GIARDIA INTESTINALIS: EVALUATION OF ELISA COPROANTIGEN IN DIAGNOSIS AND EFFECT OF NITAZOXANIDE AND METRONIDAZOLE IN TREATMENT OF GIARDIASIS IN CHILDREN

GIARDIA INTESTINALIS: EVALUATION OF ELISA COPROANTIGEN IN DIAGNOSIS AND EFFECT OF NITAZOXANIDE AND METRONIDAZOLE IN TREATMENT OF GIARDIASIS IN CHILDREN

By

Gamal A. Abo-Sheishaa, Adel O. Hafez, Tarek KH. Zalouk

and Mostafa El-SH. Mostafa*

Departments Of Parasitology, Faculty of Medicine (Cairo and Damietta*),  Al-Azhar University

ABSTRACT

Background: Giardiasis is one of the most common intestinal parasitic infections causing diarrheal illness in humans worldwide. Detection of Giardia intestinalis is traditionally performed by microscopic examination of stool specimens. Nitroimidazoles group (metronidazole and tinidazole) are conventional drugs of choice for treatment of Giardiasis with a cure rate of higher than 90%. All of these drugs may lead to numerous adverse reactions, require long duration therapy, and none of them is absolutely safe for use during pregnancy.

Objective: This study was undertaken to evaluate the performance of the ELISA coproantigen for the diagnosis of Giardiasis and to compare the efficacy and safety of Nitazoxanide versus Metronidazole in the treatment of giardiasis.

Subjects and methods: A total of 350 children, aged 6-12 years, of both sexes were randomly selected for parasitological investigation using direct wet mount and formol-ether concentration techniques. The Giardia infected cases and 20 free of parasites were subjected to ELISA coproantigen test. Eighty cases infected with Giardia intestinal is were divided randomly into 2 equal groups: Group (1) were given nitazoxanide (200 mg twice daily for 3 days respectively), and Group (2) were given metronidazole (20 mg/kg thrice daily for 7 days). To evaluate the effectiveness of the therapy, at least three stool samples from all cases were examined after completion of the treatment. A standardized questionnaire was used to record Clinical symptoms of the patients in each group prior to and after treatment.

Results: The prevalence of giardiasis in our study was 23.7% . In our study, enzyme linked immunosorbent assay for coproantigenic detection of G. intestinalis has a sensitivity of 94.9% and a specificity of 85.7% with PPV of 92.5%, and a NPV of 90 %. The two treated groups were similar with respect to sex and mean age. The cure rate was 95% and 85% for Nitazoxanide and Metronidazole respectively with  statistically significant difference.

Conclusion: The results of this study suggested that coproantigenic technique by ELISA test is suitable for use in testing a larger number of samples, especially for screening persons in regions where G. intestinalis is a common wide pathogen. Also, it confirms the efficacy and safety of nitazoxanide as a 3-day treatment of giardiasis in children.

INTRODUCTION

    Intestinal parasitic infections are most common among school age children aged 5-15 years and were attributed to poor sanitation and hygiene. These infections can affect educational achievement, reproductive health, social and economic developments (Nematian et al., 2008). The prevalence of these infections and the extent of their public health effect in Egypt are not clearly understood. The flagellated protozoan Giardia intestinalis is one of the most common intestinal parasites affecting humans worldwide. It is estimated that 200 million people in the developing countries have symptomatic giardiasis (Bilenko et al., 2004).

      The prevalence of infection varies widely depending on the sensitivity of the diagnostic method (Flanagan, 1992). Giardiasis may be asymptomatic or responsible for a broad clinical spectrum, including acute or chronic diarrhea which may be with or without dehydration and malabsorption syndrome, nausea, vomiting, abdominal pain, flatulence and weight loss are also commonly reported (Ortiz et al., 2001).

      Detection of Giardia intestinalis is traditionally performed by microscopic examination of stool specimens. Repeating this examination once or twice on additional specimens improves the sensitivity of the test due to the intermittency of cyst excretion (Ortega & Adam, 1997 and Gupta et al., 2003).

     The sensitivity of microscopy is dependent on the skill of the microscopist and the time spent scanning each preparation. Efforts have been made to improve the sensitivity of the diagnosis of Giardia. Some of the methods have been investigated for automating the detection of Giardia species, including immuno-fluorescent assay, enzyme immunoassay, counter immunoelectrophoresis and radioimmune precipitation assay (Garcia and Shimizu,  1997).

     The nitroimidazoles, metronidazole and tinidazole are conventional drugs of choice for treatment of giardiasis with a cure rate of higher than 90%. All of these drugs may lead to numerous adverse reactions, require long duration therapy and none of them is absolutely safe for use during pregnancy (Dutta et al., 1994).

