Multiplex real-time PCR for diagnosing malaria in a non-endemic setting: a prospective comparison to conventional methods

Nijhuis, Roel H. T.; Lieshout, Lisette van; Verweij, Jaco J.; Claas, Eric C. J.; Wessels, Els

doi:10.1007/s10096-018-3378-4

Cited by 14 publications

(9 citation statements)

References 16 publications

(26 reference statements)

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“…Among the total febrile study participants in this study, a larger proportion of positive cases were identified by multiplex real-time PCR than by RDT and microscopy. However, the study was conducted in non-endemic settings comparing the three methods, microscopy and RDT missed fewer cases, reported by Nijhuis et al [33]. This might be explained by the fact that the prevalence of sub-microscopic malaria cases increases in endemic settings than in non-endemic settings.…”

Section: Discussionmentioning

confidence: 88%

Evaluating performance of multiplex real time PCR for the diagnosis of malaria at elimination targeted low transmission settings of Ethiopia

Belachew

Wolde

Nega

et al. 2022

Malar J

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Background Malaria incidence has declined in Ethiopia in the past 10 years. Current malaria diagnostic tests, including light microscopy and rapid antigen-detecting diagnostic tests (RDTs) cannot reliably detect low-density infections. Studies have shown that nucleic acid amplification tests are highly sensitive and specific in detecting malaria infection. This study took place with the aim of evaluating the performance of multiplex real time PCR for the diagnosis of malaria using patient samples collected from health facilities located at malaria elimination targeted low transmission settings in Ethiopia. Methods A health facility-based, cross-sectional survey was conducted in selected malaria sentinel sites. Malaria-suspected febrile outpatients referred to laboratory for malaria testing between December 2019 and March 2020 was enrolled into this study. Sociodemographic information and capillary blood samples were collected from the study participants and tested at spot with RDTs. Additionally, five circles of dry blood spot (DBS) samples on Whatman filter paper and thick and thin smear were prepared for molecular testing and microscopic examination, respectively. Multiplex real time PCR assay was performed at Ethiopian Public Health Institute (EPHI) malaria laboratory. The performance of multiplex real time PCR assay, microscopy and RDT for the diagnosis of malaria was compared and evaluated against each other. Results Out of 271 blood samples, multiplex real time PCR identified 69 malaria cases as Plasmodium falciparum infection, 16 as Plasmodium vivax and 3 as mixed infections. Of the total samples, light microscopy detected 33 as P. falciparum, 18 as P. vivax, and RDT detected 43 as P. falciparum, 17 as P. vivax, and one mixed infection. Using light microscopy as reference test, the sensitivity and specificity of multiplex real time PCR were 100% (95% CI (93–100)) and 83.2% (95% CI (77.6–87.9)), respectively. Using multiplex real time PCR as a reference, light microscopy and RDT had sensitivity of 58% (95% CI 46.9–68.4) and 67% (95% CI 56.2–76.7); and 100% (95% CI 98–100) and 98.9% (95% CI 96–99.9), respectively. Substantial level of agreement was reported between microscopy and multiplex real time PCR results with kappa value of 0.65. Conclusions Multiplex real-time PCR had an advanced performance in parasite detection and species identification on febrile patients’ samples than did microscopy and RDT in low malaria transmission settings. It is highly sensitive malaria diagnostic method that can be used in malaria elimination programme, particularly for community based epidemiological samples. Although microscopy and RDT had reduced performance when compared to multiplex real time PCR, still had an acceptable performance in diagnosis of malaria cases on patient samples at clinical facilities.

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Section: Discussionmentioning

confidence: 88%

Evaluating performance of multiplex real time PCR for the diagnosis of malaria at elimination targeted low transmission settings of Ethiopia

Belachew

Wolde

Nega

et al. 2022

Malar J

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“…Up till now, a multiplex RDT for the simultaneous detection of more than one species of Plasmodium has not been brought to the market. However, the anticipation that it may be possible in the future is heightened from the recently developed and tested multiplex application of two other diagnostic techniques founded on nucleic acid amplification to identify species of Plasmodium [49,50]. For the first, a real-time PCR for detecting human malaria in the nonendemic setting of the Netherlands was used in a routine diagnostic laboratory to screen blood samples provided by travelers and migrants from malaria-endemic regions [50].…”

Section: Discussionmentioning

confidence: 99%

Improving Accuracy of Malaria Diagnosis in Underserved Rural and Remote Endemic Areas of Sub-Saharan Africa: A Call to Develop Multiplexing Rapid Diagnostic Tests

Makanjuola

Taylor‐Robinson

2020

Scientifica

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Clinical infection with malaria, caused by parasites of the genus Plasmodium, is considered a serious medical condition with the potential to become a life-threatening emergency. This is especially relevant to low-income countries in tropical and subtropical regions of the world where high rates of malaria-related morbidity and mortality are recorded. As a means to combat this major global public health threat, rapid and effective diagnosis remains the frontline action to initiate a timely and appropriate medical intervention. From all the approaches to parasite detection, rapid diagnostic tests, so-called RDTs, are the easiest to use and most cost-effective. However, some of the limitations inherent in this methodology could hinder effective patient treatment. A primary drawback is that the vast majority of commercially available RDTs detect only one of the five species of human malaria, P. falciparum. While this is the main cause of infection in many areas, it excludes the possibility of infection with another parasite (P. vivax, P. ovale, P. malariae, and P. knowlesi) or of mixed infections containing different species. Hence, a diagnosis of non-P. falciparum malaria is missed. In turn, in resource-constrained settings where optimal microscopy is not available, a misdiagnosis of bacterial infection based on signs and symptoms alone often results in an inappropriate prescription of antibiotics. Here, we discuss how effective diagnosis of malaria and indiscriminate use of antibiotics in sub-Saharan Africa, a hot spot for P. falciparum transmission, may both be addressed by the development of innovative multiplexing RDTs that detect two or more species of Plasmodium.

