Insights into the genetic diversity of Angiostrongylus spp. causing human angiostrongyliasis and implications for molecular identification and diagnosis.

Angiostrongylus cantonensis and Angiostrongylus costaricensis are known human pathogens responsible for eosinophilic angiostrongyliasis and abdominal angiostrongyliasis, respectively. Humans are accidental hosts, where infection occurs through the consumption of the infective larva stage 3 in intermediate or paratenic hosts. The proven method for abdominal angiostrongyliasis diagnosis is the histological examination through tissue biopsy, while the diagnosis of eosinophilic angiostrongyliasis is the detection of larva in the cerebrospinal fluid. As there is molecular evidence of cryptic species within A. cantonensis and A. costaricensis lineages, along with morphological similarities within both lineages, accurate species identification and disease diagnosis may be challenging. Moreover, species within the lineages share similar intermediate and definitive hosts and geographic distribution. For example, both A. cantonensis and Angiostrongylus malaysiensis (a closely related species in A. cantonensis lineage) overlap in their geographic distribution in Southeast Asia. Additionally, variations in the molecular makeup of A. costaricensis and A. cantonensis lineages may impact the pathogenicity, infectivity, and disease severity of angiostrongyliasis. Understanding of the genetic diversity of both lineages is a cornerstone for improved diagnosis and disease intervention, especially in a changing global environment. To shed light and provide insights into the genetic diversity of the Angiostrongylus lineages causing human angiostrongyliasis, we aim to present an up-to-date review of the studies conducted and genetic markers used for A. costaricensis and A. cantonensis lineages. The implications for accurate molecular identification and diagnosis of human angiostrongyliasis are also discussed.

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.

Case Report: The First Direct Evidence of Gnathostoma spinigerum Migration through Human Lung.

Gnathostomiasis is a helminthic infection caused by the third-stage larvae of nematodes of the genus Gnathostoma. The life cycle in humans starts with an enteric phase, with the worm perforating the gastric or intestinal mucosa to reach the peritoneal cavity and migrating through the human body. Subsequent penetration through the diaphragm may produce pleuropulmonary symptoms. We herein present a previously healthy 56-year-old Thai man from Southern Thailand who was an ex-smoker presented with chronic dry cough progressing to hemoptysis after consuming grilled swamp eels and freshwater fish. Chest computed tomography showed consolidation at the lingular segment, and the differential diagnosis was primary lung cancer and pulmonary tuberculosis. The lung tissue biopsied during bronchoscopy displayed segments of organisms with the phenotypic characteristics of Gnathostoma spp., and abundant eosinophils were seen in the alveolar tissue. Gnathostoma spinigerum infection was confirmed by a Western blot assay for G. spinigerum-specific 24-kDa reactive band. The patient received albendazole, and a follow-up chest radiograph revealed improvement in the consolidation in the lung and reduction in hemoptysis. We report the first direct evidence including pathology and immunohistochemistry of Gnathostoma invasion via the human lung, with clinical and radiographic presentations mimicking either malignancy or chronic infection.

Comparative Diagnosis of Strongyloidiasis in Immunocompromised Patients.

Strongyloides hyperinfection syndrome and disseminated strongyloidiasis frequently occur in immunocompromised persons and can lead to high complication and mortality rates. Thus, detection of Strongyloides stercolaris in those patients is crucial. The present study aimed to determine the prevalence of strongyloidiasis and compare the detection rates of different strongyloidiasis detection methods. We conducted a cross-sectional study of 135 adults with various immunocompromising conditions (corticosteroid usage, chemotherapy, hematologic malignancies, organ transplants, use of immunosuppressive agents, and symptomatic human immunodeficiency virus infection) in Phramongkutklao Hospital, Bangkok, Thailand. All patients were asked to undergo serology testing for Strongyloides IgG by indirect enzyme-linked immunosorbent assay (ELISA), and 3 days of stool collection for use in a simple smear along with formalin-ether concentration and agar plate techniques. Prevalence rates of strongyloidiasis were 5% by stool concentration technique, 5.4% by IgG-ELISA, and 6.7% by agar plate culture. Three of the eight strongyloidiasis cases in this study had hyperinfection syndrome. The tested risk factors of age, sex, occupation, and immunocompromising condition were not associated with Strongyloides infestation. Serology was only 42.9% sensitive (positive predictive value), but it was 96.3% specific (negative predictive value). In conclusion, prevalence rates of strongyloidiasis in this study were 5-7%. Although agar plate culture was the most sensitive technique, the other diagnostic methods might be alternatively used.

