Preliminary Comparison of an in-House Real-Time PCR with the Automated BD Max Enteric Parasite Panel for the Detection of Giardia intestinalis

Giardia intestinalis is a flagellated unicellular eukaryotic microorganism that commonly causes diarrheal disease and is considered one of the main non-viral causes of diarrhea in industrialized countries (Thompson et al., 2000). In developing countries, the burden of the infection is very high, and data suggest that long-term growth retardation can result from chronic giardiasis in children (Fraser et al., 2000). In Asia, Africa, and Latin America, approximately 200 million people have symptomatic giardiasis, with some 500,000 new cases being reported each year (Adam, 2001). Transmission of the infection occurs either through ingestion of water or food contaminated by cysts or through direct fecal–oral contact. Classically, diagnosis of Giardia infection relies on microscopic examination of stool samples. Sensitivity highly depends on the number of samples examined, the use of concentration techniques, and the skills and experience of the technician; as a result, the laboratory diagnosis of Giardia infections is highly variable and time consuming. Therefore, alternative techniques such as microscopy using specific fluorescent monoclonal antibodies and antibody-based antigen detection using ELISA have been introduced to increase sensitivity and reduce labor time (Garcia and Shimizu, 1997). In the last few decades, several commercial companies have developed rapid diagnostic tests that are simple to perform and can be completed in less time than traditional methods for detecting Giardia. The performance of these tests is still uncertain and highly depends on the kit used (Van den Bossche et al., 2015; Becker et al., 2017). However, more studies are needed to better establish the accuracy of antigen detection tests; indeed, one of the major problem in the diagnosis of giardiasis is that the target is emitted discontinuously (Hanson and Cartwright, 2001). In the last 20 yr, many DNA-based methods have been developed for the detection of viral, bacterial, and parasitic DNA (Perandin et al., 2018). Throughout the years, those methods have enhanced their speed and accuracy (Verweij et al., 2001). Recently, we published a paper where it has been demonstrated how molecular biology can change the classical laboratory approach for intestinal protozoan infections, including Giardia, improving the sensitivity and specificity of the diagnosis (Formenti et al., 2017). Automated procedures are also available; for instance, the BD Max enteric parasite panel (EPP) (BD Diagnostics, Sparks, Maryland), which was launched on the BD Max System (BD). The test uses an automated in vitro diagnostic panel for the direct qualitative detection of enteric parasitic pathogens. The EPP detects nucleic acids from G. intestinalis, Entamoeba histolytica, and Cryptosporidium spp. Recent studies comparing the performance of the BD Max EPP with that of microscopy demonstrated that the EPP is a good alternative to microscopy, detecting a small number of additional positives that were missed by microscopy (Batra et al., 2016; Perry et al., 2017).

This preliminary observational study aims to compare an in-house real-time PCR (Rt-PCR) for the detection of G. intestinalis with the commercial BD Max EPP.

Stool specimens of patients with available records of Rt-PCR for G. intestinalis, requested on the basis of clinical suspicion for gastrointestinal symptoms, were collected as described previously (Formenti et al., 2015), according to the routine procedure of our laboratory. All the patients included in this study provided written informed consent for the use of their biological samples.

In detail, 200 mg of stools were stored at −20 C overnight in a solution of PBS 1× with 2% polyvinylpolypyrrolidone (Sigma-Aldrich, Milan, Italy). In each sample, phocine herpesvirus type-1 (PhHV-1, kindly provided by Dr. S. Pas, Erasmus Medical Center, Department of Virology, Rotterdam, the Netherlands) was added within the S.T.A.R. buffer (Roche Molecular Systems, Branchburg, New Jersey), serving as an internal control for the isolation and amplification steps. Before starting the DNA extraction, all the samples were boiled for 10 min at 95 C. The DNA was extracted using MagnaPure LC.2 instrument (Roche Diagnostic, Monza, Italy), following the manufacturer's protocol (DNA I blood cells high performance II), using the DNA isolation kit I (Roche Diagnostics GmbH, Mannheim, Germany). The DNA was eluted in a final volume of 100 μl.

The in-house real-time assay was performed as recently described by Formenti et al. (2017). The amplification target was the small-subunit ribosomal RNA gene sequence for G. intestinalis (GenBank M54878). Primers and probe were synthesized by MWG Biotech s.r.l (Edersberg, Germany). Amplification reactions were performed in a volume of 25 μl with 2× MM (SsoFast master mix, Bio-Rad Laboratories, Milan, Italy), 2.5 μg of BSA (Sigma-Aldrich), 300 nM of each G. intestinalis specific primer, 200 nM of G. intestinalis specific probe, 80 nM of each PhHV-1 specific primer, 200 nM of PhHV-1 specific probe, and 5 μl of the DNA samples. For primer and probe sequences see Verweij et al. (2003). The Rt-PCR cycle protocol consists of 3 min at 95 C followed by 40 cycles of 15 sec at 95 C and 30 sec at 60 C, and 30 sec at 72 C. The reaction, detection, and data analysis were performed with the CFX 96 detection system (Bio-Rad Laboratories) using white plates. The Rt-PCR output consisted of a threshold cycle (Ct) value, representing the amplification cycle at which the level of the generated fluorescent signal crosses the fluorescence threshold (arbitrarily set at 200), significantly above the background fluorescence, and indicating the presence of the parasite-specific DNA load in the tested samples. Appropriate positive and negative controls were included in all experiments. As a control for PCR inhibitors and amplification, the PhHV-1 DNA was amplified with the specific primers/probe mix in the same reaction of G. intestinalis, in a multiplex setting. The analysis of all specimens by the BD Max was performed according to the manufacturer's instructions.