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“…Moreover, molecular methods such as semi-nested PCR more accurately detected P. ovale mixed infections than microscopy 28 . With more advances in molecular methods for the detection and identification of malaria parasites, real-time PCR methods using fluorescent labels for detecting and quantifying DNA targets have been developed with high sensitivity and specificity 23 , 58 , 59 . Interestingly, multiplex real-time PCR failed to detect P. falciparum and P. ovale mixed infection in a previous study because of the high difference in the ratio between P. falciparum and P. ovale (> 1000:1) 32 .…”

Section: Discussionmentioning

confidence: 99%

Misidentification of Plasmodium ovale as Plasmodium vivax malaria by a microscopic method: a meta-analysis of confirmed P. ovale cases

Kotepui

Masangkay

Kotepui

et al. 2020

Sci Rep

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Plasmodium ovale is a benign tertian malaria parasite that morphologically resembles Plasmodium vivax. P. ovale also shares similar tertian periodicity and can cause relapse in patients without a radical cure, making it easily misidentified as P. vivax in routine diagnosis. Therefore, its prevalence might be underreported worldwide. The present study aimed to quantify the prevalence of P. ovale misidentified as P. vivax malaria using data from studies reporting confirmed P. ovale cases by molecular methods. Studies reporting the misidentification of P. ovale as P. vivax malaria were identified from three databases, MEDLINE, Web of Science, and Scopus, without language restrictions, but the publication date was restricted to 1993 and 2020. The quality of the included studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS). The random-effects model was used to estimate the pooled prevalence of the misidentification of P. ovale as P. vivax malaria by the microscopic method when compared to those with the reference polymerase chain reaction method. Subgroup analysis of participants was also performed to demonstrate the difference between imported and indigenous P. ovale cases. The heterogeneity of the included studies was assessed using Cochran's Q and I2 statistics. Publication bias across the included studies was assessed using the funnel plot and Egger’s test, and if required, contour-enhanced funnel plots were used to identify the source(s) of funnel plot asymmetry. Of 641 articles retrieved from databases, 22 articles met the eligibility criteria and were included in the present study. Of the 8,297 malaria-positive cases identified by the PCR method, 453 P. ovale cases were confirmed. The pooled prevalence of misidentification of P. ovale as P. vivax malaria by the microscopic method was 11% (95% CI: 7–14%, I2: 25.46%). Subgroup analysis of the participants demonstrated a higher prevalence of misidentification in indigenous cases (13%, 95% CI: 6–21%, I2: 27.8%) than in imported cases (10%, 95% CI: 6–14%, I2: 24.1%). The pooled prevalence of misidentification of P. vivax as P. ovale malaria by the microscopic method was 1%, without heterogeneity (95% CI: 0–3%, I2: 16.8%). PCR was more sensitive in identifying P. ovale cases than the microscopic method (p < 0.00001, OR: 2.76, 95% CI: 1.83–4.15, I2: 65%). Subgroup analysis of participants demonstrated the better performance of PCR in detecting P. ovale malaria in indigenous cases (p: 0.0009, OR: 6.92, 95% CI: 2.21–21.7%, I2: 68%) than in imported cases (p: 0.0004, OR: 2.15, 95% CI: 1.41–3.29%, I2: 63%). P. ovale infections misidentified as P. vivax malaria by the microscopic method were frequent and led to underreported P. ovale cases. The molecular identification of P. ovale malaria in endemic areas is needed because a higher rate of P. ovale misidentification was found in endemic or indigenous cases than in imported cases. In addition, updated courses, enhanced training, and refreshers for microscopic examinations, particularly for P. ovale identification, are necessary to improve the microscopic identification of Plasmodium species in rural health centres where PCR is unavailable.

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Multiplex real-time PCR for diagnosing malaria in a non-endemic setting: a prospective comparison to conventional methods

Cited by 14 publications

References 16 publications

Evaluating performance of multiplex real time PCR for the diagnosis of malaria at elimination targeted low transmission settings of Ethiopia

Evaluating performance of multiplex real time PCR for the diagnosis of malaria at elimination targeted low transmission settings of Ethiopia

Improving Accuracy of Malaria Diagnosis in Underserved Rural and Remote Endemic Areas of Sub-Saharan Africa: A Call to Develop Multiplexing Rapid Diagnostic Tests

Misidentification of Plasmodium ovale as Plasmodium vivax malaria by a microscopic method: a meta-analysis of confirmed P. ovale cases

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