|Year : 2017 | Volume
| Issue : 2 | Page : 88-92
Evaluation of nanogold-beads-based enzyme-linked immunosorbent assay for detection of cryptosporidium antigen in stool samples of diarrheic patients
Mona E Naser, Mohamed S Younis, Ibrahim R Bayoumi, Maysa A Eraky, Nagwa S Aly, Rabab E.O El Attar
Parasitology Department, Benha University, Benha, Egypt
|Date of Submission||03-Jun-2017|
|Date of Acceptance||05-Jul-2017|
|Date of Web Publication||20-Nov-2017|
Rabab E.O El Attar
45 Arafa Street, Macheit El Nour, Behna, Qalyubia Governorate, 13511
Source of Support: None, Conflict of Interest: None
Background Biomedical nanotechnology is providing opportunities for rapid and simple diagnosis of many infectious diseases.
Aim The aim of this study was to evaluate nanogold-beads-based enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium antigen in stool samples of diarrheic patients.
Patients and methods Two hundred stool samples were collected from diarrheic patients attending Abo El-Resh Hospital. Parasitological examination of the collected samples was performed by direct smear, concentration method, and staining technique. Immunological examination was performed by sandwich ELISA and nanosandwich ELISA.
Results There were five (2.5%) stool samples that were positive for Cryptosporidium spp. detected by merthiolate–iodine–formaldehyde concentration technique. There were 47 (23.5%) stool samples positive for Cryptosporidium spp. detected by modified Ziehl–Neelsen staining. There were 78 (39%) stool samples positive for Cryptosporidium spp. detected by sandwich ELISA and 89 (44%) stool samples positive for Cryptosporidium spp. detected by nanosandwich ELISA. The sensitivity of sandwich ELISA and nano-ELISA for the detection of Cryptosporidium antigen in human stool samples was 68 and 85%, respectively. The specificity of sandwich ELISA was 77.3%, but the specificity of nano-ELISA was 73.4%.
Conclusion The nanosandwich ELISA improves the sensitivity and specificity of sandwich ELISA.
Keywords: Cryptosporidium spp, enzyme-linked immunosorbent assay, immunodiagnosis, nanogold
|How to cite this article:|
Naser ME, Younis MS, Bayoumi IR, Eraky MA, Aly NS, El Attar RE. Evaluation of nanogold-beads-based enzyme-linked immunosorbent assay for detection of cryptosporidium antigen in stool samples of diarrheic patients. Benha Med J 2017;34:88-92
|How to cite this URL:|
Naser ME, Younis MS, Bayoumi IR, Eraky MA, Aly NS, El Attar RE. Evaluation of nanogold-beads-based enzyme-linked immunosorbent assay for detection of cryptosporidium antigen in stool samples of diarrheic patients. Benha Med J [serial online] 2017 [cited 2018 Feb 24];34:88-92. Available from: http://www.bmfj.eg.net/text.asp?2017/34/2/88/218824
| Introduction|| |
Cryptosporidium is an apicomplexan zoonotic protozoan parasite that infects many hosts including domestic animals, birds, and humans . It has a complex life cycle with both sexual stages and invasive stages that have the characteristic apical complex from which the phylum name is derived . Cryptosporidium is an obligate intracellular parasite that infects the epithelial lining of gastrointestinal and respiratory tracts. In immunocompetent individuals, the organism is localized in the small intestine. However, in immunocompromised individuals the parasite infects the gut, biliary tract, and respiratory tract . There are 20 species of Cryptosporidium. Cryptosporidium hominis and Cryptosporidium parvum are the most detected species . The aim of this study was to evaluate nanogold-beads-based enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium antigen in stool samples of diarrheic patients.
| Patients and methods|| |
The present study was carried out on 200 cases. The study was approved by the Research Ethical Committee. Stool samples were collected after obtaining written informed consents from all patients. Stool samples were collected from diarrheic patients attending Abo El-Resh Hospital during the period from April 2015 to October 2015. All cases were subjected to the following investigations:
- Full history: age, sex, residence, complaints, history of previous contact with animals, and history of receiving immunosuppressive drugs as chemotherapy and corticosteriods were recorded.
- Stool examination:
- Fresh fecal samples were collected in clean, labeled, and wide-mouthed covered containers. Each collected sample was divided into two parts:
- The first part was frozen at −20C° for immunological analysis and nanotechniques.
- The second part was preserved in 10% formalin for direct examination and staining. Each sample was subjected to the following:
- Macroscopic examination: consistency, color, odor, blood, and mucous contents.
