Identification of medically important yeasts by sequence analysis of the internal transcribed spacer and D1/D2 region of the large ribosomal subunit.

BACKGROUND: The prevalence of opportunistic yeast infections has increased in recent decades as the result of an increasing immunocompromised patient population. AIMS: To evaluate ribosomal RNA (rRNA) gene sequence to identify medically important yeast species, to investigate the performance of both the rRNA gene internal transcribed spacer (ITS) and D1/D2 region in identifying clinically relevant yeasts, and to compare these results with those of a standard phenotypic method. METHODS: Both regions from 50 yeast strains, comprising 45 clinical isolates and 5 reference strains, were amplified using PCR and then sequenced. The sequences were compared to reference data available from the GenBank database of the National Center for Biotechnology Information using the BLASTn tool. RESULTS: Using ID32C, 88% (44/50) of all strains were identified accurately at the species level, although 6% were misidentified; two Candida eremophila isolates were identified as Candida glabrata and Candida tropicalis, and one Saprochaete clavata isolate was identified as Saprochaete capitata. Two of the four isolates identified by phenotypic methods as Trichosporon asahii were defined so by analyzing the ITS region, but the remaining two were not distinguishable from closely related species. Based on the D1/D2 region, these four isolates had 100% sequence identity with T. asahii, Trichosporon japonicum, and Trichosporon asteroides. The isolate identified as Trichosporon inkin using ID32C could not be distinguished from Trichosporon ovoides by analyzing the ITS and D1/D2 regions. CONCLUSIONS: Identifying medically important yeasts by sequencing the ITS and D1/D2 region is a rapid and reliable alternative to conventional identification methods. For a diagnostic algorithm, we suggest a two-step procedure integrating conventional methods (e.g. microscopic morphology on corn meal agar with Tween® 80 and API ID32C®) and sequence analysis of the ITS and D1/D2 region.

Identification of medically important yeasts by sequence analysis of the internal transcribed spacer and D1/D2 region of the large ribosomal subunit / Identificación de levaduras de interés médico mediante análisis de secuencia del espaciador interno transcrito y de la región D1/D2 de la subunidad ribosómica grande

Background: The prevalence of opportunistic yeast infections has increased in recent decades as the result of an increasing immunocompromised patient population. Aims: To evaluate ribosomal RNA (rRNA) gene sequence to identify medically important yeast species, to investigate the performance of both the rRNA gene internal transcribed spacer (ITS) and D1/D2 region in identifying clinically relevant yeasts, and to compare these results with those of a standard phenotypic method. Methods: Both regions from 50 yeast strains, comprising 45 clinical isolates and 5 reference strains, were amplified using PCR and then sequenced. The sequences were compared to reference data available from the GenBank database of the National Center for Biotechnology Information using the BLASTn tool. Results: Using ID32C, 88% (44/50) of all strains were identified accurately at the species level, although 6% were misidentified; two Candida eremophila isolates were identified as Candida glabrata and Candida tropicalis, and one Saprochaete clavata isolate was identified as Saprochaete capitata. Two of the four isolates identified by phenotypic methods as Trichosporon asahii were defined so by analyzing the ITS region, but the remaining two were not distinguishable from closely related species. Based on the D1/D2 region, these four isolates had 100% sequence identity with T. asahii, Trichosporon japonicum, and Trichosporon asteroides. The isolate identified as Trichosporon inkin using ID32C could not be distinguished from Trichosporon ovoides by analyzing the ITS and D1/D2 regions. Conclusions: Identifying medically important yeasts by sequencing the ITS and D1/D2 region is a rapid and reliable alternative to conventional identification methods. For a diagnostic algorithm, we suggest a two-step procedure integrating conventional methods (e.g. microscopic morphology on corn meal agar with Tween(R) 80 and API ID32C(R)) and sequence analysis of the ITS and D1/D2 region

Antecedentes: La prevalencia de infecciones oportunistas por levaduras ha aumentado en las últimas décadas como resultado de una población de pacientes inmunocomprometidos cada vez mayor. Objetivos: Evaluar la secuencia del gen del ARN ribosomal (ARNr) para identificar especies de levaduras médicamente importantes, investigar el rendimiento del espaciador transcrito interno del gen ARNr (ITS) y las regiones D1/D2 en la identificación de levaduras clínicamente relevantes, y comparar estos resultados con los de un método fenotípico estándar. Métodos: Ambas regiones del ARNr de 50 cepas de levaduras con 45 aislamientos clínicos y 5 cepas de referencia se amplificaron mediante PCR y posteriormente se secuenciaron. Las secuencias se compararon con los datos de referencia disponibles en la base de datos GenBank(R) del Centro Nacional de Información Biotecnológica mediante la herramienta BLASTn. Resultados: Mediante el método ID32C el 88% (44/50) de todas las cepas se identificaron con precisión y el 6% se identificaron erróneamente; dos aislamientos de Candida eremophila fueron identificados como Candida glabrata y Candida tropicalis, y un aislamiento de Saprochaete clavata fue identificado como Saprochaete capitata. Dos de los cuatro aislamientos identificados por métodos fenotípicos como Trichosporon asahii se catalogaron así al analizar la región ITS, pero las dos restantes no se distinguían de las especies estrechamente relacionadas. En base a la secuencia de la región D1/D2, estos cuatro aislamientos se identificaron, con un 100% de similitud, como T. asahii, Trichosporon japonicum y Trichosporon asteroides. El aislamiento identificado como Trichosporon inkin mediante ID32C no se pudo distinguir de Trichosporon ovoides al analizar las regiones ITS y D1/D2. Conclusiones: La identificación de levaduras de interés médico mediante la secuenciación de las regiones ITS y D1/D2 es una alternativa rápida y confiable a los métodos de identificación convencionales. Para un algoritmo de diagnóstico sugerimos un procedimiento de dos pasos que integre métodos convencionales (morfología microscópica en agar de harina de maíz con Tween(R) 80 y API ID32C(R)) y análisis de la secuencia de las regiones ITS y D1/D2

