Toward Molecular Parasitologic Diagnosis: Enhanced Diagnostic Sensitivity for Filarial Infections in Mobile Populations

ArticleinJournal of clinical microbiology 49(1):42-7 · October 2010with30 Reads
DOI: 10.1128/JCM.01697-10 · Source: PubMed
The diagnosis of filarial infections among individuals residing in areas where the disease is not endemic requires both strong clinical suspicion and expert training in infrequently practiced parasitological methods. Recently developed filarial molecular diagnostic assays are highly sensitive and specific but have limited availability and have not been closely evaluated for clinical use outside populations residing in areas of endemicity. In this study, we assessed the performance of a panel of real-time PCR assays for the four most common human filarial pathogens among blood and tissue samples collected from a cohort of patients undergoing evaluation for suspected filarial infections. Compared to blood filtration, real-time PCR was equally sensitive for the detection of microfilaremia due to Wuchereria bancrofti (2 of 46 samples positive by both blood filtration and PCR with no discordant results) and Loa loa (24 of 208 samples positive by both blood filtration and PCR, 4 samples positive by PCR only, and 3 samples positive by blood filtration only). Real-time PCR of skin snip samples was significantly more sensitive than microscopic examination for the detection of Onchocerca volvulus microfiladermia (2 of 218 samples positive by both microscopy and PCR and 12 samples positive by PCR only). The molecular assays required smaller amounts of blood and tissue than conventional methods and could be performed by laboratory personnel without specialized parasitology training. Taken together, these data demonstrate the utility of the molecular diagnosis of filarial infections in mobile populations.


    • "This same target (ITS1 region) was used by the same authors to develop species-specifi c polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol for the differentiation of L. loa and M. perstans from W. bancrofti . Realtime PCR assays were also developed to demonstrate their utility for the diagnosis of fi larial infections in mobile populations (Fink et al. 2011a ) and to estimate the mf burden of blood samples in comparison with calibrated microscopy (Fink et al. 2011b ). "
    [Show abstract] [Hide abstract] ABSTRACT: Loiasis, also known as African eye worm is a vector-borne parasitic disease caused by the filarial nematode Loa loa and transmitted by the bite of tabanid vectors from the genus Chrysops. The real burden of the disease is not known, but L. loa remains wrongly classified as a benign disease. Loiasis exists exclusively in Africa where it is mainly focalized in the big areas of Central Africa. The prevalence of the disease rarely exceeds 30% in endemic communities; however, the disease has become important because of the serious adverse events which occur in some heavily infected patients after treatment with Mectizan for onchocerciasis or lymphatic filariasis. The life cycle of the parasite takes about 10 days in the vector (intermediate host) and about 3 months in humans (definite host). The passage of the adult worm in the bulbar conjunctiva is the classic manifestation of loiasis. Other manifestations are common migratory transient oedema (Calabar swellings). Both of these are used in the clinical diagnosis, complemented by microscopy for microfilaria. Rare but serious manifestations of Loa loa such as encephalitis, glomerular damage, retinal damage, endomyocardial fibrosis, albuminuria and hydrocele may also occur. Both surgical removal of adult worms and chemotherapy are used for managing L. loa. Currently Diethylcarbamazine (DEC), Mectizan®, mebendazole (MBZ) and albendazole (ALB)) are effective in varying degrees against mf and/or adult L. loa , while further research is needed to improve the efficacy of ALB in the management of L. loa.
    Full-text · Chapter · Jan 2016 · PLoS Neglected Tropical Diseases
    • "The human IgG4 antibody response to O. volvulus antigen Ov16 has been identified as a specific serologic marker of exposure to Onchocerca volvulus[11] and has been accepted toTable 1. Summary statistics for temperature and humidity exposure experienced by microscopes used by APOC during (1) transit to surveillance sites and (2) during epidemiological surveys in the years monitor progress toward elimination of river blindness [29]. To address a need for a consistent, standardized, and widely accessible source of positive control, a recombinant human IgG4 with specific affinity to the Ov16 antigen was generated. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Serological assays for human IgG4 to the Onchocerca volvulus antigen Ov16 have been used to confirm elimination of onchocerciasis in much of the Americas and parts of Africa. A standardized source of positive control antibody (human anti-Ov16 IgG4) will ensure the quality of surveillance data using these tests. Methodology/principal findings: A recombinant human IgG4 antibody to Ov16 was identified by screening against a synthetic human Fab phage display library and converted into human IgG4. This antibody was developed into different positive control formulations for enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT) platforms. Variation in ELISA results and utility as a positive control of the antibody were assessed from multiple laboratories. Temperature and humidity conditions were collected across seven surveillance activities from 2011-2014 to inform stability requirements for RDTs and positive controls. The feasibility of the dried positive control for RDT was evaluated during onchocerciasis surveillance activity in Togo, in 2014. When the anti-Ov16 IgG4 antibody was used as a standard dilution in horseradish peroxidase (HRP) and alkaline phosphatase (AP) ELISAs, the detection limits were approximately 1ng/mL by HRP ELISA and 10ng/mL by AP ELISA. Positive control dilutions and spiked dried blood spots (DBS) produced similar ELISA results. Used as a simple plate normalization control, the positive control antibody may improve ELISA data comparison in the context of inter-laboratory variation. The aggregate temperature and humidity monitor data informed temperature parameters under which the dried positive control was tested and are applicable inputs for testing of diagnostics tools intended for sub-Saharan Africa. As a packaged positive control for Ov16 RDTs, stability of the antibody was demonstrated for over six months at relevant temperatures in the laboratory and for over 15 weeks under field conditions. Conclusions: The recombinant human anti-Ov16 IgG4 antibody-based positive control will benefit inter-laboratory validation of ELISA assays and serve as quality control (QC) reagents for Ov16 RDTs at different points of the supply chain from manufacturer to field use.
    Full-text · Article · Jan 2016
    • "NAATs are not widely used clinically, but may be particularly useful in settings where filarial infections are not commonly seen and expertise diagnosing infections with conventional microscopy is lacking (Fink, Fahle, et al., 2011). They may also play an important role in epidemiologic studies and large-scale filarial disease control programmes, since they can be used human as well as vector samples and thus be used for monitoring vector infection rates and asymptomatic disease in humans (Dissanayake, Min, & Piessens, 1991; Farid et al., 2001; Rao, Atkinson, et al., 2006; Takagi et al., 2011 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Molecular testing is increasingly used to supplement or replace conventional microscopy-based methods of parasite identification. Potential benefits of molecular methods such as nucleic acid amplification tests include increased sensitivity, ability to differentiate morphologically similar organisms and lack of reliance on subjective microscopic features. However, several challenges exist for widespread implementation of molecular diagnostics, including the expense of reagents and equipment, need for sophisticated facilities and lack of commercial testing options. Most molecular parasitology tests are based on non-standardised laboratory-developed methods, although new commercial options including multiplex methods for gastrointestinal pathogens have recently become available. This review will focus on the use of molecular assays for common and important parasites including Plasmodium and Babesia species, trypanosomes, filaria, Leishmania species, free-living amoebae, Trichomonas vaginalis, Toxoplasma gondii and intestinal protozoa and helminths and how testing can be integrated into patient care algorithms.
    Full-text · Article · Dec 2015 · PLoS Neglected Tropical Diseases
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