Ultra-Sensitive Detection of XXXX by Amplification of Multi-Copy Subtelomeric Targets.

@entity311 @entity281 @entity3964
BACKGROUND: Planning and evaluating @entity311 control strategies relies on accurate definition of parasite prevalence in the population. A large proportion of asymptomatic @entity281 can only be identified by surveillance with molecular methods, yet these @entity281 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 @entity281 , 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 @entity3964 , targeting the high-copy telomere-associated repetitive element 2 (TARE-2,    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/ l blood and were 10* 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 @entity281 (57%). 18S rRNA qPCR failed to identify 48 @entity281 (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 @entity3964 -positive sample, which went undetected by 18S rRNA qPCR, among nine negatives. TARE-2 and varATS qPCRs improve estimates of prevalence rates, yet other @entity281 might still remain undetected when absent in the limited blood volume sampled. CONCLUSIONS: Measured @entity311 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 @entity311 epidemiology and inform @entity311 elimination efforts.