Circulating Th17 cytokine levels are altered in Hashimoto's thyroiditis.

The disrupted autoimmune response in Hashimoto's thyroiditis (HT) has long been considered to be dominantly T helper type 1 (Th1) mediated. Recent advances in the field of immunology have introduced a new class of effector T cells, named 'Th17', which plays important roles in autoimmune disorders once thought to be merely Th1 mediated. We aimed to examine the levels of major Th17 cytokines in patients with HT in this study. We studied serum interleukin 17 (IL-17) and interleukin 23 (IL-23) levels in 46 newly diagnosed, untreated patients with HT (40 women and 6 men, aged 40.0 ± 11.8 years) divided into euthyroid (n=22) and hypothyroid (n=24) groups and compared them with age and sex matched 26 healthy euthyroid controls without HT (21 women and 5 men; aged 36.0 ± 12.9 years). Serum IL-17 and IL-23 levels were significantly different among euthyroid and hypothyroid HT patients and controls, with highest levels obtained in the euthyroid HT group (p=0.041 for IL-17 and p<0.001 for IL-23). TSH was negatively and FT4 was positively correlated with IL-17 (p=0.016 for TSH and p=0.004 for FT4) and IL-23 (p<0.001 for TSH and p=0.003 for FT4) levels. There were no correlations between thyroid volumes calculated on thyroid ultrasonography and IL-17 (p=0.630) or IL-23 (p=0.321) levels. In conclusion, the levels of IL-17, one of the major effector cytokines of the Th17 system, and IL-23, which had been implicated in the generation, survival and expansion of Th17 cells, are altered in HT. How thyroid hormone status and the course of disease affect Th17 system in chronic autoimmune thyroiditis needs to be determined with further studies.

[Investigation of some virulence factors in Trichosporon spp. strains]. / Trichosporon spp. suslarinda bazi virülans faktörlerin arastirilmasi

The frequency of fungal infections have increased recently in parallel to prolonged survival of patients with chronical infections, common use of the broad-spectrum antibiotics and cytotoxic drugs and surgical interventions. Fungi such as Trichosporon, Fusarium and Geotrichum that were previously evaluated as contaminant/colonization, become important causes of morbidity and mortality especially in neutropenic patients. The aim of this study was to investigate the presence of virulence factors such as acid proteinase, phospholipase, esterase, coagulase and hemolytic activity among Trichosporon species. A total of 40 Trichosporon strains, of them 24 (60%) were T.asahii, 6 (15%) were T.inkin and 10 (25%) were the other species (one of each of T.aquatile, T.asteroides, T.coremiiforme, T.cutaneum, T.dermatis, T.faecale, T.japonicum, T.montevideense, T.mucoides, T.ovoides) were included in the study. Identification of the isolates was performed according to microscopic morphology (blastospores, arthrospores, pseudohyphae and true hyphae) on corn meal agar media, and carbohydrate assimilation patterns (API ID32C; bioMérieux, France). Secretory acid proteinase, phospholipase and esterase activities of the strains were evaluated by 1% bovine serum albumin containing agar, by egg yolk containing solid medium, and by Tween 80 containing solid medium, respectively. Hemolytic activity of the isolates were evaluated by 5-10% sheep blood Sabouraud dextrose agar. Coagulase enzyme activity was determined by using human and rabbit plasma. In our study, all of the 40 Trichosporon spp. strains were found negative in terms of acid proteinase and phospholipase enzyme activity, however all were positive for esterase enzyme activity. Hemolytic enzyme activity were identified in a total of 15 (37.5%) strains, being "+++" in three strains (2 T.asahii, 1 T.japonicum), and "++" in 12 isolates (9 T.asahii, 1 T.inkin, 1 T.asteroides, 1 T.mentevideense). Although 11 of those 15 positive strains were T.asahii, there was no statistical difference between the species in terms of hemolytic enzyme activity (p> 0.05). Coagulase enzyme activity was detected in 5% (2/40; 1 T.asahii, 1 T.inkin) of the strains with human plasma and in 27.5% (11/40; 9 T.asahii, 1 T.inkin, 1 T.montevideense) with rabbit plasma. In conclusion, our data indicated that esterase, coagulase and hemolytic activities detected in Trichosporon spp. might play role in the pathogenesis of Trichosporon infections, however, further large-scaled clinical and mycological studies are needed to prove this relation.

