Dataset Information

Ultra-sensitive detection of Plasmodium falciparum by amplification of multi-copy subtelomeric targets.

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, ?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 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.

SUBMITTER: Hofmann N

PROVIDER: S-EPMC4348198 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Publications

Ultra-sensitive detection of Plasmodium falciparum by amplification of multi-copy subtelomeric targets.

Hofmann Natalie N Mwingira Felista F Shekalaghe Seif S Robinson Leanne J LJ Mueller Ivo I Felger Ingrid I

PLoS medicine 20150303 3

<h4>Background</h4>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. ...[more]

PMID: 25734259

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Project description:BackgroundLoop-mediated isothermal amplification (LAMP) for malaria diagnosis at the point of care (POC) depends on the detection capacity of synthesized nucleic acids and the specificity of the amplification target. To improve malaria diagnosis, new colorimetric LAMP tests were developed using multicopy targets for Plasmodium vivax and Plasmodium falciparum detection.MethodsThe cytochrome oxidase I (COX1) mitochondrial gene and the non-coding sequence Pvr47 for P. vivax, and the sub-telomeric sequence of erythrocyte membrane protein 1 (EMP1) and the non-coding sequence Pfr364 for P. falciparum were targeted to design new LAMP primers. The limit of detection (LOD) of each colorimetric LAMP was established and assessed with DNA extracted by mini spin column kit and the Boil & Spin method from 28 microscopy infections, 101 malaria submicroscopic infections detected by real-time PCR only, and 183 negatives infections by both microscopy and PCR.ResultsThe LODs for the colorimetric LAMPs were estimated between 2.4 to 3.7 parasites/µL of whole blood. For P. vivax detection, the colorimetric LAMP using the COX1 target showed a better performance than the Pvr47 target, whereas the Pfr364 target was the most specific for P. falciparum detection. All microscopic infections of P. vivax were detected by PvCOX1-LAMP using the mini spin column kit DNA extraction method and 81% (17/21) were detected using Boil & Spin sample preparation. Moreover, all microscopic infections of P. falciparum were detected by Pfr364-LAMP using both sample preparation methods. In total, PvCOX1-LAMP and Pfr364-LAMP detected 80.2% (81 samples) of the submicroscopic infections using the DNA extraction method by mini spin column kit, while 36.6% (37 samples) were detected using the Boil & Spin sample preparation method.ConclusionThe colorimetric LAMPs with multicopy targets using the COX1 target for P. vivax and the Pfr364 for P. falciparum have a high potential to improve POC malaria diagnosis detecting a greater number of submicroscopic Plasmodium infections.

Project description:BACKGROUND:The use of molecular diagnostics has revealed an unexpectedly large number of asymptomatic low-density malaria infections in many malaria endemic areas. This study compared the gains in parasite prevalence obtained by the use of ultra-sensitive (us)-qPCR as compared to standard qPCR in cross-sectional surveys conducted in Thailand, Brazil and Papua New Guinea (PNG). The compared assays differed in the copy number of qPCR targets in the parasite genome. METHODS:Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) parasites were quantified by qPCR amplifying the low-copy Pf_ and Pv_18S rRNA genes or the multi-copy targets Pf_varATS and Pv_mtCOX1. Cross-sectional surveys at the three study sites included 2252 participants of all ages and represented different transmission intensities. RESULTS:In the two low-transmission areas, P. falciparum positivity was 1.3% (10/773) (Thailand) and 0.8% (5/651) (Brazil) using standard Pf_18S rRNA qPCR. In these two countries, P. falciparum positivity by Pf_varATS us-qPCR increased to 1.9% (15/773) and 1.7% (11/651). In PNG, an area with moderate transmission intensity, P. falciparum positivity significantly increased from 8.6% (71/828) by standard qPCR to 12.2% (101/828) by us-qPCR. The proportions of P. falciparum infections not detected by standard qPCR were 33%, 55% and 30% in Thailand, Brazil and PNG. Plasmodium vivax was the predominating species in Thailand and Brazil, with 3.9% (30/773) and 4.9% (32/651) positivity by Pv_18S rRNA qPCR. In PNG, P. vivax positivity was similar to P. falciparum, at 8.0% (66/828). Use of Pv_mtCOX1 us-qPCR led to a significant increase in positivity to 5.1% (39/773), 6.4% (42/651) and 11.5% (95/828) in Thailand, Brazil, and PNG. The proportions of P. vivax infections missed by standard qPCR were similar at all three sites, with 23%, 24% and 31% in Thailand, Brazil and PNG. CONCLUSION:The proportional gains in the detection of P. falciparum and P. vivax infections by ultra-sensitive diagnostic assays were substantial at all three study sites. Thus, us-qPCR yields more precise prevalence estimates for both P. falciparum and P. vivax at all studied levels of endemicity and represents a significant diagnostic improvement. Improving sensitivity in P. vivax surveillance by us-qPCR is of particular benefit, because the additionally detected P. vivax infections signal the potential presence of hypnozoites and subsequent risk of relapse and further transmission.

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Dataset Information

Ultra-sensitive detection of Plasmodium falciparum by amplification of multi-copy subtelomeric targets.

Background

Methods and findings

Conclusions

Publications

Ultra-sensitive detection of Plasmodium falciparum by amplification of multi-copy subtelomeric targets.

Similar Datasets

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