Received: 16 May 2009

Accepted: 1^st referee: 8 June 2009

2^nd referee: 12 July 2009

 

 

Molecular Identification of Trichosporon Isolates Obtained from Different Clinical Specimens

Mohamed Taha Mahmoud, Noha El-Mashad*, Heba Allah El-Shewehy**

Departments of Microbiology (Mycology), Veterinary Medicine, Zagazig University, Clinical Pathology, Mansoura University*, Vet. Medical Science (Mycology)**, Egypt

Keywords: Trichosporon asahii, PCR, T. mucoides

                                                                                                                                                                                                         

 

 

 

 

Abstract

Background: Trichosporn is a medically important genus that includes the causative agents of both deep-seated, mucosa-associated infection and superficial infection.

Aim: In this study we aimed to present data on the phenotypic and molecular identification of Trichosporon species recovered from various clinical specimens representing both superficial and systemic infections.

Patients and methods: 397 samples (65 blood cultures, 192 nail scrapings, 140 skin scrapings) were included for isolation, phenotypic and genotypic identification of Trichosporon species.

Results: Cases of hematological malignancies, onychomycosis, and tinea pedis were positive for yeast isolates with percentage of 10.7%, 16.6%, 20.7% respectively. Based on the morphologic characters of isolated colonies on Sabouraud dextrose agar (SDA), microscopic examination of colonies on rice agar, and stained smears, yeasts identified as Trichosporon were: 9(4.68%) from nail samples, and 5(3.6%) from skin samples and 0(0.0%) from blood cultures. PCR using Trichosporn genus specific primers for 68 yeast isolates was positive in 16 samples (14 were previously identified by morphology and 2 nail scrapings were falsely diagnosed negative) at 170 bp. We performed subsequently 2 PCR runs on the identified 16 samples using specific primers for each of T. asahii and T. mucoides. Their sequences included ITS1, ITS2. They yielded specific amplification of a DNA fragment at 430 bp in 13 (of 16) samples, which is specific for T. asahii, and 0 (0.0%) sample was positive for T. mucoides specific primers.

Conclusion: Occurrence of trichosporonosis is not rare in human. T. asahii species is common in our locality. The standardization of laboratory methods for T. identification and antifungal susceptibility tests are necessary to investigate for both superficial and systemic trichosporonosis.

Introduction

Trichosporon is a medically important genus that includes the causative agents of both deep-seated, mucosa-associated infection and superficial infection⁽¹⁾.

Several methods used for Trichosporon species identification, have been reported, including morphological and biochemical tests and the use of molecular tools. Despite the fact that phenotypic methods are more suitable for routine in general microbiology laboratories, the accuracy for identification seems to be limited^(2,3).

The genus Trichosporon is characterized by the ability to form arthroconidia, blastoconidia, hyphae, and pseudohyphae. All species are able to assimilate different carbohydrates and carbon sources and to degrade urea. However significant difference in the results generated by the same organisms tested by different authors can be observed in regard to biochemical and physiological tests performed with different substrates used in yeast identification keys^(4,5).

Aim: There has been no previous study on morphologic and genotypic characterization of Trichosporon species in Egypt. In this communication our aim was to present data on the morphologic and molecular identification of Trichosporon species recovered from various clinical specimens.

Subjects and Methods

A total of 397 samples were collected over 2 years (May 2007 to May 2009). Patients of this study were of two groups, first group included 65 patients with different types of hematological malignancies selected from the Oncology Center, Mansoura University Hospitals. Their ages ranged from 21 – 45 years. They consulted for persistent fever in spite of antibiotic therapy for 7–10 days before. An oral consent was obtained from each patient. 3-5 ml of blood was obtained to perform blood culture. The second patients group included 192 patients with onychomycosis and 140 patients with Tinea pedis from the Out Patient clinic of Dermatology, Mansoura University hospital. Nail and skin scrapings were obtained, using sterile scalpels, after cleaning the area with alcohol 70%.

Methods

Isolation of Yeasts

Blood culture was performed by lysis centrifugation technique. 3-5 ml heparinaized blood collected under aseptic precautions was subjected to lysis by using sterile D.W then centrifugation. The blood concentrates were cultured on Sabouraud’s dextrose agar (SDA), incubated at 37^°C for 7 days before discarded as negative. It was mentioned by Reisner and Woods⁽⁶⁾, that the average recovery time for yeasts and molds using lysis centrifugation methods is 3.8 & 10.5 days respectively. Nail & skin collected samples were inoculated onto SDA slants with chloramphenicol (50 mg/l) and incubated at 37^°C for up to 7 days before discarded as negative.

