Development of a PCR assay and pyrosequencing for identification of important human fish-borne trematodes and its potential use for detection in fecal specimens

BACKGROUND: Small liver and minute intestinal flukes are highly prevalent in Southeast Asia. Definitive diagnosis of parasite infection is usually achieved parasitologically by finding the fluke eggs in feces. However, their eggs are difficult to differentiate morphologically in fecal samples, even for experienced technicians. The present study developed a PCR assay coupled with DNA pyrosequencing for identification of the fish-borne trematodes (FBT), Opisthorchis viverrini, Clonorchis sinensis, Haplorchis taichui, H. pumilio and Stellantchasmus falcatus, and to evaluate potential detection in fecal specimens, and identification and differentiation of cercarial and metacercarial stages. METHODS: Primers targeting the partial 28S large subunit ribosomal RNA gene were designed and about 46–47 nucleotides were selected as the target region for species identification by a PCR assay coupled with a pyrosequencing technique. RESULTS: The nucleotide variations at 24 positions, which is sufficient for the identification of the five species of FBT were selected. The method could identify O. viverrini and C. sinensis eggs in feces, cercarial and metacercarial stages of O. viverrini, and metacercarial stage of H. pumilio and H. taichui. The detection limit was as little as a single O. viverrini or C. sinensis egg artificially inoculated in 100 mg of non-infected fecal sample (equivalent to 10 eggs per gram), indicating highly sensitivity. The method was found to be superior to the traditional microscopy method and was more rapid than Sanger DNA sequencing. CONCLUSIONS: DNA pyrosequencing-based identification is a valuable tool for differentiating O. viverrini and other Opisthorchis-like eggs, and can be applied to epidemiological studies and for molecular taxonomic investigation of FBT in endemic areas

Growth and development of Gnathostoma spinigerum (Nematoda: Gnathostomatidae) larvae in Mesocyclops aspericornis (Cyclopoida: Cyclopidae)

<p>Abstract</p> <p>Background</p> <p><it>Gnathostoma spinigerum </it>larva is pathogenic, causing gnathostomiasis in humans and certain animals, and is prevalent mainly in Asia. Growth and development of <it>Gnathostoma spinigerum </it>larvae in the cyclopoid copepod <it>Mesocyclops aspericornis</it>, the first intermediate host, were examined.</p> <p>Results</p> <p>When newly hatched, ensheathed second-stage larvae (L2) were ingested by <it>M. aspericornis</it>, they immediately appeared exsheathed in the stomach of <it>M. aspericornis</it>. They then penetrated the stomach wall and entered the body cavity, where they immediately metamorphosed to a stunted form with the body length/width ratio equal to the early third-stage larvae (EL3) up to 2 h after being ingested. During metamorphosis, the anterior spine-like structure of L2 transformed into unequal transparent lips. The larvae moulted into EL3 in the body cavity of the copepod at around day 5-7 post-infection. Minute cuticular striations were seen on the whole body, with prominent single-pointed spines on the anterior part of the body. The head bulb had four rows of hooklets and two lateral trilobed lips. The size of EL3 in copepods continuously increased towards day 12 and showed a negative correlation to their density per copepod (R = -0.881, <it>P </it>< 0.05 for body length, and R = -0.906, <it>P </it>< 0.05 for body width).</p> <p>Conclusions</p> <p>The results revealed for the first time that <it>M. aspericornis</it>, one of the most abundant freshwater copepods in Thailand, is a suitable first intermediate host for <it>G. spinigerum</it>. High susceptibility of <it>M. aspericornis </it>suggests its importance for the maintenance of the life cycle of <it>G. spinigerum </it>in Thailand.</p

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts

Development and validation of a real-time SYBR green PCR method for the detection and differentiation of Babesia and Theileria species (Apicomplexa: Piroplasmida) in hard ticks and cattle blood from Thailand

