Comparison between culture and non-culture based methods for detection of Nosocomial fungal infections of Candida spp.in intensive care unit patients

The main objective of the present study was to compare between culture based method (Hicrome Candida Differential Agar) and non-culture based method (PCR) for detection and identification of Candida spp. (C. albicans, C. glabrata, C. tropicalis and C. parapsilosis) isolated from intensive care unit (ICU) patients of New Kasr El Aini Medical School Educational Hospital, Egypt. The study was carried out during the period August 2008 to September 2009. The results showed that Candida isolates that isolated from Hicrome Candida Differential Agar plates were positive with PCR technique as follow: 100, 95.0, 85.0 and 90.0 for C. albicans, C. glabrata, C. tropicalis and C. parapsilosis, respectively. This means that using of Hicrome Candida Differential Agar media for detection and enumeration of Candida spp. was highly sensitive, rapid and economic method. Key word: Nosocomial fungal infections, Candida spp INTRODUCTION Candida species are capable of causing superficial mucosal lesions in both the oral and vaginal cavity when the balance between host and fungus shifts in favour of the fungus. Candida spp. is part of the normal flora on the skin and on the mucosal membranes of the oral cavity and gastrointestinal tract. Candida spp. can be recovered from sputum in 20% of health care personnel and 55% of hospitalized patients receiving antibiotics (Baum, 1960; Meersseman et al., 2009). Candida infections are generally endogenous in origin, and prior colonization with the organism is often regarded as one of the major risk factors for candidiasis (Pfaller 1995; Pfaller and Diekema 2007). Colonization rates are higher in individuals whose mucosal immunity is impaired due to old age, diabetes mellitus or smoking (Lockhart et al., 1998; Manfredi et al., 2002). Moreover, C. albicans is by far the most common cause of mucosal yeast infection, being the sole species recovered from up to 70% of HIV infected individuals and up to 90% of cases of Candida vaginitis (Coleman et al., 1993; Sobel, 2007). Other Candida species can be recovered alone or coisolated with C. albicans from sites of mucosal infection (Coleman et al., 1995). 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 Samia Abdou Girgis et al. 38 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 costeffective 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 (CarrilloMun ̃oz et al., 2001; Ca ́rdenesa 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-acetylgalactosaminidase 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. Since Polymerase Chain Reaction (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. Several PCR methods for the detection of Candida spp. in patient materials have been published (Buchman et al., 1990; Hopfer et al., 1993; Kahn, 1993; Miyakawa et al., 1993). The main aim of this study to compare between culture based method (Hicrome Candida Differential Agar) and non-culture based method (PCR) for detection and identification of Candida spp. isolated from intensive care unit (ICU) patients of New Kasr El Aini Medical School Educational Hospital, Egypt. MATERIALS AND METHODS 1. Sampling and sampling sites: A total of 309 clinical isolates of Candida spp. were collected from different patient sources; Blood (n= 80), Urine (n= 120), Sputum (n= 73) and Vaginal discharge (n= 36) from intensive care unit patients of New Kasr El Aini Medical School Educational Hospital) during the study period (August 2008 to September 2009). Samples were obtained from patients with clinically proven or suspected systemic Candida infection. The patients’ symptoms and characteristics included persistent fever, unresponsiveness to broadspectrum antibiotic therapy, and specimen positivity by histopathology, etc... Samples were collected from patients that were isolated in ICU nits for a period not less than 48 h and have not taken any azole drugs. 