The highly sensitive and selective determination of Escherichia coli (E. coli) in urine using SYBRTM Safe loop-mediated Isothermal Amplification (LAMP) method with a distance-based paper device was developed. The new...

Asymptomatic leishmaniasis cases have continuously increased, especially among patients with HIV who are at risk to develop further symptoms of cutaneous and visceral leishmaniasis. Thus, early diagnosis using a simple, sensitive and reliable diagnostic assay is important because populations at risk mostly reside in rural communities where laboratory equipment is limited. In this study, the highly sensitive and selective determination of Leishmania infection in asymptomatic HIV patients was achieved using dual indicators (SYBR safe and gold-nanoparticle probe; AuNP-probe) in one-step LAMP method based on basic instruments. The assay can be simply evaluated under the naked eye due to clear interpretation of fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric precipitate of specific AuNP-probes. The sensitivities and specificities of fluorescent SYBR safe dye and AuNP-probe indicators were equal, which were as high as 94.1 and 97.1%, respectively. Additionally, detection limits were 102 parasites/mL (0.0147 ng/µL), ten times more sensitivity than other related studies. To empower leishmaniasis surveillance, this inexpensive one-step SYBR safe and AuNP-LAMP assay is reliably fast and simple for field diagnostics to point-of-care settings, which can be set up in all levels of health care facilities including resource limited areas, especially in low to middle income countries.

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Asymptomatic visceral leishmaniasis cases increase continuously, particularly among patients with HIV who are at risk to develop further symptoms of leishmaniasis. A simple, sensitive and reliable diagnosis is crucially needed due to risk populations mostly residing in rural communities with limited resources of laboratory equipment. In this study, a highly sensitive and selective determination of Leishmania among asymptomatic patients with Leishmania/HIV co-infection was achieved to simultaneously interpret and semi-quantify using colorimetric precipitates (gold-nanoparticle probe; AuNP-probe) and fluorescence (SYBR safe dye and distance-based paper device; dPAD) in one-step loop-mediated isothermal amplification (LAMP) assay. The sensitivities and specificities of 3 detection methods were equivalent and had reliable performances achieving as high as 95.5%. Detection limits were 102 parasites/mL (0.0147 ng/µL) which were 10 times more sensitive than other related studies. To empower leishmaniasis surveillance as well as prevention and control, this dPAD combined with SYBR safe and gold nanoparticle probe LAMP assay is reliably fast, simple, inexpensive and practical for field diagnostics to point-of-care settings in resource-limited areas which can be set up in all levels of healthcare facilities, especially in low to middle income countries.

Background Efforts to study the biology of Plasmodium vivax liver stages, particularly the latent hypnozoites, have been hampered by the limited availability of P. vivax sporozoites. Anopheles stephensi is a major urban malaria vector in Goa and elsewhere in South Asia. Using P. vivax patient blood samples, a series of standard membrane-feeding experiments were performed with An. stephensi under the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA). The goal was to understand the dynamics of parasite development in mosquitoes as well as the production of P. vivax sporozoites. To obtain a robust supply of P. vivax sporozoites, mosquito-rearing and mosquito membrane-feeding techniques were optimized, which are described here. Methods Membrane-feeding experiments were conducted using both wild and laboratory-colonized An. stephensi mosquitoes and patient-derived P. vivax collected at the Goa Medical College and Hospital. Parasite development to midgut oocysts and salivary gland sporozoites was assessed on days 7 and 14 post-feeding, respectively. The optimal conditions for mosquito rearing and feeding were evaluated to produce high-quality mosquitoes and to yield a high sporozoite rate, respectively. Results Laboratory-colonized mosquitoes could be starved for a shorter time before successful blood feeding compared with wild-caught mosquitoes. Optimizing the mosquito-rearing methods significantly increased mosquito survival. For mosquito feeding, replacing patient plasma with naïve serum increased sporozoite production > two-fold. With these changes, the sporozoite infection rate was high (> 85%) and resulted in an average of ~ 22,000 sporozoites per mosquito. Some mosquitoes reached up to 73,000 sporozoites. Sporozoite production could not be predicted from gametocyte density but could be predicted by measuring oocyst infection and oocyst load. Conclusions Optimized conditions for the production of high-quality P. vivax sporozoite-infected An. stephensi were established at a field site in South West India. This report describes techniques for producing a ready resource of P. vivax sporozoites. The improved protocols can help in future research on the biology of P. vivax liver stages, including hypnozoites, in India, as well as the development of anti-relapse interventions for vivax malaria.

