Abstract
BackgroundLoop-mediated isothermal amplification (LAMP) has been widely used to diagnose various infectious diseases. Malaria is a globally distributed infectious disease attributed to parasites in the genus Plasmodium. It is known that persons infected with Plasmodium vivax and P. ovale are prone to clinical relapse of symptomatic blood-stage infections. LAMP has not previously been specifically evaluated for its diagnostic performance in detecting P. ovale in an epidemiological study, and no commercial LAMP or rapid diagnostic test (RDT) kits are available for specifically diagnosing infections with P. ovale.MethodsAn assay was designed to target a portion of mitochondrial DNA (mtDNA) among Plasmodium spp., the five human Plasmodium species and two other assays were designed to target the nuclear 18S ribosomal DNA gene (18S rDNA) of either P. vivax or P. ovale for differentiating the two species. The sensitivity of the assays was compared to that of nested PCR using defined concentrations of plasmids containing the target sequences and using limiting dilutions prepared from clinical isolates derived from Chinese workers who had become infected in Africa or near the Chinese border with Myanmar.ResultsThe results showed that 102 copies of the mitochondrial target or 102 and 103 copies of 18S rDNA could be detected from Plasmodium spp., P. vivax and P. ovale, respectively. In 279 clinical samples, the malaria Pan mtDNA LAMP test performed well when compared with a nested PCR assay (95% confidence interval [CI] sensitivity 98.48–100%; specificity 90.75–100%). When diagnosing clinical cases of infection with P. vivax, the 18S rDNA assay demonstrated an even great sensitivity (95.85–100%) and specificity (98.1–100%). The same was true for clinical infections with P. ovale (sensitivity 90.76–99.96%; specificity 98.34–100%). Using plasmid-positive controls, the limits of detection of Malaria Pan, 18S rDNA P. vivax and 18S rDNA P. ovale LAMP were 100-, 100- and tenfold lower than those of PCR, respectively.ConclusionThe novel LAMP assays can greatly aid the rapid, reliable and highly sensitive diagnosis of infections of Plasmodium spp. transmitted among people, including P. vivax and P. ovale, cases of which are most prone to clinical relapse.Graphical
Highlights
Loop-mediated isothermal amplification (LAMP) has been widely used to diagnose various infectious diseases
Plasmodium vivax is endemic in Southeast Asia, where it is generally assumed to be the causative agent of malaria [2, 3]
We developed and tested novel LAMP assays to detect all species of all human malaria and to differentially diagnose P. vivax and P. ovale infections, using a positive control plasmid construct as well as clinical cases of malaria imported to China as a basis to evaluate sensitivity and specificity
Summary
Loop-mediated isothermal amplification (LAMP) has been widely used to diagnose various infectious diseases. Malaria is a globally distributed infectious disease attributed to parasites in the genus Plasmodium. It is known that persons infected with Plasmodium vivax and P. ovale are prone to clinical relapse of symptomatic bloodstage infections. Malaria, transmitted by female Anopheles mosquitoes, is a major global burden to human health and welfare, with an estimated 228 million cases and 405 thousand deaths reported in 2018 [1]. P. vivax is the most widespread and responsible for the majority of malaria infections outside of Africa, with 5.9 million clinical cases reported in 2018 [1]. Among the Plasmodium species of human malaria, these latter two are distinctive by sharing a dormant hypnozoite stage in hepatocytes that can cause relapse of symptomatic blood-stage infections weeks, months or even years after the primary infection [5, 6]. It is important for clinicians to identify whether either of these two species is present, so as to promptly initiate the proper therapeutic regimen
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