Please use this identifier to cite or link to this item: http://hdl.handle.net/2445/68711
Title: Ultra-Sensitive Detection of Plasmodium falciparum by Amplification of Multi-Copy Subtelomeric Targets
Author: Hofmann, Natalie E.
Mwingira, Felista
Shekalaghe, Seif
Robinson, Leanne J.
Mueller, Ivo
Felger, Ingrid
Keywords: Malària
Plasmodium falciparum
Reacció en cadena de la polimerasa
Diagnòstic
Epidemiologia
Malaria
Plasmodium falciparum
Polymerase chain reaction
Diagnosis
Epidemiology
Issue Date: 3-Mar-2015
Publisher: Public Library of Science (PLoS)
Abstract: BACKGROUND: Planning and evaluating malaria control strategies relies on accurate definition of parasite prevalence in the population. A large proportion of asymptomatic parasite infections can only be identified by surveillance with molecular methods, yet these infections also contribute to onward transmission to mosquitoes. The sensitivity of molecular detection by PCR is limited by the abundance of the target sequence in a DNA sample; thus, detection becomes imperfect at low densities. We aimed to increase PCR diagnostic sensitivity by targeting multi-copy genomic sequences for reliable detection of low-density infections, and investigated the impact of these PCR assays on community prevalence data. METHODS AND FINDINGS: Two quantitative PCR (qPCR) assays were developed for ultra-sensitive detection of Plasmodium falciparum, targeting the high-copy telomere-associated repetitive element 2 (TARE-2, approximately 250 copies/genome) and the var gene acidic terminal sequence (varATS, 59 copies/genome). Our assays reached a limit of detection of 0.03 to 0.15 parasites/mul blood and were 10x more sensitive than standard 18S rRNA qPCR. In a population cross-sectional study in Tanzania, 295/498 samples tested positive using ultra-sensitive assays. Light microscopy missed 169 infections (57%). 18S rRNA qPCR failed to identify 48 infections (16%), of which 40% carried gametocytes detected by pfs25 quantitative reverse-transcription PCR. To judge the suitability of the TARE-2 and varATS assays for high-throughput screens, their performance was tested on sample pools. Both ultra-sensitive assays correctly detected all pools containing one low-density P. falciparum-positive sample, which went undetected by 18S rRNA qPCR, among nine negatives. TARE-2 and varATS qPCRs improve estimates of prevalence rates, yet other infections might still remain undetected when absent in the limited blood volume sampled. CONCLUSIONS: Measured malaria prevalence in communities is largely determined by the sensitivity of the diagnostic tool used. Even when applying standard molecular diagnostics, prevalence in our study population was underestimated by 8% compared to the new assays. Our findings highlight the need for highly sensitive tools such as TARE-2 and varATS qPCR in community surveillance and for monitoring interventions to better describe malaria epidemiology and inform malaria elimination efforts.
Note: Reproducció del document publicat a: http://dx.doi.org/10.1371/journal.pmed.1001788
It is part of: PLoS Medicine, 2015, vol. 12, num. 3, p. e1001788
Related resource: http://dx.doi.org/10.1371/journal.pmed.1001788
URI: http://hdl.handle.net/2445/68711
ISSN: 1549-1277
Appears in Collections:Articles publicats en revistes (ISGlobal)

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