      Nitazoxanide, (2-acetyloloxy-N (5-nitro-2thiazolyl) benzamide), is the only agent that has broad coverage against both common intestinal parasitic protozoa and helminthes (Ochoa and White, 2005).

      Nitazoxanide interferes with pyruvate ferredoxin-oxidoreductase (PFOR) enzyme dependent electron transfer reaction which is important for anaerobic glucose energy metabolism resulting in cell swelling, membrane damage and vacuole injury of the trophozoites, resulting in dysfunction of the parasite (Abd el-Rahman et al., 1997).

     This study was undertaken to evaluate the performance of the RIDASCREEN^® (R-Biopharm AG, Darmstadt, Germany) Giardia kit for the prevalence of giardiasis and to compare the efficacy and safety of Nitazoxanide versus drug of choice Metronidazole in the treatment of giardiasis.

MATERIALS AND METHODS

      A randomized case- controlled  trial study, was carried out at the Department of Parasitology at Al-Azhar University between the period from March 2015 to August 2015. A total of 350 children, aged 6-12 years, of both sexes were randomly selected for parasitological investigation. The children were examined for Giardia cysts and/or trophozoites using direct wet mount and formal-ether concentration techniques (Smith and Paget, 2007). Microscopic examination consisted of two wet mount preparations for each fecal specimen; one non-stained and the other stained with iodine. Informed consent of their parents or themselves was provided.

     Sample collection: Fresh stool specimens (5-10 grams) were collected in clean plastic containers, and examined within 24 hours from the disposal of feces (Garcia, 2007). Gross examination of the sample was performed for color, consistency, mucus, blood and adult parasites. The sample was then divided into two parts: From the first part, direct wet mounts and formal-ether concentra-tion examinations were carried out. The second part was immediately stored at -20°C for performing ELISA of G. intestinalis. The Giardia infected cases were 34 males and 46 females, and mean age was 8.28±2.14 y (infected group ), and 20 subjects were 12 males and 8 females, and mean age was 8.60±2.23y (healthy control group ) were subjected to: ELISA coproantigen test using RIDASCREEN® ELISA test (r- Biopharm AG, Darmstadt, Germany) according to manufacturers method.

     Eighty cases, aged 6-12 years infected with Giardia intestinalis were divided randomly into 2 equal groups:

Group (1): Cases were given nitazoxanide 200 mg twice daily for 3 days respectively.

Group (2): Cases were given metronida-zole 20 mg/kg thrice daily for 7 days.

The criteria for inclusion were:

(1) Single infection with G. intestinalis. (2) Able to take oral medication. (3) not known to have contraindications to Nitazoxanide or Metronidazole (4) not received any anti-parasitic chemotherapy in the previous 2 months . Those who were not able to attend follow-up examinations were excluded from the study.

      To evaluate the effectiveness of the therapy, at least three stool samples from all cases were examined on the 5th, 10th and 15th day after completion of the treatment. A standardized questionnaire was used to record clinical symptoms of the patients in each group prior to and after treatment.

Statistical analysis: The collected data were organized, tabulated and statistically analyzed using SPSS, version 18 (USA). Using direct microscopy as the gold standard test for diagnosis of giardiasis. RIDASCREEN ® Giardia ELISA kit was evaluated for sensitivity, specificity, positive predictive value, and negative predictive value. For quantitative data, the mean and standard deviation were calculated. The difference between two means was statistically analyzed using the students t- test. For qualitative data, the number and percent distribution was calculated. Chi (X²) square were used for significance assossiation. The results of P < 0.05 were considered statistically significant.

RESULTS

     The prevalence of giardiasis in the present work was (23.7%) (83/350). Enzyme linked immunosorbent assay for coproantigenic detection of G. intestinalis had a sensitivity of 94.9% and a specificity of 85.7% with PPV of 92.5% and a NPV of 90 % table (1). The two treatment groups were similar with respect to sex and mean age (p>0.05) table (2). There is non-significant difference between treated groups as regard clinical manifestations abdominal pain, nausea, vomiting, constipation, distention and flatulence, steatorrhea and loss of appetite but it was significant as regard diarrhea. The children complained of more than one symptom and sign   table (3). In the present study the cure rate was 95% and 85% for Nitazoxanide and Metronidazole respectively with statistically non significant difference (p>0.05) table (4).

Table (1): Efficiency of Elisa coproantigen using microscopy as a gold standered method .

PPV: positive predictive value                                NPV: negative predictive value

Table (2): Personal data of treated groups.   

Table (3): Pre and post treatment clinical data in the treated groups .         

Table (4): Efficacy of nitazoxanide and metronidazole in treated groups.  

