Towards high-throughput molecular detection of Plasmodium: new approaches and molecular markers

Steenkeste, Nicolas; Incardona, Sandra; Chy, Sophy; Duval, Linda; Ekala, Marie-Thérèse; Lim, Pharath; Hewitt, Sean; Sochantha, Tho; Socheat, Doung; Rogier, Christophe; Mercereau‐Puijalon, Odile; Fandeur, Thierry; Ariey, Frédéric

doi:10.1186/1475-2875-8-86

Cited by 113 publications

(129 citation statements)

References 39 publications

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“…Briefly, DNA was extracted from sets of 10 pooled samples, and subsequently from all individual samples in pools testing positive. At each stage, a nested PCR reaction using primers common to the cytochrome b genes of the four major human malaria species, 8 followed by an AluI restriction digest to distinguish species, was performed (cytbPCR). 9 Duplex quantitative PCR targeting the human β tubulin gene and the plasmodial methionine transfer *Address correspondence to Kimberly A. Baltzell, Departments of Family Health Care Nursing and Global Health Sciences, University of California, San Francisco, 2 Koret Way, N-431M, San Francisco, CA 94143.…”

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Prevalence of PCR Detectable Malaria Infection among Febrile Patients with a Negative Plasmodium falciparum Specific Rapid Diagnostic Test in Zanzibar

Baltzell¹,

Shakely²,

Hsiang³

et al. 2013

The American Society of Tropical Medicine and Hygiene

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Abstract. We screened for malaria in 594 blood samples from febrile patients who tested negative by a Plasmodium falciparum-specific histidine-rich protein-2-based rapid diagnostic test at 12 health facilities in Zanzibar districts North A and Micheweni, from May to August 2010. Screening was with microscopy, polymerase chain reaction (PCR) targeting the cytochrome b gene (cytbPCR) of the four major human malaria species, and quantitative PCR (qPCR). The prevalence of cytbPCR-detectable malaria infection was 2% (12 of 594), including 8 P. falciparum, 3 Plasmodium malariae, and 1 Plasmodium vivax infections. Microscopy identified 4 of 8 P. falciparum infections. Parasite density as estimated by microscopy or qPCR was 4,000 parasites/μL in 5 of 8 cytbPCR-detectable P. falciparum infections. The infections that were missed by the rapid diagnostic test represent a particular challenge in malaria elimination settings and highlight the need for more sensitive point-of-care diagnostic tools to improve case detection of all human malaria species in febrile patients.Zanzibar has been the site of a substantial recent effort to reduce the overall burden of malaria. With these efforts, the prevalence of parasitologically confirmed malaria infection among febrile children presenting at primary health care facilities in Zanzibar has decreased from~25% in 2003 1 to 2% in 2010 (Shakely and others, unpublished data). Historically, Plasmodium falciparum has played a dominant role in malarial illness in Zanzibar, and elsewhere in sub-Saharan Africa, causing well over 90% of episodes of the disease 2,3 ; the remaining reported malaria infections in Zanzibar in recent years have been caused by Plasmodium malariae. 2Malaria rapid diagnostic tests (RDTs) that detect parasite antigens have improved the availability of parasite-based diagnosis for rural clinics in Africa. 4 However, the RDT currently used in most of sub-Saharan Africa and in Zanzibar at the time of this study detects histidine-rich protein-2 (HRP2), which is specific to P. falciparum, and does not detect other species of malaria parasites. 5The availability of highly sensitive molecular techniques provides an opportunity to better characterize the species of Plasmodia causing malaria in regions of sub-Saharan Africa experiencing decreasing P. falciparum transmission. The aim of this study was to assess the prevalence of polymerase chain reaction (PCR)-detectable malaria infection among febrile patients with a negative P. falciparum-specific RDT in Zanzibar.Samples for this study were from an RDT effectiveness trial (Shakely and others, unpublished data) performed in 12 Zanzibar primary health care facilities, six each in North A and Micheweni districts over a 12-week period during the high transmission season from May to August 2010. Inclusion criteria in this study were: age 2 months, a measured axillary temperature 37.5 C or history of fever in the preceding 24 hours, and absence of any danger signs of severe disease. 6After obtaining informed written consent, dried b...