2. Reference strains: For quality control, American Type Culture Collection (ATCC) strains were used: Candida albicans Comparison between culture and non-culture based methods for detection of Nosocomial 39 ATCC 14053, Candida glabrata ATCC 2001, Candida tropicalis ATCC 750 and Candida parapsilosis ATCC 22019 (MicroBiologics, Saint Cloud, MN, USA). 3. Detection of Candida: 3.1. Culture media: HiCrome Candida Differential Agar (Himedia, India) with composition: Peptone, special 15.0g/L, Yeast extract 4.0g/L, Dipotassium hydrogen phosphate 1.0g/L, Chromogenic mixture 7.22g/L, Chloramphenicol 0.5g/L, Agar 15.0g/L and 1L Distilled water, was used for rapid isolation and identification of Candida species from mixed cultures. 42.72 grams of the dehydrated media was suspended in 1000 ml distilled water and was heated to boiling to completely dissolve the medium. DO NOT AUTOCLAVE. It was cooled to 50°C and pour into sterile Petri plates. The clinical samples (blood, urine, sputum swabs and vaginal swabs) were directly inoculated onto the solidified Hicrome Candida differential Agar using spreading method and incubated at 30°C for 48 hours. 3.2. Non culture method (PCR): The clinical isolates were kept in Brain Heart Infusion (BHI, 37 g/l; Bio-Rad, Marnes-la-Coquette, France) with 10% glycerol (Vaz Pereira, Lisbon, Portugal), at -70°C. Prior to testing, isolates were retrieved from storage and subcultured, for 24 to 48 h, on plates of Columbia sheep blood agar (bioMérieux, Lyon, France). The PCR technique was carried out according to Huaguo et al., (2007) as follow: 3.2.1. Extraction of DNA: At least one loopful of cells was transferred into an Eppendorf tube containing 400 –800 μl of 1× TE. Then it was heated at 80 °C for 20 min to kill the Candida and then cooled to room temperature. A 50 μl 10 mg/ml lysozyme was added then, vortex and incubate for at least 1 h at 37 °C. Then add 75 μl 10% Sodium Dodecyl Sulphate (SDS)/ proteinase K solution (5 μl proteinase K, 10 mg/l and 70 μl 10% SDS) (Fermentas), vortex briefly and incubate for 10 min at 65 °C. A 100 μl 5 M NaCl and 100 μl CTAB / NaCl (4.1 g NaCl, 10 g CTAB in 100 ml distilled water) then was added (Fermentas), which is prewarmed at 65 °C, vortex until the liquid content becomes white (milky), and incubate for 10 min at 65 °C. Then a 450 μl of chloroform / isoamyl alcohol (24:1) was added, vortex for 10 s, and then centrifuge at room temperature for 5 min at 12,000 g. Transfer the aqueous supernatant to a fresh microcentrifuge tube (Labfuge, 460). Add 450 μl isopropanol, incubate for 10 min on ice, centrifuge for 15 min at room temperature. Discard the supernatant and wash the pellet with 500 μl of 70% ethanol and centrifuge for approximately 5 min at room temperature. Discard the supernatant and dry the pellet. Redissolve the pellet in 25 μl 1× TE buffer; the DNA can be stored at 4°C until required. 3.2.2. Primer design: One set of primers targeting the ITS2 region of Candida were used, CN1 with sequence 427 GCATCGATGAAGAACGCAGC 446 and CN2 with sequence 834 TTGATATGCTTAAGTTCAGCGGG T 811. A PCR marker (HaeIII-digested ΦX174 replicative-form DNA) was used. 3.2.3. PCR amplification: The reaction mixture (25 μl) contained 2.5 μl of 10× buffer (750 mM Tris–HCl, 200 mM (NH4)2SO4, 0.1% Tween 20), 2 μl of 10 mM dNTP mixture (2.5 mM eachdNTP), 1.5 μl of 25 mol/LMgCl2, 0.2 μl of each primer (50 pmol), 1 U of Taq DNA Samia Abdou Girgis et al. 40 polymerase (MBI America), 5 μl of template DNA, and sterile distilled water to bring the total volume to 25 μl. PCR amplification conditions were 5 min of denaturation at 95 °C, followed by 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, with final extension step of 72 °C for 5 min. Appropriate positive and negative controls were included. All PCR amplifications were performed by using (BIOER; model TC25/H & BIORAD; model PTC 196). 3.2.4. Agarose gel electrophoresis. Electrophoresis was conducted in TBE (0.1 M Tris, 0.09 M boric acid, 0.001 M EDTA [pH 8.4]) buffer at 76 V for approximately 1 h in gels composed of 1% (wt/vol) agarose (Boehringer Mannheim) and


