Evaluation of a real-time polymerase chain reaction assay for the laboratory diagnosis of giardiasis

Abstract

A real-time polymerase chain reaction (PCR) assay was evaluated in comparison with the combination of conventional methods (microscopic examination and antigen detection assay) during the period 2006 to 2008 on 771 fecal samples belonging to 386 patients to assess its usefulness for an accurate laboratory diagnosis of giardiasis. The real-time PCR assay detected Giardia intestinalis DNA in 195 samples (106 patients), including 26 samples (21 patients) negative by the conventional assays. Among the 21 patients, in 8 cases, giardiasis was previously diagnosed also by conventional methods in additional samples of the same patients, whereas in 13, it would have been undiagnosed if real-time PCR assay was not used. The real-time PCR assay demonstrated a detection limit of 2 cysts per reaction and 100% specificity and sensitivity compared to conventional methods. A genotype analysis targeting the β-giardin gene allowed to identify 53 samples (23 patients) containing genotype A and 59 samples (45 patients) containing genotype B.

Introduction

Giardia intestinalis, the only species belonging to the genus Giardia found in humans, is a pathogenic protozoan with a worldwide distribution having a more relevant prevalence in warm climate and in children (http://www.cdc.gov). Studies about the spread of human giardiasis have been published since 1960 in Italy where subjects at risk include residents in psychiatric institutions and subjects infected by human immunodeficiency virus (HIV) (Giangaspero et al., 2007). Imported giardiasis is also documented in Italy (Giangaspero et al., 2007), and an outbreak of G. intestinalis infection associated with Entamoeba histolytica was identified in travelers returning from Thailand (de Lalla et al., 1992).

Clinical features of giardiasis range from acute or chronic diarrhea, abdominal pain, nausea, flatulence, vomiting, weight loss, to absence of symptoms and signs (Cacciò and Ryan, 2008, Faubert, 2000). The diagnosis of giardiasis is done by light microscopy on fresh and concentrated fecal samples, antigen detection by immunoenzymatic and immunochromatographic (IC) assays (Garcia and Bruckner, 1997), and direct immunofluorescence (IF) assay that is still considered the gold standard method (http://www.cdc.gov). Recently, polymerase chain reaction (PCR)-based methods for the laboratory diagnosis of giardiasis were developed and showed excellent specificity and sensitivity, compared with antigen detection, and an increased sensitivity compared with microscopy (Verweij et al., 2003, Verweij et al., 2004).

PCR-based methods were also developed to detect different genotypes of G. intestinalis. These techniques allowed to distinguish 7 different genotypes, also named assemblages from A to G. Only assemblages A and B were found in humans and in other mammals, including pets and livestocks (Cacciò et al., 2002, Smith et al., 2007, Thompson et al., 2000). In Italy, assemblage A seems to be more widespread than assemblage B, in contrast with the results observed in other countries where assemblage B seems to be the most represented (Giangaspero et al., 2007). Nowadays, there is still an open debate on a possible correlation between G. intestinalis genotypes and clinical manifestations (Haque et al., 2005, Homan and Mank, 2001, Read et al., 2002, Sahagún et al., 2008) and on a potential zoonotic transmission in situations of close contact among humans and animals (Cacciò et al., 2005, Cacciò and Ryan, 2008). To focus parasite genotyping on investigating aspects such as host specificity and transmission patterns, the use of multiple gene markers including β-giardin was suggested (Faubert, 2000, Cacciò and Ryan, 2008, Cacciò et al., 2008).

The aim of this study was to evaluate the performance of a real-time PCR assay based on a previously described target sequence (Verweij et al., 2003, Verweij et al., 2004) during a 3-year period (2006–2008) comparing the results with those obtained by the combination of conventional methods (microscopic examination including light microscopy and direct IF assay and an IC assay) currently used in our laboratory for the diagnosis of giardiasis.

Our laboratory is in a developed country but receives a consistent number of samples belonging to subjects from high-prevalence areas for fecal–oral infections, as previously reported (Calderaro et al., 2006). Therefore, to assess the usefulness of the real-time PCR assay in diagnostic practice, this study was performed on a more extensive number of samples and patients than those analyzed in previous published reports using a real-time PCR targeting the same DNA sequence (Schuurman et al., 2007, Verweij et al., 2003, Verweij et al., 2004). Moreover, in the present study, the distribution of the genotypes A and B of G. intestinalis among the infected subjects was investigated.