Prototheca bovis, a unicellular achlorophyllous trebouxiophyte green alga in the healthy human intestine

Introduction: Prototheca species are non-photosynthetic trebouxiophyte algae ubiquitously distributed in nature and can be found in sewage and soil. This microbial eukaryote causes human protothecosis in immunocompromised individuals. Thus, Prototheca presence in the stool of individuals without gastrointestinal symptoms has been reported only rarely. Hypothesis/Gap statement: There is an absence of detailed characterization of human Prototheca isolates. Aim: The aim of this study was to perform morphological and molecular characterization of Prototheca isolates obtained from human stool. Methodology: Prototheca was isolated from fecal samples of four individuals living in a rural area in Thailand. A combination of bioimaging along with molecular and bioinformatics tools was used to characterize the four strains. The growth rate was tested using four media and three temperature conditions. Phylogenetic analysis using the small subunit ribosomal RNA (SSU rRNA) and cytochrome b (cytb) was also performed. Results: Static and live microscopy demonstrated the various life stages of Prototheca and its major defining cellular characteristics. An optimized DNA extraction methodology that improves DNA yield is provided. Partial fragments of the SSU rRNA and cytb genes were obtained. Phylogenetic analysis placed all four strains in the clade with Prototheca bovis. More broadly, Prototheca was not monophyletic but split into at least two distinct clades instead. Conclusion: The results represent the first molecular characterization of Prototheca in Thailand. The study provides insight into transmission dynamics of the organism and potential caveats in estimating the global prevalence of Prototheca. These will spearhead further investigations on Prototheca occurrence in rural areas of both industrialized and developing nations

A comprehensive epidemiological study of opisthorchiasis in a rural community of Thailand

Opisthorchiasis is a zoonotic parasitic infection caused by human liver flukes. Opisthorchis viverrini (OV) is endemic in Southeast Asia along Mekong basin which estimated 9 million people are infected. Transmission to humans occurs through the consumption of uncooked fish; cyprinoid or white-scale freshwater fish containing infective stage metacercariae. The infection is mainly asymptomatic. Adult flukes could live up in the bile duct in the absence of treatment. Chronic infection is strongly related with bile duct cancer or cholangiocarcinoma. The International Agency for Research on Cancer has declared that Opisthorchis viverrini is a member of group 1 agent, carcinogenic to humans. Thailand has the highest incidence of cholangiocarcinoma in the world. However, opisthorchiasis is acknowledged as a neglected and underestimated disease on the global scale. In the present day, the epidemiological data on opisthorchiasis from OV infection in Thailand are considered outdated. An extensive cross-sectional study was undertaken from 1984 to 2001 under the National Control Program, but data on incidence is very limited. Moreover, infection dynamics are still poorly understood. This project aimed to comprehensively study the infection dynamics of OV infection using multiple tools in order to explore the infection in various aspects. Statistical models were developed to explore epidemiological data; prevalence, incidence and infection intensity, with risk factors for acquiring the infection. Infection dynamics will be described using mathematical modelling. The qualitative technique, by interviewing and group discussions, will be used to explain the cause of uncontrolled infection rate in bio-psycho-social aspect. Integrated study results will be used to develop community intervention strategies under the framework of the public health planning model. The overall outcome will be valuable for Thailand National Health Policy and epidemiological data will provide the basis for further rigorous academic research.Open Acces

Molecular discrimination of Opisthorchis-like eggs from residents in a rural community of central Thailand.

Opisthorchis viverrini infection is a major public health problem in northern and northeastern Thailand. The chronic infection of O. viverrini is related to cholangiocarcinoma which causes high mortality in endemic areas. Therefore, the diagnosis, treatment, control and prevention of O. viverrini infection are necessary. The morphology of the egg is very similar to that of other species of human liver flukes (Opisthorchis felineus and Clonorchis sinensis) as well as that of small intestinal flukes in the family Heterophyidae. Thus, molecular characterization is crucially required to discriminate species of Opisthorchis-like eggs in fecal examination.We aimed to determine the prevalence of O. viverrini infection among villagers living in Sanamchaikate District, Chachoengsao Province, in central Thailand, where O. viverrini infection has previously been reported. A total of 2,609 fecal samples were examined for Opisthorchis-like eggs using microscopic examination. PCR-RFLP analysis of the ITS2 region was used to discriminate Opisthorchis-like eggs. The genetic structure of O. viverrini infection was demonstrated using nucleotide sequencing of cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit 1 (nad1). Testing of evolutionary neutrality of the cox1 and nad1 sequences of O. viverrini was performed using Tajima's D tests and Fu's Fs tests. Moreover, the haplotype networks and phylogenetic trees were constructed to study the relationships of O. viverrini isolated from different endemic areas. A high prevalence of O. viverrini infection is still observed in a rural community of Chachoengsao Province, central Thailand. The overall prevalence of Opisthorchis-like eggs using microscopic examination was 16.8%. PCR-RFLP profiles showed the predominant infection of O. viverrini (9.6%) including very low infections of other small intestinal flukes, Haplorchis taichui (0.08%) and Euparyphium albuferensis (0.08%). The genetic structure of O. viverrini populations in central Thailand was also described and revealed a non-significant difference in genetic diversity. In addition, the genetic background of the O. viverrini populations was closely related to the isolate from Lao PDR.Our study highlighted the prevalence of O. viverrini infection in central Thailand indicating that control programs and health education regarding opisthorchiasis is still required in this endemic area. Additionally, the study demonstrated the genetic structure of O. viverrini, in central Thailand which could provide information on the molecular epidemiology of this parasite