First case of neurognathostomiasis in Taiwan--A Thai laborer presenting with eosinophilic meningitis and intracranial hemorrhage.

We report a case of neurognathostomiasis in a Thai laborer for the first time in Taiwan. For patients with eosinophilic meningitis, neurognathostomiasis should be considered when brain image discloses subarachnoid or intracranial hemorrhage and when an appropriate exposure risk is available, especially a history of raw freshwater fish consumption in endemic areas, even a long time ago.

Diagnosis of gnathostomiasis by skin testing using partially purified specific antigen and total IgE levels.

BACKGROUND: A finding of antibodies to Gnathostoma spinigerum 24-kDa antigen by immunoblot analysis is currently used to confirm a diagnosis of gnathostomiasis. A simple skin test for the diagnosis of gnathostomiasis was developed, and the results were evaluated and compared with the standard Western blot (WB) test. METHODS: This cross-sectional study was conducted at the Hospital for Tropical Diseases, Bangkok, Thailand, in 2008-2011. All eligible patients were tested with partially purified proteins of mAb-detected fractions pooled and sterilized by 0.2 µm diameter syringe filter, with a phenol saline solution of 1:10 w/v. RESULTS: A total of 69 cases, 39 gnathostomiasis cases and 30 controls, were enrolled into the study; the median age (IQR) was 40 (30.5-52.5) years. The most common presenting symptom was edema (56/69, 81%). Gnathostomiasis cases having strong cutaneous reactions to the intradermal test (81%) were also positive by immunoblot. A significant correlation between skin and immunoblot tests was detected (p<0.001). The difference in total IgE levels between cases and controls was not statistically significant (p=0.51). Logistic regression models showed that positive WB and skin-test results were significantly associated with gnathostomiasis (p=0.001 and p=0.007, respectively). CONCLUSION: Gnathostoma skin testing, using prepared fractionated antigen solution of Gnathostoma spinigerum, yields good reactivity and significantly correlates with the results of immunoblot testing.

Severe form of radiculo - myelo - neuropathy with meningo - encephalitis secondary to Angiostrongylus cantonensis infection: unusual corpus callosal lesions and serial magnetic resonance imaging findings.

A 43-year-old man presented with the symptoms of recurrent lower abdominal pain, malaise and loss of appetite of 3-week duration, followed by acute onset of generalized paresthesias, fever and headache which progressed over few days to quadriparesis, altered sensorium, urinary and fecal incontinence. He had consumed raw tongue, liver, gall bladder and testicles of monitor lizard (Varanus bengalensis). Blood picture showed eosinophilia and cerebrospinal fluid (CSF) analysis revealed elevated protein and eosinophilia. Serum and CSF serology was positive for angiostrongyliasis. Magnetic resonance imaging showed focal hyperintense lesions in the corpus callosum and brainstem and an enhancing lesion in the cerebellum. Post-contrast T1-weighted axial images of spine showed evidence of cervical cord hyperintense lesions and root enhancement. Susceptibility weighted images/phase images showed unusual feature of multiple hemorrhagic lesions in the posterior fossa and supratentorial areas. Diffusion showed no restriction of corpus callosal lesions. Patient was treated with the high dose parenteral steroids with albendazole and at 6-month follow-up and had a remarkable recovery.

Population structure of Angiostrongylus cantonensis (Nematoda: Metastrongylidae) in Thailand based on PCR-RAPD markers.