- Microscopic examination: direct smear, concentration technique [merthiolate–iodine–formaldehyde concentration (MIFc) technique], and staining methods [modified Ziehl–Neelsen (MZN)] for parasitic detection.
The collected samples were examined by the following methods:
- Direct smear : About 2 mg of stool was taken with a wooden stick and emulsified in normal saline on a clean microscopic slide, a coverslip was placed, and then the slide was examined by ×10 and ×40 magnifications of the ordinary light microscope.
- Concentration method: MIFc was used .
- Staining method: MZN  staining was used. The oocyst of Cryptosporidium was tested by using modified acid-fast staining method, which was sensitive and specific for the identification of Cryptosporidium spp.
Sandwich ELISA and nanosandwich ELISA were used for immunological examinations.
Technique of sandwich enzyme-linked immunosorbent assay 
Microtitration plates were coated with purified anti-purified G. lamblia cysts antigen (PGA) and then blocked by adding FCS; next, fecal supernatant samples were added to each well. Plates were washed and peroxidase-conjugated pAbs were added, and then the plates were washed again. O-phenylenediamine substrate solution was added. The reaction was stopped by adding H2SO4, and the plates were read using an ELISA microplate reader.
Technique of nanosandwich enzyme-linked immunosorbent assay 
Anti-PGA conjugated with nanogold beads nanoparticles was used as the capture antibody and anti-PGA conjugated with horseradish peroxidase was used as the conjugated antibody. Microtiter plates were coated with capture antibody. Next, fecal supernatant samples were added to wells. Plates were washed and peroxidase-conjugated anti-PGA antibody was added. The reaction was visualized by the addition of O-phenylenediamine substrate solution. The reaction was stopped by adding H2SO4 and the plates were read using an ELISA microplate reader.
Specificity and sensitivity can be selected and adjusted to meet the needs of a clinician for the diagnosis and monitoring of a disease. This may be accomplished by changing the selection of the reference value (i.e. cutoff or upper limit of normal) for the particular test .
Stool samples were collected after obtaining written informed consent from all patients.
| Results|| |
Cryptosporidium spp. was not detected by direct smear. Cryptosporidium spp. was detected in five (2.5%) stool samples by MIF, but by MZN Cryptosporidium spp. was detected in 47 (23.5%) stool samples. P value is highly significant ([Table 1]). Cryptosporidium spp. was detected in 78 stool samples by sandwich ELISA. Cryptosporidium spp. was mixed with other parasites (36 stool samples). Pure positive samples for Cryptosporidium spp. were 42 ([Table 2]). Cryptosporidium spp. was detected in 89 stool samples by nanogold-beads-based sandwich ELISA. Cryptosporidium spp. was mixed with other parasites (42 stool samples) but pure samples positive for Cryptosporidium spp. were 47 ([Table 3]). Cryptosporidium spp. was not detected by direct smear. Cryptosporidium spp. was detected by MIF (2.5%). Cryptosporidium spp. was detected by MZN (23.5%). It was detected by sandwich ELISA (39%) and by nanosandwich ELISA (44%). The positivity of nano-ELISA is higher than sandwich ELISA. P value is highly significant ([Table 4]). Sensitivity of nanogold-beads-based sandwich ELISA is higher than traditional sandwich ELISA ([Table 5]).
|Table 1 Comparison between direct parasitological techniques in detection of Cryptosporidium spp. infection|
Click here to view
|Table 2 Pure and mixed Cryptosporidium spp. infection by sandwich enzyme-linked immunosorbent assay|
Click here to view
|Table 3 Pure and mixed Cryptosporidium spp. infection by nanogold-beads-based sandwich enzyme-linked immunosorbent assay|
Click here to view
|Table 4 Comparison between different techniques in detection of Cryptosporidium spp. infection|
Click here to view
|Table 5 Sensitivity, specificity, positive predictive value, and negative predictive value of sandwich enzyme-linked immunosorbent assay versus nanogold-beads-based sandwich enzyme-linked immunosorbent assay for detection of Cryptosporidium. antigen in stool samples|
Click here to view
| Discussion|| |
Cryptosporidium is an important food-borne pathogen causing a disease of socioeconomic significance worldwide . It causes gastrointestinal disease in humans . It is also a waterborne parasite present worldwide . Infection by Cryptosporidium spp. occurs through person-to-person contact (anthroponotic), animal contact (zoonotic), ingestion of water, or through food . The resistance of Cryptosporidium oocysts to disinfectants is high, which enables them to survive for long periods and still remain infective . The infection is self-limited in immunocompetent persons. However, in immunocopromised persons, it can cause life-threatening diarrhea . The aim of this study was to evaluate nanogold-beads-based ELISA for detection of Cryptosporidium antigen in stool samples of diarrheic patients. In the current study, 200 stool samples were collected from diarrheic patients attending Abo El-Resh Hospital during the period from April 2015 to October 2015. All stool samples included in this study were subjected to direct parasitological examination for detection of Cryptosporidium oocysts or other parasites using direct smear, MIFc, MZN stain, and immunological examination by sandwich ELISA and nanosandwich ELISA.
