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.

Enterocytozoon bieneusi genotypes in people with gastrointestinal disorders in Queensland and Western Australia.

Enterocytozoon bieneusi is the commonest pathogenic microsporidian found in humans and animals in many countries, but there is scant information on this pathogen in Australia. Here, we conducted the first molecular epidemiological investigation of E. bieneusi in humans with gastrointestinal disorders in Queensland and Western Australia. Genomic DNAs derived from 605 individual faecal samples from children (n = 279) and adults (n = 326) were extracted, and then subjected to nested PCR-based sequencing of the internal transcribed spacer (ITS) of nuclear ribosomal DNA to detect and characterise E. bieneusi. Enterocytozoon bieneusi was detected in eight of 605 human faecal samples (1.3%), including five children (≤3 years of age) and one adult (58 years) in Queensland, and two children (≤3 years) in Western Australia. Analysis of ITS sequence data revealed two known zoonotic (ALP1 and Ind4) and three novel (Hum_q1-3) genotypes of E. bieneusi. Genotype ALP1 identified here in humans has been found previously in farmed alpacas in Australia. Phylogenetic analysis showed that genotypes ALP1, Hum_q1-2 and Ind4 belonged to E. bieneusi Group 1 (with zoonotic potential), whereas genotype Hum_q3 clustered within E. bieneusi Group 10, suggesting that some genotypes within Group 10 might have zoonotic potential. Further investigations of humans, alpacas, marsupials and other animals in Australia will be significant to understand the epidemiology of E. bieneusi in Australia, to identify possible reservoirs of human infection, and to assist in the prevention and control of human microsporidiosis.

Application of PCR-Based Tools to Explore Strongyloides Infection in People in Parts of Northern Australia.

Strongyloidiasis, which is caused by infection with the nematode Strongyloides stercoralis, is endemic to areas of northern Australia. Diagnosis in this region remains difficult due to the distances between endemic communities and diagnostic laboratories, leading to lengthy delays in stool processing for microscopy and culture. PCR represents a viable solution to this difficulty, having potential for high sensitivity detection of S. stercoralis, even in older, unpreserved faecal samples. We prospectively collected 695 faecal specimens that were submitted to The Townsville Hospital Microbiology Laboratory from the North Queensland region for routine parasitological examination, and subjected them to a Strongyloides sp. real-time (q)PCR. Results were confirmed with a novel nested conventional PCR assay targeting the 18S rRNA gene, followed by single-strand conformation polymorphism analysis (SSCP). Of the 695 specimens tested, S. stercoralis was detected in three specimens (0.4%) by classical parasitological methods (direct microscopy and formyl-ether acetate concentration), whereas 42 positives were detected by qPCR (6.0%). Conventional PCR confirmed the real-time PCR results in 24 of the samples (3.5%). Several apparent false-positive results occurred at higher cycle times (Ct) in the qPCR. Use of real-time PCR in these populations is promising for the enhanced detection of disease and to support eradication efforts.