الفهرس 
INTRODUCTION AND AIM OF THE STUDYOnly 14 pages are availabe for public view
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Abstract
Opportunistic parasitic diarrhea has gained plenty of importance with the rise of various immunosuppressive states and the improvement of various highly sensitive diagnostic techniques for detection of the causative agent. Enteric protozoa have emerged as important opportunistic parasites that can cause disseminated and fatal infections in immunosuppressed individuals and among these organisms, intestinal microsporidia are taken into account as a crucial reason of opportunistic parasitic diarrhea (Tuli et al., 2010).
Cases of microsporidiosis in immunocompromised persons who are not infected with HIV as well as immunocompetent subjects including travelers, the elderly and children also have been reported.
Proper diagnosis and treatment of intestinal microsporidiosis are not only important for the patients’ health status but also it forms a pivotal role in the prevention and control measures.
Identification of intestinal microsporidia to the species level is especially important as this plays an important role in the management of the disease, since infection with Encephalitozoon spp. can be treated with albendazole successfully, whereas those caused by Ent. bieneusi are known to be resistant to the same drug but can be treated by fumagillin or its synthetic derivatives. Also, some species of the genus Encephalitozoon can be disseminated to distant tissues in immunosuppressed individuals, and this is another important point in terms of diagnosis and treatment modalities.
The aim of the present work was to diagnose intestinal microsporidiosis and to identify Microsporidia species in children with hematological malignancies using SYBR green real-time PCR and melting curve analysis.
The study was implemented over a period of 23 months from March 2018 to February 2020. It enrolled 145 patients attending the Department of Pediatric Hematology, and Department of Clinical Oncology, Smouha and Borg Al-Arab University Hospitals in Alexandria.
Samples were stained and examined microscopically in the laboratory of Tropical Health Department, High Institute of Public Health in Alexandria. Whilst the molecular study using SYBR green real time PCR technique and melting curve analysis was done in the Molecular Epidemiology laboratory, Faculty of Medicine, Alexandria University.
An interviewing questionnaire was designed to collect data (sociodemographic, hygienic practices and medical data) from the caregiver of the patient, health care workers and hospital records.
The results of the present study revealed that:
• Using molecular methods, out of 145 samples, microsporidia was found in 52 samples (35.9%); Enc. cuniculi was the most common species; it was found in 23 samples (15.9%). Enc. intestinalis was found in 12 samples (8.3%), Ent. bieneusi was found in 5 samples (3.4%), and Enc. hellem was found in 4 samples (2.8%). Ent.beineusi and Enc. cuniculi mixed infection was found in 2 patients (1.4%), while infection with other species of microsporidia was found in 6 samples (4.1%).
• Using coproscopic techniques, the prevalence of parasitic infections among the studied children were 64.8%. The most common parasitic infection was Microsporidia spp. (28.3%), followed by Cryptosporidium parvum (24.8%). Blastocystis hominis was found in (20.7%) of samples, Giardia lamblia was present in (7.6%) of samples. The prevalence of Entamoeba histolytica/dispar and Iodoamoeba butchlii was (1.4%) while Entamoeba coli, Cystoisospora belli and Ascaris lumbricoides were the least common infections as they were only found in 0.7% (each) of samples.
• There was significant reverse association between intestinal microsporidiosis and going to school or nursery as this variable was associated with lower microsporidiosis rate (17.1%). However, the infection rate was 41.8% among those who weren’t attending school or nursery.
• Dealing with animals was statistically related to intestinal microsporidiosis; children who had no history of animal contact had a significantly lower infection rate as only 30% of them were infected. On the other hand, dealing with cats or dogs was significantly associated with microsporidiosis as 70% of children who were dealing with dogs or cats were infected. In addition, 54% of children who were dealing with livestock were infected but this was not statistically significant.
• There was significant association between intestinal microsporidiosis and the season during which stool samples were collected as more than half (53.7%) of the children whose samples were collected during spring and summer seasons were infected. However, only 28.8% of samples that were collected during the autumn and winter seasons were positive for microsporidia.
• There was no significant association between intestinal microsporidiosis and either of the following variables: age, sex, residency, father/mother education and occupation.
