الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Infection with Cryptosporidium spp. has attracted much attention as an emerging
foodborne and waterborne disease as well as an opportunistic infection in HIV individuals.
Cryptosporidium infection is acquired through ingestion of oocysts with contaminated water
or food. The source of infection can either be anthroponotic or zoonotic.
Cryptosporidiosis is either asymptomatic or associated with mild and self-limiting diarrhea
in patients with good immunity. However, in HIV patients the infection is more serious; it
can become chronic and sometimes fatal.
Based on the characteristic morphology of the oocysts, microscopic techniques are
commonly used for Cryptosporidium detection in faeces. Molecular techniques allow
accurate identification of Cryptosporidium at the species level. The application of
genotyping has provided a better understanding of the epidemiology of cryptosporidiosis.
Among the 16 currently known species, C. hominis and C. parvum are the most
frequently associated with human infections. However, zoonotic species, such as C.
meleagridis and some genotypes of C. parvum, adapted to varied animal species, have been
isolated from patients with cryptosporidiosis.
The present work aimed to determine Cryptosporidium genotypes in stool samples of
HIV patients and to compare the performance of MZN stain and nested PCR for the
detection of Cryptosporidium infection. The genotypes of the Cryptosporidium isolates
were identified after PCR amplification and analysis by RFLP and DNA sequencing of the
COWB gene.
A cross-sectional study was carried out on adult HIV infected patients. They were
recruited from patients attending the out-patient clinic or admitted to Alexandria Fever
Hospital. A sample of 100 HIV patients was selected randomly and was invited to
participate in the study. The study was approved by the Ethical Committee of the Medical
Research Institute, Alexandria University, and the Ministry of health. All procedures were
explained to eligible participants and informed consent was obtained before enrollment in
the study.
A questionnaire sheet was completed through an interview with each HIV infected
patient to collect socio-demographic data, manifestations of Cryptosporidium infection,
and the suspected mode of transmission of HIV infection. Fresh stool specimens were
collected from each participant. A portion of each sample was examined after processing
by saline and iodine smears, FEA concentration, and MZN staining. Another portion was
kept at -20oC for DNA extraction and nested PCR amplification. A RFLP protocol with
RsaI restriction enzyme and sequencing were used to characterize the detected
Cryptosporidium isolates.
Summary, Conclusion & Recommendations
79
Among the 100 HIV patients studied, parasitologic examination of faeces revealed
that the overall prevalence of intestinal parasitic infections was 29% as diagnosed by direct
wet mounts and FEA concentration techniques. The distribution of different parasitic
infections diagnosed in the examined patients showed that Blastocystis hominis was the most
prevalent parasite (22%), followed by G. intestinalis (4%). The percentage of
Cryptosporidium infection was 15% as determined by MZN stain.
Amplicons with the expected size were generated from eleven samples using the
COWP Nested-PCR. A total of 16 Cryptosporidium positive cases were detected, ten cases
were positive by both MZN and PCR, five cases were positive by MZN only and one case
was positive by PCR only. Statistical analysis revealed good agreement between both
techniques in detection of Cryptosporidium infection.
Among the 16 Cryptosporidium positive cases, abdominal pain was the most
frequent symptom (68.8%), followed by diarrhea (43.8%) and loss of appetite (25%). Less
frequent symptoms were nausea, vomiting, and fever (each in 12.5%). Asymptomatic
infection was recorded in two patients (12.5%). The duration of diarrhea in infected cases
ranged from 1-5 days with a mean of 2.06 ± 1.01 days. It was found that 73.3% of infected
patients had soft stool and 26.7% had formed stool.
Cryptosporidium infected patients had significantly lower CD4+ count (median 202.0,
IQR 143.5 – 425.0 cells/mm3), compared to Cryptosporidium negative patients (median 454.5,
IQR 377.5 – 600 cells/mm3) (P<0.001). There was no significant association between diarrhea
and other GIT symptoms in Cryptosporidium positive patients. The CD4+ counts in diarrheic
and non-diarrheic Cryptosporidium infected patients were not significantly different.
