Comparison of multiplexed-tandem real-time PCR panel with reference real-time PCR molecular diagnostic assays for detection of Giardia intestinalis and Tritrichomonas foetus in cats

Abstract

Giardia intestinalis and Tritrichomonas foetus are frequent enteric protozoan parasites of the gastrointestinal track of domestic cats. Because of different treatment options for the parasites, confirmation of presence of one or both pathogens is necessary. The PCR based assays are suitable for differential diagnosis. We evaluated the performance of Small Animal Diarrhoea panel, a multiplexed-tandem real-time PCR (MT-PCR) assay, that detects DNA of both G. intestinalis and T. foetus. The sensitivity and specificity were compared to reference real-time PCR assays using 105 faecal samples, 39.05% (n = 41) positive for G. intestinalis and 30.48% (n = 32) were positive for T. foetus. The faecal samples positive for T. foetus had a high proportion of late amplifiers, determined by an arbitrary threshold of C t -values > 35. On the other hand, only one G. intestinalis positive sample was considered a late amplifier. For G. intestinalis DNA, the MT-PCR assay had 95.1% sensitivity and 92.1% specificity. For T. foetus DNA, the MT-PCR assay had 41.9% sensitivity and 100.0% specificity. To evaluate the interlaboratory reproducibility of the MT-PCR assay, results were compared in two different laboratories and found to be in a very good agreement (Kappa = 0.9). Further analysis of the DNA using conventional PCR determined presence of G. intestinalis Assemblage F and T. foetus genotype 'feline'. In conclusion, the MT-PCR Small Animal Diarrhoea panel had a good and poor performance against reference assays for G. intestinalis and T. foetus, respectively. The assay is suitable for detection and differential diagnosis of G. intestinalis and moderate to high burdens of T. foetus in small animal clinical practice.

Prospective case‐control study of enterovirus detection differences in children’s cerebrospinal fluid between multiplex PCR and real‐time RT‐PCR assay

“…The clinical application of single‐targeted NAATs is limited due to the insufficient CSF volume and small number of detection channels. On the other hand, methodological studies indicated that multiplex PCR may be less sensitive than the corresponding single‐targeted real‐time PCR due to the imbalance in amplification efficiency between diverse targets 8 . Therefore, it is necessary to use CSF from children with viral encephalitis to compare the differences between the two methods.…”

“…At the same time, several other groups have also designed single‐targeted real‐time PCR for early detection of DNA or RNA of common viruses in CSF 7 . However, it has been reported that the sensitivity of multiplex PCR is inferior than that of single‐targeted real‐time PCR 8 . In addition, the role of molecular diagnostic testing in clinical applications remains unclear, as early studies focused solely on patients with confirmed infection, while the control group was not included 9 …”

Molecular detection of Tritrichomonas foetus in bovine samples: a novel real-time polymerase chain reaction (PCR) assay targeting EF1-alpha-Tf1 and a comparative study of published PCR techniques

“…The validation data from the original studies (and subsequent studies), which assessed the specificity of each PCR assay, were taken into account for the analytical specificity test (Gookin et al 2002(Gookin et al , 2005Nickel et al 2002;McMillen and Lew 2006;Effinger et al 2014;Mueller et al 2015;Ginter Summarell et al 2018;Dąbrowska et al 2019a;Meggiolaro et al 2019). In addition, information about the specificity of the genetic target on which the novel PCR protocol was based was considered (Oyhenart 2018).…”

“…The OIE cited real-time PCR, mentioning the work of McMillen and Lew (McMillen and Lew 2006) (here called the M06 protocol) as a suitable method for the detection of T. foetus in clinical samples (OIE 2018). This widely used PCR assay (Effinger et al 2014;Dąbrowska et al 2019a;Meggiolaro et al 2019) is based on the genomic region comprising the 18S, 5.8S, and 28S rRNA genes and internal transcribed spacers 1 and 2 (rRNA-ITS region), a multicopy target (Chakrabarti et al 1992) used for the design of various published PCR assays (Felleisen et al 1998;Gookin et al 2002Gookin et al , 2005Nickel et al 2002;McMillen and Lew 2006;Mueller et al 2015;Ginter Summarell et al 2018;Dąbrowska et al 2019a), most of them used for the diagnosis of clinical samples (Köster et al 2015;Casteriano et al 2016;Li et al 2016;Dąbrowska et al 2020). Despite the value of this diagnostic approach, to our knowledge, no systematic comparative study of all the PCR assays for the detection of T. foetus has been published.…”

Comparative Evaluation of Real-Time Screening PCR Assays for Giardia duodenalis and of Assays Discriminating the Assemblages A and B

“…Various DNA sequence targets have been tested and applied for real-time PCR-based screening for G. duodenalis as well as for diagnostic discrimination of the etiologically relevant assemblages A and B in both validation studies and epidemiological assessments, including genes encoding beta-giardin ( bg ), triose phosphate isomerase ( tpi ), 18S rRNA (=the small sub-unit of ribosomal RNA), and glutamate dehydrogenase ( gdh ) [ 19 , 20 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. In this study, three previously described real-time PCR screening assays for G. duodenalis targeting the 18S rRNA gene, the bg gene and the gdh gene, respectively [ 25 , 26 , 27 ], as well as three described real-time PCR differentiation assays targeting the bg gene ( n = 2 assays) and the tpi gene [ 25 , 28 , 29 ] were compared in head-to-head test comparisons without a reference standard applying latent class analysis [ 16 , 37 ].…”