INTRODUCTION
Candida species are capable of causing superficial mucosal lesions in both the oral and vaginal cavity when the balance between host and fungus shifts in favour of the fungus.Candida spp. is part of the normal flora on the skin and on the mucosal membranes of the oral cavity and gastrointestinal tract.Candida spp.can be recovered from sputum in 20% of health care personnel and 55% of hospitalized patients receiving antibiotics (Baum, 1960;Meersseman et al., 2009).
Candida infections are generally endogenous in origin, and prior colonization with the organism is often regarded as one of the major risk factors for candidiasis (Pfaller 1995;Pfaller and Diekema 2007).Colonization rates are higher in individuals whose mucosal immunity is impaired due to old age, diabetes mellitus or smoking (Lockhart et al., 1998;Manfredi et al., 2002).Moreover, C. albicans is by far the most common cause of mucosal yeast infection, being the sole species recovered from up to 70% of HIV infected individuals and up to 90% of cases of Candida vaginitis (Coleman et al., 1993;Sobel, 2007).Other Candida species can be recovered alone or coisolated with C. albicans from sites of mucosal infection (Coleman et al., 1995).
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 costeffective 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´rdenesa 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.

Since
Polymerase Chain Reaction (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.Several PCR methods for the detection of Candida spp. in patient materials have been published (Buchman et al., 1990;Hopfer et al., 1993;Kahn, 1993;Miyakawa et al., 1993).The main aim of this study to compare between culture based method (Hicrome Candida Differential Agar) and non-culture based method (PCR) for detection and identification of Candida spp.isolated from intensive care unit (ICU) patients of New Kasr El Aini Medical School Educational Hospital, Egypt.

MATERIALS AND METHODS 1. Sampling and sampling sites:
A total of 309 clinical isolates of Candida spp.were collected from different patient sources; Blood (n= 80), Urine (n= 120), Sputum (n= 73) and Vaginal discharge (n= 36) from intensive care unit patients of New Kasr El Aini Medical School Educational Hospital) during the study period (August 2008to September 2009).
Samples were obtained from patients with clinically proven or suspected systemic Candida infection.The patients' symptoms and characteristics included persistent fever, unresponsiveness to broadspectrum antibiotic therapy, and specimen positivity by histopathology, etc... Samples were collected from patients that were isolated in ICU nits for a period not less than 48 h and have not taken any azole drugs.

Reference strains:
For quality control, American Type Culture Collection (ATCC) strains were used: Candida albicans The clinical samples (blood, urine, sputum swabs and vaginal swabs) were directly inoculated onto the solidified Hicrome Candida differential Agar using spreading method and incubated at 30°C for 48 hours.

Extraction of DNA:
At least one loopful of cells was transferred into an Eppendorf tube containing 400 -800 μl of 1× TE.Then it was heated at 80 °C for 20 min to kill the Candida and then cooled to room temperature.
Then a 450 μl of chloroform / isoamyl alcohol (24:1) was added, vortex for 10 s, and then centrifuge at room temperature for 5 min at 12,000 g.Transfer the aqueous supernatant to a fresh microcentrifuge tube (Labfuge, 460).Add 450 μl isopropanol, incubate for 10 min on ice, centrifuge for 15 min at room temperature.
Discard the supernatant and wash the pellet with 500 μl of 70% ethanol and centrifuge for approximately 5 min at room temperature.Discard the supernatant and dry the pellet.Redissolve the pellet in 25 μl 1× TE buffer; the DNA can be stored at 4°C until required.

Primer design:
One set of primers targeting the ITS2 region of Candida were used, CN1 with sequence 427 GCATCGATGAAGAACGCAGC 446 and CN2 with sequence 834 TTGATATGCTTAAGTTCAGCGGG T 811.A PCR marker (HaeIII-digested ΦX174 replicative-form DNA) was used.
PCR amplification conditions were 5 min of denaturation at 95 °C, followed by 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, with final extension step of 72 °C for 5 min.Appropriate positive and negative controls were included.

Agarose gel electrophoresis.
Electrophoresis was conducted in TBE (0.1 M Tris, 0.09 M boric acid, 0.001 M EDTA [pH 8.4]) buffer at 76 V for approximately 1 h in gels composed of 1% (wt/vol) agarose (Boehringer Mannheim) and 1% (wt/vol) NuSieve (FMC Bioproducts, Rockland, Maine).Gels were stained with 0.5 mg of ethidium bromide per ml of deionized water for 30 min, followed by a 30-min wash in deionized water.DNA bands confirming a positive PCR were visualized with a UV transilluminator and photographed.

Culture method results (HiCrome Candida Differential Agar).
The results in Tables 1, 2, 3 and 4 showed the average counts of Candida spp.isolated from blood, urine, sputum and vaginal swabs samples, respectively using HiCrome Candida Differential Agar in Intensive Care Unit (ICU) of New Kasr El Aini Medical School Educational Hospital.
The obtained results in Table 1 showed that, the average counts of Candida spp.isolated from blood samples were 13.42, 10, 7.71 and 5.57 CFU/ml for Candida albicans, Candida glabrata, Candida tropicalis and Candida parapsilosis, respectively during the sampling period.The minimum counts were 10, 5, 4 and 4 CFU/ml for Candida albicans, Candida glabrata, Candida tropicalis and Candida parapsilosis, respectively.While the maximum counts were 18, 13, 12 and 7 CFU/ml for Candida albicans, Candida glabrata, Candida tropicalis and Candida parapsilosis, respectively.
The highest counts of isolated Candida spp.were Candida albicans in blood samples, while the lowest counts were Candida parapsilosis during the sampling months.

