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


RESULTS
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/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.


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.
Swiss Tropical and Public Health Institute, Basel, Switzerland; University of Basel, Basel, Switzerland.
Natalie
N
Hofmann
Swiss Tropical and Public Health Institute, Basel, Switzerland; University of Basel, Basel, Switzerland; Biological Sciences Department, Dar es Salaam University College of Education, Dar es Salaam, Tanzania.
Felista
F
Mwingira
Ifakara Health Institute, Bagamoyo, Tanzania.
Seif
S
Shekalaghe
Papua New Guinea Institute of Medical Research, Madang and Maprik, Papua New Guinea; Walter and Eliza Hall Institute, Parkville, Victoria, Australia; Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia.
Leanne J
LJ
Robinson
Walter and Eliza Hall Institute, Parkville, Victoria, Australia; Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia; Centre de Recerca en Salut Internacional de Barcelona, Barcelona, Spain.
Ivo
I
Mueller
Swiss Tropical and Public Health Institute, Basel, Switzerland; University of Basel, Basel, Switzerland.
Ingrid
I
Felger
D003430:Cross-Sectional Studies; D016054:DNA, Protozoan / Q000097:blood*; D006801:Humans; D016778:Malaria, Falciparum / Q000175:diagnosis* / Q000453:epidemiology / Q000469:parasitology / Q000635:transmission; D008853:Microscopy; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D017720:Molecular Epidemiology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D010963:Plasmodium falciparum / Q000235:genetics*; D015995:Prevalence; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D013636:Tanzania / Q000453:epidemiology; D016615:Telomere
D016054:DNA, Protozoan; D012337:RNA, Ribosomal, 18S