DISCUSSION

     Giardiasis is one of the most common intestinl parasitic infections causing diarrheal illness in humans worldwide. The infection rate is 2-7% in developed countries and 20-30% in developing countries (Bilenko et al., 2004). The prevalence of giardiasis in the present work was 23.7 % in accordance with that reported in other studies from Egypt which varied between 14.8 and 30.8 % (El-Kadi et al., 2006 and Sabry et al., 2009). Also, it was somewhat similar to the 24.7% recorded in the Behera Governorate (Curtale et al., 1998), lower than 33% among a sample of Cairo residents (Shukry et al., 1986), and (Elswaifi et al., 2014) who recorded that the prevalence was 38 % in Dakahlia Governorate .

     In the present study, enzyme linked immunosorbent assay for coproantigenic detection of G. intestinalis has a sensitivity of 94.9% and a specificity of 85.7% with PPV of 92.5% and a NPV of 90 %. It was quick and convenient method for screening tests. This was in agreement with Selim, et al. (2009) who reported that ELISA technique for detection of Giardia copro-antigen had a sensitivity of 97.3% and a specificity of 82.6% with PPV of 80.4% and a NPV of 97.7%.

     It was comparable to studies performed by Duque-Beltron et al. (2002) , Guimarães & Sogayar (2002) and Ozekinci et al. (2005) where the sensitivity of ELISA for Giardia was 100%, 96.4% and 82%, respectively, and the specificity was 95%, 80.8% and 39%, respectively. Of the 360 cases, 17.2% samples were positive for Giardia by direct microscopy and 23.6% were found to be positive by ELISA (sensitivity ~97%), but specificity was ~92% only (Singhal et al., 2015). Also, Jahan et al. (2014) detected that the sensitivity and specificity of ELISA test in comparison with direct wet mount microscopy was found to be 100% and 91.5% respectively. In another study sensitivity and specificity of ELISA test was found to be 76.4% and 100% respectively (Al-Saeed and Issa, 2010).

       In the present work, ELISA had a high sensitivity (94.9%) but a compara-tively low specificity (85.7%). It was a very good diagnostic test at finding the disease because it was sensitive, but because of its lower specificity, it can give positive results when the disease is not actually present. Accordingly, false positive cases can be present because it is not very specific. This may be due to some cross-reactions with other intestinal parasites and some past infection with giardiasis. However, if the ELISA result is negative, we can be fairly certain that the patient does not have giardiasis.

    A patient was only considered to be cured if no Giardia trophozoites or cysts could be found in any of the three post-treatment fecal specimens. The two treatment groups were similar with respect to sex and mean age. The cure rate reached 95% and 85% for nitazoxanide and metronidazole respectively with non statistically significant difference. The frequency of parasitological cure after the nitazoxanide was a little higher than that obtained with metronidazole, but the difference was not statistically significant (Canete et al., 2010). 

     These results were similar to the results of Ortiz et al. (2001) who made a randomized clinical study of nitazoxanide compared to metronidazole in the treatment of symptomatic giardiasis in children from Northern Peru. Also, Ali et al. (2014) reported that the proportions of children resolving diarrhea (had no parasites in their stool) in the nitazoxanide group was higher than metronidazole group in giardiasis. The parasitological cure after the nitazoxanide in the present study was 95% higher than the 80.4% reported by Rodríguez-García et al. (1999) in Mexican children, but similar to the 94% reported by Abaza et al. (1998) in Egypt.

      Sadjadi et al. (2001) treated Giardia lambilia infected cases (7-12 years old) either with 200 mg mebendazole three times a day for 5 days or metronidazole with a daily 15mg/kg for 7 days and reported cure rates of 86% and 90% for mebendazole and metronidazole, respec-tively. Cure rate was 60%, 57.1%, 42.1%, 52% for albendazole, nitazoxanide, nitazoxanide-albendazole combination and placebo respectively for giardiasis (Speich et al., 2013). Both treatment schedules were well accepted and well tolerated, with only mild, transient and self-limited side-effects reported (Escobedo et al., 2008).

      Although metronidazole has been a common and effective treatment for giardiasis, it has some disadvantages, such as long duration of treatment, a multiple-dose regimen and frequent side effects, such as a metallic taste, nausea, vomiting, abdominal cramps, headache, anorexia and neurological side effects. All of these features may result in poor compliance in a significant number of patients, especially  children (Raether and Hanel, 2003).

CONCLUSION

      ELISA test for detection of Giardia coproantigen is an alternative diagnostic method for microscopy and the efficacy and safety of nitazoxanide as a 3-day treatment of giardiasis in children. Further studies are  needed on a larger sample size using other molecular tests in order to get more accurate estimations.

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