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Acremonium spp. (previously known as Cephalosporium) and Pseudomonas mesophilica were isolated from a patient who had infective keratitis caused by corneal trauma. In this case, clinical and microbiological findings of Acremonium spp. and P. mesophilica are presented.

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.

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 (p0.05) 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, p0.001) between CF staining and MTS, while there was strong and significant (r= 0.719, p0.001) 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.

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 (Tıb 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 (p0.05), 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.

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.

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.

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.

This study was planned to determine the effect of building constructions in and around our hospital, on the development of opportunistic mold infections in immunocompromised patients hospitalized in bone marrow and kidney transplantation units and haematology and oncology units. Samples were collected from high risk units by an air sampler (Air Ideal) from indoors and outdoors of a total of 43 patient rooms. The most commonly isolated species from indoor air cultures of our hospital were Penicillium spp. (50.6%), Cladosporium spp. (20%), Chrysonilia spp. (11%) and Aspergillus (10.6%) species. When outdoor samples were considered, Penicillium spp. (38.8%) was still in the first line, followed by Cladosporium spp. (24.3%), Paecillomyces spp. (10.7%) and Aspergillus (8.7%) species. There was no statistically significant difference of total colony and spore numbers between the samples obtained from indoor and outdoor air (p0.05), indicating the close relation with the construction studies in and around the hospital. Clinical samples including bronchoalveoler lavage (BAL) fluid, sputum, endotracheal aspirate and sinus tissue were collected from the total of 43 patients staying at these air sampled rooms, and eight of them (18.6%) yielded positivity for the growth of molds. Of them four were identified as Penicillium chrysogenum (sputum isolates), two as Aspergillus fumigatus (sputum and BAL isolates), one as Aspergillus flavus (BAL isolate), and one as Valsa sordida (sinus tissue) which is considered as a plant pathogen. A total of 53 sera, BAL, and tissue supernatant samples were screened by ELISA for the presence of galactomannan antigen, and five of the eight patients whose cultures were positive were also found positive for galactomannan antigen. One patient has died due to invasive aspergillosis whose BAL specimen and indoor air sample were positive for A. fumigatus. In evaluation of indoor air samples before and after the change of HEPA filters, statistically significant decrease was detected in total colony and spore numbers between the samples taken before and after the filter changes (p0.005). This study has emphasized the importance of examination of mold flora of indoor air and clinical samples of high risk group patients intermittantly, in order to prevent opportunistic mold infections in crucial units especially during hospital constructions.

Since nosocomial candidemiae is mainly evolved from the endogenous flora of the patients, the detection of colonization indices may guide for the risk of infection especially in intensive care unit (ICU) patients. The aims of this study were the detection of colonization rates of ICU patients with Candida spp., establishment of C. albicans colonization index (CI), and investigation of the relationship between the presence of C. albicans IgM and IgG antibodies and colonization indices. A total of 191 swab specimens collected from at least five different body sites of 37 patients, together with 29 serum samples were included to the study. The rate of patients colonized with Candida spp. was found 70.3% (26/37). C. albicans were isolated from 43 samples of 22 patients, whereas C. tropicalis, C. glabrata, C. krusei and C. parapsilosis were isolated from one each patient's single samples. In seven (27%) of 26 colonized patients, CI was found high (0.5), and all of them were found to be colonized with C. albicans. Five of the seven patients with CI0.5 were detected as IgM + IgG positive, and one was IgG positive, while one patient's serum could not be obtained. Nineteen patients yielded low CI (0.5), of which 15 were found to be colonized with C. albicans. Twelve serum samples could be obtained from these patients, and three were found positive for IgM + IgG, six were positive for IgG alone, whereas three were negative for anti-C. albicans. Seven serum samples could be collected from 11 non-colonized patients, and only two (18.2%) have yielded IgG positivity. A statistically significant difference was detected in IgM positivity (p0.05), although there was no significance in IgG positivity (p0.05) between the patients with high and low colonization indices. In the follow-up of the patients, no candidemiae developed and this was thought to be due to the preventive measures which were taken especially in ICU patients with CI0.5. As a result, the follow-up of the ICU patients in terms of C. albicans CI and IgM would be effective for the prevention of serious Candida infections.