• Regarding housing conditions, microsporidiosis was not statistically associated with source of water, water storage, type of latrines, sanitary system, solid waste disposal or presence of animals’ shelter. However, most of children (71.4%) who had cats or dogs in their homes were infected. Moreover, 60% of children who had sheep and goats in their houses were infected.
• Regarding children aging less than two years, only 20% of the breastfed children were infected and 100% of the children who were additionally formula-fed were infected. 50% of those children who were drinking tap water were infected. However, these results were not statistically significant.
• Microsporidiosis was not statistically associated with hygienic practices of the studied children and their mothers.
• There was insignificant association between intestinal microsporidiosis and symptomatology among the studied children one week before their enrollment in the study. However, among those who suffered from diarrhea 30.4% of children were infected by microsporidia. Among children who suffered from colic, 41.9% of them had microsporidiosis. Moreover, only two children were suffering from tenesmus, and they were infected (100%).
• There was no statistical association between type of hematological malignancy and microsporidiosis, but the infection rate was higher among those having lymphoma.
• Considering the real time PCR technique as a gold standard for diagnosis of intestinal microsporidia in stool samples the MZN showed a sensitivity of 55.77%, the specificity was 98.92%, the positive predictive value was 96.67%, the negative predictive value was 80 % and the accuracy was 83.45%. The MTS showed a sensitivity of 44.23%, the specificity was 95.7%, the positive predictive value was 85.19%, the negative predictive value was 75.42% and the accuracy was 77.24% i.e., in our study the use of MZN stain was better in diagnosis of microsporidia.
6.2 Conclusions:
1. Microsporidia may infect children with hematological malignancies. Diarrhea and colic may occur due to their deficient immunity with increased morbidity.
2. Microsporidia was the most common parasite detected coproscopically among the studied children followed by Cryptosporidium spp. and the least was Ascaris lumbricoides. Enc. cuniculi was the most commonly identified species of microsporidia by real-time PCR followed by Enc. intestinalis, Ent. bieneusi, while Enc. hellem was the least common species.
3. Real time PCR has been proven to be a sensitive and reliable technique for detection and quantitation of parasites in stool specimens. It greatly improves the sensitivity and accuracy of detection of microsporidia over those of traditional staining methods.
4. Using the real-time PCR, there is no need for post-PCR handling so, it has a much lower risk of false-positive results that may result from contamination by previously amplified products as conventional PCR and by the use of the MsRTf1/MsRTr1 primer set and SYBR green dye it has one more important advantage that it provides simultaneous detection and species identification of microsporidia in one step via melting curve analysis.
5. In relation to the efficiency of MTS compared to the SYBR green real time PCR in the present study, the stain by being a cheaper and simpler method for diagnosis than the molecular method, makes staining and microscopic examination more feasible providing that the laboratory workers have appropriate training and experience to avoid false positive or false negative results.
6.3 Recommendations
from the results of the present study, the following recommendations are suggested:
1. Being an important cause of diarrhea among children with hematological malignancies, microsporidial infection should be considered in the diagnosis of any patient with diarrhea or colic. It is important to raise the concern of physician about its proper management.
2. There is an insistent need to raise the patients’ personal hygiene regarding proper hand wash and proper washing of raw vegetables to reduce the risk of transmission of microsporidian spores.
3. Patients with hematological malignancies should avoid contact with animals due to zoonotic potential of intestinal microsporidia.
4. Drinking mineral or filtered water especially in the hot seasons to reduce the risk of transmission of the microsporidian spores.
5. Diagnosis of the microsporidian infection to the species level is recommended for the choice of the appropriate drug for treatment, thus commercial availability of the kits for routine use in clinical laboratories will be effective and provides a one-step diagnosis technique with high sensitivity and specificity. This also will help in protecting the patients from the systemic complications of multiple drugs especially if they are immunocompromised.
6. Treatment of the patients with the drug of choice for the infecting species in those immunocompromised patients has an important role in prevention of dissemination of infection to extraintestinal tissues - which can occur with the species of the genus Encephalitozoon - and its hazardous consequences.
7. Further studies are required to determine seasonality and implication of drinking water in microsporidian spores’ transmission in Egypt.
8. More research are also needed to study microsporidian phylogenetics to ascertain whether it is related to fungi.