The prevalence of Cryptosporidium infection among studied diarrheic patients by MIFc technique was 2.5%, whereas 23.5% of stool samples were positive for Cryptosporidium spp. using MZN stain. These high results agreed with those of Abdel-Maboud et al. , who detected the prevalence of Cryptosporidium spp. in diarrheal patients of all age groups attending the outpatient clinics in Qaluibia province to be 19.5% using MZN. Al-Hindi et al.  performed a study to determine the prevalence of cryptosporidiosis among children attending Al-Nasser Pediatric Hospital in Giza and found that Cryptosporidium oocysts were detected in 14.9% of the tested specimens using acid-fast stain. These results were also in agreement with those of Shoukry et al. , who detected the prevalence of Cryptosporidium spp. in children in Ismailia province to be 33.3%.
In the present study, it was found that 78 (39%) stool samples were positive for Cryptosporidium spp. These results were in agreement with those of El-Helaly et al. . In the study group (n=177), cryptosporidiosis was revealed in 27 (15.3%) samples using the antigen detection method and in 20 (11.3%) samples using the acid-fast stain. No Cryptosporidium spp. infection was detected in the control group. ELISA technique is considered the best method used for the detection of cryptosporidial infection, as its sensitivity is 90% . These results disagreed with the results of El-Sweify and El-Moamly , who recorded similar efficacies for comparison of an acid-fast stain and ELISA, and this may be due to low antigens found in the stool because of dilution. These results also disagreed with the results of examination of 250 stool samples, which recorded that the higher rate of infection was 15.2% by MZN stain, whereas by using ELISA test it was 6.8% .
In the present study, it was found that 89 (44%) stool samples were positive for Cryptosporidium spp. by nano-ELISA. In our study, the sensitivity of sandwich ELISA and nano-ELISA for the detection of Cryptosporidium antigen in human stool samples was 68 and 85%, respectively. The specificity of sandwich ELISA was 77.3% but that of nano-ELISA was 73.4%. This indicates that nanosandwich ELISA improves the sensitivity of sandwich ELISA. In India, the sensitivity and specificity of ELISA for the detection of cryptosporidial coproantigen in stool samples were 90.9 and 98.7%, respectively . Although ELISA test is expensive and has the possibility of false-positive results , it is simple, rapid, reliable, and specific test and may be useful for large-scale epidemiological studies of cryptosporidiosis . ELISA proved to be a good positive and good negative test with a sensitivity of 90% . A cross-sectional study was carried out from September 2012 to November 2013 on patients admitted to Tropical Medicine Department, Menoufia University Hospitals, and outpatients of Theodor Bilharz Research Institute, Giza. The study aimed to detect the potential specificity and sensitivity of nanogold-beads-based ELISA in the diagnosis of giardiasis in stool of asymptomatic and symptomatic infected individuals. Giardia lamblia antigen in stool samples was detected by using the conjugated anti-purified G. lamblia cysts antigen with nanoparticle-sandwich ELISA, compared with the traditional sandwich ELISA. Sandwich ELISA achieved sensitivity of 93%, specificity of 92.5%, positive predictive value of 95.7%, and negative predictive value of 88%, whereas nanosandwich ELISA achieved sensitivity, specificity, positive predictive value, and negative predictive value of 95.8, 95, 97.2, and 92.6%, respectively .
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Abu Samra N, Thompson PN, Jori F, Frean J, Poonsam B, Plessis D et al.
Genetic characterization of Cryptosporidium spp. in diarrhoeic children from four provinces in South Africa. Zoonoses Public Health 2012; 60:154–159.
Leitch GJ, He O. Cryptosporidiosis: an overview. J Biomed Res 2012; 25:1–16.
Mumtaz S, Ahmed J, Ali L. Frequency of Cryptosporidium
infection in children under five years of age having diarrhea in north west of Pakistan. Afr J Biotech 2010; 9:1230–1235.
El-Helaly NS, Aly MM, Attia SS. Detection of Cryptosporidium
infection among children with diarrhea. New York Sci J 2012; 5:68–76.
Michael W, Dryden DVM, Patricia A, Payne DVM. Fecal Examination Technique. Kansas State University, Diagnostics Parasitology; 2010. NAVC Clinician’s Brief.