Due to the increase in the number of immunosuppressive patients, an increase in the frequency of Candida albicans infections is recorded during the recent years. C. albicans strains can be grouped into three genotypes (genotypes A, B and C) by 25S intron analysis according to the presence of a transposable group-1 intron. Genotype A isolates were found to be associated with increased resistance to flucytosine. The aim of this study was to determine the genotypic distribution of C. albicans isolates and investigate the relationship between the genotypes and antifungal susceptibility patterns. Seventy clinical C. albicans isolates were included in the study. The strains were identified by API ID 32C (bioMerieux, France), and antifungal susceptibilities were determined by ATB Fungus 2 (bioMerieux, France) system. Following DNA extraction from the isolates, polymerase chain reaction (PCR) was performed as indicated in the literature. The genotypes were determined according to the size of the amplified PCR product. For the statistical analysis of the relationship between the genotypes and antifungal susceptibility patterns, Pearson's khi square and Fisher's exact tests were performed. Among the 70 strains investigated, 35 (50%) were found as genotype A, nine (12.9%) were genotype B and 26 (37.1%) were genotype C. Nystatin, miconazole and ketoconazole susceptibilities were significantly different among the genotypes, genotype B being more resistant to these agents (p values were 0.032, 0.035 and 0.035, respectively). When the susceptibility of the strains were compared according to the presence of the transposable intron, no significant difference was observed. There was also no statistically significant difference between the genotype distribution of the isolates and flucytosine, amphotericin B and econazole susceptibilities (p values were 0.357, 0.602 and 0.051, respectively). As a result, in order to clarify the resistance mechanisms of different genotypes of C. albicans isolates, more sophisticated and large-scale studies should be performed.

The aim of this study was to evaluate the value of the direct fluorescent antibody (DFA) techniques reported to have high sensitivity and specificity and the enzyme linked immunosorbent assay (ELISA) test used to determine antigens in stool samples in the routine diagnosis of Giardia intestinalis. When 44 stool samples in which G. intestinalis cysts and/or trophozoites had been seen during native Lugol examination were investigated, positivity detected with the trichrome staining method, monoclonal ELISA method and monoclonal DFA method was found to be 37 (84.0%), 39 (88.6%) and 35 (79.5%) respectively. DFA detected Crytosporidium parvum cysts in addition to G. intestinalis in one sample. Twenty-seven (61.4%) of the samples were positive with all three methods. When compared with the DFA method, the ELISA method had a sensitivity of 88.6%, a specificity of 88.8%, a positive predictive value of 79.5% and a negative predictive value of 20.0% while the trichrome staining method had a sensitivity of 85.7%, a specificity of 77.8%, a positive predictive value of 81.1% and a negative predictive value of 22.2%. There was no statistically significant difference between the DFA and ELISA tests and between the DFA test and the trichrome staining method for diagnosing G. intestinalis (p0.05).

Acquisition of infectious agents during blood transfusions is an important complication. The choice of screening tests is of crucial importance since it is difficult to detect the markers of the infection in blood donors, especially before the window period when seroconversion has not taken place. In this study the blood samples of 150 volunteer donors were screened for anti-HIV 1/2, anti-HCV, and HBsAg by chemiluminescence method. Samples found to be positive for anti-HIV 1/2 and anti-HCV were confirmed by Western Blot and Inno-Lia HCV Ab III tests, respectively. HBsAg, anti-HCV and anti-HIV 1/2 antibodies were also investigated by the Simul-Tec immunoassay in all of the samples, and the results were compared. As a result, 4.7% of the donors were positive for HBsAg and confirmatory tests showed that the positivity rate was 2.7% for anti-HCV, 0.7% for anti-HIV 1/ 2. Although the results obtained with the Simul-Tec immunoassay were similar to the screening test results obtained for HBsAg, discrepant results were obtained for anti-HCV and anti-HIV 1/ 2. The results of this study indicated that Simul-Tec immunassay method is not yet ready to meet the demands in terms of anti-HCV and anti-HIV detection.

Pneumocystis carinii is one of the most common causative agents of pneumonia in immunocompromised patients, but the problems in the laboratory diagnosis of the disease frequently leads to diagnosis according to the response to medical treatment. In this study, the presence of P. carinii was investigated in immunocompromised patients who were presenting with the clinical symptoms of atypical pneumonia, by Gomori methenamine silver staining (GMS), direct fluorescent antibody (DFA) test and nested-polymerase chain reaction (nPCR) methods. Fifty-three samples of 49 patients were included in the study. Twelve of the samples (22.6%) were found to be positive by nPCR, 6 of them (11.3%) were found to be positive by DFA, while only one of them (1.8%) was positive by GMS staining method. As a result, for the appropriate treatment and prophylaxis of P. carinii infections, PCR which is a rapid and reliable diagnostic test should be used for diagnosis.

A 49-year-old woman was admitted to Hacettepe Medical Faculty Hospital with the complaints of headache, nausea, vomiting, lethargy, and weakness on her right side. She revealed a history of pulmonary Cryptococcus infection 5 years before and she had been treated with amphotericin B. After clinical and laboratory investigation she was thought to have an intracranial mass, but her deteriorating situation did not allow any surgical intervention; she died within 7 days. On necropsy, hard, gray-white nodular pulmonary lesions, ranging 0.1-4 cm in diameter, basal meningitis, infarcts, and a nodular lesion 1.5 cm in diameter similar to those of the lung were present in the white matter of the right hemisphere of the brain. Microscopic examination revealed granulomatous inflammation caused by Cladosporium, which had brown pigment and septate hyphae.