CASE PRESENTATION: An army officer who returned from Malaysia in October 2016 was found to be positive for Plasmodium both by microscopy and rapid diagnostic test (RDT) by the Anti Malaria Campaign Sri Lanka (AMC) during his third visit to a health care provider. Microscopy findings were suspicious of P. knowlesi infection as the smears showed parasite stages similar to both Plasmodium malariae and Plasmodium falciparum. Nested PCR at AMC confirmed Plasmodium genus, but not the species. In the absence of species confirmation, the patient was treated as a case of P. falciparum. The presence of P. knowlesi was later confirmed by a semi-nested PCR assay performed at the Environmental Health Institute, National Environmental Agency in Singapore. The parasite strain was also characterized by sequencing the circumsporozoite gene. Extensive case investigation including parasitological and entomological surveillance was carried out.
CONCLUSIONS: Plasmodium knowlesi should be suspected in patients returning from countries in the South Asian region where the parasite is prevalent and when blood smear results are inconclusive.
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 Plasmodium vivax, 21 Plasmodium falciparum, and 10 Plasmodium 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.
METHODS: Ten RDTs were evaluated: nine to detect clinical P. knowlesi infections from Malaysia, and nine assessing limit of detection (LoD) for P. knowlesi (PkA1-H.1) and P. falciparum (Pf3D7) cultures. Targets included Plasmodium-genus parasite lactate dehydrogenase (pan-pLDH) and P. vivax (Pv)-pLDH.
RESULTS: Samples were collected prior to antimalarial treatment from 127 patients with microscopy-positive PCR-confirmed P. knowlesi mono-infections. Median parasitaemia was 788/µL (IQR 247-5,565/µL). Pan-pLDH sensitivities ranged from 50.6% (95% CI 39.6-61.5) (SD BIOLINE) to 87.0% (95% CI 75.1-94.6) (First Response® and CareStart™ PAN) compared to reference PCR. Pv-pLDH RDTs detected P. knowlesi with up to 92.0% (95% CI 84.3-96.7%) sensitivity (Biocredit™). For parasite counts ≥200/µL, pan-pLDH (Standard Q) and Pv-pLDH RDTs exceeded 95% sensitivity. Specificity of RDTs against 26 PCR-confirmed negative controls was 100%. Sensitivity of six highest performing RDTs were not significantly different when comparing samples taken before and after (median 3 hours) antimalarial treatment. Parasite ring stages were present in 30% of pre-treatment samples, with ring stage proportions (mean 1.9%) demonstrating inverse correlation with test positivity of Biocredit™ and two CareStart™ RDTs.For cultured P. knowlesi, CareStart™ PAN demonstrated the lowest LoD at 25 parasites/µL; LoDs of other pan-pLDH ranged from 98 to >2000 parasites/µL. Pv-pLDH LoD for P. knowlesi was 49 parasites/µL. No false-positive results were observed in either P. falciparum-pLDH or histidine-rich-protein-2 channels.
CONCLUSION: Selected RDTs demonstrate sufficient performance for detection of major human malaria species including P. knowlesi in co-endemic areas where microscopy is not available, particularly for higher parasite counts, although cannot reliably differentiate among non-falciparum malaria.
RESULTS: A noticeable variation between the RDT (Alltest Biotech, China) and nPCR results was observed, for RDT 78% (46/59) were P. falciparum positive, 6.8% (4/59) were co-infected with both P. falciparum and Plasmodium vivax, 15.3% (9/59) were negative by the RDT. However, when the nPCR was applied only 44.1% (26/59) and 55.9% (33/59) was P. falciparum positive and negative respectively. The pfhrp2 was further amplified form all nPCR positive samples. Only 17 DNA samples were positive from the 26 positive P. falciparum, interestingly, variation in band sizes was observed and further confirmed by DNA sequencing, and sequencing analysis revealed a high-level of genetic diversity of the pfhrp2 gene in the parasite population from the study area. However, despite extreme sequence variation, diversity of PfHRP2 does not appear to affect RDT performance.