Identification of isolated yeast

·         Macromorphology: isolated colonies were described considering the size, consistency and pigmentation if present. Isolated Trichosporon characterized by being flat, creamy, moistened, wrinkled in the centre.

·         Microscopic examination by each of wet preparation and gram stained smears showing yeast cells. Trichosporon species are characterized by hyphae, and arthrospores which are gram positive in reaction.

·         Subculture on rice agar plates: 20 gm of rice were added to 400 ml water and boiled then simmered over a low flame for 45 min. The liquid was filtered and 20 gm of agar were added to the filtrate and completed to I liter of distilled water, then the mixture was autoclaved at 121°C for 15 min. The poured plates of rice agar were inoculated with yeast to be identified in short 3–4 streaks and covered with sterile cover slip, incubated at 25°C for 48 h, then examined microscopically for detection of blastospores, hyphae, pseudohyphae, and arthrospores. In this study, 14 isolates were identified phenotypically as Trichosporon species. They were subjected to PCR.

·         PCR: Genomic DNA of isolated yeast species was extracted.

DNA extraction⁽⁷⁾

Briefly, one loop of yeast cells was suspended in lysing solution (100 mM Tris-HCl [pH 8.0], 30 mM EDTA, and 0.5% sodium dodecyl sulfate) and then heated at 100°C for 15 min. The solution was extracted with phenol-chloroform-isoamyl alcohol (25:24:1 [vol/vol]). DNA was precipitated with cold isopropanol.

PCR amplification: Three different PCR protocols were performed using 3 different primer pairs. The first primer pairs⁽⁷⁾ would specifically amplify only Trichosporon species. They were chosen to align with regions on small subunit (SSU) of ribosomal DNA (rDNA) which were not conserved in other medically important yeasts. The amplification reactions were performed according to authors instructions.

Sixty eight yeast isolates were subjected to PCR using T. genus specific primers. Positive bands were detected at 170 bp were obtained in 16 (23.5) samples while the remaining 52 (76.5%) yeast isolates were negative. The 14 (of 16) positive PCR cases were previously identified phenotypically as T. species while the two cases were falsely diagnosed negative.

 

Primer	Primer Design
Forward	5́ – AGAGGCCTACCATGGTATCA-3́
Reverse	5́ – TAAGACCCAATAGAGCCCTA-3́

 

The positively identified 16 isolates were subjected to subsequent two PCR runs using 2 different primer pairs that would specifically amplify T. asahii or T. mucoides DNA.

The variable interspacer region (ITS) located between 18 S and 26 S r RNA genes and comprising ITS-I (between 18 S and 5.8 S rRNA genes) and ITS-2 (between 5.8 S and 26 S rRNA), showing the location of the T. asahii-specific primers (TASF & TASR) and T. mucoides primers (TMF & TMR)⁽⁸⁾. The amplification reactions were performed according to authors instructions^(8,9).

 

Species	Primer	Primer Design	Amplicon size%
T. asahii	TASF	5́-GGATCATTAGTGATTGCCTTTATA-3́	340bp
	TASR	5́-AGCACGCTTCAACACAATGGAC-3́
T. mucoides	TMF	5́-GGATCATTAGTGAATTGCTCTTTGA-3́	416bp
	TMR	5́-TTAGAAGCGCACTTCTCAAGTCT-3́

 

 

 

 

Results

There were 7 (10.7%) yeast isolates out of 65 cases suffering from persistent fever on top of hematologic malignancies. 32 (16.67%) yeast isolates were recovered out of 192 cases of onychomycosis, and 29(20.7%) out of 140 cases of tinea pedis.

A total of 14(3.5%) isolates were identified phenotypically in this study as Trichosporon species. Phenotypic identification was based on macromorphology on SDA, gram stained smears, subculture and microscopic characters on rice agar plates. There were 0 (0.0%), 9 (4.68%) and 5 (3.6%) isolates from cases with hematologic malignancies onchomycosis and tinea pedis respectively.

Photos 1 & 2 represent genotypic identification of Trichospon by PCR.

 

Table 1.    Frequency of isolated yeasts in the examined clinical samples

 

 

 

Table 2.    Frequency of phenotypically identified Trichosporon in the examined specimens.

 

 

 

 

 

Photo 1.         PCR of the genus T. was positive in 16 cases (16/68) at 170 bp

 

 

 

 

 

 

 

 

Photo 2.         PCR for T. asahii positive in 13/16

 

 

 

 

 

 

 

 

Discussion

It is important to emphasize that emergent fungal infections are usually difficult to diagnose, refractory to conventional antifungal drugs and associated with high mortality rates^(10,3).

