A Novel, Sensitive Assay for High-Throughput Molecular Detection of Plasmodia for Active Screening of Malaria for Elimination

Cheng, Zhibin; Sun, Xin; Yang, Yiying; Wang, Heng; Zheng, Zhi

doi:10.1128/jcm.02010-12

Cited by 16 publications

(6 citation statements)

References 28 publications

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“…Of the current molecular detection methods available for malaria diagnosis (summarized in Table 1 ), RNA-based techniques such as quantitative reverse transcription PCR (qRT-PCR) [ 10 – 12 ], nucleic acid sequence-based amplification (NASBA) [ 13 – 15 ], and ELISA-like hybridization assays [ 16 ] reach the highest sensitivities by targeting the highly abundant 18S small subunit ribosomal RNA (18S rRNA). However, because of the unstable nature of RNA, these assays require dedicated and controlled sample collection and storage, and thus have only a limited application in field settings.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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“…Several NATs, including quantitative PCR (qPCR) combined with microfluidics [ 24 ], and those linked to enzyme-linked immunosorbent assays [ 25 – 27 ] as well as non-nucleic acid techniques [ 28 – 30 ] are in development for the purposes of malaria diagnosis but are not discussed further here. However, for even the most promising molecular technologies to be useful as point of care (POC) tests, significant challenges of portability, sample preparation, power supply and high throughput [ 31 ] have yet to be overcome.…”

Section: Introductionmentioning

confidence: 99%

Novel molecular diagnostic tools for malaria elimination: a review of options from the point of view of high-throughput and applicability in resource limited settings

Britton

Cheng²,

McCarthy

2016

Malar J

121

105

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As malaria transmission continues to decrease, an increasing number of countries will enter pre-elimination and elimination. To interrupt transmission, changes in control strategies are likely to require more accurate identification of all carriers of Plasmodium parasites, both symptomatic and asymptomatic, using diagnostic tools that are highly sensitive, high throughput and with fast turnaround times preferably performed in local health service settings. Currently available immunochromatographic lateral flow rapid diagnostic tests and field microscopy are unlikely to consistently detect infections at parasite densities less than 100 parasites/µL making them insufficiently sensitive for detecting all carriers. Molecular diagnostic platforms, such as PCR and LAMP, are currently available in reference laboratories, but at a cost both financially and in turnaround time. This review describes the recent progress in developing molecular diagnostic tools in terms of their capacity for high throughput and potential for performance in non-reference laboratories for malaria elimination.

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“…However, most require specialized equipment, reference laboratory availability, and are technically demanding with unsatisfactory turn-around-times. Several recently reported techniques with more satisfactory characteristics for malaria testing are PET-PCR [ 20 , 21 ], RNA Hybridization Assay [ 22 ], Ligase Detection Reaction-Fluorescent Microsphere (LDR-FM) [ 23 ], Loop Mediated Isothermal Amplification (LAMP) [ 24 ] and a malaria biosensor [ 25 ]. Reported sensitivities and specificities are up to 100 % with some having more advantageous turn-around-times.…”

Section: Introductionmentioning

confidence: 99%

Malaria elimination in Haiti by the year 2020: an achievable goal?

Boncy¹,

Adrien

Lemoine³

et al. 2015

Malar J

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Haiti and the Dominican Republic, which share the island of Hispaniola, are the last locations in the Caribbean where malaria still persists. Malaria is an important public health concern in Haiti with 17,094 reported cases in 2014. Further, on January 12, 2010, a record earthquake devastated densely populated areas in Haiti including many healthcare and laboratory facilities. Weakened infrastructure provided fertile reservoirs for uncontrolled transmission of infectious pathogens. This situation results in unique challenges for malaria epidemiology and elimination efforts. To help Haiti achieve its malaria elimination goals by year 2020, the Laboratoire National de Santé Publique and Henry Ford Health System, in close collaboration with the Direction d’Épidémiologie, de Laboratoire et de Recherches and the Programme National de Contrôle de la Malaria, hosted a scientific meeting on “Elimination Strategies for Malaria in Haiti” on January 29-30, 2015 at the National Laboratory in Port-au-Prince, Haiti. The meeting brought together laboratory personnel, researchers, clinicians, academics, public health professionals, and other stakeholders to discuss main stakes and perspectives on malaria elimination. Several themes and recommendations emerged during discussions at this meeting. First, more information and research on malaria transmission in Haiti are needed including information from active surveillance of cases and vectors. Second, many healthcare personnel need additional training and critical resources on how to properly identify malaria cases so as to improve accurate and timely case reporting. Third, it is necessary to continue studies genotyping strains of Plasmodium falciparum in different sites with active transmission to evaluate for drug resistance and impacts on health. Fourth, elimination strategies outlined in this report will continue to incorporate use of primaquine in addition to chloroquine and active surveillance of cases. Elimination of malaria in Haiti will require collaborative multidisciplinary approaches, sound strategic planning, and strong ownership of strategies by the Haiti Ministère de la Santé Publique et de la Population.

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A Novel, Sensitive Assay for High-Throughput Molecular Detection of Plasmodia for Active Screening of Malaria for Elimination

Cited by 16 publications

References 28 publications

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

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

Novel molecular diagnostic tools for malaria elimination: a review of options from the point of view of high-throughput and applicability in resource limited settings

Malaria elimination in Haiti by the year 2020: an achievable goal?

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