[Determination of anti-echinococcus IgG antibodies by ELISA in patients with suspected hydatid cyst]. / Kist Hidatik Süpheli Hastalarda Anti-Echinococcus IgG Seropozitifliginin ELISA Yöntemiyle Belirlenmesi.

OBJECTIVE: Cystic echinococcosis (hydatid cyst) is one of the most important parasitic zoonoses that affect both humans and animals and has been known since prehistoric times. The cystic echinoccosis is a major health problem in our country as well as in many countries worldwide, and serological methods, in addition to imaging techniques, are used in the diagnosis of hydatid cyst. In the present study, anti- Echinococcus antibodies were investigated by ELISA in patient's serum samples, which were routinely delivered to the Medical Microbiology Laboratory of Gazi University Faculty of Medicine due to suspected hydatid cyst. METHODS: A total of 186 patients with suspected hydatid cysts from several outpatient clinics and departments of Gazi University Hospital were included in the present study. IgG antibodies in serum samples of patients with anti-Echinococcus were investigated by ELISA (Novalisa Echinococcus IgG, NovaTec, Germany). RESULTS: Anti-Echinococcus IgG seropositivity was determined as 35.5% in patients with suspected hydatid cyst. There are no statistical differences in ELISA positive results relating to gender, adult/child age group and associated clinics. CONCLUSION: Hydatid cyst seropositivity was higher among patients admitted to our hospital.

[The rare genes related to resistance to macrolide-lincosamide and streptogramin B group antibiotics among coagulase-negative staphylococci]. / Koagülaz-Negatif Stafilokoklarda Makrolid-Linkozamid-Streptogramin B Grubu Antibiyotiklere Karsi Nadir Direnç Genlerinin Arastirilmasi

Macrolide-lincosamide-streptogramin B (MLSB) group antibiotics are recommended as first choice in the treatment of staphylococcal infections. All of those drugs bind to the 50S subunit of bacterial ribosomes, thus cross-resistance is a major concern in this group of drugs. The mechanisms associated to resistance are (a) ribosomal methylation due to the methylases encoded by erm genes, (b) active drug efflux due to msrA, msrB, vga, vgb gene activity, (c) enzymatic inactivation of the drug due to the activity of linA, vat, vatB genes. While the most common resistance genes are ermA, ermB, ermC, msrA and msrB genes; linA, vga, vgb, vat and vatB genes have also been found in some studies. In this study it was aimed to investigate the presence of the rare MLSB resistance genes and their coexistence with erm and msr genes in 454 clinical isolates of coagulase-negative staphylococci (CNS). Of them 46.5% (n= 211) were S.hominis, 30.8% (n= 140) were S.epidermidis, 12.1% (n= 55) were S.haemolyticus, 3.5% (n= 16) were S.warnerii and 7% (n= 32) were the other coagulase-negative staphylococcal species. Resistance phenotypes were determined by using D-test method according to the recommendation of Clinical and Laboratory Standards Institute (CLSI). With the D-test 107 (23.6%) strains were determined as M phenotype (resistant to erythromycin and inducible clindamycin resistance was not detected), 92 (20.3%) were iMLSB phenotype (inducible clindamycin resistance was detected by the D-test) and 110 (24.2%) were cMLSB phenotype (constitutive erythromycin and clindamycin resistance was detected). linA, vga, vgb, vat, vatB, ermA, ermB, ermC, msrA, msrB genes were investigated by polymerase chain reaction in all strains showing iMLSB (n= 92) and cMLSB (n= 110) phenotypes and 46 randomly selected strains among 107 strains exhibiting the M phenotype. linA gene was found in 91 (20%) strains as single gene or in combination with erm or msr genes, and vga gene was found in 19 (4.2%) strains. linA gene was found in 52% of iMLSB phenotype, in 26% of cMLSB phenotype and 13% of M phenotype while vga gene was found in 5.4% of iMLSB phenotype, in 12% of cMLSB phenotype and in 0.9% of M phenotype. The most common resistance gene among iMLSB and cMLSB phenotypes was ermC (32.6% and 42.7%, respectively), followed by ermC + linA gene combination (31.5% and 14.5%, respectively). The most frequent gene combination was msrA and msrB in M phenotype (34.8%) and it was followed by a combination of msrA + msrB + linA genes (19.1%). None of the strains revealed presence of vgb, vat and vatB genes. There were no previous reports about the rarely detected resistance genes against MLSB antibiotics in our country. This was the first study which reported the frequency of linA, vga, vgb, vat and vatB genes in MLSB resistant CNS. In conclusion, since linA and vga genes were detected in high frequency in MLSB resistant CNS in this study, it was thought that the investigation of these genes should be included in the further related epidemiologic gene research.