Tick-borne pathogens, particularly Babesia and Theileria species, are major threats to cattle production, causing economically significant diseases such as babesiosis and theileriosis. In this study, a real-time SYBR Green PCR assay was developed to detect Babesia and Theileria species in hard ticks (N = 65) and cattle blood samples (N = 143) from Thailand. Using primers targeting the mitochondrial cytochrome b gene for Babesia and the nuclear 18S rRNA gene for Theileria, the assay measured specific melting temperatures (Tm) for each species. The results showed distinct Tm values for Babesia bigemina (74.38 ± 0.04 °C), Babesia bovis (75.7 ± 0.06 °C), Theileria orientalis (74.61 ± 0.03 °C), Theileria sinensis (75.84 ± 0.03 °C), and Theileria annulata (74.06 ± 0.03 °C). The assay demonstrated high specificity, with a cutoff cycle threshold of < 35 cycles and a minimum detectable concentration of 10 copies/μL. Significant species differences in melting curves were confirmed using Tukey’s HSD test (p < 0.05). Theileria orientalis was detected in 8.4% of cattle blood samples, while T. sinensis was found in 25.9%, and B. bigemina in 0.7%. Theileria orientalis was also detected in 7.7% of tick samples, T. sinensis in 16.9%, and B. bigemina in 6.1%. The assay returned negative results for all non-target blood and tissue pathogens tested for specificity. This robust, high-throughput assay is highly effective for monitoring Babesia and Theileria infections, facilitating close surveillance and intervention efforts against tick-borne diseases in cattle

Development of a Machine Learning Model for the Classification of Enterobius vermicularis Egg

Enterobius vermicularis (pinworm) infections are a significant global health issue, affecting children predominantly in environments like schools and daycares. Traditional diagnosis using the scotch tape technique involves examining E. vermicularis eggs under a microscope. This method is time-consuming and depends heavily on the examiner&rsquo;s expertise. To improve this, convolutional neural networks (CNNs) have been used to automate the detection of pinworm eggs from microscopic images. In our study, we enhanced E. vermicularis egg detection using a CNN benchmarked against leading models. We digitized and augmented 40,000 images of E. vermicularis eggs (class 1) and artifacts (class 0) for comprehensive training, using an 80:20 training&ndash;validation and a five-fold cross-validation. The proposed CNN model showed limited initial performance but achieved 90.0% accuracy, precision, recall, and F1-score after data augmentation. It also demonstrated improved stability with an ROC-AUC metric increase from 0.77 to 0.97. Despite its smaller file size, our CNN model performed comparably to larger models. Notably, the Xception model achieved 99.0% accuracy, precision, recall, and F1-score. These findings highlight the effectiveness of data augmentation and advanced CNN architectures in improving diagnostic accuracy and efficiency for E. vermicularis infections

Molecular identification of Trichuris species in long-tailed macaques from Dong Ling Don Chao Pu Park and Kumphawapi Monkey Garden, Northeast Thailand: First report suggesting possible Trichuris ovis infection in non-human primates

This study aimed to molecularly identify Trichuris spp. in long-tailed macaques from two key habitats in Northeast Thailand: Dong Ling Don Chao Pu Park, Amnat Charoen Province, and Kumphawapi Monkey Garden, Udon Thani Province. Genomic DNA was extracted from 13 Trichuris spp. egg samples collected from 13 infected long-tailed macaques, and PCR amplification targeting partial sequences of the 18S rRNA gene and ITS2 region was performed for phylogenetic analysis. Of the 13 Trichuris spp. egg samples, the partial 18S rRNA gene was successfully amplified from six, while ITS2 amplification was unsuccessful. Phylogenetic analysis indicated that four specimens sequenced from Dong Ling Don Chao Pu Park were T. trichiura. In contrast, two specimens sequenced from Kumphawapi Monkey Garden clustered with the only confirmed T. ovis from goat, as well as unconfirmed Trichuris spp. from other ruminant hosts. These findings suggest that the Trichuris spp. in macaques are likely T. ovis; however, the evidence remains inconclusive. Therefore, accurate species identification in this region requires further molecular analysis using additional genetic markers. This study provides the first molecular identification of T. trichiura in long-tailed macaques from Northeast Thailand. Additionally, it is the first report suggesting the possible T. ovis infection in non-human primates. These findings highlight the potential for Trichuris spp. transmission across diverse host species, underscoring the need for enhanced surveillance of parasitic infections in wildlife and livestock, particularly in regions with close human-animal interactions. Continued molecular investigations are essential to elucidate Trichuris spp. transmission dynamics and zoonotic potential, aiding in public health risk mitigation