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confidence: 99%

Prevalence of PCR Detectable Malaria Infection among Febrile Patients with a Negative Plasmodium falciparum Specific Rapid Diagnostic Test in Zanzibar

Baltzell¹,

Shakely²,

Hsiang³

et al. 2013

The American Society of Tropical Medicine and Hygiene

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“…This may be an effect of low malaria parasite presented in the samples, and it may be possible that the DNA templates of these 5 samples have some sequence variation, so the primer was not annealed with the DNA templates. In addition, several studies were described inconsistencies in malaria positive when reaction was performed using the nested PCR including lower of DNA yield from blood spot and presence of blood inhibitors such as hemoglobin, hem, immunoglobulin G, and lactoferrin [27,28].…”

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confidence: 99%

Evaluation of Two Nested PCR-Based Diagnostic Assays for Plasmodium Falciparum Infection

Kritsiriwuthinan

Choosang

Sunantaraporn

2017

Asian J Pharm Clin Res

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Objective: The majority of malaria cases and deaths are caused by Plasmodium falciparum. The rapid and accurate diagnosis is very important for malaria treatment and control. The aim of this study was to evaluate two nested polymerase chain reaction (PCR)-based methods (protocol A and protocol B) for P. falciparum infection, diagnosis in Thailand.Methods: A total of 90 dried blood spot samples were investigated. The samples composed of P. falciparum-, Plasmodium vivax-infected blood and normal human blood samples. The microscopic examination was used as gold standard. Results:The results showed the sensitivity of 100/83.33%, specificity of 100/100%, and accuracy of 100/94.44% for protocol A and protocol B, respectively. The analytical sensitivity of protocol A and protocol B was 0.625 and 6.25 parasites/µl, respectively. The comparison among microscopic examination, protocol A and protocol B by statistical analysis, found that they were not a significant difference. The agreements between each method were good. The kappa value between protocol A and protocol B was 0.87, protocol A and microscopy was 1.00, and protocol B and microscopy was 0.87. Conclusion:The results demonstrated that protocol A should be used for further development of P. falciparum diagnosis in Thailand, especially in case of low parasitemia such as asymptomatic infection and for screening blood donors.

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“…Due to the recent advancements in technology, several malaria diagnostic techniques such as microarray [17], PCR [18], loop-mediated isothermal amplification (LAMP) [19], flow cytometry [20], hemozoin detection using automated hematology analyser [21] have been developed for efficient malaria diagnosis. Diagnostic methods leveraging PCR, 18s-rRNA detection [22], mitochondrial cytochrome b activity [23,24], PgMt19 and PfMT869 mitochondrial regions [25], and the Pvr47 and Pfr364 genes [26,27], have been used for detecting Plasmodium species. However, quantitative amplification of genes demands careful processing of blood to remove the inhibitors of amplification and thermal cycling, making it a cumbersome procedure.…”

Section: Introductionmentioning

confidence: 99%

<strong>A portable image-based cytometer for rapid malaria detection and quantification</strong>

Yang

Subramanian

Duan

et al. 2017

Proceedings of the 7th International Multidisciplinary Conference on Optofluidics 2017

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Increasing resistance by malaria parasites to currently used antimalarials across the developing world warrants timely detection and classification so that appropriate drug combinations can be administered before clinical complications arise. However, this is often challenged by low levels of infection (referred to as parasitemia) and presence of predominantly young parasitic forms in the patients' peripheral blood. Herein, we developed a simple, inexpensive and portable image-based cytometer that detects and numerically counts Plasmodium falciparum infected red blood cells (iRBCs) from Giemsa-stained smears derived from infected blood. Our cytometer is able to classify all parasitic subpopulations by quantifying the area occupied by the parasites within iRBCs, with high specificity, sensitivity and negligible false positives (~0.0025%). Moreover, we demonstrate the application of our image-based cytometer in testing anti-malarial efficacy against a commercial flow cytometer and demonstrate comparable results between the two methods. Collectively, these results highlight the possibility to use our image-based cytometer as a cheap, rapid and accurate alternative for antimalarial testing without compromising on efficiency and minimal processing time. With appropriate filters applied into the algorithm, to rule out leukocytes and reticulocytes, our cytometer may also be used for field diagnosis of malaria.

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Towards high-throughput molecular detection of Plasmodium: new approaches and molecular markers

Cited by 113 publications

References 39 publications

Prevalence of PCR Detectable Malaria Infection among Febrile Patients with a Negative Plasmodium falciparum Specific Rapid Diagnostic Test in Zanzibar

Prevalence of PCR Detectable Malaria Infection among Febrile Patients with a Negative Plasmodium falciparum Specific Rapid Diagnostic Test in Zanzibar

Evaluation of Two Nested PCR-Based Diagnostic Assays for Plasmodium Falciparum Infection

<strong>A portable image-based cytometer for rapid malaria detection and quantification</strong>

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