Quality assurance in rapid diagnostic tests for accurate diagnosis of malaria

BackgroundMalaria remains a significant public health challenge in Nigeria, where diagnostic accuracy is essential for effective treatment and management. This systematic review and meta-analysis aimed to compare the diagnostic performance of malaria rapid diagnostic tests (RDTs) with that of microscopy, the gold standard for malaria diagnosis, in Nigeria.MethodsA systematic search of Medline (PubMed), Scopus, Web of Science, and Google Scholar and other manually searched articles yielded 502 records. After removing duplicates and screening, 15 studies met the inclusion criteria for the meta-analysis. The risk of bias and applicability were assessed via QUADAS-2. A random effects model was used to pool sensitivity, specificity, positive and negative likelihood ratios (LR + , LR–), diagnostic odds ratios (DORs), and summary receiver operating characteristic (SROC) curves, with analysis conducted via Meta-Disc 1.4 and the metafor package (version 4.6–0) in R (version 4.4.1). Publication bias was assessed via funnel plots and Egger’s test. Leave-one-out sensitivity analyses were conducted to assess the robustness of pooled estimates.ResultsThe pooled sensitivity of RDTs was 69.7% [95% confidence interval (CI)]: 67.7–71.6%, and the specificity was 81.5% (95% CI 79.9–83.1%). The DOR was 21.87 (95% CI 7.45–64.21). The SROC curve was 0.902, though heterogeneity was high (I2 = 96.8%). Subgroup analysis showed regional variability, with better performance in urban settings. Funnel plot asymmetry and Egger’s test suggested publication bias. Leave-one-out analyses confirmed robustness, with pooled estimates ranging 68–75% for sensitivity, 88–91% for specificity, and 17.7–27.6 for DOR.ConclusionMalaria RDTs in Nigeria show suboptimal sensitivity and specificity compared with WHO benchmarks. While RDTs remain the most practical frontline tool due to their accessibility and ease of use, their limitations highlight the need for strengthened diagnostic algorithms that integrate RDTs with microscopy or molecular methods where feasible. Leave-one-out sensitivity analyses confirmed the robustness of the pooled estimates, reinforcing the validity of the conclusions. Advancing RDT technology, improving microscopy practices, and ensuring consistent study reporting are essential to enhance malaria diagnosis and control efforts.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12936-025-05680-4.

Rapid diagnostic tests (RDTs) have revolutionized malaria diagnosis, playing a crucial role in improving timely treatment and supporting surveillance efforts, especially in resource-limited settings. However, the performance of RDTs can vary widely due to factors such as parasite genetic diversity, environmental conditions, and operational challenges. Understanding these variations is essential to ensuring accurate and reliable malaria diagnosis. This systematic review and meta-analysis critically evaluate the diagnostic performance of malaria RDTs across sub-Saharan Africa, identifying key gaps and proposing strategies for developing novel tests. By pooling data from 48 studies, the analysis quantifies the sensitivity and specificity of various RDT brands in different settings. The results reveal considerable variability, influenced by factors such as antigen persistence, cross-reactivity with other infections, and genetic polymorphism in the HRP2 gene, which can lead to false positives and negatives. The findings underscore the need for region-specific diagnostic strategies and the development of advanced diagnostic tools capable of detecting low-level parasitemia and differentiating between Plasmodium species. Emerging technologies and multi-platform approaches are recommended to enhance the accuracy and reliability of malaria diagnosis, ultimately contributing to more effective malaria control and elimination efforts in sub-Saharan Africa.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-97259-x.

