Molecular insights versus morphological traits: rethinking identification of the closely related Angiostrongylus cantonensis and Angiostrongylus malaysiensis.

BACKGROUND: The closely related Angiostrongylus cantonensis and Angiostrongylus malaysiensis have been reported to coexist in Thailand and share similar hosts and life cycles. Recently, in an angiostrongyliasis outbreak in Thailand, both A. cantonensis and A. malaysiensis were found in the cerebrospinal fluid of affected patients. Morphological similarities, overlapping distribution, shared hosts and habitats, and the close genetics of the two Angiostrongylus species can complicate accurate species identification. Addressing these challenges, this study aims to evaluate whether a correlation between the morphological and genetic identities of A. cantonensis and A. malaysiensis can improve species identification accuracy. METHODS: Angiostrongylus spp. specimens from five zoogeographical regions in Thailand were subjected to morphological and molecular identification using the mitochondrial cytochrome b gene and the nuclear internal transcribed spacer 2 region (ITS2). The morphological characters for males and females were then validated using the species identity obtained from the nuclear ITS2 region. RESULTS: The results revealed that morphological misidentifications between these two closely related species are common due to overlapping morphological characters. Although certain male traits such as body length and width aided species differentiation, female traits were found to be less reliable. Furthermore, hybrid forms (8.2%) were revealed through the ITS2 results, which can further complicate morphological identification. Mito-nuclear discordance was also present in 1.9% of the Angiostrongylus specimens from Thailand, suggesting a complex historical interbreeding between the species. CONCLUSIONS: Based on our findings, we suggest that nuclear ITS2 is a reliable marker for species identification of A. cantonensis and A. malaysiensis, especially in regions where both species coexist. Additionally, the scope and consequences of hybridization between the two closely related Angiostrongylus species should be further investigated in Thailand.

Discovery of Encyclometra bungara (Digenea: Encyclometridae) in a new host (Enhydris enhydris) from Thailand and Cambodia through morphological and molecular identification.

The genus Encyclometra is one of the two genera in family Encyclometridae, known for parasitising the oesophagus, stomach and intestine of snakes. Among Encyclometra, the species present are: Encyclometra colubrimurorum, Encyclometra japonica, Encyclometra asymmetrica and Encyclometra bungara. Species discrimination within Encyclometra has predominantly relied on morphological differences, such as the length of the caeca and the position of the testes. Morphological overlaps exist among these species making species discrimination challenging. Additionally, the use of molecular information has been limited for Encyclometra. To determine the Encyclometra species infecting Enhydris enhydris from Thailand and Cambodia, morphological and molecular identification was conducted. Morphological characters and measurements were obtained from 30 Encyclometra adults, and they were compared with previous studies of other Encyclometra species. Novel sequences of E. bungara were generated using the nuclear 18S and 28S ribosomal RNA genes, and the mitochondrial cytochrome c oxidase subunit 1 gene. Our results revealed that the specimens could be morphologically identified as E. bungara, with support from molecular information obtained from the phylogenies of the 3 genetic markers employed. Molecular analysis showed that the Encyclometra specimens were distinct from E. colubrimurorum and E. japonica. Through morphological and molecular identification of the Encyclometra specimens found in E. enhydris from Thailand and Cambodia, we describe and provide a record of E. bungara in a new host and new locality. Additionally, novel molecular sequences were generated, revealing the phylogenetic position of E. bungara within the superfamily Gorgoderoidea.

Prevalence of Strongyloides in Southeast Asia: a systematic review and meta-analysis with implications for public health and sustainable control strategies.

BACKGROUND: Strongyloidiasis, caused by the nematodes Strongyloides stercoralis and Strongyloides fuelleborni, is estimated to affect over 600 million individuals worldwide. The disease is endemic in Southeast Asia, where a warm-humid climate and socio-economic conditions maintain the parasite's life cycle and transmission. However, the current diagnostic methods may not be sufficiently sensitive, suggesting that the true prevalence of strongyloidiasis could be seriously underestimated in this. This study aims to determine the prevalence of strongyloidiasis in Southeast Asia through a systematic review and meta-analysis and to discuss the implications of the estimated prevalence on diagnostic approaches and control strategies. METHODS: Following PRISMA guidelines, we conducted a systematic literature search in PubMed and Google Scholar databases to identify studies reporting Strongyloides prevalence data in the 11 Southeast Asian countries up to December 2022. A random effects model was employed to estimate the pooled prevalence of S. stercoralis at both regional and country levels. RESULTS: Out of 3722 articles identified, 224 met our inclusion criteria. For S. stercoralis specifically, we found 187 articles, of which 52.4% were from Thailand. All Southeast Asian countries, except Brunei, had at least one study on Strongyloides prevalence. The estimated pooled prevalence of S. stercoralis regionally was 12.7% (95% CI 10.70-14.80%), ranging from 0.4 to 24.9% at the country level. Cambodia had the highest pooled prevalence (24.9%, 95% CI 15.65-35.38%), followed by Lao PDR (16.5%, 95% CI 9.50-24.95%). Moreover, we obtained a pooled prevalence of 10% (95% CI 7.06-13.52%) in a group comprising immigrants, workers, and veterans from Southeast Asian countries. S. stercoralis infects various host types, including nonhuman primates, domestic dogs and cats, rodents, and transport carriers such as cockroaches and vegetables. CONCLUSIONS: A high prevalence of strongyloidiasis in Southeast Asia was revealed, highlighting the importance of the region's ongoing research, surveillance, and control efforts. Factors contributing to the strongyloidiasis transmission include the role of animal hosts, the impact of global connectivity, and the significance of the co-endemicity of other Strongyloides species. Based on these findings, a multi-pronged One-Health approach is essential for sustainable intervention and control.

