Nosocomial Diarrhea Distribution by Cryptosporidium in Isfahan

Abstract

Background:

Cryptosporidium spp. is a parasite that causes diarrheal disease in humans and animals, known as cryptosporidiosis. It is commonly transmitted through contaminated water, food, and surfaces contaminated with feces from infected humans or animals. Cryptosporidiosis is a severe public health and environmental challenge, so most studies focus on vulnerable groups, including immunocompromised individuals, children, livestock, and water.

Materials and Methods:

In this study, 217 patients admitted to the hospital who experienced symptoms such as acute or chronic diarrhea within 48-72 hours of admission were examined by the modified Ziehl Neelsen (MZN) stain for microscopically, and the amplification of 18S rRNA gene for the nested polymerase chain reaction (PCR) survey.

Results:

Cryptosporidium spp. prevalence was estimated to be 0.00% (0/217) by microscopy; however, PCR results indicate that 0.92% (2/217) were positive fecal samples.

Conclusions:

The distribution of nosocomial diarrhea specifically caused by Cryptosporidium is uncommon. It is imperative to note that healthcare settings, including hospitals, can be potential sources of transmission of various infectious diseases, including cryptosporidiosis. Understanding the impact of nosocomial diarrhea caused by various pathogens in developing countries is necessary for hygiene management.

INTRODUCTION

Cryptosporidium spp. are obligate intracellular apicomplexan parasites recognized as opportunistic pathogens since 1907, ranking fifth among significant food-borne parasites. Infective oocysts containing four sporozoites are excreted in the feces of infected hosts. They can be transmitted through the oral-fecal route, ingesting contaminated water (including surface, drinking, and recreational water), and food (such as chicken salad, fruits, and vegetables). Additionally, they can spread through person-to-person contact, leading to community and hospital infections.[1] These parasites cause gastroenteritis in various vertebrates and require a host cell for replication. In healthy individuals, cryptosporidiosis generally leads to self-limiting diarrhea that resolves within 1–2 weeks. Molecular diagnostics have identified 19 species and four genotypes of Cryptosporidium in humans across over 90 countries, with C. hominis and C. parvum accounting for 95% of cases. Global prevalence rates indicate approximately 4.3% infection in high-income countries and 10.4% in low-income countries, with higher rates in children and HIV-positive individuals.[1] In Iran, infection rates vary significantly, ranging from 1.9% to 46.8% in children and 1% to 51.4% in immunocompromised patients, suggesting potential underreporting of cryptosporidiosis due to limited awareness.[2] Various diagnostic methods exist, with PCR being the gold standard for its sensitivity and ability to identify species and genotypes.[3]

Nosocomial diarrhea is diarrhea that occurs more than 72 hours after admission to a hospital.[4] Cryptosporidium can spread among patients, hospital staff, and healthcare professionals during hospitalization, leading to nosocomial outbreaks.[5,6] Contamination of water, food, and houseflies or insufficient hand hygiene by an infected individual may transmit infection.[7,8,9,10] However, detailed data on nosocomial infections still need to be included. Patients must be informed about the importance of handwashing in hospitals. In addition, they should avoid contact with young pets, infected people, and surface waters to reduce infection risk.[11] The low infectious dose and prolonged survival in moist environments enhance the spread of Cryptosporidium. Oocysts resist chlorine disinfection and are effectively eradicated only by hydrogen peroxide.[12] Cryptosporidium spp. pose a significant health risk due to their environmental resilience and diverse transmission routes, making them a critical focus for public health efforts to control food-borne illnesses. This study investigates the distribution of nosocomial diarrhea caused by Cryptosporidium in Isfahan, central Iran.

MATERIALS AND METHODS

Patients and feces sampling

A cross-sectional study was conducted on fecal samples from patients in Al-Zahra Hospital in Isfahan City, Isfahan Province, Iran, between 2016 and 2020. The sample estimate was calculated as 215 samples, based on a previous study and a probable prevalence of 4.3%, with a confidence interval of 95% and an error rate of 5%.[5] Patients were admitted to the hospital for reasons other than diarrhea, and 217 cases at least 18 years old who experienced symptoms such as acute or chronic diarrhea within 48–72 hours of admission were examined. Patients under 18 years of age, pregnant women, mental illnesses, and Incomplete completion of the questionnaire were excluded:

Purification of oocysts and microscopy

All 217 samples were purified using concentrated sucrose (1 M) density as an effective and economical method to increase sensitivity, as described previously. Briefly, one to two grams of feces were dissolved in 30 mL of physiological saline for sieving fecal samples. The suspension was filtered through a two-layer gauze using a funnel. This filtered suspension was washed twice at 1500 rpm for 5 minutes, and the resulting pellet was resuspended in physiological saline to a volume of 5 mL. The obtained suspension was gently overlaid over 20 mL of 1 M sucrose solution and centrifuged at 3500 rpm for 20 minutes. The tip of a pipette was used to swirl the water layer gently, and the cloudy layer formed between the sucrose layer below and the fecal suspension above was collected and washed twice with physiological saline. A smear was prepared from the sediment obtained for acid-fast staining, and the remaining pellet was stored in 10 ml of isopropanol in a new tube at −20°C for PCR procedures.[13]