Angiostrongylus cantonensis is the causative agent of angiostrongyliasis, which is widely distributed throughout the world. It can specifically infect many species of intermediate and definitive hosts. This study examined the genetic differentiation and population structure using the RAPD-PCR method of parasites obtained from 8 different geographical areas of Thailand. Based on 8 primers, high levels of genetic diversity and low levels of gene flow among populations were found. Using genetic distance and neighbor-joining dendrogram methods, A. cantonensis in Thailand could be divided into two groups with statistically significant genetic differentiation of the two populations. However, genotypic variations and haplotype relationships need to be further elucidated using other markers.

Genetic differentiation of strongyloides stercoralis from two different climate zones revealed by 18S ribosomal DNA sequence comparison.

Over 70 countries in tropical and subtropical zones are endemic areas for Strongyloides stercoralis, with a higher prevalence of the parasite often occurring in tropical regions compared to subtropical ones. In order to explore genetic variations of S. stercoralis form different climate zones, 18S ribosomal DNA of parasite specimens obtained from Thailand were sequenced and compared with those from Japan. The maximum likelihood indicates that S. stercoralis populations from these two different climate zones have genetically diverged. The genetic relationship between S. stercoralis populations is not related to the host species, but rather to moisture and temperature. These factors may directly drive genetic differentiation among isolated populations of S. stercoralis.

Rapid and simple identification of human pathogenic heterophyid intestinal fluke metacercariae by PCR-RFLP.

Six species of heterophyid intestinal flukes (HIFs) constitute the major endemic zoonotic fish-borne pathogens in Asia: Haplorchis taichui, H. pumilio, H. yokogawai, Procerovum varium, Stellantchasmus falcatus, and Centrocestus formosanus. Several different species of these parasites are often found co-infecting the same second intermediate fish host. Because of their morphological similarities, differentiating between species of HIF metacercariae is difficult, time-consuming, and frequently results in misidentification. In this study, we aimed to develop an efficient and accurate method of identifying metacercariae of these 6 HIFs. Metacercariae were roughly grouped together, based on morphological characteristics seen under a stereomicroscope. Then, PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the exact species of each metacercaria, using the 28S ribosomal RNA gene as the genetic marker and MboII as the restriction enzyme. Using a combination of morphological and molecular methods eliminates the need for DNA sequencing and infecting animal subjects to obtain adult worms, increases accuracy, and decreases the need for laborious morphological identification. Because the method is simple, rapid, and relatively cheap compared with PCR-sequencing, it may be an effective tool for epidemiological studies of HIFs in endemic areas.

Confirmation of the paraphyletic relationship between families Opisthorchiidae and Heterophyidae using small and large subunit ribosomal DNA sequences.

Opisthorchiidae and Heterophyidae are classified into different families based on morphological identification. However, recent molecular phylogenetic studies suggested the possible paraphyletic relationship between these two families. In this study, the paraphyletic relationship between these two families was confirmed further by maximum likelihood and Bayesian inference analyses using the combined sequences of SSU and LSU rDNA.

Iso-electricfocusing of Bithynia snail antigens for IgG- and IgG(1-4)-ELISA detection of human opisthorchiasis.

Diagnosis of opisthorchiasis is confirmed by the presence of characteristic eggs and worms. However, misdiagnosis may occur in light infections, and also due to the morphological similarity of opisthorchid eggs to other species. A finding of specific immune mediators can help confirm infection. This study used indirect ELISA to detect total IgG and IgG(1-4) with selected antigens of Bithynia siamensis goniomphalos extract, which were derived by liquid-phase iso-electricfocusing (IFE). Antigens (Iso-AgF) from 20 IEF fractionated fractions were selected based on a high ELISA-OD ratio between pooled-positive and pooled-negative sera. Iso-AgF 7, 7, 6, 2, and 10 resulted in high OD-ratio to total IgG, IgG1, 2, 3, and 4, respectively. A full-scale ELISA was conducted with sera from 50 opisthorchiasis cases, 196 from other parasitic-disease cases, and 35 healthy controls. Iso-AgF7 to IgG1 showed the best result, with sensitivity, specificity, positive and negative predictive value of 100, 96, 86, and 100%, respectively, at a cut-off 0.221. Low cross-reactivity to IgG1 was found in one case each of gnathostomiasis, trichinellosis, toxocariasis, angiostrongyliasis, bancroftian filariasis, enterobiasis, neurocysticercosis, and taeniasis. Thus, Iso-AgF7 to IgG1 was a good candidate antigen to be developed for detection of antibodies against Opisthorchis viverrini.

