Use of universal primers for the 18S ribosomal RNA gene and whole soil DNAs to reveal the taxonomic structures of soil nematodes by high-throughput amplicon sequencing

Nematodes are abundant metazoans that play crucial roles in nutrient recycle in the pedosphere. Although high-throughput amplicon sequencing is a powerful tool for the taxonomic profiling of soil nematodes, polymerase chain reaction (PCR) primers for amplification of the 18S ribosomal RNA (SSU) gene and preparation of template DNAs have not been sufficiently evaluated. We investigated nematode community structure in copse soil using four nematode-specific (regions 1–4) and two universal (regions U1 and U2) primer sets for the SSU gene regions with two DNAs prepared from copse-derived mixed nematodes and whole soil. The major nematode-derived sequence variants (SVs) identified in each region was detected in both template DNAs. Order level taxonomy and feeding type of identified nematode-derived SVs were distantly related between the two DNA preparations, and the region U2 was closely related to region 4 in the non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarity. Thus, the universal primers for region U2 could be used to analyze soil nematode communities. We further applied this method to analyze the nematodes living in two sampling sites of a sweet potato-cultivated field, where the plants were differently growing. The structure of nematode-derived SVs from the two sites was distantly related in the principal coordinate analysis (PCoA) with weighted unifrac distances, suggesting their distinct soil environments. The resultant ecophysiological status of the nematode communities in the copse and field on the basis of feeding behavior and maturity indices was fairly consistent with those of the copse- and the cultivated house garden-derived nematodes in prior studies. These findings will be useful for the DNA metabarcoding of soil eukaryotes, including nematodes, using soil DNAs.

A retrospective observational study of the impact of 16s and 18s ribosomal RNA PCR on antimicrobial treatment over seven years: A tertiary hospital experience

Background Although culture-based methods remain a staple element of microbiology analysis, advanced molecular methods increasingly supplement the testing repertoire. Since the advent of 16s and 18s ribosomal RNA PCR in the 2000s, there has been interest in its utility for pathogen detection. Nonetheless, studies assessing the impact on antimicrobial prescribing are limited. We report a single-centre experience of the influence of 16s and 18s PCR testing on antimicrobial treatment, including a cost-analysis. Methods Data were collected retrospectively for all samples sent for 16s and 18s PCR testing between January 2014 and December 2020. Results were compared to any culture-based result. Assessment focused on any change of antimicrobial treatment based on PCR result, or use of the result as supportive evidence for microbiological diagnosis. Results 310 samples relevant to 268 patients were referred for 16s/18s rRNA PCR testing during the period. Culture was performed for 234 samples. Enrichment culture was performed for 83 samples. 82 of 300 samples sent for 16s PCR had positive results (20.8%). When culture was performed, enrichment reduced the outcome of 16s PCR only positive results (4/36 [11.1%] versus 14/35 [40.0%], p = 0.030 where a pathogen found). 18s PCR yielded 9 positive results from 67 samples. The 16s PCR result influenced antimicrobial change for 6 patients (2.2%). We estimated the cost for 16s PCR testing to result in one significant change in antimicrobial therapy to be €3,340. 18s PCR did not alter antimicrobial treatment. Conclusion There was limited impact of 16s PCR results on antimicrobial treatments. Relevance to practice was affected by relatively long turn-around-time for results. Utility may be increased in specialised surgical centres, or by reducing turn-around-time. Enrichment culture should be considered on samples where 16s PCR is requested. There remains limited evidence for use of 18s PCR in clinical management, and further studies in this area are likely warranted.

Myositis caused by Trachipleistophora hominis in a person with HIV: the first case in Thailand

Background To date, the cases of extraintestinal microsporidiosis have been increasingly reported in both otherwise healthy and immunocompromised individuals. Among them, microsporidial myositis is very rare (2,3). To the best of our knowledge, this is the first report of microsporidial myositis caused by Trachipleistophora hominis in a patient with HIV in Thailand. Case presentation A Thai male with HIV presented with fever and muscle pain at both anterior thighs and left arm for 3 months. Muscle biopsy was performed, and pathology exhibited neutrophils infiltrates and focal aggregations of microsporidial spores. The 18S ribosomal RNA sequence revealed the species of this microsporidium as Trachipleistophora hominis (T. hominis), and albendazole of 800 mg/day was initiated. He gradually improved, and was discharged home 6 weeks after hospitalization. Conclusion to the best of our knowledge, this is the first report of microsporidial myositis caused by Trachipleistophora hominis in a person with HIV in Thailand.

