Comparison of parasitological, immunological and molecular methods for evaluation of fecal samples of immunosuppressed rats experimentally infected withStrongyloides venezuelensis

Abstract

Definitive diagnosis of strongyloidiasis in humans is typically achieved by detection of larvae in fecal samples. However, limitations on sensitivity of parasitological methods emphasize the need for more robust diagnostic methods. The aim of this study was to compare the diagnostic value of three methods: eggs per gram of feces (EPG), coproantigen detection by enzyme linked immunosorbent assay (ELISA), and DNA detection by conventional polymerase chain reaction (PCR). The assays were performed at 0 and 5, 8, 13, 21 and 39 days post-infection (dpi) using fecal samples from experimentally infected immunocompetent and immunosuppressed rats. In immunocompetent rats, eggs were detected in feces on days 5, 8 and 13 dpi; coproantigen detection and PCR amplification were successful at all post-infection time points (5, 8, 13, 21 and 39 dpi). In immunosuppressed rats, eggs were detected at 5, 8, 13 and 21; coproantigen detection and PCR amplification were successful at all post-infection time points. In conclusion, these results suggest that coproantigen detection and PCR may be more sensitive alternatives to traditional methods such as EPG for diagnosis of Strongyloides venezuelensis infection.

Strongyloidiasis Current Status with Emphasis in Diagnosis and Drug Research

“…Routine diagnosis usually consists of parasitological and/or serological methods, though their performance is far from great [ 60 ]. Several parasitological tests have been used to detect larvae in stool samples, including Baermann method, formalin-ethyl acetate, Harada-Mori culture, and Agar Plate Culture (ACP); however, these tests have low sensitivity (21 to 89%) due to low parasite load and irregular larvae elimination, making it necessary to collect several stool samples throughout several days [ 7 , 60 , 63 ]. Among these methods, ACP is considered the most efficient but also the most laborious one, taking 2 to 3 days, requiring fresh stool samples and an experienced technician [ 7 , 63 – 65 ].…”

“…Among diagnostic techniques, the utilization of polymerase chain reaction (PCR) (conventional PCR (cPCR), nested-PCR, or real-time-PCR (qPCR)) [ 7 , 8 , 62 , 79 – 91 ] has been considered as a valuable method. PCR methodologies show superior sensitivity when compared with parasitological or immunological methods even studies indicating that sensitivity can vary, possibly as a result of the extraction method [ 7 , 62 , 79 , 80 , 82 , 86 , 88 , 89 ]. Thus, optimizing and standardizing DNA extraction methodology are extremely important to increase molecular diagnostic sensitivity.…”

“…Better diagnostic tools for S. stercoralis infections are needed. Parasitological methods show an unsatisfactory sensitivity, even when APC is performed and although serological diagnosis shows a good sensitivity, there are several concerns regarding cross-reactions [ 6 , 7 , 60 , 67 – 73 , 75 , 76 ]. Molecular diagnosis can be considered a promising tool that has been employed in some research and clinical laboratories, but with no standardized methodology so far [ 12 , 40 , 92 – 95 ].…”

Microscopic and molecular evaluation of Strongyloides venezuelensis in an experimental life cycle using Wistar rats

“…The methods of parasitic detection we detailed could be used for the diagnosis of homologous parasites such as S. stercoralis in humans. From the different qualitative tests, such as direct swabbing and coproparasitic methods with Lugol staining, and the quantitative assays, such as McMaster quantification, serodiagnostic tests (ELISA, IFAT, and immunoblot), or molecular amplification (10,11,(20)(21)(22), we chose the Kato-Katz method as an immediate quantitative measurement technique given that S. venezuelensis eggs present an easily degradable thin chitinous membrane common in the Rhabditidae family (8,23). This technique allowed us to observe eggs in the feces as of the 6 th .…”

“…Thus, S. venezuelensis can be used in inference-based studies to improve the strategies for strongyloidiasis control. A detailed experimental study of the S. venezuelensis biological cycle can optimize molecular biology analyses, as well as the knowledge on parasite-host interactions for therapeutic assays to obtain heterologous antigens and develop immunological techniques (10)(11)(12)(13).…”