The 73 stool samples were independently tested in 2 laboratories: the in-house Rt-PCR was performed at the Centre for Tropical Diseases, in Negrar, whereas the BD Max EPP was performed at the Ospedale Maggiore Cà Granda, in Milan.

The in-house Rt-PCR provided 34 (46.6%) positive and 39 (54.4%) negative results, whereas the BD Max provided 27 (37%) positive and 46 (67%) negative results for G. intestinalis (Fig. 1). There were no samples positive at the BD Max, which resulted in a negative with the in-house Rt-PCR. Among the 7 (9.6%) discordant results (positive with the Rt-PCR and negative with the BD), 6 of 7 (86%) showed a Ct value between 35.27 and 39.62, with an average Ct of 36.51, thus indicating that these samples probably contained lower amounts of the targeted nucleic acid. To confirm G. intestinalis presence in these discordant samples, Sanger sequencing analysis was performed on all 7 specimens. Conventional PCR was conducted using forward Giardia-F80 (5′-GACGGCTCAGGACAACGGTT-3′) and reverse Giardia-R181 (5′-CTGCGTCACGCTGCTCGC-3′) primers, resulting in a 171-bp amplicon. The reaction was carried out with HotStarTaq DNA polymerase (Qiagen, Milan, Italy) in a 25-μl volume, containing 2.5 μl of BSA (Sigma-Aldrich) and 5 μl of DNA sample and consisted of an initial heating at 95 C for 15 min, followed by 50 amplification cycles of 1 min at 94 C, 1 min at 58 C, 15 sec at 72 C, and 10 min at 72 C. PCR products were resolved on a 2% agarose gel, using CSL-runSafe loading buffer on RunVIEW system (Cleaver Scientific, Rugby, U.K.). All analyzed samples showed the presence of the expected amplicon (data not shown). The 171-bp bands were excised and purified by QIAquick gel extraction kit (Qiagen) and sequenced using BigDye terminator sequencing_3.1 kit (Thermo Fisher Scientific, Austin, Texas), following the manufacturer's instructions. The obtained sequences were compared with sequence databases by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). All DNA samples were identified as G. intestinalis sequences. One sample presented a mixed sequence, thus resulting in a lower percentage of identity (73% compared with 100%) with G. intestinalis sequence. To further confirm the presence of G. intestinalis in this sample, microscopy and an antigen test (Oxoid ProSpecT Giardia) were checked. Microscopy analysis revealed the presence of rare cysts of G. intestinalis and the antigen test confirmed the positive result.

The comparison between the in-house Rt-PCR and BD Max EPP resulted in a K = 0.805 (95% CI 0.670–0.940) (Table I). These preliminary results suggest that the Rt-PCR could possibly demonstrate higher sensitivity than BD Max EPP, which tended to miss infections of low intensity. This finding was in agreement with the paper of Mölling et al. (2016), which showed that the BD Max missed 4 of 12 samples that were positive with the in-house Rt-PCR for G. intestinalis.

Among the positive samples, we noticed a wide range of Ct values in both Rt-PCR and BD Max assays. Causes for this variability are not clear, although stool samples are by definition not homogenous and this can partly explain the different results, as well as differences in the DNA sequence target of the amplification (unknown for the BD Max EPP) and in the DNA extraction protocols. Unfortunately, the lack of a gold standard for the diagnosis of Giardia spp. limits the comparison and standardization of molecular techniques. The choice between the 2 methods depends mostly on the available human and economic resources, and on the preference for either an open (in-house Rt-PCR) or a closed (BD Max EPP) system. In fact, the commercial BD Max assay has the advantage to be fully automated (DNA extraction and DNA amplification); hence it does not require skilled personnel, and in addition, it can be performed in either 10% formalin-preserved or unpreserved stool samples. On the other hand, an in-house Rt-PCR is cheaper than a commercial kit, but requires high expertise, availability of several samples for an internal validation, and is more time consuming.

In our opinion the big advantage of an in-house Rt-PCR is in the knowledge of how the system works, making it possible to adjust the protocol if needed to improve test accuracy. The authors are aware that more studies with a larger sample size are required to get a higher confidence for the utility of the new assay.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.