Microscopy was performed on the supernatant obtained after centrifuging the phosphate-buffered saline (PBS) sample at 10,000 rpm for five minutes. The supernatant was stained using the modified Ziehl–Neelsen technique (mZN) and examined using light microscopy at 1000× magnification. The supernatant was smeared on a microscope slide, fixed with methanol for 20 minutes, stained with Carbol-Fuchsin, destained with 10% sulfuric acid for five minutes, and washed under running tap water for five minutes. It was then counterstained with methylene blue for 30 seconds, rinsed under running tap water for 5 minutes, drained, and air-dried. A sample was considered Cryptosporidium-positive if typical 4–6 μm diameter oocysts were visible. The purified oocysts were washed and resuspended in phosphate-buffered saline to increase the PCR reaction sensitivity.[13]

Molecular characterization

DNA extraction method

The presence of inhibitors, the quality and quantity of feces, and preservative fluids create challenges for DNA isolation methods, making commercial kits efficient but expensive alternatives. Cetyltrimethylammonium bromide (CTAB) is a common surfactant used for DNA extraction, and several modifications have been developed to extract Cryptosporidium genomic DNA from fecal samples.[14] This study optimized CTAB with a specific protocol: 1 µL of 1% potassium was added to 400 µL of purified feces, mixed, and incubated at 60°C for 30 minutes. The sample was washed with PBS three times, followed by adding guanidine hydrochloride and Tris–EDTA buffer, and incubated again. The mixture was incubated for 20 minutes at 60°C and washed with PBS as in the previous step. Next, 200 µL of TE buffer and 100 µL of 10% sodium dodecyl sulfate were combined. To disrupt the oocysts, the samples were frozen in liquid nitrogen for 3 minutes and thawed at 37°C for 3 minutes, three times. Then, 400 µL of TE buffer and 15 µL of proteinase K were added, and digestion was performed overnight at 60°C. In the subsequent step, 200 µL of 6 M sodium chloride and 100 µL of CTAB extraction buffer (previously heated to 60°C). This was incubated for 10 minutes at 60°C, after which 600 µL of chloroform isoamyl alcohol (24:1) was added to each tube and mixed vigorously until a milky emulsion formed. The samples were centrifuged at 11,000 rpm for 8 minutes to remove particulates. The supernatants were collected, and cold isopropanol in equal volume was added to each tube and mixed gently to precipitate the DNA. This was followed by incubation for 24 hours at −20°C or one hour at −70°C. The samples centrifuged for 15 minutes at 13,000 rpm. The supernatant was gently discarded, and equal volumes (3 ml) of cold 70% ethanol were added before centrifuging again at 13,000 rpm for 5 minutes. The supernatants were discarded, and the resulting pellets were allowed to dry for 30 minutes at RT to evaporate any remaining alcohol. Finally, the dried colorless DNA pellets were dissolved in 30–50 μl Tris–EDTA buffer. The quality and quantity of DNA before storage at −20°C until further use was assessed using an ultraviolet spectrophotometer (NanoDrop) at 260 nm and 280 nm. Additionally, high molecular weight DNA was verified through agarose gel electrophoresis at a concentration of 1%.

Nested PCR of Cryptosporidium spp.

Positive microscopic cases and positive and negative controls were molecularly evaluated using an amplified part of the 18S rRNA gene of the Cryptosporidium genus, and nested PCR was performed. Two pairs of specific primers were used to detect Cryptosporidium spp., resulting in PCR products of approximately 750 bp and 590 bp in the primary and secondary PCR, respectively, using a Thermal Cycler (BioRad).

Cryptosporidium species were screened using the partial sequence of a small subunit ribosomal RNA gene (18S rRNA), previously designed by Silva. A two-step nested PCR protocol was employed to amplify the 18S rRNA gene (590 bp). For the first amplification, the SHP1 (5′ ACC TAT CAG CTT TAG ACG GTA GGG TAT 3′) and SHP2 (5′ TTC TCA TAA GGT GCT GAA GGA GTA AGG 3′) primers were used. For the second amplification (nPCR-SH), the SHP3 primer (5′ ACA GGG AGG TAG TGA CAA GAA ATA ACA 3′) and the SSU-R3 primer (5′ AAG GAG TAA GGA ACA ACC TCC A 3′) were utilized.[15]

For the primary amplification, a solution of 25 µL was prepared containing 2 µL of DNA solution, 2.5 µL of PCR buffer solution (20 mM Tris–HCl pH 8.4 and 50 mM KCl), 200 µM of each dNTP, 1.5 mM MgCl2, 0.4 µM of each primer, 1.25 units of Taq DNA polymerase (Invitrogen Life Technologies), and distilled water. The cycling protocol included an initial denaturation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 45 s, annealing at 50°C for 45 s, and extension at 72°C for 1 min, with a final extension at 72°C for 10 min. The secondary PCR conditions consisted of an initial denaturation at 94°C for 5 min, followed by 40 cycles comprising 45 s at 94°C, 45 s at 61°C, and 1 min at 72°C, concluding with a final extension step of 10 min at 72°C.[15]

Following amplification, secondary PCR products were subjected to electrophoresis and stained with safe stain dye (CinnaGen, Iran) on a 1.5% agarose gel. The length of each band was determined using a 100-bp ladder. After approximately 30–45 min of electrophoresis, the gel was visualized using a conventional UV transilluminator as a documentation system.