In patients with malignant hematological diseases, genus Trichosporon has been reported as the second most common agent of yeast disseminated infections; only behind the genus Candida⁽¹¹⁾ .

In this study, it was found out of 397 samples (65 blood cultures, 192 nail scrapings, 140 skin scrapings), yeast isolates were obtained in 68 (17.1%) (Table 1).

Cases of hematological malignancies, onychomycosis, and those with tinea pedis were positive for yeast isolates with percentage of 10.7%, 16.6%, 20.7%, respectively. Based on the morphologic characters of isolated colonies on SDA, microscopic examination of colonies on rice agar, and stained smears, yeasts identified as Trichosporon were: 9 (4.68%) from nail samples, and 5 (3.6%) from skin samples and 0 (0.0%) from blood cultures (Table 2).

Trichosporon species are mostly associated with benign superficial lesions⁽¹²⁾. Some Mexican authors have documented that the isolation of Trichosporon species from tinea pedis and onchyomycosis ranged from 2.8 to 42.8% of cases⁽¹³⁾ .

Trichosporon species, for example T. asahii and T. mucoides have been described as emergent opportunistic pathogens related to disseminated infections in immunocompromised patients^(14,3).

However, Rastogi and Nirwan⁽¹⁵⁾ reported a rare case of meningoencephalitis and pneumonia due to T. asahii in an immunocompetent patient. This fact demonstrates the pathogenic role of T. species to cause human diseases.

Several methods used for Trichosporon species identification have been reported, including morphological and biochemical tests and the use of molecular tools⁽³⁾. Phenotypic methods are based on the characterization of micromorphological aspects of the colonies as well as the biochemical profiling. Performing a slide microculture to search for arthroconidia is a very useful tool for identification. However the accuracy of the phenotypic identification of Trichosporon species seems to be limited⁽⁴⁾.

Some authors suggest that the evaluation of specific nucleotide sequences can be a precise method to resolve taxonomic and epidemiological problems generated by the phenotypic identification of T. species⁽⁷⁾. Taj-Aldeen et al.⁽¹⁶⁾, stated that molecular biologic technique (PCR) is more precise for Trichosporon identification.

In this study, PCR using Trichosporon genus specific primers for 68 yeast isolates was positive in 16 samples (14 were previously identified by morphology and 2 nail scrapings were falsely diagnosed  negative by morphology) at 170 bp. It was stated by Sugita et al.⁽⁷⁾, that to detect T. species, they designed genus specific primers for PCR, based on small subunit (SSU) rDNA sequences that produced a 170 bp (photo 1) fragment for positive samples. They concluded that the method presented in their study can specifically detect the DNA of T. species.

Molecular methods are more precise for identification but are still costly for routine laboratory^(17,3).

In our study, we performed subsequently 2 PCR runs on the identified 16 samples using specific primers for each of T. asahii and T. mucoides. Their sequences included ITS1, ITS2. They yielded specific amplification of a DNA fragment at 430 bp in 13/16 (photo 2) samples, which is specific for T. asahii, and 0 (0.0%) sample was positive for T. mucoides specific primers.

Sugita et al.⁽⁷⁾, constructed a phylogenetic tree with the small subunit (SSU) region sequences of rDNA for specific identification of the genus Trichosporon. Subsequently Sugita et al.^(8,1), have sequenced and analyzed the interspacer regions (ITS-1 and ITS-2) genes of DNA from T. species . The ITS-1 and ITS-2 regions are highly conserved among Trichosporon species and sequencing of those two regions is sufficient to establish the identity of Trichosporon or other fungal organisms⁽¹⁶⁾.

In our study, the amplification of DNA (TISI & ITS2) from 13 (72.2%) of 16 clinical isolates with T. asahii specific primers, suggested that the 13 isolates were actually T. asahii strains, while the remaining 3 isolates remained unidentified.

It seems that, it is the first time to isolate and identify Trichosporon species in Egypt. This study suggests that T. asahii is the most common species associated with human clinical trichosporonosis in our locality.

Conclusion

Occurrence of trichosporonosis is not rare in human. T. asahii species is common in our locality. Therefore the standardization of laboratory methods for T. identification and antifungal susceptibility tests are necessary to investigate for both superficial and systemic trichosporonosis.

Reference

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2.       Sugita T, Nishikawa A, Shinoda T (1994): Reclassification of Trichosporon cutaneum by DNA relatedness by using the spectrophotometric method and chemiluminometric method. J Gen Appl Microbiol.40:397–408.

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