There were no significant age or gender differences in Cryptosporidium infection. It was
also found that the place of residence had no significant association with infection (P=0.844).
Also, neglecting hand washing before eating, contact with animals, and the type of drinking
water (tap, filtered, or bottled water) had no significant effect on Cryptosporidium infection.
RFLP indicated the presence of C. hominis in five samples, C. parvum in three
samples, C. meleagridis in two samples, and mixed infection with C. hominis and C.
meleagridis in one sample. Eight samples were successfully sequenced and the results
confirmed the RFLP classification.
In the younger age group (<40years), C. hominis was the most common (three out of seven
samples) followed by C. meleagridis (two out of seven samples). In the older age group, C.
parvum and C. hominis were equally detected (two cases each). The most common species in
males was C. hominis, followed by C. meleagridis. Among females, C. parvum was detected.
There was a significant association between infection by different species and patient’s residence;
five out of six samples in urban areas were C. hominis while in rural areas, C. parvum and C.
meleagridis were detected (two cases each). The patient with mixed infection was from a rural
area.
The relation between Cryptosporidium spp. and clinical manifestations showed the
occurrence of abdominal colic in two out of five cases infected with C. hominis, two out of three
C. parvum cases, the two C. meleagridis infected patients and the patient with mixed infection.
In addition, one case of C. hominis and one case of C. parvum suffered from loss of appetite.
Fever was detected in only one case of C. hominis infection. Diarrhea was recorded in 3 patients
Summary, Conclusion & Recommendations
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with C. hominis and one patient with mixed infection. Asymptomatic Cryptosporidium infection
was found in one C. hominis infected patient and one C. parvum infected patient.
Among patients neglecting regular hand washing before eating, the only detected
species was C. hominis. Contact with animals was recorded in one case of C. parvum
infection but not in infection with other species. The type of drinking water was not related
to infection with a particular Cryptosporidium species. The three identified species were
found in patients using tap or filter water for drinking.
The majority of cases in all species have CD4+ count less than 200 cells/mm3. CD4+
count > 200 cells/mm3 was found only in one C. parvum and two C. hominis cases.
6.2. Conclusion and recommendations
from the present study, it could be concluded and recommended that:
Infection with Cryptosporidium spp. is common in HIV patients in Egypt. Few
infections were asymptomatic. Clinicians should not overlook the possibility of
occurrence of cryptosporidiosis in these patients.
MZN detected a higher Cryptosporidium infection rate than PCR which failed to
diagnose some positive cases identified by MZN technique. Because of PCR cost,
and unavailability in most laboratories and hospitals, MZN staining of smears prepared
by concentration method and examined by 1000X power seems to be useful showing
enough accuracy for detection of Cryptosporidium spp. in HIV patients. Technicians
should be well trained on the use of these staining techniques for detection of
Cryptosporidium oocysts in fecal samples.
There was no significant association between different epidemiologic variables and
cryptosporidiosis in HIV patients. Low CD4+ count was the single most important
predisposing factor for Cryptosporidium infection in these patients.
PCR-RFLP targeting the COWP gene is an effective tool for genetic characterization
of Cryptosporidium isolates.
C. hominis, C. parvum and C. meleagridis were the predominant species among the
examined HIV patients as determined by RFLP analysis and sequencing of COWP
gene. More studies on a larger scale are needed to confirm these results.
C. hominis, which is mainly anthroponotic, was the most common species in patients
residing in urban areas while C. parvum was found in both urban and rural residents.
Improved hygiene and avoidance of animal contact among HIV patients should be
advocated to reduce the occurrence of Cryptosporidium infections. Further
genotyping studies of Cryptosporidium spp. isolated from human and animal
samples are needed to assess their zoonotic/or anthroponotic transmission potential.
Species and subtype data should routinely be incorporated into national surveillance
programs, which would facilitate epidemiological interpretation of genotype
occurrence and distribution trends in both sporadic and outbreak cases.