Comparison of prevalence estimates of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum determined by conventional PCR and multiplex qPCR and implications for surveillance and monitoring.

OBJECTIVES: The accuracy of malaria rapid diagnostic tests is threatened by Plasmodium falciparum with pfhrp2/3 deletions. This study compares gene deletion prevalence determined by multiplex real time polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR) using existing samples with clonality previously determined by microsatellite genotyping. METHODS: Multiplex qPCR was used to estimate prevalence of pfhrp2/3 deletions in three sets of previously collected patient samples from Eritrea and Peru. The qPCR was validated by multiplex digital polymerase chain reaction. Sample classification was compared with cPCR, and receiver operating characteristic curve analysis was used to determine the optimal ΔCq threshold that aligned the results of the two assays. RESULTS: qPCR classified 75% (637 of 849) of samples as single, and 212 as mixed-pfhrp2/3 genotypes, with a positive association between clonality and proportion of mixed-pfhrp2/3 genotype samples. The sample classification agreement between cPCR and qPCR was 75.1% (95% confidence interval [CI] 68.6-80.7%) and 47.8% (95% CI 38.9-56.9%) for monoclonal and polyclonal infections. The qPCR prevalence estimates of pfhrp2/3 deletions showed almost perfect (κ = 0.804, 95% CI 0.714-0.895) and substantial agreement (κ = 0.717, 95% CI 0.562-0.872) with cPCR for Peru and 2016 Eritrean samples, respectively. For 2019 Eritrean samples, the prevalence of double pfhrp2/3 deletions was approximately two-fold higher using qPCR. The optimal threshold for matching the assay results was ΔCq = 3. CONCLUSIONS: Multiplex qPCR and cPCR produce comparable estimates of gene deletion prevalence when monoclonal infections dominate; however, qPCR provides higher estimates where multi-clonal infections are common.

A global comparative evaluation of commercial immunochromatographic rapid diagnostic tests for visceral leishmaniasis.

BACKGROUND: Poor access to diagnosis stymies control of visceral leishmaniasis (VL). Antibody-detecting rapid diagnostic tests (RDTs) can be performed in peripheral health settings. However, there are many brands available and published reports of variable accuracy. METHODS: Commercial VL RDTs containing bound rK39 or rKE16 antigen were evaluated using archived human sera from confirmed VL cases (n = 750) and endemic non-VL controls (n = 754) in the Indian subcontinent (ISC), Brazil, and East Africa to assess sensitivity and specificity with 95% confidence intervals. A subset of RDTs were also evaluated after 60 days' heat incubation (37°C, 45°C). Interlot and interobserver variability was assessed. RESULTS: All test brands performed well against ISC panels (sensitivity range, 92.8%-100%; specificity range, 96%-100%); however, sensitivity was lower against Brazil and East African panels (61.5%-91% and 36.8%-87.2%, respectively). Specificity was consistently > 95% in Brazil and ranged between 90.8% and 98% in East Africa. Performance of some products was adversely affected by high temperatures. Agreement between lots and readers was good to excellent (κ > 0.73-0.99). CONCLUSIONS: Diagnostic accuracy of VL RDTs varies between the major endemic regions. Many tests performed well and showed good heat stability in the ISC; however, reduced sensitivity against Brazilian and East African panels suggests that in these regions, used alone, several RDTs are inadequate for excluding a VL diagnosis. More research is needed to assess ease of use and to compare performance using whole blood instead of serum and in patients coinfected with human immunodeficiency virus.

Cost-effectiveness analysis of introduction of rapid, alternative methods to identify multidrug-resistant tuberculosis in middle-income countries.

BACKGROUND: Resistance to commonly used antituberculosis drugs is emerging worldwide. Conventional drug-susceptibility testing (DST) methods are slow and demanding. Alternative, rapid DST methods would permit the early detection of drug resistance and, in turn, arrest tuberculosis transmission. METHODS: A cost-effectiveness analysis of 5 DST methods was performed in the context of a clinical trial that compared rapid with conventional DST methods. The methods under investigation were direct phage-replication assay (FASTPlaque-Response; Biotech), direct amplification and reverse hybridization of the rpoB gene (INNO-LiPA; Innogenetics), indirect colorimetric minimum inhibitory concentration assay (MTT; ICN Biomedicals), and direct proportion method on Löwenstein-Jensen medium. These were compared with the widely used indirect proportion method on Löwenstein-Jensen medium. RESULTS: All alternative DST methods were found to be cost-effective, compared with other health care interventions. DST methods also generate substantial cost savings in settings of high prevalence of multidrug-resistant tuberculosis. Excluding the effects of transmission, the direct proportion method on Löwenstein-Jensen medium was the most cost-effective alternative DST method for patient groups with prevalences of multidrug-resistant tuberculosis of 2%, 5%, 20%, and 50% (cost in US$2004, $94, $36, $8, and $2 per disability-adjusted life year, respectively). CONCLUSION: Alternative, rapid methods for DST are cost-effective and should be considered for use by national tuberculosis programs in middle-income countries.