Rate of Kidney Function Decline and Factors Predicting Progression of Kidney Disease in Type 2 Diabetes Mellitus Patients with Reduced Kidney Function: A Nationwide Retrospective Cohort Study.

OBJECTIVES Currently, the data on independent risk factors for the progression of kidney disease in type 2 diabetes mellitus (T2DM) patients with CKD were limited. This study aimed to investigate CKD progression in T2DM patients who have reduced kidney function with baseline eGFRs between 15 and 59 mL/min/1.73 m . METHODS This study was composed of a nationwide retrospective cohort of adult T2DM patients from 831 public hospitals in Thailand during the year 2015. T2DM patients with CKD stage 3-4 were followed up until development of CKD stage 5, requirement of chronic dialysis, loss to follow-up, death, or May 31st, 2018, whichever came first. Cox proportional hazard regression was utilized to analysis. RESULTS A total of 8464 participants were included. 30.4% were male. The mean age was 69 ± 10 years. The mean eGFR was 45 ± 11 mL/min/1.73 m . The incidence of CKD stage 5 or need for chronic dialysis was 16.4 per 1000 person-years. The annual rate of eGFR decline during a mean follow-up of 29 months was -2.3 mL/min/1.73 m . 14.4% had a rapid decline in eGFR. The risk factors associated with progression to CKD stage 5 or need for chronic dialysis were diabetes duration, systolic blood pressure, serum uric acid, albuminuria, and baseline eGFR. Conversely, older age and the use of renin-angiotensin aldosterone system blockades were associated with decreased risks for rapid CKD progression and incidence CKD stage 5 or dialysis. CONCLUSION This study identifies multiple predictive risk factors that support a multifaceted approach to prevent progression of advanced CKD

The indicators of genetic diversity and neutrality tests in the populations of <i>O</i>. <i>viverrini</i> from three villages using <i>cox1</i> sequences.

<p>The indicators of genetic diversity and neutrality tests in the populations of <i>O</i>. <i>viverrini</i> from three villages using <i>cox1</i> sequences.</p

A flow diagram illustrating the methods used for fecal examination, <i>Opisthorchis</i>-like egg positive samples and available fecal samples for DNA extraction.

<p>A flow diagram illustrating the methods used for fecal examination, <i>Opisthorchis</i>-like egg positive samples and available fecal samples for DNA extraction.</p

The Randomized Axelerated Maximum Likelihood tree based on the <i>nad1</i> sequences of <i>O</i>. <i>viverrini</i>.

<p>The alignment of 688 nucleotide sequences without gaps and 18 variants from three villages (NY = Na-Yao, NN = Na-Ngam, TH = Thoong-Heang) were analyzed. <i>O</i>. <i>viverrini</i> Thai isolates (BR, KS, LP, NP, KPv, MS, KLp, KBp, SK, NADH1), Lao PDR isolates (CP, NG, SV, VT, KM, <i>O</i>. <i>viverrini</i> mitochondrion partial genome), Vietnamese isolates (BD, OvBD1, OvL, PY3, OvPY3, QN, DL3) and a Cambodian isolate (KD) were used as the reference isolates. The percentages of 1,000 replications (bootstraps) of more than 50% are shown at the nodes. The number of samples is in parentheses.</p

The Randomized Axelerated Maximum Likelihood tree based on the <i>cox1</i> sequences of <i>O</i>. <i>viverrini</i>.

<p>The alignment of 393 nucleotide sequences without gaps and 27 variants from three villages (NY = Na-Yao, NN = Na-Ngam, TH = Thoong-Heang) were analyzed. <i>O</i>. <i>viverrini</i> Thai isolates (CP, BR, KBp, KPv, SK, NG, MS, KLp, KS) and Lao PDR isolates (SV, VT, <i>O</i>. <i>viverrini</i> mitochondrion partial genome) were used as the reference isolates. The percentages of 1,000 replications (bootstraps) of more than 50% are shown at the nodes. The number of samples is in parentheses.</p

The prevalences of <i>Opisthorchis</i>-like egg, ITS2-PCR sensitivities and PCR-RFLP profiles for detection <i>Opisthorchis</i>-like eggs in Chachoengsao Province, central Thailand.

<p>The prevalences of <i>Opisthorchis</i>-like egg, ITS2-PCR sensitivities and PCR-RFLP profiles for detection <i>Opisthorchis</i>-like eggs in Chachoengsao Province, central Thailand.</p