Comparison of Loop-Mediated Isothermal Amplification and Real-Time PCR Assays for Detection of Strongyloides Larvae in Different Specimen Matrices.

Strongyloides stercoralis can cause disease that ranges from asymptomatic chronic infection to fatal hyperinfection. Diagnosis from stool can be challenging because the most sensitive conventional tests require live larvae to be effective and there can be low larval output in chronic infection. Nucleic acid amplification tests (NAAT) have been developed to complement existing diagnostic methods. We compared a recently developed loop-mediated isothermal amplification (LAMP) assay with a real-time PCR that has previously been validated with larval microscopy. The limits of detection-quantified using serial dilutions of DNA extracts from single Strongyloides ratti third-stage (L3) larvae spiked into approximately 250 µl of 5 different S. stercoralis-negative stool specimens-were 10-3 (1/5 replicates) and 10-2 (1/5 replicates) dilutions for PCR and LAMP, respectively. PCR was positive for 4/5 replicates at 10-2 LAMP was compared to PCR using extracts from 396 stool specimens collected in Bangladesh and Australia, of which 53 were positive and 343 were negative by PCR. The positive percentage agreement of LAMP was 77.3% (95% score confidence interval [CI], 64.5 to 86.6). The negative percentage agreement was 100% (95% CI, 98.9 to 100). In a preliminary investigation, PCR and LAMP assays were positive using DNA extracted from serum (PCR, 3/16 extracts; LAMP, 2/16 extracts) and bronchoalveolar lavage fluid (PCR and LAMP, 2/2 extracts), demonstrating proof of concept. Compared to PCR, the lower number of positive results using the LAMP assay may have been due to reaction inhibitors and DNA degradation, and strategies to improve the LAMP assay are discussed.