From past to present: opportunities and trends in the molecular detection and diagnosis of Strongyloides stercoralis.

Strongyloides stercoralis is a soil-transmitted helminth that is mainly found in the tropical and subtropical regions and affects approximately 600 million people globally. The medical importance of strongyloidiasis lies in its capacity to remain asymptomatic and chronically unnoticed until the host is immunocompromised. Additionally, in severe strongyloidiasis, hyperinfection syndrome and larva dissemination to various organs can occur. Parasitological techniques such as Baermann-Moraes and agar plate culture to detect larvae in stool samples are the current gold standard. However, the sensitivity might be inadequate, especially with reduced worm burden. Complementing parasitological techniques, immunological techniques including immunoblot and immunosorbent assays are employed, with higher sensitivity. However, cross-reactivity to other parasites may occur, hampering the assay's specificity. Recently, advances in molecular techniques such as polymerase chain reaction and next-generation sequencing technology have provided the opportunity to detect parasite DNA in stool, blood, and environmental samples. Molecular techniques, known for their high sensitivity and specificity, have the potential to circumvent some of the challenges associated with chronicity and intermittent larval output for increased detection. Here, as S. stercoralis was recently included by the World Health Organization as another soil-transmitted helminth targeted for control from 2021 to 2030, we aimed to present a review of the current molecular techniques for detecting and diagnosing S. stercoralis in a bid to consolidate the molecular studies that have been performed. Upcoming molecular trends, especially next-generation sequencing technologies, are also discussed to increase the awareness of its potential for diagnosis and detection. Improved and novel detection methods can aid in making accurate and informed choices, especially in this era where infectious and non-infectious diseases are increasingly commonplace.

Sensitive and accurate DNA metabarcoding of parasitic helminth mock communities using the mitochondrial rRNA genes.

Next-generation sequencing technologies have accelerated the pace of helminth DNA metabarcoding research, enabling species detection in bulk community samples. However, finding suitable genetic markers with robust species-level resolution and primers targeting a broad species range among parasitic helminths are some of the challenges faced. This study aimed to demonstrate the potential use of the mitochondrial 12S and 16S rRNA genes for parasitic helminth (nematodes, trematodes, cestodes) DNA metabarcoding. To demonstrate the robustness of the 12S and 16S rRNA genes for DNA metabarcoding, we determined the proportion of species successfully recovered using mock helminth communities without environment matrix and mock helminth communities artificially spiked with environmental matrices. The environmental matrices are human fecal material, garden soil, tissue, and pond water. Our results revealed the robustness of the mitochondrial rRNA genes, through the high sensitivity of the 12S rRNA gene, and the effectiveness of the 12S and 16S primers targeting platyhelminths. With the mitochondrial rRNA genes, a broad range of parasitc helminths were successfully detected to the species level. The potential of the mitochondrial rRNA genes for helminth DNA metabarcoding was demonstrated, providing a valuable gateway for future helminth DNA metabarcoding applications like helminth detection and biodiversity studies.

The potential use of mitochondrial ribosomal genes (12S and 16S) in DNA barcoding and phylogenetic analysis of trematodes.