Leptospirosis is an endemic zoonotic disease with protean clinical manifestations caused by pathogenic spirochetes of the Leptospira genus. The microscopic agglutination test (MAT) is the gold standard for leptospirosis diagnosis and can only be conducted in a reference laboratory. Therefore, alternative tests, such as the IgM anti-Leptospira rapid diagnostic test (RDT), are preferred for general use. In this study, we aimed to compare the accuracy of four products of anti-Leptospira IgM detection RDTs, which are available in Indonesia, against the gold standard of Leptospira MAT. This study was a diagnostic validation test using bioarchived serum from 364 human serum samples tested by MAT from August to September 2020 in Demak, Central Java, Indonesia. The four products were (i) Fokus Leptospira, (ii) Answer Leptospira, (iii) SD Bioline Leptospira IgG/IgM, and (iv) Uji Leptospira IgM, sequentially renamed RDT-1 to RDT-4. Interobserver agreements were analyzed using the kappa value. The diagnostic performance of the four RDTs were compared against MAT results as the gold standard. We also evaluated the combination of two RDTs' performance, which were RDT (1 + 2), RDT (1 + 3), RDT (1 + 4), RDT (2 + 4), and RDT (3 + 4). We found that the kappa coefficients of RDT-1, RDT-2, and RDT-4 were greater than 80%, while RDT-3 had a moderate kappa value of 69.1%. RDT-1, RDT-2, and RDT-4 had moderate to good sensitivities of 78.2%, 74.3%, and 83.6%, respectively, while RDT-3 had the lowest sensitivity at 30.9%. RDT-3 demonstrated the highest specificity. RDT-2 showed the highest predictive value at 75.9%, while RDT-4 showed the highest negative predictive value at 96.9%. In addition, the combination of two RTDs provided better diagnostic performances. The four RDTs performed varied in their ability to diagnose leptospirosis, but only RDT-4 showed a sensitivity of more than 80%. We recommend caution in diagnosing only one RDT result. Testing by other RDTs and confirmation by MAT are strongly recommended.IMPORTANCEThe performance of the anti-Leptospira IgM antibody rapid diagnostic tests (RDTs) has yet to be evaluated. In this study, we compare the accuracy of these four RDTs available in Indonesia against the gold standard of Leptospira microscopic agglutination test (MAT). Utilizing the best performance of RDT in the point of care with limited facilities will be effective since MAT is complicated, laborious, and time-consuming if done at public health centers. The MAT requires maintaining the culture of each live Leptospira strain circulating in the region and needs trained personnel.

The recent COVID-19 pandemic has profoundly impacted global malaria elimination programs, resulting in a sharp increase in malaria morbidity and mortality. To reduce this impact, unmet needs in malaria diagnostics must be addressed while resuming malaria elimination activities. Rapid diagnostic tests (RDTs), the unsung hero in malaria diagnosis, work to eliminate the prevalence of Plasmodium falciparum malaria through their efficient, cost-effective, and user-friendly qualities in detecting the antigen HRP2 (histidine-rich protein 2), among other proteins. However, the testing mechanism and management of malaria with RDTs presents a variety of limitations. This paper discusses the numerous factors (including parasitic, host, and environmental) that limit the performance of RDTs. Additionally, the paper explores outside factors that can hinder RDT performance. By understanding these factors that affect the performance of HRP2-based RDTs in the field, researchers can work toward creating and implementing more effective and accurate HRP2-based diagnostic tools. Further research is required to understand the extent of these factors, as the rapidly changing interplay between parasite and host directly hinders the effectiveness of the tool.

Background:Current malaria control strategies are based on early diagnosis and appropriate treatment of malaria cases. The study aimed at comparing the performance of blood film microscopy and rapid diagnostic test (RDT) in Plasmodium falciparum detection in patients ≥6 years of age.Materials and Methods:A total of 154 consecutive pyretic patients aged 6-62 years were enrolled, sampled, and tested for malaria using RDT (first response) and microscopy by Giemsa staining. Genomic DNA was extracted after saponin hemolysis and nested polymerase chain reaction (PCR) was used to detect Plasmodium falciparum. The endpoints were sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV).Results:Of the 154 patients, 80 (51.9%) had fever of ≥37.5°C. 106 (68.8%) were positive by First response®, 132 (85.7%) by microscopy, and 121 (78.6%) by PCR. The sensitivity, specificity, PPV, and NPV of first response compared to microscopic method were 82.2%, 100.0%, 100.0%, and 34.3%, respectively, while it was 75.4%, 75.0%, 95.3%, and 31.2%, respectively, when compared to PCR. The sensitivity, specificity, PPV, and NPV of the microscopic method compared to PCR were 92.3%, 50.0%, 90.91%, and 54.5%, respectively. There was a significant difference in the performance of RDT and film microscopy methods (P ≤ 0.05).Conclusion:Microscopy performed better and is more reliable than first response (RDT) in areas with low parasite density among patients ≥6 years of age. Rapid diagnostic tests could be useful in aareas with high parasite density as an alternative to smear microscopy