Blagg W, Schloegel El, Mansour NS, Khalaf GI. A new concentration technique for the demonstration of protozoa and helminth eggs in feces. Am J Trop Med Hyg 1955; 4:23–28.
Henriksen SA, Pohlens J. Staining of Cryptosporidium
by modified Ziehl-Neelsen technique. Acta Vet Scand 1981; 22:594–596.
Tijssen P, Kurstak P. Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugate for enzyme immunoassays. Anal Biochem 1984; 136:451–457
Demerdash ZA, Mohamed SH, Shaker ZA, Hassan SI, El Attar GM, Saad-El Din A et al.
Detection of circulating Schistosome antigens in serum and urine of schistosomiasis patients and assessment of cure by monoclonal antibody. J Egypt Soc Parasitol 1995; 25:471–484.
Zane HD. Laboratory safety and test quality assurance. In: Immunology: theoretical and practical concepts in laboratory medicine. Philadelphia, PA: Saunders WB Company; 2001. pp.193–207.
Putignani L, Menichella D. Global distribution, public health and clinical impact of the protozoan pathogen cryptosporidium. Interdiscip Perspect Infect Dis 2010; 2010:pii:753512.
Oyibo WA, Okangba CC, Obi RK, Nwanebu FC, Ojuromi T. Diagnosis of intestinal cryptosporidiosis in Africa: prospects and challenges. PDR. For herbal medicines, 1st ed. Montvale, NJ: Medical Economics Company; 2011. pp.501–1273.
Chalmers RM, Campbell BM, Crouch N, Charlett A, Davies AP. Comparison of diagnostic sensitivity and specificity of seven Cryptosporidium
assays used in the UK. J Med Microbiol 2011; 60:1598–1604.
Carpenter C, Fayer R, Trout J, Beach M. Chlorine disinfection of recreational water for Cryptosporidium parvum
. Emerg Infect Dis 1999; 5:579–584.
Youssef FG, Adib I, Riddle MS, Schlett CD. A review of cryptosporidiosis in Egypt. J Egypt Soc Parasitol 2008; 38:9–28.
Abdel-Maboud AI, Rossignol JF, El-Kady MS, Mostafa MS, Kabil SM. Cryptosporidiosis in Benha, study of some recent modalities in diagnosis and treatment. J Egypt Soc Parasitol 2000; 30:717–725.
Al Hindi AI, El Manama AA, Elnabris K. Cryptosporidiosis among children attending Al-Nasser Pediatric Hospital, Gaza, Palestine. Turk J Med Sci 2007; 37:367–372.
Shoukry NM, Dawoud HA, Haridy FM. Studies on zoonotic cryptosporidiosis parvum in Ismailia Governorate, Egypt. J Egypt Soc Parasitol 2009; l39:479–488.
Gabr NS, Abdellatif MZ, Abd El-Hafeez E, Abd Rabou RA. Comparison between ELISA various stains techniques in laboratory diagnosis of cryptosporidiosis. J Egypt Soc Parasitol 2014; 44:509–516.
El-Sweify MA, El-Moamly A. ImmunoCard STAT! cartridge antigen detection assay compared to microplate enzyme immunoassay and modified Kinyoun’s acid-fast staining technique for detection of Cryptosporidium
in fecal specimens. Parasitol Res 2011; 110:1037–1041.
Ali FM, Ali AS. Cryptosporidiosis in Sulaimani Pediatric Teaching Hospital and comparison of different diagnostic methods for its detection. Eur Sci J 2013; 36:1857–7881.
Sarkar R, Ajjampur SS, Prabakaran AD, Geetha JC, Sowmyanarayanan TV. Cryptosporidiosis among children in an endemic semi urban community in southern India: does a protected drinking water source decrease infection? Clin Infect Dis 2013; 57:398–406.
Vohra P, Sharma M, Chaudhary U. A comprehensive review of diagnostic techniques for detection of Cryptosporidium parvum
in stool samples. IOSR J Pharma 2012; 2:15–26.
Jayalakshmi J, Appalaraju B, Mahadevan K. Evaluation of an enzyme-linked immunoassay for the detection of Cryptosporidium
antigen in fecal specimens of HIV/AIDS patients. Indian J Pathol Microbiol 2008; 51:137–138.
] [Full text]
Moharm MI, El Enain G, El-Deen BW, El Aswad MA, Hendawy M, Aly I. Evaluation of nano-gold beads based-ELISA for detection of Giardia lamblia antigen in stool samples of infected patients. Middle East J Sci Res 2014; 21:2264–2273.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]