BACKGROUND: Genetic markers like the nuclear ribosomal RNA (rRNA) genes, internal transcribed spacer regions, mitochondrial protein-coding genes, and genomes have been utilized for molecular identification of parasitic trematodes. However, challenges such as the design of broadly applicable primers for the vast number of species within Digenea and the genetic markers' ability to provide sufficient species-level resolution limited their utility. This study presented novel and broadly applicable primers using the mitochondrial 12S and 16S rRNA genes for Digenea and aimed to show their suitability as alternative genetic markers for molecular identification of orders Plagiorchiida, Echinostomida, and Strigeida. RESULTS: Our results revealed that the mitochondrial 12S and 16S rRNA genes are suitable for trematode molecular identification, with sufficient resolution to discriminate closely related species and achieve accurate species identification through phylogenetic placements. Moreover, the robustness of our newly designed primers to amplify medically important parasitic trematodes encompassing three orders was demonstrated through successful amplification. The convenience and applicability of the newly designed primers and adequate genetic variation of the mitochondrial rRNA genes can be useful as complementary markers for trematode molecular-based studies. CONCLUSIONS: We demonstrated that the mitochondrial rRNA genes could be alternative genetic markers robust for trematode molecular identification and potentially helpful for DNA barcoding where our primers can be widely applied across the major Digenea orders. Furthermore, the potential of the mitochondrial rRNA genes for molecular systematics can be explored, enhancing their appeal for trematode molecular-based studies. The novelty of utilizing the mitochondrial rRNA genes and the designed primers in this study can potentially open avenues for species identification, discovery, and systematics in the future.

Co-occurrence of Angiostrongylus malaysiensis and Angiostrongylus cantonensis DNA in cerebrospinal fluid: Evidence from human eosinophilic meningitis after ingestion of raw snail dish in Thailand.

Angiostrongylus cantonensis, the main causative agent of human neuroangiostrongyliasis, is a food-borne parasitic zoonosis, particularly in Southeast Asia and Mainland China. Angiostrongylus malaysiensis, a cryptic species, has not been unequivocally identified as a causative agent for human angiostrongyliasis. Here, we investigated a local incidence of human angiostrongyliasis in Kalasin Province, northeastern part of Thailand. Field and laboratory investigations, clinical symptoms, and treatment of the disease are also discussed. Five sera and three cerebrospinal fluid samples were taken from each patient who displayed clinical symptoms of mild or severe headache without neck stiffness after ingesting a local dish containing Pila virescens. With molecular evidence using PCR and DNA sequencing approaches, we confirmed the presence of A. malaysiensis and A. cantonensis DNA in the patient samples. In addition, P. virescens and Pomacea canaliculata collected in the vicinity were also examined for the existence of angistrongylid larvae. The rate of infection in the snail population was 33.3% (18 infection out of 54 examined), with A. cantonensis as the predominant species. Notably, two snails were found to be co-infected with both A. malaysiensis and A. cantonensis. This discovery comes after several years of suspicion that it could be a zoonotic pathogen. Therefore, our findings are important for public health and clinical diagnosis since clinicians are not aware of the zoonotic potential of A. malaysiensis in humans.

Assessing the suitability of mitochondrial and nuclear DNA genetic markers for molecular systematics and species identification of helminths.

BACKGROUND: Genetic markers are employed widely in molecular studies, and their utility depends on the degree of sequence variation, which dictates the type of application for which they are suited. Consequently, the suitability of a genetic marker for any specific application is complicated by its properties and usage across studies. To provide a yardstick for future users, in this study we assess the suitability of genetic markers for molecular systematics and species identification in helminths and provide an estimate of the cut-off genetic distances per taxonomic level. METHODS: We assessed four classes of genetic markers, namely nuclear ribosomal internal transcribed spacers, nuclear rRNA, mitochondrial rRNA and mitochondrial protein-coding genes, based on certain properties that are important for species identification and molecular systematics. For molecular identification, these properties are inter-species sequence variation; length of reference sequences; easy alignment of sequences; and easy to design universal primers. For molecular systematics, the properties are: average genetic distance from order/suborder to species level; the number of monophyletic clades at the order/suborder level; length of reference sequences; easy alignment of sequences; easy to design universal primers; and absence of nucleotide substitution saturation. Estimation of the cut-off genetic distances was performed using the 'K-means' clustering algorithm. RESULTS: The nuclear rRNA genes exhibited the lowest sequence variation, whereas the mitochondrial genes exhibited relatively higher variation across the three groups of helminths. Also, the nuclear and mitochondrial rRNA genes were the best possible genetic markers for helminth molecular systematics, whereas the mitochondrial protein-coding and rRNA genes were suitable for molecular identification. We also revealed that a general gauge of genetic distances might not be adequate, using evidence from the wide range of genetic distances among nematodes. CONCLUSION: This study assessed the suitability of DNA genetic markers for application in molecular systematics and molecular identification of helminths. We provide a novel way of analyzing genetic distances to generate suitable cut-off values for each taxonomic level using the 'K-means' clustering algorithm. The estimated cut-off genetic distance values, together with the summary of the utility and limitations of each class of genetic markers, are useful information that can benefit researchers conducting molecular studies on helminths.

