Development of High Resolution Melting Analysis for the Diagnosis of Human Malaria

Chua, Kek Heng; Lim, Siew Chee; Ng, Ching Ching; Lee, Ping Chin; Lim, Yvonne Ai Lian; Lau, Tze Pheng; Chai, Hwa Chia

doi:10.1038/srep15671

Cited by 54 publications

(69 citation statements)

References 54 publications

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“…The rnl real-time PCR-HRM delivered the same results as ITS sequencing (Figure 2 and Table S4). Real-time PCR-HRM assays for species identification have previously been established for bacteria [46,47], protozoans [48], and other fungi [38,[49][50][51][52][53][54][55]. Bialek et al (2005) [56] published an alternative PCR-HRM approach using 18 S rDNA primers and an intercalating dye, similar to our study.…”

Section: Discussionmentioning

confidence: 54%

Evaluation of a Novel Mitochondrial Pan-Mucorales Marker for the Detection, Identification, Quantification, and Growth Stage Determination of Mucormycetes

Caramalho

Madl

Rosam

et al. 2019

JoF

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Mucormycosis infections are infrequent yet aggressive and serious fungal infections. Early diagnosis of mucormycosis and its discrimination from other fungal infections is required for targeted treatment and more favorable patient outcomes. The majority of the molecular assays use 18 S rDNA. In the current study, we aimed to explore the potential of the mitochondrial rnl (encoding for large-subunit-ribosomal-RNA) gene as a novel molecular marker suitable for research and diagnostics. Rnl was evaluated as a marker for: (1) the Mucorales family, (2) species identification (Rhizopus arrhizus, R. microsporus, Mucor circinelloides, and Lichtheimia species complexes), (3) growth stage, and (4) quantification. Sensitivity, specificity, discriminatory power, the limit of detection (LoD), and cross-reactivity were evaluated. Assays were tested using pure cultures, spiked clinical samples, murine organs, and human paraffin-embedded-tissue (FFPE) samples. Mitochondrial markers were found to be superior to nuclear markers for degraded samples. Rnl outperformed the UMD universal ® (Molyzm) marker in FFPE (71.5% positive samples versus 50%). Spiked blood samples highlighted the potential of rnl as a pan-Mucorales screening test. Fungal burden was reproducibly quantified in murine organs using standard curves. Identification of pure cultures gave a perfect (100%) correlation with the detected internal transcribed spacer (ITS) sequence. In conclusion, mitochondrial genes, such as rnl, provide an alternative to the nuclear 18 S rDNA genes and deserve further evaluation.

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Section: Discussionmentioning

confidence: 54%

Evaluation of a Novel Mitochondrial Pan-Mucorales Marker for the Detection, Identification, Quantification, and Growth Stage Determination of Mucormycetes

Caramalho

Madl

Rosam

et al. 2019

JoF

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“…malariae result for a P. malariae monoinfection of 224 parasites/µL, the corresponding abTES™ result was positive for P. malariae only. This anomaly may have been caused by unintended annealing of P. knowlesi-speci c probes onto P. malariae genomic DNA [46]; a plausible scenario given primers used for the QuantiFast™ P. knowlesi monoplex are not Plasmodium species-speci c. The QuantiFast™ assay also demonstrated a false-positive mixed P. falciparum/P. vivax result from a P. vivax monoinfection during the LOD analysis at low-level parasitaemia (2 and 20 parasites/µL).…”

Section: Discussionmentioning

confidence: 99%

Comparative evaluation of two commercial real-time PCR kits (QuantiFast™ and abTES™) for the detection of Plasmodium knowlesi and other Plasmodium species in Sabah, Malaysia