[Investigation of Microsporidia prevalence by different staining methods in cases of diarrhea]. / Ishalli Olgularda Microsporidia Sikliginin Farkli Boyama Yöntemleriyle Arastirilmasi

Microsporidia, depending on their different species, generally lead to self-limited, sporadic and mild infections such as diarrhea, corneal ulcer and myositis. They are considered as opportunistic pathogens in HIV-positive patients however in recent years Microsporidia have been detected also in immunocompetent individuals as a cause of diarrhea. Diagnosis of Microsporidia depends on the detection of spores or different developmental stages of protozoon in stool, urine, sinus aspirates, nasal discharge, bronchoalveolar lavage or tissue biopsies. Diagnosis of Microsporidia infections is usually achieved by the use of different staining methods, serological tests, polymerase chain reaction, and electron microscopic methods. The aims of this study were to detect the incidence of microsporidia in patients with diarrhea by using three different staining methods and to compare the performance of these methods. A total of 225 stool samples from diarrheal patients (84 were children, 141 were adults; 103 were female, 122 were male) admitted to Gazi University Medical Faculty Hospital between March-June 2009, have been evaluated in the laboratory of Medical Microbiology Department. Stool samples were examined in terms of the presence of Microsporidia spores by Weber's modified trichrom staining (MTS), calcofluor (CF) and acridine orange (AO) staining methods. Microsporidia positivity rate was 9.8% (22/225) in the diarrheal patients, the rate being 9.5% (8/84) in children and 9.9% (14/141) in adults. There was no statistically significant difference between age and gender groups (p> 0.05) regarding Microsporidia detection. When MTS was considered as the reference method, sensitivity, specifity and consistency of AO staining were estimated as 100%, 91.6% and 92%, respectively, while those rates for CF staining were 95.4%, 99.5% and 99%, respectively. There was very strong and significant correlation (r= 0.950, p< 0.001) between CF staining and MTS, while there was strong and significant (r= 0.719, p< 0.001) correlation between AO staining and MTS. Although AO staining is rapid and convenient, the positive predictive value was measured very low (56.4%) and the interpretation of stained slides was very difficult since background of the slides was stained orange and there were a lot of dye artefacts. In conclusion, screening Microsporidia in all diarrheal stool samples is of diagnostic value. To increase sensitivity and reliability in the detection of Microsporidia spores in diarrheal samples, initial application of calcofluor staining should be followed by the confirmatory MTS method.

Yeast vaginitis during pregnancy: susceptibility testing of 13 antifungal drugs and boric acid and the detection of four virulence factors.

A higher prevalence of vulvovaginal candidiasis (VVC) is seen in pregnant women compared with those who are not pregnant. Recurrence is also more common in pregnant women, and therapeutic responses are reduced. In this investigation, 207 vaginal yeast isolates recovered from pregnant women were tested for susceptibility to 13 antifungal drugs and boric acid and through these studies four virulence factors were also determined. The isolates were recovered from vaginal samples of patients with acute VVC [AVVC, (n = 73)], symptomatic recurrent VVC [RVVC, (n = 89)], asymptomatic RVVC (n = 27), and those without signs and symptoms (n = 18). Candida albicans was the most common species found (59.9%), followed by C. glabrata (19.8%), other Candida spp., (19.8%), and Saccharomyces cerevisiae (0.5%). Antifungal susceptibility testing was performed as described in CLSI document M27-A3. Additionally, we examined phospholipase and proteinase production, adhesion to vaginal epithelial cells and hemolytic activity. Notably, the MIC values of Candida spp. isolates derived from patients with VVC were no different from those of the controls (P > 0.05). In addition, Candida isolates derived from patients with AVVC or RVVC produced significantly higher amounts of phospholipase and proteinase compared with the controls (P < 0.05). Antifungal testing and the determination of virulence factors may lead to the effective and prompt treatment of VVC, particularly in pregnant women.

Cytokine profile in murine toxoplasmosis.

OBJECTIVE: To investigate which cytokines are produced after acute infection of mice with Toxoplasma gondii (T. Gondii) RH strain. METHODS: Mus domesticus domesticus mice in infected group were inoculated with with highly virulent T. Gondii RH strain by intraperitoneally. Serum samples were obtained from infected and non-infected mice for cytokine levels for ELISA assay. RESULTS: The concentrations of tumor necrosis factor-α, interferon-γ, interleukin (IL)-10 and IL-12 in the cardiac blood sample of the infected mice were significantly higher than those in uninfected controls (P<0.05). The levels of transforming growth factor-1ß decreased in mice infected with T. gondii compared to those of the controls, the decrease was statistically significant (P<0.05). No significant difference was observed in levels of IL-4 between infected and healty control groups (P>0.05). CONCLUSIONS: According to our findings, immune response into T helper type 1 was predominant during acute T. gondii infection. Further characterization and purification of Toxoplasma molecule(s) implicated in the regulation of cytokines could lead to the development of new drug prospects to control Toxoplasma infection.