Newly developed SYBR Green-based quantitative real-time PCRs revealed coinfection evidence of Angiostrongylus cantonensis and A. malaysiensis in Achatina fulica existing in Bangkok Metropolitan, Thailand.

Angiostrongylus cantonensis is a well-known pathogen causing eosinophilic meningitis associated with angiostrongyliasis. Humans, as accidental hosts, are infected by consuming undercooked snails containing third-stage larvae. A. malaysiensis is closely related to A. cantonensis and has been described as a potential human pathogen. The two species distribution was recently reported to overlap in the same endemic area, particularly in the Indochina Peninsula. Similar morphological characteristics of the third-stage larva in the snail-intermediate host often lead to misidentification of the two species. Thus, we aimed to develop a sensitive and specific method to detect and discriminate Angiostrongylus third-stage larva by designing species-specific primers based on the mitochondrial cytochrome b gene. We developed the SYBR Green quantitative real-time PCR (qPCR) method for two species-specific detection assays, which could be conducted simultaneously. The method was subsequently employed to detect and identify third-stage larvae of Angiostrongylus isolated from infected Achatina fulica collected from six public parks in Bangkok Metropolitan, Thailand. The method was also a preliminary applied to detect parasite tissue debris in the patients' cerebrospinal fluid (CSF). SYBR Green qPCRs quantitatively detected approximately 10-4 ng of genomic DNA from one larva, facilitating species-specific detection. Based on the pools of third-stage larvae isolated individually from the tissue of each infected A. fulica collected from the public parks, the qPCR results revealed that A. malaysiensis was the predominant species infecting 5.26% of the collected snails. In comparison, coinfection between A. malaysiensis and A. cantonensis was 5.97%, and no single infection of A. cantonensis was detected in A. fulica. Our SYBR Green qPCR method is a useful and inexpensive technique for A. cantonensis and A. malaysiensis discrimination, and the method has sufficient sensitivity to detect isolated larvae from a snail-intermediate host. The ratio of A. cantonensis and A. malaysiensis larvae infecting the snails can also be estimated simultaneously. Our qPCRs can be employed in a molecular survey of A. cantonensis and A. malaysiensis within intermediate hosts and for clinical diagnosis of angiostrongyliasis with CSF specimens in future studies.

Evaluation and utility of mitochondrial ribosomal genes for molecular systematics of parasitic nematodes.

BACKGROUND: Molecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics. METHODS: To study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms. RESULTS: The phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers. CONCLUSIONS: This study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.

Mitochondrial ribosomal genes as novel genetic markers for discrimination of closely related species in the Angiostrongylus cantonensis lineage.

The Angiostrongylus cantonensis lineage (Nematoda: Metastrongyloidea) consists of the closely related species A. cantonensis, Angiostrongylus malaysiensis, and Angiostrongylus mackerrasae. Various genetic markers have been used for species discrimination in molecular phylogenetic studies of this lineage. However, despite showing potential in other organisms, mitochondrial 12S and 16S ribosomal RNA (rRNA) genes have not been used for Angiostrongylus species discrimination. Therefore, this study assessed these genes' suitability for inter- and intraspecies discrimination in the A. cantonensis lineage. The ultimate aim was to provide a novel genetic marker to support existing phylogenies. Sixty adult Angiostrongylus spp. worms from four geographic locations in Thailand were identified morphologically before molecular identification with 12S and 16S rRNA genes. Neighbor-joining and maximum likelihood algorithms were used for phylogenetic analyzes, and sequence variation was calculated to determine whether the genes could be used to discriminate among species. Furthermore, sequence variation was compared among previously used genetic markers to evaluate the robustness of the 12S and 16S rRNA genes as markers. Using both markers, the A. cantonensis lineage formed a monophyletic clade with a clear separation between A. cantonensis, A. malaysiensis, and A. mackerrasae. From our representative A. cantonensis and A. malaysiensis specimens, the genetic distance between the two clades was 6.8% -7.9% and 7.9% -10.0% for 12S and 16S rRNA genes, respectively, which is sufficient interspecific genetic variation for species discrimination. Higher levels of genetic variation were observed for the 16S rRNA gene, with 12 haplotypes and an intraspecific variation ≤2.2%. Thus, as a genetic marker, the 16S rRNA gene is comparable to mitochondrial protein-coding genes, which are commonly used in intra-level Angiostrongylus spp. studies. In conclusion, mitochondrial 12S and 16S rRNA genes can discriminate among closely related species in the A. cantonensis lineage, and they represent novel genetic markers for supporting existing phylogenies and verifying the phylogenetic position of A. mackerrasae.