A cost-effective strategy for identifying Angiostrongylus spp. larvae in Achatina fulica: combined morphological and molecular biology.

Abstract

Background: The 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.

Methods: In 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.

Results: The 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.

Conclusions: This 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.