[Investigation of interleukin-10, tumor necrosis factor-alpha and interferon-gamma expression in experimental model of pulmonary aspergillosis]. / Deneysel Pulmoner Aspergilloz Modelinde Interlökin-10, Tümör Nekroz Faktörü-alfa ve Interferon-gama Ifadelenmesinin Arastirilmasi

Pulmonary aspergillosis which is an important opportunistic infection in neutropenic patients, is usually caused by Aspergillus fumigatus. Since the pathogenesis of disease is not well understood, the main proposed mechanism is thought to be cell-mediated immunity and cytokine response. The aim of this study was to investigate the local production of cytokines in the lung tissues of rats with experimentally developed aspergillosis, by reverse transcriptase-polymerase chain reaction (RT-PCR). A total of 33 Wistar albino type rats were included in the study with the consent of Experimental Animal Ethics Committee. Twenty-five of the rats were infected with A.fumigatus by intratracheal way, while 8 animals were used as controls. The presence of A.fumigatus in the lung tissues of infected rats was confirmed with the use of quantitative culture and histologic staining methods. RNA isolation from the lung tissue samples of both groups were performed by a commercial kit (Qiagen, Germany). After obtaining complementary DNAs from the genomic RNAs, in-house qualitative and quantitative (real-time) PCR methods were used to amplify the target regions for interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-?) and interferon-gamma (IFN-?) by using specific primers (Tib Molbiol, Germany). Mean mRNA levels achieved by real-time PCR for IL-10, TNF-? and IFN-? in aspergillosis group were 6.5 x 106 copies/ml, 7.9 x 105 copies/ml and 2.2 x 103 copies/ml, respectively, while those values in control group were 4.3 x 102 copies/ml, 5.6 x 103 copies/ml and 1.3 x 102 copies/ml, respectively. Our data indicated that rat model of aspergillosis was associated with significantly increased expression of mRNA encoding IL-10 and TNF-? than controls (p< 0.05), however there was no statistically significant difference between the groups with respect to IFN-? expression (p= 0.53). In conclusion, the production of proinflammatory cytokines which mediate the influx of phagocytic cells might account for the localization of Aspergillus infection to the upper respiratory tract. The up-regulation of the expression of the immunomodulatory cytokine TNF-? and IL-10 in lung tissue from infected rats might be important to limit the extent of local tissue destruction, but might also account for the fact that infected rats are generally unable to clear the infection spontaneously.

Evaluation of DNA extraction kits for molecular diagnosis of human Blastocystis subtypes from fecal samples.

Blastocystis sp. is now recognized as one of the most common intestinal parasite in human fecal examinations. Recently, PCR-based diagnostic methods of Blastocystis infection using direct DNA extraction from fresh fecal samples with commercially available kits are reported. Several kits have been developed, but little has been done in comparing the detective sensitivity between PCR methods using the commercial kits. In this study, we compared the detective sensitivity among five commercially available kits (MagNA Pure LC DNA Isolation Kit I, Roche; QuickGene SP Kit DNA, FujiFilm; NucleoSpin Plant II, Macherey-Nagel; QIAamp DNA Stool Mini Kit, Qiagen; ZR Fecal DNA Kit, Zymo Research) and fecal culture method. In a preliminary test, the DNA isolated with two kits (FujiFilm and Macherey-Nagel) showed negative PCR, while the other three kits showed positive PCR. Then, DNA from 50 clinical samples that was Blastocystis-positive in the examination of fecal culture method were isolated with the three kits and 1.1 kbp SSU rRNA gene was detected with PCR. The positive rates of the three kits (Roche, Qiagen, and Zymo Research) were 10, 48 and 94%, respectively. The present study indicated that there is different detective sensitivity among the commercial kits, and fecal culture method is superior in detection rate and cost performance than DNA-elution kits for diagnosis of Blastocystis sp. subtypes.

[DNA extraction and identification of Trichophyton rubrum by real-time polymerase chain reaction from direct nail scraping specimens of patients with onycomycosis]. / Onikomikozlu Olgularin Direkt Tirnak Kazinti Örneklerinden DNA Izolasyonu ve Gerçek Zamanli Polimeraz Zincir Reaksiyonu ile Trichophyton rubrum Tanimlanmasi