PCR results:
Forty isolates of Candida albicans, 20 isolates of Candida glabrata, 20 isolates of Candida tropicalis and 10 isolates of Candida parapsilosis were isolated randomly from HiCrome Candida Differential Agar Petri dishes and examined using PCR technique.From these 40 isolates of C. albicans, a fifty isolates the obtained results showed that all isolates of C. albicans were positive using PCR (100%).
However, 20 isolates of Candida glabrata the results showed that 19 isolates were positive (95%).In addition to that, 20 isolates of Candida tropicalis the results showed that 17 isolates were positive (85%).
Moreover, 10 isolates of Candida parapsilosis showed that 9 isolates were positive (90%) as showed in Table 5 and Fig. 1.

DISCUSSION
Invasive fungal infections represent a major public health concern.In particular, systemic candidiasis remains an increasing source of morbidity and mortality especially in immunocompromised patients such as neutropenic patients undergoing antiblastic chemotherapy or bone marrow transplants (Jarvis, 1995;Pfaller et al., 1998;La Valle et al., 2000).
Candidemia has been estimated as the fourth most common nosocomial infection with an attributable mortality rate of about 50% (Edmond et al., 1999;Gudlaugsson et al., 2003).
Candida species may cause severe opportunistic infections, particularly in hospitalized patients.The predominant species remains C. albicans, comprising 54-67% of the clinical isolates of Candida (Milde et al., 2000;Carvalho et al., 2007).
Candida albicans is the most common and clinically relevant pathogen of the genus.However, there has been a significant trend in the emergence of species other than C. albicans, with a particular increase in Candida glabrata and Candida krusei frequency (Pfaller and Diekema, 2002;Tortorano et al., 2004) and to a lesser extent, Candida parapsilosis and Candida tropicalis (Kao et al., 1999).
During the study period, we obtained 309 Candida isolates from different body sites (blood, urine, sputum and vaginal swabs) of the hospitalized intensive care and cardiac unit's patients.The urinary tract being the most involved body site among the nosocomial fungal infections, Chen et al. (1997), indicated that bloodstream and urinary tract infections were the most frequently seen nosocomial fungal infections.
The results obtained in the study 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.
These results were similar to those obtained by Findik and Tuncer, (2002)  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).However, the diagnostic usefulness of PCR remains to be established because of the reported limitations of the technique when whole blood is used (Hopfer et al., 1993;Kahn, 1993), the lack of verification with a speciesspecific probe for the amplified product (Buchman et al., 1990), and the inability to detect medically important Candida spp.other than C. albicans (Miyakawa et al., 1992;Van Deventer et al., 1995).
The PCR according to its sensitivity and specificity seem promising.The advantages of PCR are the relatively short processing time and its high sensitivity and specificity.The amplification feature of the PCR assay make it ideal for detecting low yeast levels from minimal volume of clinical samples.DNA based diagnosis tests have also the potential to decrease the time taken for the laboratory identification of pathogens that are growing slowly or difficult to culture (Khlif et al., 2007).
Moreover, rapid identification of the fungal pathogens such as Candida spp.directly from clinical samples and institution of early therapy may help to reduce the hospital stay and high overall costs associated with management of candidemia.It is also of great value in epidemiological studies.
This study has showed that using of Hichrome Candida Differential Agar was useful for detection and identification of Candida spp.isolated from different clinical samples.The isolated strains were confirmed by PCR technique which showed that most presumptive Candida strains (isolated from Hichrome Candida Differential Agar) were as suspected according to its growth on chromogenic media (Hichrome Candida Differential Agar).

3. Detection of Candida: 3.1. Culture media:
Candida species from mixed cultures.42.72 grams of the dehydrated media was suspended in 1000 ml distilled water and was heated to boiling to completely dissolve the medium.DO NOT AUTOCLAVE.It was cooled to 50°C and pour into sterile Petri plates.

Table 1 :
Average counts of Candida spp.isolated from blood samples from ICU patients of New Kasr El Aini Hospital (n =80).

Table 2
showed that, the average counts of Candida spp.isolated from urine samples were 21.14, 14.14, 15.14 and 8.35 CFU/ml for Candida albicans, Candida

Table 3 :
Average counts of Candida spp.isolated from sputum swabs samples from ICU patients of New Kasr El Aini Hospital (n =73).

Table 4 :
Average counts of Candida spp.isolated from vaginal swabs samples from ICU patients of New Kasr El Aini Hospital (n =36).

Table 5 :
PCR results of Candida spp.isolates from sampling sites during the study period.

Comparison between culture and non-culture based methods for detection of Nosocomial 43
who found that, 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