الفهرس 
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Abstract
The majority of nosocomial fungal infections are reported to be caused by Candida spp. All Candida spp. may cause a similar spectrum of diseases, ranging from thrush to invasive diseases such as arthritis, endophthalmitis, meningitis or fungemia. However, there may be differences in severity and therapeutic options worth noting. C. albicans is by far the most common Candida spp. causing infection in humans (Bodey, 1988; Chen et al., 1997).
Reports indicate that C. tropicalis, C. parapsilosis and Cryptococcus gastricus also produce structures, which resemble germ tubes (Perry et al., 1990; Lipperheide et al., 1993; Freydie´re & Guinet, 1997; Campbell et al., 1998). Moreover, 5% of routine isolates of C. albicans have recently been shown to be germ tube negative (Lipperheide et al., 1993).
The presumptive clinical identification of C. albicans is usually made on the basis of its ability to generate germ tubes when incubated at 30 to 37°C for 2–4 h in serum. This germ tube production in C. albicans is affected by various environmental conditions. Although the germ tube (GT) test is an economic, easy, and rapid method available for screening for C. albicans, up to 5% of C. albicans strains are germ tube-negative (Perry and Miller, 1987) and false positive results can occur with certain non-albicans yeasts, such us Candida tropicalis or Candida parapsilosis (Freydie´re and Guinet, 1997).
These problems imply that a well-trained laboratory staff in clinical mycology is strongly required. There is an obvious need for rapid and cost-effective differentiation of C. albicans from other sometimes drug-resistant Candida species in clinical microbiology (Odds, 1993)
Also, reliable detection of mixed cultures might improve therapeutic intervention (Ainscough & Kibbler, 1998). In response to this increased need, several commercial systems are now available. Albicans ID2, Chromalbicans Agar, and CHROM agar Candida contain chromogenic or fluorogenic substrates hydrolyzed by the hexosaminidase of C. albicans. This leads to a rapid identification of C. albicans on the basis of colony colour. These substrates offer rapid identification directly upon primary culture (Carrillo-Mun˜oz et al., 2001; Ca´rdenes et al., 2002).
There are several chromogenic media available for the isolation and presumptive identification of C. albicans based on the pigmentation of the developing colonies, which is due to different enzyme activities among Candida species (Baumgartner et al., 1996; Odds & Bernaerts, 1994; Quindo´s et al., 2001).
HiCrome Candida Differential Agar (Himedia, India) is recommended for rapid isolation and identification of Candida species from mixed cultures. Perry and Miller (1987) reported that Candida albicans produces an enzyme β -N-acetyl- galactosaminidase and according to Rousselle et al (1994) incorporation of chromogenic or fluorogenic hexosaminidase substrates into the growth medium helps in identification of C. albicans isolates directly on primary isolation.
According to the results showed that :The samples were taken from the I.C.U. and C.C.U.units of the five largest institutional hospitals in Cairo and the samples were cultures by Cuture based chromogenic media (Hicrome media) from the following hospitals:
1- New Kasr El Aini Hospital
2- Old Kasr El Aini Hospital
3- National Cancer Institute
4- Ain - Shams University Specialized Hospital.
5- Ain -Shams University Hospital(El- Demerdash Hospital).
1-Culture based technique results
I-New Kasr El Aini Hospital
The average Percentage of Candida spp. isolated from blood, urine, sputum and vaginal swabs samples samples from ICU patients of New Kasr El Aini Hospital were (36.17%, 25.07% , 23.52% and15.14% )for C. albicans, C. glabrata, C. tropicalis and C. parapsilosis, respectively on Hicrome media
II Old Kasr El Aini Hospital
The average Percentage of Candida spp. isolated from blood ,urine,sputum and vaginal swabs samples were (35.25%, 27.66%, 22.4% and 14.66%)for C. albicans, C. glabrata, C. tropicalis and C. parapsilosis, respectively on Hicrome media.
III National Cancer Institute:
The average Percentage of Candida spp. isolated from blood ,urine,sputum and vaginal swabs samples were (35.05%, 27.15 %, 22.7% and 15.82%)for C. albicans, C. glabrata, C. tropicalis and C. parapsilosis, respectively on Hicrome media.
IV- Ain - Shams University Specialized Hospital
The average Percentage of Candida spp. isolated from blood ,urine,sputum and vaginal swabs samples were (36.87%, 28.03 %, 20.93% and 14.23%)for C. albicans, C. glabrata, C. tropicalis and C. parapsilosis, respectively on Hicrome media
V- Ain -Shams University Hospital(El- Demerdash Hospital).
The average Percentage of Candida spp. isolated from blood ,urine,sputum and vaginal swabs samples were (36.83%, 27.44 %, 21.74% and 13.95%)for C. albicans, C. glabrata, C. tropicalis and C. parapsilosis, respectively on Hicrome media
General notes on results of culture technique:
1-The highest counts of isolated Candida spp. from all hospitals were Candida albicans in Blood samples, Urine Samples, sputum swabs samples and also vaginal swabs samples, while the lowest counts were Candida parapsilosis during the sampling months.
