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

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Journal of clinical microbiology (Impact Factor: 3.99). 10/2010; 49(1):42-7. 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.

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    • "A dipstick test to detect O. volvulus-specific antigen has been developed, with a sensitivity of 100% in urine and 92% in tears and a specificity of 100% in both (Stingl 2009). PCR amplifying repetitive nematode DNA sequences taken from skin snips reveals greater sensitivity in patients with low infection level (Fink et al. 2011). Diethylcarbamazine containing cream can be applied on a very limited skin area expected to be microfilaria-loaded. "

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    • "If the threshold is set at 53 HhaI copies, the likelihood to detect exposed but not yet infected individuals is higher. The results show that PCR cannot replace individual diagnostics for the determination of infection status, but could help mapping endemic areas which is important for monitoring mass drug administration (MDA) programs [9,18,19]. "
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    ABSTRACT: Lymphatic filariasis caused by Wuchereria bancrofti or Brugia spp. is a public health problem in developing countries. To monitor bancroftian filariasis infections, Circulating Filarial Antigen (CFA) test is commonly used, but for brugian infections only microfilariae (Mf) microscopy and indirect IgG4 antibody analyses are available. Improved diagnostics for detecting latent infections are required. An optimized real-time PCR targeting the brugian HhaI repeat was validated with plasma from microfilariae negative Mongolian gerbils (jirds) infected with B. malayi. Plasma samples from microfilaremic patients infected with B. malayi or W. bancrofti were used as positive and negative controls, respectively. PCR results of plasma samples from a transmigrant population in a B. malayi endemic area were compared to those of life-long residents in the same endemic area; and to IgG4 serology results from the same population. To discriminate between active infections and larval exposure a threshold was determined by correlation and Receiver-Operating Characteristics (ROC) curve analyses. The PCR detected HhaI in pre-patent (56 dpi) B. malayi infected jirds and B. malayi Mf-positive patients from Central Sulawesi, Indonesia. HhaI was also detected in 9/9 elephantiasis patients. In South Sulawesi 87.4% of the transmigrants and life-long residents (94% Mf-negative) were HhaI PCR positive. Based on ROC-curve analysis a threshold for active infections was set to >53 HhaI copies/mul (AUC: 0.854). The results demonstrate that the HhaI PCR detects brugian infections with greater sensitivity than the IgG4 test, most notably in Mf-negative patients (i.e. pre-patent or latent infections).
    Parasites & Vectors 03/2014; 7(1):146. DOI:10.1186/1756-3305-7-146 · 3.43 Impact Factor
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    • "Skin snips combined with microscopy are the gold standard, but are relatively insensitive when microfilarial (MF) skin densities are low. Polymerase chain reaction of the skin snips using the O-150 repeat sequence as the target provides significantly greater sensitivity [10], [11] but is not, at the moment, suitable for either surveillance or point of care. As areas get close to transmission interruption and elimination and the disease burden is no longer significant within a community, reduced acceptability by the community members of skin-snip testing and the inadequate sensitivity of such testing become significant problems. "
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    ABSTRACT: Onchocerciasis is a neglected tropical disease caused by infection with the parasite Onchocerca volvulus (Ov). An estimated 180 million people are at risk for Ov infection, and 37 million people are infected, mostly in Africa. A lateral flow-based assay to detect human IgG4 antibodies to the Ov-specific antigen Ov-16 was developed as a rapid tool to detect exposure to Ov. The test, when performed on 449 sera specimens from patients with microfiladermia and Ov-negative patients, has a sensitivity of 89.1% (95% confidence interval: 86.2%-92.0%), and specificity of 97% (95% confidence interval: 95.4%-98.6%). Because the intended use of the test is for surveillance, it is highly desirable to have a stable, long-lasting result. An extended read window is thus desirable for a high-volume, busy workflow and facilitates post-surveillance quality assurance. The main restriction on achieving an extended read window for this assay was the erythrocyte lysis that can alter the signal-to-noise ratio, especially in those with low IgG4 levels (weak positives). We describe a test housing that incorporates a user-independent feature driven by assay fluid and an expanding wick that detaches the blood separation membrane from the nitrocellulose used in the assay, but before hemolysis occurs. We demonstrated material functionality at extreme operational conditions (37°C, 80% relative humidity) and a read window of a minimum of 70 days. The fluid-driven assay device performs equally as well with whole blood as with plasma, as demonstrated with 100 spiked clinical specimens (with a correlation coefficient of 0.96). We show a novel, inexpensive, and simple approach to actuating the detachment of the blood separation membrane from the nitrocellulose test with no impact on the performance characteristics of the test.
    PLoS ONE 07/2013; 8(7):e69231. DOI:10.1371/journal.pone.0069231 · 3.23 Impact Factor
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