METHODS: A high-throughput LAMP assay targeting a P. vivax mitochondrial gene and deploying colorimetric detection in a 96-well plate format was developed and evaluated in the laboratory. Diagnostic accuracy was compared against microscopy, antigen detection tests and PCR and validated in samples from malaria patients and community controls in a district hospital setting in Sabah, Malaysia.
RESULTS: The high throughput LAMP-P. vivax assay (HtLAMP-Pv) performed with an estimated limit of detection of 1.4 parasites/ μL. Assay primers demonstrated cross-reactivity with P. knowlesi but not with other Plasmodium spp. Field testing of HtLAMP-Pv was conducted using 149 samples from symptomatic malaria patients (64 P. vivax, 17 P. falciparum, 56 P. knowlesi, 7 P. malariae, 1 mixed P. knowlesi/P. vivax, with 4 excluded). When compared against multiplex PCR, HtLAMP-Pv demonstrated a sensitivity for P. vivax of 95% (95% CI 87-99%); 61/64), and specificity of 100% (95% CI 86-100%); 25/25) when P. knowlesi samples were excluded. HtLAMP-Pv testing of 112 samples from asymptomatic community controls, 7 of which had submicroscopic P. vivax infections by PCR, showed a sensitivity of 71% (95% CI 29-96%; 5/7) and specificity of 93% (95% CI87-97%; 98/105).
CONCLUSION: This novel HtLAMP-P. vivax assay has the potential to be a useful field applicable molecular diagnostic test for P. vivax infection in elimination settings.
GOALS: The goal was to ascertain whether urine testing could be used as screening method to detect C. trachomatis infections in commercial sex workers, patients at sexually transmitted diseases clinic, and asymptomatic patients in Kuala Lumpur, Malaysia.
METHODS: First-void urine specimens from 300 men and 300 women were tested by LCR, as well as by a commercially available enzyme immunoassay. The LCR assay amplifies specific sequences within the chlamydial plasmid with ligand-labeled probes, and the resultant amplicons are detected by an automated immunoassay. Specimens with discrepant results were confirmed by another LCR of the specimen that targeted the gene for the major outer membrane protein (OMP1).
RESULTS: There were 31 LCR-positive male urine and 37 LCR-positive female urine specimens. The resolved sensitivity and specificity for the LCR of the male urine specimens were 100% and 99.6%, respectively, whereas for female urine specimens, the sensitivity and specificity were 100% and 98.5%, respectively. After resolution of discrepant test results by OMP1 LCR, the prevalence was 10% for men and 11% for women. The urine enzyme immunoassay was not useful in diagnosing C. trachomatis infections in either men or women, as the resolved sensitivities were 10% and 15.2%, respectively. The specificities were 99.6% for men and 98.9% for women.
CONCLUSIONS: Testing first-void urine specimens by LCR is a highly sensitive and specific method to diagnose C. trachomatis infections in men and women, providing health care workers and public health officials with a new molecular amplification assay that uses noninvasive urine specimens for population-based screening purposes.
METHODS: Study participants included 73 uncomplicated malaria patients with PCR species confirmation: 50 P. knowlesi, 20 P. falciparum and 3 P. vivax. Nineteen malaria-negative, non-endemic area controls were also included. The sensitivity of the Eiken Loopamp™ MALARIA Pan Detection kit (Pan LAMP) for detecting each Plasmodium species was evaluated. Sensitivity and specificity of the Eiken Loopamp™ MALARIA Pf Detection kit (Pf LAMP) for P. falciparum were also determined. The limit of detection for each LAMP assay was evaluated, with results compared to PCR. All P. knowlesi patients were also tested by CareStart™ (Pf/VOM) and OptiMAL-IT™ (Pan/Pf) RDTs.
RESULTS: The sensitivity of the Pan LAMP assay was 100% for P. knowlesi (95% CI 92.9-100), P. falciparum (95% CI 83.2-100), and P. vivax (95% CI 29.2-100). The Pf LAMP was 100% sensitive and specific for P. falciparum detection, with all P. knowlesi samples having a negative reaction. LAMP sensitivity was superior to both RDTs, with only 10 and 28% of P. knowlesi samples testing positive to CareStart™ and OptiMAL-IT™, respectively. Limit of detection using the Pan LAMP for both P. knowlesi and P. vivax was 2 parasites/μL, comparable to PCR. For P. falciparum both the Pan LAMP and Pf LAMP demonstrated a limit of detection of 20 parasites/μL.
CONCLUSIONS: The Eiken Loopamp™ MALARIA Pan Detection kit is sensitive for detection of P. knowlesi in low parasitaemia clinical infections, as well as P. falciparum and P. vivax. However, a P. knowlesi-specific field assay in a simpler format would assist correct species identification and initiation of optimal treatment for all malaria patients.