Developing a multiplex loop-mediated isothermal amplification assay (LAMP) to determine severe fever with thrombocytopenia syndrome (SFTS) and scrub typhus.

Severe fever with thrombocytopenia syndrome (SFTS) and scrub typhus are endemic zoonotic diseases that pose significant public health threats in East Asia. As these two diseases share common clinical features, as well as overlapping disease regions, it is difficult to differentiate between SFTS and scrub typhus. A multiplex reverse-transcription loop­mediated isothermal amplification (RT-LAMP) assay was developed to detect large segments and GroES genes for SFTS virus (SFTSV) and Orientia tsutsugamushi (OT). The performance of the RT-LAMP assay was compared and evaluated with those of commercial PowerChek™ SFTSV real-time PCR and LiliF™ TSUTSU nested PCR for 23 SFTS and 12 scrub typhus clinical samples, respectively. The multiplex SFTSV/OT/Internal control (IC) RT-LAMP assay showed comparable sensitivity (91.3%) with that of commercial PowerChek™ SFTSV Real-time PCR (95.6%) and higher sensitivity (91.6%) than that of LiliF™ TSUTSU nested PCR (75%). In addition, the multiplex SFTSV/OT RT-LAMP assay showed 100% specificity and no cross-reactivity for blood from uninfected healthy patients and samples from patients infected with other fever viruses. Thus, the multiplex SFTSV/OT/IC RT-LAMP assay could serve as a useful point-of-care molecular diagnostic test for SFTS and scrub typhus.

An in vitro study on the differentiated metabolic mechanism of chloroquine-resistant Plasmodium falciparum using high-resolution metabolomics.

Chloroquine (CQ) is an important drug used therapeutically for treatment of malaria. However, due to limited number of studies on metabolic targets of chloroquine (CQ), it is difficult to attribute mechanisms underlying resistance associated with usage of this drug. The present study aimed to investigate the metabolic signatures of CQ-resistant Plasmodium falciparum (PfDd2) compared to CQ-sensitive Plasmodium falciparum (Pf3D7). Both Pf3D7 and PfDd2 were treated with CQ at 200 nM for 48 hr; thereafter, the harvested red blood cells (RBCs) and media were subjected to microscopy and high-resolution metabolomics (HRM). Glutathione, γ-L-glutamyl-L-cysteine, spermidine, inosine monophosphate, alanine, and fructose-1,6-bisphosphate were markedly altered in PfDd2 of RBC. In the media, cysteine, cysteic acid, spermidine, phenylacetaldehyde, and phenylacetic acid were significantly altered in PfDd2. These differential metabolic signatures related signaling pathways of PfDd2, such as oxidative stress pathway and glycolysis may provide evidence for understanding the resistance mechanism and pathogenesis of the CQ-resistant parasite.

Discovery of metabolic alterations in the serum of patients infected with Plasmodium spp. by high-resolution metabolomics.

INTRODUCTION: Despite the advances in diagnosis and treatment, malaria has still not been eradicated. Metabolic interactions between the host and Plasmodium may present novel targets for malaria control, but such interactions are yet to be deciphered. An exploration of metabolic interactions between humans and two Plasmodium species by high-resolution metabolomics may provide fundamental insights that can aid the development of a new strategy for the control of malaria. OBJECTIVES: This study aimed at exploring the metabolic changes in the sera of patients infected with Plasmodium falciparum and Plasmodium vivax. METHODS: Uni- and multivariate metabolomic analyses were performed on the sera of four groups of patients, namely normal control (N, n = 100), P. falciparum-infected patients (PF, n = 21), P. vivax-infected patients (PV, n = 74), and non-malarial pyretic patients (Pyr, n = 25). RESULTS: Univariate and multivariate analyses of N, PF, and PV groups showed differential metabolic phenotypes and subsequent comparisons in pairs revealed significant features. Pathway enrichment test with significant features showed the affected pathways, namely glycolysis/gluconeogenesis for PF and retinol metabolism for PV. The metabolites belonging to the affected pathways included significantly low 2,3-diphosphoglycerate and glyceraldehyde-3-phosphate in the sera of PF. The sera of PV had significantly low levels of retinol but high levels of retinoic acid. CONCLUSION: Our study reveals metabolic alterations induced by Plasmodium spp. in human serum and would serve as a milestone in the development of novel anti-malarial strategies.