Trichophyton rubrum is the most frequently encountered dermatophyte species causing onichomycosis. The routine diagnosis of dermatophytes depends on the direct microscopic examination (DME) and culture methods, however due to the phenotypic identification problems related to those agents, the molecular methods come into question. The aim of this study was to evaluate the diagnostic performance of real-time polymerase chain reaction (RT-PCR) for the identification of T.rubrum by comparing to DME and culture methods, from nail samples of patients with the complaints of onychomycosis. A total of 90 patients of whom 58 were male who were admitted to the dermatology outpatients clinics of our hospital with the complaints of color/shape changes in the nails and thickening of the nail, were included in the study, together with the 20 healthy volunteer subjects as controls. The nail scraping samples obtained from the patients and controls were examined with direct microscopy using 15% potassium hydroxide, dimethyl sulphoxide and chlorazole black mixture and cultivated onto Sabouraud dextrose agar with and without cycloheximide. For DNA isolation, after the disruption of nail samples with a steel tool, phenol-chloroform-isoamyl alcohol purification method were used. The amplification and demonstration of the T.rubrum DNA have been performed by using specific primers and probes following TaqMan protocol of RT-PCR (LightCycler-Roche, USA) method. Seventy-two of the patients yielded positive and 18 yielded negative results with DME. Growth of molds was detected in the cultures of 20 (27.8%) of the 72 DME positive patients and all of the isolates were identified as T.rubrum. No fungal growth was seen in the samples of 18 patients who were DME negative. In DME positive group, 67 (93%) patients were found to be positive in RT-PCR, while 8 (44.4%) patients were RT-PCR positive in DME negative group. All of the culture positive samples (n= 20) were also found positive in RT-PCR. All of the samples from the control group with healthy nails yielded negative results in DME, culture and RT-PCR methods. The performance of PCR method were compared to direct microscopy that had higher sensitivity than culture and the sensitivity, specificity, positive and negative predictive values of RTPCR assay were estimated as 93%, 56%, 89% and 67%, respectively. In conclusion RT-PCR was thought to be an efficient and rapid assay in the diagnosis of onichomycosis. Although RT-PCR seems more expensive than culture, for the centres which already have support for the molecular methods, the difference in total cost doesn't count much. In conclusion, by the use of molecular methods DNA isolation was successfully done from a relatively difficult clinical specimen, namely nail scraping, a protocole that could easily be applied in routine laboratory was established and species-level identification in a short time was accomplished in this study.

Comparison of methods for detection of Blastocystis infection in routinely submitted stool samples, and also in IBS/IBD Patients in Ankara, Turkey.

BACKGROUND: This study compared diagnostic methods for identifying Blastocystis in stool samples, and evaluated the frequency of detection of Blastocystis in patients with irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). RESULTS AND DISCUSSION: From a set of 105 stool specimens submitted for routine parasitological analysis, 30 were identified as positive for Blastocystis by the culture method. From that group of 30 positives, Lugol's stain, trichrome staining, and an immunofluorescence assay identified 11, 15, and 26 samples as positive respectively. Using culture as a standard, the sensitivity of Lugol's stain was 36.7%, trichrome staining was 50%, and the IFA stain was 86.7%. The specificity of Lugol's stain was 91%, trichrome staining was 100%, and the IFA stain was 97.3%. In the group of 27 IBS and IBD patients, using all methods combined, we detected Blastocystis in 67% (18/27) of the patients. Blastocystis was detected in 33% (2/6) of IBD patients and 76% (16/21) of IBS patients. For comparison, trichrome staining alone, the method most frequently used in many countries, would have only identified Blastocystis infection in 29% (6/21) of the IBS patients. No parasitic co-infections were identified in the IBS/IBD patients. Most Blastocystis-positive IBS/IBD patients were over 36 with an average length of illness of 4.9 years. CONCLUSIONS: Most IBS patients in this study were infected with Blastocystis. IFA staining may be a useful alternative to stool culture, especially if stool specimens have been chemically preserved.

[Comparison of glucan and galactomannan tests with real-time PCR for diagnosis of invasive aspergillosis in a neutropenic rat model]. / Invazif aspergilloz olusturulan siçanlarda glukan ve galaktomannan testleri ile gerçek zamanli polimeraz zincir reaksiyonu sonuçlarinin karsilastirilmasi

