Evaluation of an immunochromatographic assay: Giardia-Strip® (Coris BioConcept) for detection of Giardia intestinalis in human fecal specimens


Y Khoa Huế 13 junior ! Thế hệ thứ 2 tiếp nối sự nghiệp khoa học…


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

Introduction

Giardiasis is a ubiquitous intestinal parasitic disease that affects up to 30% of the population in developing countries [1]. It has also been reported to be the leading cause of outbreaks of waterborne disease in the USA [2]. Infection is transmitted by the ingestion of cysts, which are parasitic stages that are resistant to chlorination and can remain viable for several weeks [3]. 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 [4]. Most infected people remain asymptomatic, which contributes to the spread of the disease [5]. 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 [6]. Since excretion of Giardia intestinalis cysts may be intermittent, the sensitivity of this method is rather low [7]. 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 [8]. 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. [9]. 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).

Results

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%

Microscopic examination
Positive Negative Total
Giardia-Strip® Positive 77 7 84
Negative 3 309 312
Total 80 316 396

Discussion

As cysts are excreted intermittently and their numbers may vary from day to day, the sensitivity of microscopic examination is rather low [7]. Assays that detect the presence of small amounts of antigens in the feces have been proven to be useful [10]. Garcia et al. tested another solid-phase qualitative immunochromatographic assay that produced a sensitivity of 97.2% and a specificity of 100% [11]. Previous studies had already reported good scores for a test based on the detection of 65-kDa antigens [7]. More recently, PCR yielded 100% specificity and sensitivity compared with conventional methods [12]. Moreover, the use of multiplex tandem real-time PCR led to the detection of four common pathogenic parasites in stools [13]. 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 [14].

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.

References

1. Farthing MJ (1993) Diarrhoeal disease: current concepts and future challenges. Pathogenesis of giardiasis. Trans R Soc Trop Med Hyg 87(Suppl 3):17–21
2. Lengerich EJ, Addiss DG, Juranek DD (1994) Severe giardiasis in the United States. Clin Infect Dis 18:760–763
3. Ali SA, Hill DR (2003) Giardia intestinalis. Curr Opin Infect Dis 16:453–460
4. Theron J, Cloete TE (2002) Emerging waterborne infections: contributing factors, agents, and detection tools. Crit Rev Microbiol 28:1–26
5. Escobedo A, Cimerman S (2007) Giardiasis: a pharmacotherapy review. Expert Opin Pharmacother 8:1885–1902
6. Farthing M, Cevallos AM, Kelly P (2009) Intestinal protozoa. In: Cook and Gordon (ed) Tropical diseases, 22nd edn. Saunders-Elsevier, Amsterdam, pp 1375–1406
7. Faubert G (2000) Immune response to Giardia duodenalis. Clin Microbiol Rev 13:35–54
8. Guimaraes S, Sogayar MI, de Franco MF (1999) Giardia duodenalis: inter-strain variability of proteins, antigens, proteases, isoenzymes and nucleic acids. Rev Inst Med Trop Sao Paulo 41:45–58
9. Verweij JJ, Schinkel J, Laeijendecker D, van Rooyen MA, van Lieshout L, Polderman AM (2003) Real-time PCR for the detection of Giardia lamblia. Mol Cell Probes 17:223–225
10. Garcia LS, Shimizu RY (1997) Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens. J Clin Microbiol 35:1526–1529
11. Garcia LS, Garcia JP (2006) Detection of Giardia lamblia antigens in human fecal specimens by a solid-phase qualitative immunochromatographic assay. J Clin Microbiol 44:4587–4588
12. Calderaro A, Gorrini C, Montecchini S, Peruzzi S, Piccolo G, Rossi S, Gargiulo F, Manca N, Dettori G, Chezzi C (2010) Evaluation of a real-time polymerase chain reaction assay for the laboratory diagnosis of giardiasis. Diagn Microbiol Infect Dis 66:261–267
13. Stark D, Al-Qassab SE, Barratt JL, Stanley K, Roberts T, Marriott D, Harkness J, Ellis JT (2011) Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples. J Clin Microbiol 49:257–262
14. Vidal AM, Catapani WR (2005) Enzyme-linked immunosorbent assay (ELISA) immunoassaying versus microscopy: advantages and drawbacks for diagnosing giardiasis. Sao Paulo Med J 123:282–285


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
e-mail: francois.peyron@chu-lyon.fr

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