Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples

Taylor, Brian J.; Martin, Kimberly; Arango, Eliana; Agudelo, Olga; Maestre, Amanda; Yanow, Stephanie K.

doi:10.1186/1475-2875-10-244

Cited by 44 publications

(47 citation statements)

References 20 publications

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“…Molecular methods, such as single-step PCR (13), loopmediated isothermal amplification (LAMP) assay (14), nested PCR (15,16), multiplex PCR (17,18), multiplex real-time PCR (19), and real time PCR (20)(21)(22)(23) basically use conventional PCR techniques to detect and identify malaria parasites. Even though PCR assay initially requires expensive equipment (a thermal cycler and gel electrophoresis tools and documentation), it is considered reasonably cheap in the long run for large-scale malaria detection and speciation, and it is highly sensitive in terms of detection (17), especially in regions where malaria is endemic.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of new multiplex PCR primers for the identification ofPlasmodium species found in Sabah, Malaysia

et al. 2016

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Background/aim: Malaria is a major public health problem, especially in the Southeast Asia region, caused by 5 species of Plasmodium (P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi). The aim of this study was to compare parasite species identification methods using the new multiplex polymerase chain reaction (PCR) against nested PCR and microscopy.Materials and methods: Blood samples on filter papers were subject to conventional PCR methods using primers designed by us in multiplex PCR and previously designed primers of nested PCR. Both sets of results were compared with microscopic identification.Results: Of the 129 samples identified as malaria-positive by microscopy, 15 samples were positive for P. falciparum, 14 for P. vivax, 6 for P. knowlesi, 72 for P. malariae, and 2 for mixed infection of P. falciparum/P. malariae. Both multiplex and nested PCR identified 12 P. falciparum single infections. For P. vivax, 9 were identified by multiplex and 12 by nested PCR. For 72 P. malariae cases, multiplex PCR identified 58 as P. knowlesi and 10 as P. malariae compared to nested PCR, which identified 59 as P. knowlesi and 7 as P. malariae. Conclusion:Multiplex PCR could be used as alternative molecular diagnosis for the identification of all Plasmodium species as it requires a shorter time to screen a large number of samples.

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Section: Introductionmentioning

confidence: 99%

Evaluation of new multiplex PCR primers for the identification ofPlasmodium species found in Sabah, Malaysia

et al. 2016

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“…Although real-time PCR and LAMP were recently reported to be able to detect plasmodia directly from whole blood (17,31), the sensitivities were compromised compared with those obtained using purified DNA. For example, the direct qPCR missed 5 positive samples out of 74 compared with conventional qPCR (31), and the detection limit of LAMP, when used directly on heated blood instead of DNA, changed from 1 to 10 parasites/l to 40 parasites/l (17), a sensitivity not significantly better than that of microscopy or RDT.…”

Section: Discussionmentioning

confidence: 96%

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

et al. 2013

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bAlthough malaria remains one of the leading infectious diseases in the world, the decline in malaria transmission in some area makes it possible to consider elimination of the disease. As countries approach elimination, malaria diagnosis needs to change from diagnosing ill patients to actively detecting infections in all carriers, including asymptomatic and low-parasite-load patients. However, few of the current diagnostic methods have both the throughput and the sensitivity required. We adopted a sandwich RNA hybridization assay to detect genus Plasmodium 18S rRNA directly from whole-blood samples from Plasmodium falciparum and Plasmodium vivax patients without RNA isolation. We tested the assay with 202 febrile patients from areas where malaria is endemic, using 20 l of each blood sample in a 96-well plate format with a 2-day enzyme-linked immunosorbent assay (ELISA)-like work flow. The results were compared with diagnoses obtained using microscopy, a rapid diagnostic test (RDT), and genus-specific real-time PCR. Our assay identified all 66 positive samples diagnosed by microscopy, including 49 poorly stored samples that underwent multiple freeze-thaw cycles due to resource limitation. The assay uncovered three falsenegative samples by microscopy and four false-negative samples by RDT and agreed completely with real-time PCR diagnosis. There was no negative sample by our assay that would show a positive result when tested with other methods. The detection limit of our assay for P. falciparum was 0.04 parasite/l. The assay's simple work flow, high throughput, and sensitivity make it suitable for active malaria screening.

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“…Currently, the detection of P. falciparum infection has required the method that has high sensitivity and specificity, especially for a screening of the asymptomatic patients and blood donation [21]. PCR has been developed to detect the malaria parasite for increasing the sensitivity and specificity, including semi-nested PCR [17], nested PCR [13,23,15], and real-time PCR [18].…”

Section: Discussionmentioning

confidence: 99%

“…Several PCR-based assays include conventional PCR [10], multiplex PCR [11], loop-mediated isothermal amplification (LAMP) [12], nested PCR [13][14][15][16], semi-nested PCR [17], and real-time PCR [18]. PCR has shown to be good alternative methods, because of the high sensitivity and specificity.…”

Section: Introductionmentioning

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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Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples

Cited by 44 publications

References 20 publications

Evaluation of new multiplex PCR primers for the identification ofPlasmodium species found in Sabah, Malaysia

Evaluation of new multiplex PCR primers for the identification ofPlasmodium species found in Sabah, Malaysia

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

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

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