Quality laboratory services are a requisite to guide rational case management of malaria. Using a pre-tested, standardized assessment tool, we assessed laboratory diagnostic capacity in 69 primary, secondary and tertiary health facilities as well as specialized laboratories in five administrative zones in Oromia Regional State, Ethiopia, during February and March 2009. There was marked variability in laboratory diagnostic capacity among the facilities assessed. Of 69 facilities surveyed, 53 provided both comprehensive malaria laboratory diagnosis and outpatient treatment services, five provided malaria microscopy services (referring elsewhere for treatment), and 11 primary care health posts provided rapid diagnostic testing and outpatient malaria treatment. The facilities' median catchment population was 39, 562 and 3581 people for secondary/tertiary and primary health facilities, respectively. Depending on facility type, facilities provided services 24 hrs a day, had inpatient capacity, and access to water and electricity. Facilities were staffed by general practitioners, health officers, nurses or health extension workers. Of the 58 facilities providing laboratory services, 24% of the 159 laboratory staff had received malaria microscopy training in the year prior to this survey, and 72% of the facilities had at least one functional electric binocular microscope. Facilities had variable levels of equipment, materials and biosafety procedures necessary for laboratory diagnosis of malaria. The mean monthly number of malaria blood films processed at secondary/tertiary facilities was 225, with a mean monthly 56 confirmed parasitologically. In primary facilities, the mean monthly number of clinical malaria cases seen was 75, of which 57 were tested by rapid diagnostic test (RDTs). None of the surveyed laboratory facilities had formal quality assurance/quality control protocols for either microscopy or RDTs. This is the first published report on malaria diagnostic capacity in Ethiopia. While our assessment indicated that malaria laboratory diagnosis was available in most facilities surveyed, we observed significant gaps in laboratory services which could significantly impact quality and accessibility of malaria diagnosis, including laboratory infrastructure, equipment, laboratory supplies and human resources.

BackgroundMalaria rapid diagnostic tests (RDTs) are relatively simple to perform and provide results quickly for making treatment decisions. However, the accuracy and application of RDT results depends on several factors such as quality of the RDT, storage, transport and end user performance. A cross sectional survey to explore factors that affect the performance and use of RDTs was conducted in the primary care facilities in South Africa.MethodsThis study was conducted in three malaria risk sub-districts of the Limpopo Province, in South Africa. Twenty nurses were randomly selected from 17 primary health care facilities, three nurses from hospitals serving the study area and 10 other key informants, representing the managers of the malaria control programmes, routine and research laboratories, were interviewed, using semi-structured questionnaires.ResultsThere was a high degree of efficiency in ordering and distribution of RDTs, however only 13/20 (65%) of the health facilities had appropriate air-conditioning and monitoring of room temperatures. Sixty percent (12/20) of the nurses did not receive any external training on conducting and interpreting RDT. Fifty percent of nurses (10/20) reported RDT stock-outs. Only 3/20 nurses mentioned that they periodically checked quality of RDT. Fifteen percent of nurses reported giving antimalarial drugs even if the RDT was negative.ConclusionStorage, quality assurance, end user training and use of RDT results for clinical decision making in primary care facilities in South Africa need to be improved. Further studies of the factors influencing the quality control of RDTs, their performance of RDTs and the ways to improve their use of RDTs are needed.

Prompt and rapid diagnosis of malaria has gained importance in health programmes in endemic countries, and recognition of the importance of early, correct treatment to the reduction in malaria morbidity and mortality. Blood-based diagnosis using lateral-flow immunochromatographic tests, commonly called rapid diagnostics tests (RDTs), offer great promise in extending rapid diagnosis to areas where traditional microscopy is not accessible. Large-scale operational use has raised questions about the accuracy of current RDT technology in tropical conditions. As utilization of RDTs has increased rapidly in the last few years, there is a clear and urgent need to address issues on quality performance and appropriateness of use, particularly in remote endemic areas. The need for Quality Assurance (QA) systems to maintain the quality of microscopy diagnosis of malaria is well established but the extent of implementation varies widely. Quality assurance process must become an integral part of RDT budgets, procurement and implementation plans. Responsibility for overseeing QA processes, extending from post purchase testing of RDTs to training and supervision of users and control of storage and transport, should be clearly defined and coordinated from a central level. A system of regional and referral laboratories, based on standard operating procedures would test RDTs after purchase and for the duration of shelf-life using quality control (QC) panels prepared from wild-type parasites. The use of positive control wells (PCW) containing recombinant antigens would assist in assuring the performance of RDTs in the field and this would assist in the parasite-base confirmation and in the case management of malaria. Quality assurance processes must be transparent, and good communication with manufacturers and end-users during QA development is necessary.

