T. K. T. Nguyen & H. Kherouf & V Blanc-Pattin & E. Allais & Y. Chevalier & A. Richez & C. Ramade & F. Peyron
Published online: 06 july 2011, Springer-Verlag 2011
Giardiasis is a ubiquitous intestinal parasitic disease that affects up to 30% of the population in developing countries . It has also been reported to be the leading cause of outbreaks of waterborne disease in the USA . Infection is transmitted by the ingestion of cysts, which are parasitic stages that are resistant to chlorination and can remain viable for several weeks . The following routes of infection have been identified: travel in endemic countries, consumption of tap water, consumption of raw vegetables, and swimming in rivers and lakes . Most infected people remain asymptomatic, which contributes to the spread of the disease . In some cases, especially in children, infection can lead to a wide range of symptoms, including abdominal discomfort and watery diarrhea; in severe cases, malnutrition, malabsorption and disruption of the weight curve may be observed.
Giardiasis is usually diagnosed in fecal specimens by light microscopy, which remains the gold standard method . Since excretion of Giardia intestinalis cysts may be intermittent, the sensitivity of this method is rather low . In order to increase this sensitivity, tests that identify a soluble Giardia antigen have been developed.
Materials and methods
We report here the performance of Giardia-Strip®, which is an immunochromatographic assay that is based on the detection of a 65-kDa coproantigen, a glycoprotein that is present in the cysts and trophozoites of G. intestinalis . Small amounts of diluted fecal samples were mixed with anti-65 antibody conjugated with colloidal gold migrated onto a nitrocellulose band. Specific antibodies revealed the presence of coproantigens that were fixed on the strip by another specific antibody. Tests were read after 15 min and positivity was denoted as a pink line. Internal controls that appeared as a strong pink line were used to validate the test.
A pilot study was first carried out on 17 and 20 microscopically positive and negative frozen stools respectively. A prospective study was then performed on 359 consecutive samples from 214 patients referred to our laboratory. Medical history, clinical conditions, travel in endemic countries and prescriptions were recorded for all patients included. Microscopic examination was performed on fresh unfixed samples, as soon as they reached the laboratory.
Wet mounts were read blind at a magnification of ×400 either directly or after formalin–ether concentration.
Discrepant results were retested using both methods. When the discrepancy persisted, a G. intestinalis-specific polymerase chain reaction (PCR) was performed on the samples according to the protocol published by Verweij et al. . Briefly, primers and a detection probe were chosen using Primer Express software (Applied Biosystems) on the basis of the known SSU RNA gene sequence for G. intestinalis (GenBank accession no. M54878), and consisted of forward primer Giardia-80F, reverse primer Giardia-127R, and the G. intestinalis-specific double-labeled probe Giardia-105 T (Biolegio, Malden, The Netherlands).
Among 80 samples (from 38 patients) that were positive for G. intestinalis by microscopic examination, Giardia-Strip® scored positive for 77 samples (Table 1). Three samples two patients) who initially yielded negative results on microscopic examination and positive results with the Giardia-Strip® were found to be positive with few G. intestinalis cysts after retrospective examination of the slides. PCR confirmed the positivity of the three falsenegative samples. Seven samples (4 patients) gave negative results under microscopy, but gave positive results with the Giardia-Strip®. PCR testing performed on only 4 samples was negative. These patients presented ulcerative colitis and digestive cancer and were passing blood in their stools. When negative fecal samples were contaminated with blood, the test did not give positive results. The overall results for the performance of Giardia-Strip® were: 96.2% (confidence interval [CI], 88.6–99%) sensitivity; 97.7% (CI, 95.2–99%) specificity; 91.6% (CI, 83–96.2%) positive predictive value; and 99% (CI, 96.9–99.7%) negative predictive value. No cross-reaction was observed with other intestinal parasites (68 samples; tested for Escherichia coli, Entamoeba histoliyica/dispar, Entamoeba hartmanni, Entamoeba nanus, Chilomastix mesnili, Blastocystis hominis, Strongyloides stercoralis, Hymenolepis nana, Isospora belli, and Enterobius vermicularis). All patients with positive results were given a 5-day course of metronidazole. Sixteen of them were re-tested for the presence of parasites 1 month after cessation of treatment. For 7 of them, both tests yielded negative results; 9 had positive results with both tests, indicating a failure of treatment.
Table 1 Comparison of results between microscopy and Giardia-Strip® test for the diagnosis of Giardia intestinalis infection in 396 fecal samples. The sensitivity was 96.2%, the specificity was 97.7%, the positive predictive value was 91.6%, and the negative predictive value was 99.0%
As cysts are excreted intermittently and their numbers may vary from day to day, the sensitivity of microscopic examination is rather low . Assays that detect the presence of small amounts of antigens in the feces have been proven to be useful . Garcia et al. tested another solid-phase qualitative immunochromatographic assay that produced a sensitivity of 97.2% and a specificity of 100% . Previous studies had already reported good scores for a test based on the detection of 65-kDa antigens . More recently, PCR yielded 100% specificity and sensitivity compared with conventional methods . Moreover, the use of multiplex tandem real-time PCR led to the detection of four common pathogenic parasites in stools . Molecular methods will certainly play a major role in the future diagnoses of parasites; at present, however, they are expensive and are not carried out routinely in all laboratories. Therefore, the recommended method for routine parasitological examination of stools is still the microscopic examination of three fecal samples .
The Giardia-Strip® test is simple, rapid, and easy to read even in cases of low parasitic load. However, it cannot replace conventional methods as it displayed false-negative results and did not detect other intestinal parasites. Nevertheless, in settings where microscopists lack experience or in epidemiological studies, this test could also be useful for diagnosing the disease and monitoring the efficacy of treatment.
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T. K. T. Nguyen H. Kherouf V Blanc-Pattin E. Allais Y Chevalier A. Richez C. Ramade F. Peyron (*)
Laboratory of Parasitology, Croix-Rousse Hospital, Lyon, France