The incidence of aspergillosis which has high mortality rates, has increased gradually. Since invasive aspergillosis (IA) is one of the leading causes of death in immunocompromized and neutropenic patients, early and accurate diagnosis of IA is of crucial importance. The aims of this study were to compare the results of culture, real-time polymerase chain reaction (RtPCR), galactomannan (GM) antigen and glucan (GC) antigen detection tests and to evaluate their performances in view of rapid and accurate diagnosis of IA in neutropenic rat model. Female Wistar albino rats were included in the study with the consent of Animal Searching Ethical Committee and classified into three groups as healthy controls (n= 6), neutropenic controls (n= 10) and pulmonary aspergillosis (n= 35) groups. Rats were immunosuppressed with 5-flourourasil and then Aspergillus fumigatus conidia were inoculated intranasally. On the seventh day of the infection, blood, bronchoalveolar lavage (BAL) and lung tissue samples were collected from the animals, and control and aspergillosis groups were compared in terms of infection markers. All of the tests (culture, RtPCR, GM and BG tests) were found to be negative in controls. At the end of the study 22 rats in aspergillosis group survived. Lung tissue samples from those 22 animals were all positive for the presence of hypha on pathological preparations, while 20 (91%) yielded Aspergillus colonies on the cultures. Aspergillus DNA was detected in 7 of the 12 BAL samples (58.3%), 7 of 19 blood samples (36.8%) and 4 of 22 lung tissue samples (18%) using RtPCR method. GM antigen was detected in 7 of 20 serum samples (35%) with a commercial kit (Platelia® Aspergillus ELISA, BioRad, France). Quantitative detection of betalucan levels were investigated by using a commercial kit (Fungitell™, Cape Cod, USA) in serum and BAL samples and positive results were obtained in 11 of 22 serum (50%) and 9 of 17 BAL (52.9%) samples. In this study it was demonstrated that PCR performed in BAL samples is the most sensitive method compared to the others, for the diagnosis of IA in the rat model. The sensitivity rates were as follows when culture method accepted as the gold standard: 58.3% for BAL-PCR, 41.2% for blood-PCR, 20% for tissue-PCR, 38.9% for serum GM, 55% for serum GC and 52.9% for BAL-GC. It was also concluded that detection of GC activity in serum was more sensitive than GM detection in serum (sensitivity of GM was %38.9, sensitivity of GC was %55, while specificities were 100% for both of the tests), for laboratory diagnosis of IA. The BAL samples were evaluated as the most valuable clinical samples to screen the suspected patients. However, even in proven cases, 41.7% of BAL samples were found negative with PCR, 50% of serum samples were found negative with GC test, and 65% of serum samples were found negative with GM test. Since the pathogenesis of IA has not been completely clarified, the performance of non-culture based diagnostic tests exhibit great variability. Future clinical studies are required to compare the performance of different nonculture based diagnostic methods and the optimal combination of these tests for the most accurate laboratory diagnosis of IA.

[In vitro synergistic effect of moxifloxacin and amphotericin B combination against Candida strains]. / Moksifloksasin ve amfoterisin b kombinasyonunun Candida türleri uzerine in vitro sinerjistik etkisi.

Contradictory results such as synergy or indifferent effect, have been reported about the interactions between quinolones and antifungal drugs in different studies. The aim of this study was to investigate the in vitro susceptibilities of Candida spp. to moxifloxacin (MOX) alone and MOX + amphotericin B (AmB) combination. A total of 20 strains were included to the study, of which 19 were clinical isolates (10 Candida albicans, 4 Candida glabrata, 2 Candida parapsilosis, 1 Candida tropicalis, 1 Candida pelliculosa ve 1 Candida sake) and 1 was a standard strain (C. albicans ATCC 90028). In vitro susceptibilities of the strains to MOX with AmB were investigated by broth microdilution method according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI), and in vitro interaction of these drugs were determined by a chequerboard titration method. Minimal inhibitory concentration (MIC) values of Candida spp. for MOX were found > or = 400 microg/ml indicating that MOX, by itself has no antifungal activity. AmB MIC values were found 1 microg/ml in 11 of the clinical isolates, and < or = 0.5 microg/ml in the other 8 clinical isolates and 1 standard strain. The inhibitor activity of AmB was slightly enhanced when combined with MOX, there being a decrease of 1-4 fold dilutions in the AmB MICs against all isolates tested. Synergistic effect between MOX and AmB, defined as a fractional inhibitory concentration (FIC) index as < or = 0.5, was observed in 90% (18/20; all were clinical isolates) of the strains, whereas indifferent effect (FIC = 1) was detected in 10% (2/20; 1 was clinical and 1 was standard strain) of the strains. Antagonistic effect was not observed for this combination even at 48th hours. It was concluded that these preliminary results should be confirmed by large-scaled in vitro and in vivo studies to evaluate MOX + AmB combination as a therapeutic option for the treatment of Candida infections.

Nosocomial transmission of Candida pelliculosa fungemia in a pediatric intensive care unit and review of the literature.

Horizontal transmission of Candida species in the hospital environment and the fungemia rates have increased in the past decade. We describe a nosocomial cluster of fungemia caused by Candida pelliculosa (teleomorph Pichia anomala) in four infants hospitalized in the pediatric intensive care unit. Candida isolates had strictly related fingerprints, as generated by randomly amplified polymorphic DNA analysis using five different primer sets. The four babies were all treated successfully and recovered. All of the isolates were susceptible to the antifungals tested including amphotericin B, flucytosine, fluconazole, miconazole, micafungin, itraconazole, and voriconazole. Infection control procedures were adapted in the unit and no relapse was detected. In addition, 30 publications presenting 450 pediatric and 28 adult cases are reviewed.

Molecular identification, genotyping, and drug susceptibility of the basidiomycetous yeast pathogen Trichosporon isolated from Turkish patients.

