Ultra-Sensitive Detection of Plasmodium falciparum by Amplification of Multi-Copy Subtelomeric Targets

Abstract

BackgroundPlanning 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 FindingsTwo quantitative PCR (qPCR) assays were developed for ultra-sensitive detection of Plasmodium falciparum, 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/μl 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 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.ConclusionsMeasured 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.

Treatment of Chronic Asymptomatic Plasmodium falciparum Infection Does Not Increase the Risk of Clinical Malaria Upon Reinfection

“…Recent studies in endemic areas have shown that more sensitive detection methods reveal larger reservoirs of asymptomatic P. falciparum infection than previously appreciated [44,45]. Therefore, it is possible that some subjects in this study had parasite densities below the detection limit of our PCR assay.…”

Assessing the impact of next-generation rapid diagnostic tests on Plasmodium falciparum malaria elimination strategies

“…This may be due to degradation of nucleic acids that disproportionally affect RNA-based diagnostics 3 or to a limitation in sensitivity regardless of sample handling. Gametocytaemic individuals with sub-PCR infection were also found in a cross-sectional survey in Tanzania using a new ultra-sensitive PCR technique 12 . These findings illustrate that the available diagnostics do not detect all infections that are present in populations and that onward malaria transmission is possible (although with a low probability) from apparently parasite-negative individuals.…”

“…The advent of highly sensitive molecular methods that detect parasite DNA or RNA such as polymerase chain reaction (PCR) 12 and quantitative nucleic acid sequence based amplification (QT-NASBA) has highlighted that many infected individuals have parasite densities that are too low to be detected either by microscopy or RDTs 13,14 . However, even these methods do not detect all the infections that are present in infected human hosts 12,15 and they are currently not available as routine field diagnostic tests because they require high-level laboratory facilities for nucleic acid extraction, amplification and detection that are unavailable in many resource-poor endemic settings. Even the most operationally attractive molecular diagnostic available -loop-mediated isothermal amplification For each value of the x-axis we calculate the proportion of each density distribution (from a) that would be detected.…”

“…The lower thresholds were chosen to reflect log orders of difference, while remaining a feasible target product profile for a new diagnostic test based on current technologies. A diagnostic threshold of 2 parasites per microlitre is currently achieved by LAMP and nested PCR 12 .…”

Improving the Specificity of Plasmodium falciparum Malaria Diagnosis in High-Transmission Settings with a Two-Step Rapid Diagnostic Test and Microscopy Algorithm

“…After confirmation of successful extraction via Qubit fluorescence (Thermo Fisher Scientific, Waltham, MA, USA), quantitative PCR was attempted to detect the P. falciparum LDH (PfLDH) loci (37). Upon amplification of positive controls but failure of experimental samples, the protocol was adjusted to accommodate an increase from the minimum 1 l of DNA to a maximum of 5 l. Following a second amplification failure, we changed assays to the ultrasensitive protocol targeting the var ATS sequence for qualitative detection to confirm the presence of P. falciparum DNA (38).…”