A field trial of a PCR-based Mansonella ozzardi diagnosis assay detects high-levels of submicroscopic M. ozzardi infections in both venous blood samples and FTA® card dried blood spots

Abstract

BackgroundMansonella ozzardi is a poorly understood human filarial parasite with a broad distribution throughout Latin America. Most of what is known about its parasitism has come from epidemiological studies that have estimated parasite incidence using light microscopy. Light microscopy can, however, miss lighter, submicroscopic, infections. In this study we have compared M. ozzardi incidence estimates made using light microscopy, with estimates made using PCR.Methods214 DNA extracts made from Large Volume Venous Blood Samples (LVVBS) were taken from volunteers from two study sites in the Rio Solimões region: Codajás [n = 109] and Tefé [n = 105] and were subsequently assayed for M. ozzardi parasitism using a diagnostic PCR (Mo-dPCR). Peripheral finger-prick blood samples were taken from the same individuals and used for microscopic examination. Finger-prick blood, taken from individuals from Tefé, was also used for the creation of FTA®card dried blood spots (DBS) that were subsequently subjected to Mo-dPCR.ResultsOverall M. ozzardi incidence estimates made with LVVBS PCRs were 1.8 times higher than those made using microscopy (44.9 % [96/214] compared with 24.3 % [52/214]) and 1.5 times higher than the PCR estimates made from FTA®card DBS (48/105 versus 31/105). PCR-based detection of FTA®card DBS proved 1.3 times more sensitive at diagnosing infections from peripheral blood samples than light microscopy did: detecting 24/105 compared with 31/105. PCR of LVVBS reported the fewest number of false negatives, detecting: 44 of 52 (84.6 %) individuals diagnosed by microscopy; 27 of 31 (87.1 %) of those diagnosed positive from DBSs and 17 out of 18 (94.4 %) of those diagnosed as positive by both alternative methodologies.ConclusionsIn this study, Mo-dPCR of LVVBS was by far the most sensitive method of detecting M. ozzardi infections and detected submicroscopic infections. Mo-dPCR FTA®card DBS also provided a more sensitive test for M. ozzardi diagnosis than light microscopy based diagnosis did and thus in settings where only finger-prick assays can be carried-out, it may be a more reliable method of detection. Most existing M. ozzardi incidence estimates, which are often based on light microscope diagnosis, are likely to dramatically underestimate true M. ozzardi parasitism incidence levels.

Mansonella ozzardi: a neglected New World filarial nematode

“…87 The PCR method described by Tang and colleagues 19 was 1.5 × more sensitive when DNA templates were prepared from 2.5-μl of venous blood, compared with those isolated from 0.6 μl of dried blood from FTA membranes. 87 Moreover, venous blood-based PCR was 1.8 × more sensitive than conventional thick smear microscopy (one smear examined per subject).…”

“…PCR-based amplification of species-specific target sequences allows for increased diagnostic sensitivity, compared with microscopic methods, and reliable differentiation between M. ozzardi and co-endemic filarial species such as O. volvulus and M. perstans. 19,20,86,87 Key factors that determine the diagnostic sensitivity of PCR include sample storage conditions prior to DNA extraction, sample volume, and methods used to isolate DNA. 87 The PCR method described by Tang and colleagues 19 was 1.5 × more sensitive when DNA templates were prepared from 2.5-μl of venous blood, compared with those isolated from 0.6 μl of dried blood from FTA membranes.…”

“…19,20,86,87 Key factors that determine the diagnostic sensitivity of PCR include sample storage conditions prior to DNA extraction, sample volume, and methods used to isolate DNA. 87 The PCR method described by Tang and colleagues 19 was 1.5 × more sensitive when DNA templates were prepared from 2.5-μl of venous blood, compared with those isolated from 0.6 μl of dried blood from FTA membranes. 87 Moreover, venous blood-based PCR was 1.8 × more sensitive than conventional thick smear microscopy (one smear examined per subject).…”

<i>Mansonella ozzardi</i>: uma filaria negligenciada que pode modular a resposta imune.

“…Uma técnica de concentração amplamente utilizada foi originalmente descrita por Knott em 1939(KNOTT, 1939RACCURT et al, 1982). Trata-se de misturar 5 ml de sangue venoso anticoagulado com 50 ml de uma solução de formalina a 2% em um tubo de poliestireno e recuperar microfilárias no sedimento após uma centrifugação breve a 400 g. (MEDEIROS et al, 2015;MORALES-HOJAS et al, 2001;TANG et al, 2010;VERA et al, 2011).…”

“…Fatores como o armazenamento da amostra sanguínea antes da extração do DNA, o volume da amostra e os métodos utilizados no isolamento do DNA podem alterar consideravelmente a sensibilidade diagnóstica da PCR (MEDEIROS et al, 2015;MORALES-HOJAS et al, 2001). …”