Development and evaluation of a loop-mediated isothermal amplification (LAMP) technique for rapid, accurate, and specific detection of Blastocystis spp. in AIDS patients.

Abstract

Blastocystis spp. is one of the most prevalent intestinal parasites with worldwide distribution. Various diagnostic methods with different sensitivities and specificities have been used to detect Blastocystis in clinical samples. The present study aims to develop and evaluate a LAMP assay to detect Blastocystis spp. in AIDS patients for the first time. In this cross-sectional study, 98 AIDS patients with an average CD4 + T lymphocyte count lower than 150 cells/mm3 participated in the study. The presence of Blastocystis spp. in the stool samples of AIDS patients was examined by parasitology (direct wet mount and concentration assays) and molecular (PCR and LAMP) methods. The 18 SSU rRNA genomic target was used to design the specific primers for the PCR and LAMP assays. The specificity of designed primers for the LAMP assay was evaluated using the sequencing of a conventional PCR product by the external LAMP primers. The data were analyzed using the SPSS software and chi-square test and Fischer's exact tests were used and Cohen's Kappa calculates the agreement of the molecular tests. Associations were tested using odd ratios (OR) and 95% confidence intervals (CI) after adjustments. Out of 98 stool samples from patients with AIDS, 9 (9.18%), 13 (13.26%), and 15 (15.30%) samples were detected positive for Blastocystis spp. by parasitology, PCR, and LAMP techniques, respectively. PCR amplification and subsequent sequencing of the product sequences revealed that the obtained partial sequences were identical to the corresponding 18 SSU rRNA sequences reported in GenBank. The higher positivity rate for Blastocystis spp. among studied AIDS patients by LAMP technique compared to other diagnostic methods showed the higher potential and effectiveness of this relatively new described molecular assay for the detection of Blastocystis spp. in AIDS patients. The accurate and rapid detection of emerging intestinal protozoa such as Blastocystis is of clinical importance for better prevention and timely treatment of the disease, especially in immunocompromised patients. The results obtained for the first time showed that the sensitivity and accuracy of the LAMP technique in the diagnosis of Blastocystis spp. in AIDS patients is very high.