STANDARDIZATION AND APPLICATION OF PCR TARGETING CHLORELLA SPECIES ISOLATED FROM ENVIRONMENTAL SAMPLES

Objective: Identification of Chlorella species from the environment through 18s ribosomal RNA sequencing. This study was aimed to design primer targeting Chlorella and other closely related algal species targeting 18s ribosomal RNA, ITS1 region. Methods: Sanger sequencing was carried out for the identification of algae up to the genus and species level using an in-house designed primer and optimized PCR conditions. Results: Out of 2 algae samples identified phenotypically, one isolate identified as Chlorella vulgaris and other one identified as Chlorella sorokiniana based on the results of Basic Alignment Search Tool (BLAST). Conclusion: To conclude, this study provided primers with PCR conditions to characterize algal samples through molecular identification with 100% accuracy than the phenotypic method.

Molecular Identification of Trypanosoma evansi Isolated from Arabian Camels (Camelus dromedarius) in Riyadh and Al-Qassim, Saudi Arabia

We analyzed the blood from 400 one-humped camels, Camelus dromedarius (C. dromedarius), in Riyadh and Al-Qassim, Saudi Arabia to determine if they were infected with the parasite Trypanosoma spp. Polymerase chain reaction (PCR) targeting the internal transcribed spacer 1 (ITS1) gene was used to detect the prevalence of Trypanosoma spp. in the camels. Trypanosoma evansi (T. evansi) was detected in 79 of 200 camels in Riyadh, an infection rate of 39.5%, and in 92 of 200 camels in Al-Qassim, an infection rate of 46%. Sequence and phylogenetic analyses revealed that the isolated T. evansi was closely related to the T. evansi that was detected in C. dromedarius in Egypt and the T. evansi strain B15.1 18S ribosomal RNA gene identified from buffalo in Thailand. A BLAST search revealed that the sequences are also similar to those of T. evansi from beef cattle in Thailand and to T. brucei B8/18 18S ribosomal RNA from pigs in Nigeria.

Molecular Detection and Genetic Analysis of Theileria equi Detected in Apparently Healthy Horses in Nigeria

Equine theileriosis, an apicomplexan debilitating tick-borne parasitic disease of horses has caused considerable havoc to equine production all over the world. There is a dearth of information on the molecular characteristic of the parasites, Theileria equi Laveran, 1901, in Nigeria. Thus, in this study microscopy techniques and PCR were used to detect the T. equi of horses in Ogun, Oyo and Lagos States of Nigeria. We also characterized the partial region of 18S ribosomal RNA gene by sequencing and sequences analysis. One hundred and two horses consisting of Argentine 34 (33.3 %), Sudanese 21 (20.6 %) and local breeds 47 (46.1 %) including 2 females and 100 males were randomly sampled from the Polo Clubs in Ibadan, Lagos and from privately owned horse stables in Abeokuta. Blood samples were collected from the jugular vein, thin smears were prepared and stained with a field stain. The DNA was extracted from the blood and a partial region of the 18S ribosomal RNA gene was amplified. The amplified products were sequenced unidirectionally and subjected to phylogenetic analysis with those sequences obtained from the Gen-Bank. Of the 102 horses tested, 12 (11.7 %) were positive for T. equi by microscopy which included 9 (19.1 %) local breeds, 2 (5.8 %) Argentine breed and 1 (4.8 %) Sudanese breed. In contrast, 7 (6.8 %) were positive by the PCR method; out of which 5 (10.6 %) of these samples were from the local breed of horses while the remaining 2 (5.8 %) were from the Argentine breed. The Packed Cell Volume (PCV) of the infected and non-infected horses did not show any significant (P < 0.05) difference. The sequences lengths obtained were 311 bp and they had 97.43—98.07 % homologies with available sequences in the GenBank. The phylogenetic analysis of the sequences suggested that the strain of T. equi detected in the study area formed a new genotype different from the established genotypes around the world. In conclusion, the prevalence of T. equi was very low in the study area and one strain of the parasite may be in circulation among the studied horses.