Deep-seated infections due to Trichosporon species are emerging mycoses that have a very poor prognosis in patients with persistent neutropenia. This study elucidated the mycological characteristics of Trichosporon strains obtained from deep-seated infections in Turkish patients and identified by DNA sequence analysis of intergenic spacer (IGS) region 1 of the rDNA locus. In addition, we genotyped the major causative agent, T. asahii, and evaluated the in vitro drug susceptibility of the isolates. While 87 (81.3%) of the 107 isolates were T. asahii, the remaining 20 were T. faecale (14.0%), T. asteroids (0.9%), T. coremiiforme (0.9%), T. japonicum, (0.9%), T. lactis (0.9%), and a new species (0.9%). In addition to the eight known T. asahii genotypes, one novel genotype was identified. The distribution of the T. asahii genotypes in this study were genotype 1 (79.3%), followed by 5 (8.0%), 3 (6.9%), 6 (3.4%), 4 (1.1%), and 9 (1.1%). Turkish isolates showed low susceptibility to amphotericin B, 5-flucytosine, and fluconazole. Although relatively low minimum inhibitory concentrations (MICs) were found with all drugs, voriconazole appeared to be the most active. The MICs of the non-Trichosporon asahiiTrichosporon species were similar to those of the T. asahii strains. Our findings suggest that Trichosporon species isolated from Turkish patients are more diverse than those reported from other countries.

High incidence of Candida parapsilosis candidaemia in non-neutropenic critically ill patients: epidemiology and antifungal susceptibility.

The epidemiological and antifungal susceptibility data for 35 episodes of candidemia in intensive care units (ICU) in 2007 were evaluated by prospective active surveillance. The incidence of fungaemia was 39.1 cases per 1000 ICU admissions and 2.85 cases per 1000 patient-days. The crude mortality was 65.7%; 70.8% of the fatalities occurred within 7 days of admission to the ICU. Only 2 species were isolated, Candida parapsilosis (77.1%) and Candida albicans (22.9%). There was no association between mortality and patient characteristics, prior antifungal usage, Candida subspecies or antifungal resistance (p > 0.05). Of the isolates, 5.7% were resistant to fluconazole and caspofungin, and 3.4% to voriconazole and amphotericin B. In molecular analysis of the isolates, 2 clusters of C. parapsilosis in the neurology and anaesthesiology ICUs were detected by randomly amplified polymorphic DNA (RAPD), suggesting a nosocomial transmission. In conclusion, a high incidence and high mortality rate of C. parapsilosis candidaemia were found in the ICUs. An excessive use of invasive procedures, total parenteral nutrition and broad-spectrum antibiotics in the ICUs, combined with a lack of proper infection control measures, may possibly explain the high incidence of C. parapsilosis candidaemia in our hospital.

Blastocystis subtypes in irritable bowel syndrome and inflammatory bowel disease in Ankara, Turkey.

Blastocystis infection has been reported to be associated with irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) and chronic diarrhoea. The availability of data on the subtypes of Blastocystis found in these patient groups would be of interest in understanding the significance of Blastocystis infection in chronic illness. In this study, we identify Blastocystis subtypes found in patients presenting with IBS, IBD, chronic diarrhoea and asymptomatic patients in Ankara, Turkey. Blastocystis was detected in 11 symptomatic patients by microscopy and 19 by stool culture. Stool culture was more sensitive than microscopy in identifying Blastocystis. Using standard nomenclature adopted in 2007, Blastocystis sp. subtype 3 was the most common in all groups, followed by Blastocystis sp. subtype 2. Identical subtypes of Blastocystis are found in patients with IBS, IBD and chronic diarrhoea. These particular subtypes show low host specificity and are carried by humans and some farm animals. The subtypes of Blastocystis that are commonly found in rodents and certain wild birds were not found in these patients. We suggest a model in which the severity of enteric protozoan infection may be mediated by host factors.

PCR-based subtyping of Blastocystis isolates from symptomatic and asymptomatic individuals in a major hospital in Ankara, Turkey.

The stool samples obtained from 94 patients with gastrointestinal symptoms and 109 asymptomatic individuals, who checked in due to other reasons, admitted at a major hospital in Ankara, Turkey were examined with native Lugol's iodine, trichrome, and Kinyoun's acid-fast stainings for parasitology examinations and with in vitro culture method for detection of Blastocystis. In a total of 203 stool samples tested, native Lugol's iodine and trichrome stainings could detect 12 (5.9%) and 20 (9.9%) positive samples for Blastocystis, respectively. Conversely, culture method could detect 66 (32.5%) positive samples, and this method was more sensitive compared to the both microscopic examinations (p < 0.001). Among 66 positive samples for Blastocystis, 27 were from symptomatic patients and 39 were from asymptomatic group. Subtypes (STs) were determined by PCR using seven different sequence-tagged site primers. ST3 was the most dominant in both symptomatic and asymptomatic groups and followed by ST1 or ST2. There were mixed infections with STs 1 and 2 or STs 1 and 3 in nine isolates. There was no statistical significance of the distribution of Blastocystis sp. subtypes between symptomatic and asymptomatic individuals (p > 0.05).