciasis prevalence in this zone. Sustained support from the national program and appropriate communication and health education strategies to reinforce participation in CDTI activities are essential to ensure progress towards onchocerciasis elimination in the country.</p></div

Laboratory assessments, Bioko Island, January 2014.

<p>Laboratory assessments, Bioko Island, January 2014.</p

Clinical characteristics of onchocerciasis of the study population, by age Bioko Island, Equatorial Guinea, January 2014.

<p>Clinical characteristics of onchocerciasis of the study population, by age Bioko Island, Equatorial Guinea, January 2014.</p

House-holds distribution in the study area, Bioko Island, Equatorial Guinea.

<p>House-holds distribution in the study area, Bioko Island, Equatorial Guinea.</p

Preventive treatment by age Bioko Island, Equatorial Guinea, January 2014.

<p>Preventive treatment by age Bioko Island, Equatorial Guinea, January 2014.</p