Predictive factors for Gnathostoma seropositivity in patients visiting the Gnathostomiasis Clinic at the Hospital for Tropical Diseases, Thailand during 2000-2005.

This was a retrospective study of patients having Gnathostoma antibody testing at the Hospital for Tropical Diseases, Bangkok during 2000-2005 to investigate predictive factors for Gnathostoma seropositivity in patients attending the Gnathostomiasis Clinic. Out of 849 patients tested, 531 (62.5%) were Gnathostoma seropositive. The median absolute eosinophil counts were 464 (0-16,796) and 326.5 (0-10,971) cells/mm3 in seropositive and seronegative patients, respectively (p<0.001). Differences in a history of cutaneous swelling, the habit of eating raw meat, eosinophilia (>500 cells/mm3), and the frequency of cutaneous swellings between seropositive and seronegative patients were all statistically significant. Patients with a history of eating raw meat and a history of cutaneous swelling were at 2.1 and 1.8 times more likely to be Gnathostoma seropositive, respectively. Logistic regression analysis showed eosinophilia was not a predictive factor for Gnathostoma seropositivity.

Double-dose ivermectin vs albendazole for the treatment of gnathostomiasis.

A comparative study was performed for the treatment of gnathostomiasis patients with ivermectin 0.2 mg/kg for 2 days in 15 patients vs albendazole 400 mg twice daily for 21 days in 14 patients. The ivermectin and albendazole gave cure rates of 100% and 78.5%, respectively, however the difference was not statistically significant between the two drugs (Fisher's exact, p=0.0996). One year after treatment, the patients who had no migratory swellings and a drop in ELISA titers or a negative immunoblot test were considered to be cured. The side effect of ivermectin for two days was dizziness. The side effects of albendazole were nausea, dizziness, and an increased alkaline phosphatase.

Negative-ELISA using native and filtrated cystic fluid antigens to rule out cystic echinococcosis.

An increasing number of cases of echinococcosis in Thailand have been imported, probably native infections and medical transfers. Serodiagnosis is one diagnostic choice for interpreting infections before a further step is done. Due to limited antigen, indirect ELISA has been used as a negative screening test for IgG-detection to rule out echinococcosis. Native hydatid cystic fluid (HCF) antigen from Belgium was used for such testing, in which the ODs-ELISA of samples were compared with those of two positive controls. Subsequently, hydatid cyst fluid from a Thai patient was obtained and the filtrated cyst fluid antigen [(<30)-(>10) kDa, HCF30.10] was prepared to develop negative screening results for the serum samples. By using HCF, three of twenty-four samples resulted in higher ODs-ELISA than the controls. In an attempt to observe the cross-reactivity of this native antigen, IgG-antibodies from many helminthiases cross-reacted and showed high ODs-ELISA. The HCF30.10 Ag was used to develop the test and analyze IgG-antibodies from 5 positive controls (2 parasite-confirmed and 3 positive-serodiagnosed), 183 heterologous cases of 29 diseases and 50 healthy control sera. At a cut-off value of 0.484, the test had 100% sensitivity and 42% specificity. Only Malayan filariasis, onchocercosis, fascioliasis, amebiasis, giardiasis and blastocystosis gave true negatives. Antibodies from nematodiases strongly cross-reacted with HCF30.10 Ag. Nine of fifty (18%) healthy serum controls produced higher OD-values than the cut-off. The routine ELISA uses the HCF30.10 Ag to produce a negative result to echinococcosis, because limited cystic fluid antigen (Thai patient) for test improvement, a lot of cross-reactions and only two protoscolex-positive controls are available.