RESULTS

Microscopically, no Cryptosporidium spp. oocysts were detected in stool samples of patients with a modified Ziehl–Neelsen stain, except for one positive control [Figure 1]. By nested PCR of all samples, Cryptosporidium spp. were detected in 2 out of 217 (0.92%) diarrhea stool samples collected from humans [Figure 2].

Figure 1.

Light microscopy of Cryptosporidium. Modified Ziehl–Neelsen staining of oocysts (red, circular bodies) in a positive control stool sample

Figure 2.

Representative simply safe stained agarose gel showing nested polymerase chain reaction (PCR) products of Cryptosporidium DNA extracts from fecal samples. Lane 1: 100 bp DNA ladder, Lanes 2, 3, 5, 6, and 8–12: Negative samples, Lanes 4 and 7: Positive samples, Lane 13: Positive control, Lane 14: Negative control

DISCUSSION

This study was conducted on fecal samples from 217 patients admitted to the hospital for reasons other than diarrhea that experienced diarrhea symptoms within 48–72 hours of admission. No Cryptosporidium oocysts were detected in stool samples with a modified Ziehl–Neelsen stain by microscopic survey. Using nested PCR, Cryptosporidium spp. were detected in 2 out of 217 (0.92%) diarrhea samples. While cryptosporidiosis was initially thought to affect immunocompromised individuals, it is now also believed to threaten immunocompetent individuals. These infections have been acquired in institutional settings.[11] In this regard, several cases of nosocomial cryptosporidiosis have been reported.[16] Also, in veterinary clinics, transmission has been described from animals to humans.[17] Several cases of Cryptosporidium spp. contamination have been reported among elderly patients with chronic illnesses.[18,19] Cryptosporidium infection in hospitalized children is most likely to occur in malnourished children and can be fatal.[20,21]

Several studies have been conducted on the epidemiology and molecular epidemiology of cryptosporidiosis in human society in different countries, including Iran. Based on the available data, it is estimated that cryptosporidiosis prevalence rates in Iran are 3.8% in children, 2.94% in healthy adults, 1.29% in patients with gastrointestinal problems, and 8% in patients with immunodeficiency.[22,23] In this study, Cryptosporidium could not be identified on any sample slides. Two readers viewed the slides independently, and a third reader resolved discrepancies. Microscopy may underestimate the prevalence of Cryptosporidium infection due to its low sensitivity. We must note that we collected only one stool sample per participant, which may result in an underestimation of the true prevalence of the disease. Parasites are not routinely examined in stool specimens collected from nosocomial diarrhea patients. Nevertheless, further research is needed to determine the role played by parasites as risk factors for hospital infections in developing countries.[7,24] Cryptosporidium should be tested and isolated in patients with prolonged diarrhea.[11]

Izadi et al.[25] investigated the prevalence of cryptosporidiosis in 183 patients (6.01%) with immune system disorders, including HIV-positive patients, those with blood malignancies, and organ transplant recipients in Isfahan, using staining and PCR methods. Pestechian et al.,[26] in their microscopic evaluation of Cryptosporidium in 187 cancer patients, reported an infection rate of 4.3%. Additionally, 3.9%, 4.8%, and 4.8% contamination rates were reported among patients with immune system problems from Isfahan, Yazd, and Chaharmahal provinces.[27] Furthermore, the prevalence of Cryptosporidium was investigated in dialysis patients in Isfahan compared to a control group; it was found that the prevalence was significantly higher in these patients (11.53%) than in the control group (3.9%).[28] In Isfahan, diabetes patients were found to have a contamination rate of 2%.[22] Other studies in Isfahan focused on children using staining methods; in 2002, 2005, 2008, and 2010, contamination rates of 3.75%, 22.77%, 4.61%, and 4.62% were reported.[29,30,31,32] In a recent cross-sectional study of immunocompromised patients attending Baqiyatallah Hospital in Tehran, a Cryptosporidium infection rate of 0.9% was observed.[33] Comparing the findings of the present study with other studies conducted in this region, it can be concluded that the rate of Cryptosporidium identified in related studies and considering the studied group is reasonable. The study results indicate the appropriate health status of Al-Zahra Hospital in Isfahan in terms of transmission of Cryptosporidium as an opportunistic disease that can be transmitted from person to person, surfaces, water, and food.

Considering the high quality of Al-Zahra Hospital’s medical training center, it is suggested that this study be conducted in health centers with lower health facilities in more diverse dimensions of hospital departments.

Ethics approval and consent to participate

The study is a part of research conducted by our institute which has been approved by The Research Ethical Committee of Isfahan University of Medical Sciences reviewed and approved the study protocol with Ethics Code number IR.MUI.MED.REC.1400.124.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This research was supported by the vice-chancellor of research and technology at Isfahan University of Medical Sciences, Grant Number: 140023.