Nuin¹,

Tan

Lew³

et al. 2020

Preprint

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Background : The monkey parasite Plasmodium knowlesi is an emerging public health issue in Southeast Asia. In Sabah, Malaysia, P. knowlesi is now the dominant cause of human malaria. Molecular detection methods for P. knowlesi are essential for accurate diagnosis and in monitoring progress towards malaria elimination of other Plasmodium species. However, recent commercially available PCR malaria kits have unpublished P. knowlesi gene targets or have not been evaluated against clinical samples. Methods : Two real-time PCR methods currently used in Sabah for confirmatory malaria diagnosis and surveillance reporting were evaluated: the QuantiFast™ Multiplex PCR kit (Qiagen, Germany) targeting the P. knowlesi 18S SSU rRNA; and the abTES™ Malaria 5 qPCR II kit (AITbiotech, Singapore), with an undisclosed P. knowlesi gene target. Diagnostic accuracy was evaluated using 52 P. knowlesi, 25 P. vivax , 21 P. falciparum , and 10 P. malariae clinical isolates, and 26 malaria negative controls, and compared against a validated reference nested PCR assay. The limit of detection (LOD) for each PCR method and Plasmodium species was also evaluated. Results : The sensitivity of the QuantiFast™ and abTES™ assays for detecting P. knowlesi was comparable at 98.1% (95%CI 89.7-100) and 100% (95%CI 93.2-100) respectively. Specificity of the QuantiFast™ and abTES™ for P. knowlesi was high at 98.8% (95%CI 93.4-100) for both assays. The QuantiFast™ assay demonstrated falsely-positive mixed Plasmodium species at low parasitaemias in both the primary and LOD analysis. Diagnostic accuracy of both PCR kits for detecting P. vivax , P. falciparum , and P. malariae was comparable to P. knowlesi . The abTES™ assay demonstrated a lower LOD for P. knowlesi of ≤0.125 parasites/µL compared to QuantiFast™ with a LOD of 20 parasites/µL. Hospital microscopy demonstrated a sensitivity of 78.8% (95%CI 65.3-88.9) and specificity of 80.4% (95%CI 67.6-89.8) compared to reference PCR for detecting P. knowlesi . Conclusion : The QuantiFast™ and abTES™ commercial PCR kits performed well for the accurate detection of P. knowlesi infections. Although the QuantiFast™ kit is cheaper, the abTES™ kit demonstrated a lower LOD, supporting its use as a second-line referral-laboratory diagnostic tool in Sabah, Malaysia.

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“…namely P. malariae or P. knowlesi. Furthermore, of the 28 studies, ten studies (12,24,25,28,31,34,41,52,61,63) conducted PCR in the whole samples regardless of the microscopy results in order to trace the sub-microscopic infections. In addition, 18 studies utilised only one method of detection for malaria.…”

Section: Description Of Included Studiesmentioning

confidence: 99%

Malaria distribution and performance of malaria diagnostic methods in Malaysia (1980-2019): a systematic review

Rahim

Munajat

Idris

2020

Preprint

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Background: Malaysia has already achieved remarkable accomplishments in reaching zero indigenous human malaria cases in 2018. Prompt malaria diagnosis, surveillance and treatment played a key role in the country’s elimination success. Looking at the dynamics of malaria distribution during the last decades might provide important information regarding the potential challenges of such an elimination strategy. This study was performed to gather all data available in term of prevalence or incidence on Plasmodium infections in Malaysia over the last four decades. Methods: A systematic review of the published English literature was conducted to identify malaria distribution from 1980 to June 2019 in Malaysia. Two investigators independently extracted data from PubMed, Scopus, Web of Science and Elsevier databases for original papers.Results: The review identified 46 epidemiological studies in Malaysia over the 39-year study period, on which sufficient information was available. The majority of studies were conducted in Malaysia Borneo (31/46; 67.4%), followed by Peninsular Malaysia (13/46; 28.3%) and in both areas (2/46; 4.3%). More than half of all studies (28/46; 60.9%) were assessed by both microscopy and PCR. Furthermore, there was a clear trend of decreases of all human malaria species with increasing P. knowlesi cases throughout the year of sampling period. The summary estimates of sensitivity were higher for P. knowlesi than other malaria species for both microscopy and PCR. Nevertheless, the specificities of summary estimates were similar for microscopy (40 – 43%) but varied for PCR (2 – 34%).Conclusions: This study outlined the epidemiological changes in Plasmodium species distribution in Malaysia. Malaria cases shifted from predominantly caused by human malaria to simian malaria, which accounted for the majority of indigenous cases particularly in Malaysia Borneo. Therefore, malaria case notification and prompt malaria diagnosis in regions where health services are limited in Malaysia should be strengthened and reinforced to achieving the final goal of malaria elimination in the country.

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Development of High Resolution Melting Analysis for the Diagnosis of Human Malaria

Cited by 54 publications

References 54 publications

Evaluation of a Novel Mitochondrial Pan-Mucorales Marker for the Detection, Identification, Quantification, and Growth Stage Determination of Mucormycetes

Evaluation of a Novel Mitochondrial Pan-Mucorales Marker for the Detection, Identification, Quantification, and Growth Stage Determination of Mucormycetes

Comparative evaluation of two commercial real-time PCR kits (QuantiFast™ and abTES™) for the detection of Plasmodium knowlesi and other Plasmodium species in Sabah, Malaysia

Malaria distribution and performance of malaria diagnostic methods in Malaysia (1980-2019): a systematic review

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