Liver fluke infection caused by Opisthorchis viverrini is recognized as a potential risk factor for cholangiocarcinoma (CCA). The National Strategic Plan to Control Liver Fluke Infection and Cholangiocarcinoma has implemented microscopic-based stool examination screening. However, eggs of O. viverrini and minute intestinal flukes (MIFs) are nearly morphologically similar and could result in inaccurate O. viverrini diagnosis. Stool specimens were collected from eight districts of Chiang Mai Province in northern Thailand. Opisthorchis-like eggs were identified with the Kato-Katz technique and differentiated for O. viverrini and MIFs using molecular study by PCR and PCR-restriction fragment length polymorphism targeting the internal transcribed spacer 2 (ITS2) gene. Prevalence of Opisthorchis-like eggs was 5.9% from a total of 9,570 specimens. From PCR assays, all liver flukes were O. viverrini and all MIFs were Haplorchis taichui. The distribution of species was H. taichui (38.2%), O. viverrini (10.5%), coinfection of H. taichui and O. viverrini (37.2%), and 14.1% were negative from PCR. Totally, H. taichui was found in 75.4% of infections from Opisthorchis-like specimens. ITS2 nucleotide sequencing analysis showed a single variant of O. viverrini with no variation and two variants of H. taichui. This study first revealed the genetic background of Opisthorchis-like eggs in northern Thailand. Minute intestinal flukes are occasionally misdiagnosed as O. viverrini leading to misinterpretation and overestimation of the burden of O. viverrini infection. Molecular diagnosis such as PCR could effectively discriminate species of Opisthorchis-like eggs and help shape the robustness of epidemiological data to control liver fluke infection and raise awareness of other risk factors for CCA.

Background Primaquine and tafenoquine are the only licensed drugs that effectively kill the hypnozoite stage and are used to prevent Plasmodium vivax malaria relapse. However, both primaquine and tafenoquine can cause acute hemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient people with varying degrees of severity depending on G6PD variants. Additionally, primaquine efficacy against malaria parasites was decreased in individuals with impaired cytochrome P450 2D6 (CYP2D6) activity due to genetic polymorphisms. This study aimed to characterize G6PD and CYP2D6 genetic variations in vivax malaria patients from Yala province, a malaria-endemic area along the Thai–Malaysian border, and determine the biochemical properties of identified G6PD variants. Methodology/Principle findings Multiplexed high-resolution melting assay and DNA sequencing detected five G6PD variants, including G6PD Kaiping, G6PD Vanua Lava, G6PD Coimbra, G6PD Mahidol, and G6PD Kerala-Kalyan. Biochemical and structural characterization revealed that G6PD Coimbra markedly reduced catalytic activity and structural stability, indicating a high susceptibility to drug-induced hemolysis. While Kerala-Kalyan had minor effects, it is possible to develop mild adverse effects when receiving radical treatment. CYP2D6 genotyping was performed using long-range PCR and DNA sequencing, and the phenotypes were predicted using the combination of allelic variants. Decreased and no-function alleles were detected at frequencies of 53.4% and 14.2%, respectively. The most common alleles were CYP2D*36+*10 (25.6%), *10 (23.9%), and *1 (22.2%). Additionally, 51.1% of the intermediate metabolizers showed CYP2D6*10/*36+*10 as the predominant genotype (15.9%). Conclusions/Significance Our findings provide insights about genetic variations of G6PD and CYP2D6 in 88 vivax malaria patients from Yala, which may influence the safety and effectiveness of radical treatment. Optimization of 8-aminoquinoline administration may be required for safe and effective treatment in the studied population, which could be a significant challenge in achieving the goal of eliminating malaria.