ABSTRACT
We propose a protocol suitable for point-of-care diagnosis of malaria utilizing a simple and purification-free DNA extraction method with the combination of loop-mediated isothermal amplification assay and lateral flow (LAMP-LF). The multiplex LAMP-LF platform developed here can simultaneously detect Plasmodium knowlesi, P. vivax, P. falciparum, and Plasmodium genus (for P. malariae and P. ovale). Through the capillary effect, the results can be observed by the red band signal on the test and control lines within 5 min. The developed multiplex LAMP-LF was tested with 86 clinical blood samples on-site at Hospital Kapit, Sarawak, Malaysia. By using microscopy as the reference method, the multiplex LAMP-LF showed 100% sensitivity (95% confidence interval (CI): 91.4 to 100.00%) and 97.8% specificity (95% CI: 88.2% to 99.9%). The high sensitivity and specificity of multiplex LAMP-LF make it ideal for use as a point-of-care diagnostic tool. The simple and purification-free DNA extraction protocol can be employed as an alternative DNA extraction method for malaria diagnosis in resource-limited settings. By combining the simple DNA extraction protocol and multiplex LAMP-LF approach, we aim to develop a simple-to-handle and easy-to-read molecular diagnostic tool for malaria in both laboratory and on-site settings.
ABSTRACT
BACKGROUND: Leptospirosis diagnoses have increased in Sarawak, Malaysia in recent years. METHODS: To better understand the burden of disease and associated risk factors, we evaluated 147 patients presenting with clinical leptospirosis to local hospitals in Sarawak, Malaysia for the presence of Leptospira and associated antibodies. Sera and urine specimens collected during the acute illness phase were assessed via a commercially available rapid diagnostic test (Leptorapide, Linnodee Ltd., Antrim, Northern Ireland), an ELISA IgM assay (Leptospira IgM ELISA, PanBio, Queensland, Australia) and a pan-Leptospira real-time PCR (qPCR) assay to estimate disease prevalence and diagnostic accuracy of each method. Microagglutination testing was performed on a subset of samples. RESULTS: Overall, 45 out of 147 patients (30.6%) showed evidence of leptospires through qPCR in either one or both sera (20 patients) or urine (33 patients), and an additional ten (6.8%) were considered positive through serological testing, for an overall prevalence of 37.4% within the study population. However, each diagnostic method individually yielded disparate prevalence estimates: rapid test 42.2% for sera and 30.5% for urine, ELISA 15.0% for sera, qPCR 13.8% for sera and 23.4% for urine. Molecular characterization of a subset of positive samples by conventional PCR identified the bacterial species as Leptospira interrogans in 4 specimens. A multivariate risk factor analysis for the outcome of leptospirosis identified having completed primary school (OR = 2.5; 95 CI% 1.0-6.4) and weekly clothes-washing in local rivers (OR = 10.6; 95 CI% 1.4-214.8) with increased likelihood of leptospirosis when compared with those who had not. CONCLUSION: Overall, the data suggest a relatively high prevalence of leptospirosis in the study population. The low sensitivities of the rapid diagnostic test and ELISA assay against qPCR highlight a need for better screening tools.
ABSTRACT
Burkholderia pseudomallei infections are prevalent in Southeast Asia and northern Australia and often misdiagnosed. Diagnostics are often neither sensitive nor rapid, contributing up to 50% mortality rate. In this 2018 pilot study, we enrolled 100 patients aged 6 months-79 years from Kapit Hospital in Sarawak, Malaysia, with symptoms of B. pseudomallei infection. We used three different methods for the detection of B. pseudomallei: a real-time polymerase chain reaction (PCR) assay, a rapid lateral flow immunoassay, and the standard-of-care bacterial culture-the gold standard. Among the 100 participants, 24 (24%) were positive for B. pseudomallei by one or more of the detection methods. Comparing the two individual diagnostic methods against the gold standard-bacterial culture-of any positive test, there was low sensitivity for each test (25-44%) but high specificity (93-98%). It seems clear that more sensitive diagnostics or a sensitive screening diagnostic followed by specific confirmatory diagnostic is needed for this disease.
