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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.

ABSTRACT: 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.

SUBMITTER: Jakkul W

PROVIDER: S-EPMC8005753 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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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.

Jakkul Wallop W Chaisiri Kittipong K Saralamba Naowarat N Limpanont Yanin Y Dusitsittipon Sirilak S Charoennitiwat Vachirapong V Chan Abigail Hui En AHE Thaenkham Urusa U

Food and waterborne parasitology 20210315

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 morpholog ...[more]

PMID: 33817357

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Project description:Angiostrongyliasis is a parasitic disease caused by nematodes of the genus Angiostrongylus. Distribution of this worm corresponds to the dispersal of its main intermediate host, the giant African land snail Achatina fulica. Genetic characterization can help identify parasitic pathogens and control the spreading of disease. The present study describes infection of A. fulica by Angiostrongylus, and provides a genetic outlook based on sequencing of specific regions. We collected 343 land snails from 22 provinces across six regions of Thailand between May 2017 and July 2018. Artificial digestion and Baermann's technique were employed to isolate Angiostrongylus larvae. The worm and its intermediate host were identified by sequencing with specific nucleotide regions. Phylogenetic tree was constructed to evaluate the relationship with other isolates. A. fulica from Chaiyaphum province was infected with A. cantonensis, whereas snails collected from Phrae and Chiang Rai provinces were infected with A. malaysiensis. The maximum likelihood tree based on 74 A. fulica COI sequences revealed monophyletic groups and identified two haplotypes: AF1 and AF2. Only AF1, which is distributed in all regions of Thailand, harbored the larvae of A. cantonensis and A. malaysiensis. Two mitochondrial genes (COI and cytb) and two nuclear regions (ITS2 and SSU rRNA) were sequenced in 41 Angiostrongylus specimens. The COI gene indicated that A. cantonensis was closely related to the AC10 haplotype; whereas the cytb gene revealed two new haplotypes: AC19 and AC20. SSU rRNA was useful for the identification of A. cantonensis; whereas ITS2 was a good genetic marker for differentiating between A. cantonensis and A. malaysiensis. This study provides genetic information about the parasite Angiostrongylus and its snail intermediate host. The data in this work may be useful for further study on the identification of Angiostrongylus spp., the genetic relationship between intermediate host and parasite, and control of parasites.

Project description:BackgroundThe detection of Angiostrongylus spp. larvae in intermediate host snails is a critical epidemiological investigation, essential for the effective control of disease outbreaks. Compared to molecular biological detection methods, lung microscopy, a traditional pathogen morphological detection approach, is susceptible to oversights and exhibits relatively lower sensitivity. However, we posit that lung microscopy offers irreplaceable advantages in the context of large-scale field surveys and can serve as a vital foundation for use in conjunction with other diagnostic technologies.MethodsIn this study, 348 Achatina fulica samples were examined using lung microscopy, PCR, and AcanITS1 qPCR. Statistical analysis was conducted to compare detection rates and sensitivities among these methods. DNA from a snail confirmed positive by lung microscopy was diluted and tested using PCR and AcanITS1 qPCR to assess the diagnostic efficacy of the molecular assays. Finally, we combined the highly sensitive AcanITS1 qPCR with lung microscopy for identifying Angiostrongylus spp. larvae in Achatina fulica for the first time to our knowledge and compared its diagnostic efficacy with that of individual testing methods.ResultsThe lung microscopy, PCR, AcanITS1 qPCR, and combined test yielded detection rates of 29.31%, 32.18%, 38.22%, and 38.51%, respectively. These differences were statistically significant (X2 = 9.565, p < 0.05). Notably, AcanITS1 qPCR demonstrated superior sensitivity with a detection threshold of 10 pg/μl, outperforming the PCR with a threshold of 10 ng/μl. When PCR was utilized as the gold standard, the sensitivities for lung microscopy, AcanITS1 qPCR, and the combined test were determined to be 88.39%, 97.32%, and 98.21%, respectively. Correspondingly, the specificities were 98.73%, 89.83%, and 89.83%, respectively.ConclusionsThis novel straregy, the combined test for the detection of Angiostrongylus spp. larvae in Achatina fulica exhibited superior positive detection rates and sensitivity compared to each of the three individual methods. We believe that this novel strategy is not only applicable to large-scale field investigations of Achatina fulica and Pomacea canaliculata but also has potential application value for monitoring the infection of these snails sold at the local farmers' markets with Angiostrongylus spp. larvae. Of course, while qPCR is exceptionally sensitive, the potential for false negatives remains a consideration. Repeated experimentation is also essential to maximize the reliability and accuracy of the outcomes.

Dataset Information

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.

Publications

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

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