Background While rapid diagnostic tests are an alternative diagnostic tests for microscopy in the diagnosis of malaria in rural settings, their performance has been inconsistent. Performance of rapid diagnostic tests might be affected by manufacturing process, transportation and storage, parasitemia level, and skill of personnel who perform the tests. Therefore, periodic evaluation of the local field performance of rapid diagnostic tests is mandatory in order to make early corrections in case of decreased performance. Methods A facility-based cross-sectional study was conducted from January to May 2020 among 257 malaria-suspected patients attending selected health centers in Bahir Dar Zuria district. Capillary blood was collected from each participant and tested for Plasmodium infection by CareStart™ rapid diagnostic test kit and thin and thick blood film microscopy. Data were analyzed using statistical software for social sciences version 20 and MedCalc software version 19.3. Sensitivity, specificity, positive and negative predictive values, and kappa value were calculated to evaluate the performance of rapid diagnostic tests against microscopy. Results Among 257 study participants, 47 (18.3%) were tested positive for Plasmodium infection by at least one of the diagnostic methods. Rapid diagnostic tests revealed 3 false positive and 3 false negative results. The sensitivity and specificity of CareStart Malaria Pf/Pv Combo test were 93.2% and 98.6%, respectively (kappa = 0.918). Conclusion CareStart™ rapid diagnostic test has comparable performance with microscopy for malaria diagnosis. We recommend continued use of CareStart Malaria Pf/Pv Combo test at health posts in Ethiopia where microscopy is not available.

To determine the performance of rapid diagnostic tests for dengue and leptospirosis that rely on detecting antibodies that may not be produced when patients present for medical treatment. We prospectively enrolled 723 patients with undifferentiated febrile illness presenting to rural hospitals in northern and northeastern Thailand over a 1-year period. We evaluated rapid antibody detection diagnostic tests for dengue and leptospirosis on these patients. Sensitivity of the tests was low at the acute visit (7.6-21.5%). Sensitivity at the convalescent visit ranged from 25.8% to 81.5% and was significantly higher than at the acute visit for all tests (chi(2), P < 0.001). Low sensitivity of the rapid tests at presentation suggests that their utility in the acute phase of dengue and leptospirosis is limited.

Aims: To firstly determine the incidence of acute renal failure (ARF) related to malaria in a series of 11 cases among admitted patients at the pediatric hospitals in Dakar, Senegal and to lastly measure the performance of rapid diagnostic test (RDT) and microscopy in malaria diagnosis using polymerase chain reaction (PCR) as a gold standard.&#x0D; Study Design: A bi-centric and descriptive study was carried out.&#x0D; Place and Duration of Study: From June 2018 to January 2019 in two university hospitals of Dakar, Senegal: Aristide Le Dantec university hospital and Albert Royer university hospital.&#x0D; Methodology: Pediatric patients aged under 18 years with confirmed malaria by RDT or microscopy and ARF defined by anuria or oligo-anuria and a decrease in glomerular filtration rate were included. Nested PCR was performed to confirm malaria diagnosis and Plasmodium species typing.&#x0D; Results: In total, 11 ARF cases (8.5%) related to malaria among 130 children infected with malaria parasites were included out of 4,474 hospitalized. Affected children were aged between 2 and 16 years with a mean age of 11 years and a sex ratio of 0.57. For malaria diagnosis, RDTs were positive for all patients while microscopy only revealed 6 cases (54.5%) as well as PCR. However, microscopy and PCR presented two discrepancies. Considering PCR as the gold standard tool, RDT showed a relative high sensitivity (100%) and zero specificity with a positive predictive value (PPV) of 54.6% while microscopy respectively showed a sensitivity and a specificity of 66.7 and 60%.&#x0D; Conclusion: This study showed the relatedness between ARF and P. falciparum malaria. Even though microscopy remains the gold standard for the diagnosis of malaria but microscopists must be regularly trained. In addition, RDT should always be confirmed by microscopy and preferably by PCR.

Background: The first-line diagnosis of malaria in Mali is based on the use of rapid diagnostic tests (RDT) that detect the Histidin Rich Protein 2 (HRP2) antigen specific to Plasmodium falciparum. Our study, based on a real-time polymerase chain reaction (qPCR) gold standard, aimed to describe the distribution of the Plasmodium species in each administrative region of Mali and to assess the performance of RDTs. Methods: We randomly selected 150 malaria-negative and up to 30 malaria-positive RDTs in 41 sites distributed in 9 regions of Mali. DNA extracted from the RDT nitrocellulose strip was assayed with a pan-Plasmodium qPCR. Positive samples were then analyzed with P. falciparum-, P. malariae-, P. vivax-, or P. ovale-specific qPCRs. Results: Of the 1496 RDTs, 258 (18.6%) were positive for Plasmodium spp., of which 96.9% were P. falciparum. The P. vivax prevalence reached 21.1% in the north. RDT displayed acceptable diagnostic indices; the lower CI95% bounds of Youden indices were all ≥0.50, except in the north (Youden index 0.66 (95% CI [0.44–0.82]) and 0.63 (95% CI [0.33–0.83]. Conclusions: Overall, RDT diagnostic indices are adequate for the biological diagnosis of malaria in Mali. We recommend the use of RDTs detecting P. vivax-specific antigens in the north.

