The Development of an External Quality Assessment System Using Inter-Laboratory Comparison for Real-Time PCR-Based Malaria Diagnosis in Regional Reference Laboratories under the Department of Disease Control, Thailand

Abstract

        Malaria remains a major global public health challenge, particularly in countries pursuing malaria elimination. Real-time polymerase chain reaction (Real-time PCR) is a highly sensitive and specific diagnostic method capable of detecting low-density and asymptomatic infections. However, a standardized External Quality Assessment (EQA) system for malaria diagnosis using Real-time PCR has not previously been established for the network of regional reference laboratories under Thailand’s Department of Disease Control (DDC). This study aimed to develop an External Quality Assessment system using an Inter-laboratory Comparison (ILC) approach and to evaluate the performance of regional reference laboratories for malaria diagnosis by Real-time PCR.

        A descriptive study was conducted between September 2022 and October 2025 involving 10 regional reference laboratories under the DDC. Dried blood spot (DBS) panels were developed from residual malaria-positive blood specimens representing five human malaria species (Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi) and negative controls. The DBS panels were evaluated for homogeneity and stability before being distributed for two rounds of ILC assessment. Participating laboratories performed malaria detection using their routine Real-time PCR protocols and reported species identification and cycle threshold (Ct) values. Laboratory performance was assessed using concordance rates, Z-scores, and diagnostic performance indicators.

        The developed DBS panels demonstrated acceptable homogeneity and stability, with Ct coefficient of variation values below 5% and Ct variation within ±1.0 cycle. Across two ILC rounds, the participating laboratories achieved a mean performance score of 99% (SD = 3.16). All laboratories obtained 100% accuracy in the first round, while nine laboratories achieved 100% accuracy in the second round and one laboratory obtained 80% due to a single discrepant result involving a low-concentration P. knowlesi specimen. Overall agreement among laboratories was excellent, with a concordance coefficient (Fleiss' Kappa) of 0.976 (p < 0.001), indicating almost perfect agreement. Most laboratories showed acceptable Z-scores ( ≤ 2), and Ct deviations remained within the predefined acceptance limits. Diagnostic performance analysis demonstrated 100% sensitivity, 95.24% specificity, 98.75% positive predictive value, and 100% negative predictive value.

        The ILC-based EQA system developed in this study is suitable for evaluatingthe performance of malaria Real-time PCR laboratories. The findings demonstrate a high level of inter-laboratory agreement and confirm the capability of Thailand’s regional reference laboratory network to provide reliable molecular diagnosis for malaria surveillance and elimination activities. This system also supports compliance with ISO 15189 requirements and strengthens confidence in laboratory data used for malaria control and elimination programs in Thailand.