.2- C. albicans was the most frequently isolated species in patients with nosocomial fungal infection (75.4%). It was followed by C. glabrata (8.2%), C. tropicalis (4.8%), C. parapsilosis (2.5%),
2 - Non-Cultural based method (PCR):
Two hundreds isolates of Candida albicans, 160 isolates of Candida glabrata, 100 isolates of Candida tropicalis and 100 isolates of Candida parapsilosis were isolated randomly from the five sampling sites using HiCrome Candida Differential Agar medium during the study period (August 2008 to September 2009).
from these 200 two hundreds isolates of C. albicans, 50 a fifty isolates (50/200) were tested for confirmation using polymerase chain reaction (PCR) technique.
( PCR ) Results:
- The obtained results showed that all isolates of C. albicans were positive using PCR (100%).
However, thirty isolates of Candida glabrata (40/160) were tested for confirmation using PCR; the results showed that 39 isolates were positive (97.5%).
In addition to that, 30 thirty isolates of Candida tropicalis (30/100) were tested using PCR; the results showed that 27 isolates were positive (90%).
Moreover,30 thirty isolates of Candida parapsilosis (30/100) were tested using PCR; the results showed that 29 isolates were positive (96.6%)
Several commercial systems have been developed and compared for yeast identification (Fenn et al., 1994; Gutierrez et al., 1994; Fricker-Hidalgo et al., 1995; Verweij et al., 1999; Hata et al., 2007), such as the manual Auxacolor (colorimetric) and ID32 (turbidimetric) systems, and the automated Vitek 2 YST (colorimetric) system (Campbell et al., 1999; Verweij et al., 1999; Ana et al., 2010).
HiCrome Candida Differential Agar is a selective and differential medium, which facilitates rapid isolation of yeasts from mixed cultures and allows differentiation of Candida species namely C. albicans, C. parapsilosis, C. tropicalis and C. glabrata on the basis of coloration and colony morphology. On this medium results are obtained within 48 hours and it is useful for the rapid and presumptive identification of common yeasts in Mycology and Clinical Microbiology Laboratory.
C. albicans appear as light green colored smooth colonies, C. tropicalis appear as blue to metallic blue colored raised colonies. C. glabrata colonies appear as cream to white smooth colonies, while C. parapsilosis as off-white to cream colored colonies.
Traditional means of identification of pure cultures of Candida spp. include laborious and slow morphological and assimilation tests that can take several days to identify the isolates in a culture (Warren & Hazen, 1999), and clinical yeast isolates are sometimes misidentified when automated biochemical systems are used (Dooley et al., 1994).
The development of techniques and strategies that can accurately differentiate clinical isolates is of great relevance. These techniques should provide the ability to differentiate among strains responsible for clinical infections, as well as to trace their epidemiological pathways. Several molecular methods have been used to differentiate C. albicans strains, including electrophoretic karyotyping, the use of species-specific probes such as Ca3 or 27A in restriction enzyme analysis (Magee et al., 1987; Pujol et al., 1997; Pfaller et al., 1998; Schmid et al., 1999), PCR-based methods (Graser et al., 1996; Thanos et al., 1996; Barchiesi et al., 1997; Correia et al., 2004).
Since PCR has proven to be a powerful tool in the early diagnosis of several infectious diseases, it might also be a more sensitive alternative assay in the diagnosis of invasive candidiasis.
However, the main aim of this study was to compare the culture based method (HiCrome Candida Differential Agar) with non-culture based method (PCR) for detection and enumeration of Candida spp. from different medical specimens (blood- urine- sputum- vaginal discharge) of the hospitalized intensive care units(I.C.U) and (C.C.U.)cardiac care unit’s patients.
The results showed that, C. albicans was the most frequently isolated species in patients with nosocomial fungal infection. It was followed by C. glabrata, C. tropicalis and C. parapsilosis.
Chromogenic media such as HiCrome Candida Differential Agar which facilitates rapid isolation of yeasts from mixed cultures and allows differentiation of Candida species namely C. albicans (light green), C. parapsilosis (off-white to cream), C. tropicalis (blue to metallic blue) and C. glabrata (cream to white) on the basis of coloration in less than 48 hours.
Although detailed cost-benefit survey were not carried out, it seems clear that these chromogenic media are economical in terms of labor and time. Moreover, their cost would be more than offset by the decreased need for secondary biochemical tests (Baumgartner et al., 1996).
Also, results of PCR showed that from these two hundreds isolates of C. albicans, a fifty isolates (50/200) were tested for confirmation using polymerase chain reaction (PCR) technique.
The obtained results showed that all isolates of C. albicans were positive using PCR (100%).
However, thirty isolates of Candida glabrata (40/160) were tested for confirmation using PCR; the results showed that 39 isolates were positive (97.5%).
In addition to that, thirty isolates of Candida tropicalis (30/100) were tested using PCR; the results showed that 27 isolates were positive (90%).
Moreover, thirty isolates of Candida parapsilosis (30/100) were tested using PCR; the results showed that 29 isolates were positive (96.6%).
However, we conclude that, using of chromogenic media such as Hichrome Candida Selective Agar was useful for rapid detection, enumeration and identification of Candida spp. (Candida albicans, Candida glabrata, Candida tropicalis and Candida parapsilosis) from different clinical samples (blood, urine, sputum and vaginal discharge).