BackgroundRapid accurate diagnosis followed by effective treatment is very important for malaria control. Light microscopy remains the “golden standard” method for malaria diagnosis. Diagnostic test method must have sufficient level of accuracy for detecting malaria parasites. Therefore, this study aimed to investigate the diagnostic accuracy of rapid diagnostic tests (RDTs), microscopy, loop-mediated isothermal amplification (LAMP) and/or polymerase chain reaction (PCR) for the malaria diagnosis in Ethiopia.MethodsData bases such as PubMed, PubMed central, Science direct databases, Google scholar, and Scopus were searched from September to October, 2020 for studies assessing the diagnostic accuracy of RDTs, microscopy, LAMP and PCR methods for malaria diagnosis.ResultsA total of 29 studies published between 2001 and 2020 were analysed using review manager, Midas (Stata) and Meta-disc. The sensitivity and specificity of studies comparing RDT with microscopy varies from 79%–100% to 80%–100%, respectively. The sensitivity of LAMP (731 tests) was 100% and its specificity was varies from 85 to 99% when compared with microscopy and PCR. Considerable heterogeneity was observed between studies included in this meta-analysis. Meta-regression showed that blinding status and target antigens were the major sources of heterogeneity (P < 0.05). RDT had an excellent diagnostic accuracy (Area under the ROC Curve = 0.99) when compared with microscopy. Its specificity was quite good (93%–100%) except for one outlier (28%), but lower “sensitivity” was observed when PCR is a reference test. This indicates RDT had a good diagnostic accuracy (AUC = 0.83). Microscopy showed a very good diagnostic accuracy when compared with PCR.ConclusionsThe present study showed that microscopy and RDTs had high efficiency for diagnosing febrile malaria patients. The diagnostic accuracy of RDT was excellent when compared with microscopy. This indicates RDTs have acceptable sensitivities and specificities to be used in resource poor settings as an alternative for microscopy. In this study, LAMP showed an excellent sensitivities and specificities. Furthermore, the need of minimum equipment and relatively short time for obtaining results can made LAMP one of the best alternatives especially for accurate diagnosis of asymptomatic malaria.

How Do We Best Diagnose Malaria in Africa?

The emergence of pfhrp2/3-deleted parasites threatens histidine-rich protein 2 (HRP2)-based malaria rapid diagnostic test (RDT) performance. RDTs targeting Plasmodium falciparum (Pf) lactate dehydrogenase (LDH) may address current product limitations and improve case management. To evaluate the performance and usability of three LDH-based RDTs in febrile patients. A cross-sectional diagnostic accuracy study was conducted in Kédougou, Senegal. Capillary blood was tested using the SD Bioline Ag Pf (#05FK50) and three LDH-based RDTs: BIOCREDIT Malaria Ag Pf (pLDH), BIOCREDIT Pf (pLDH/HRPII), and BIOCREDIT Pf/Pv (pLDH/pLDH) (Rapigen Inc., Republic of Korea). Venous blood was collected to repeat the BIOCREDIT RDTs and conduct microscopy. Frozen venous specimens were tested using a reference PCR assay. A quantitative multiplex malaria antigen assay measured antigen concentration. RDT performance was determined and analyzed as a function of antigen concentration distribution. Usability of the Pf-only BIOCREDIT tests was evaluated using a questionnaire. 154/220 participants (70%) were Pf-positive by PCR. The Pf (pLDH/HRPII) test demonstrated the highest sensitivity at 78% (70.9%-84.5%); specificity was 89% (79.4%-95.6%). All RDTs performed better than microscopy (53% sensitivity). RDTs performed better when compared to antigen concentration over PCR results. Improved sensitivity of the Pf (pLDH/HRPII) test was driven by the HRP2 line. Line intensity correlated with antigen concentration. Predicted sensitivity using the analytical limit of detection (LOD) was comparable to observed sensitivity. RDTs demonstrated acceptable usability. Both HRP2 and LDH contributed to the sensitivity of the best-performing Pf-RDT. RDT analytical LODs can be used to predict performance in populations with known antigen concentrations.