[25S intron analysis followed by restriction enzyme digestion performed for genotyping Candida albicans isolates]. / Candida albicans Suslarinin Genotiplendirmesinde 25S Intron Analizini Takiben Restriksiyon Enzim Analizi Uygulanmasi

Candida albicans is the most frequently encountered fungal pathogen especially in the immunocompromised hosts. Genotyping clinical microbial isolates is important for obtaining epidemiological data and for establishing appropriate infection control strategies in the hospital setting. 25S intron analysis is an easy and reliable method used for genotyping C.albicans strains. As it has a low discriminatory power, its use is limited in epidemiological studies. In this study, our aim was to genotype clinical C.albicans isolates by using 25S intron analysis followed by restriction enzyme digestion in order to develop a more discriminative genotyping system for C.albicans. A total of 260 clinical C.albicans strains isolated from various infection sites (121 blood, 69 sputum, 36 vaginal discharge, 26 wound, 8 urine samples) were genotyped by 25S intron analysis, and all the products obtained by polymerase chain reaction (PCR) were digested with HaeIII restriction enzyme. Discriminatory power of each method was calculated. Among the isolates 184 (70.8%) were classified as genotype A, 42 (16.2%) as genotype B, and 34 (13%) as genotype C by 25S intron analysis. Discriminatory power of the method was calculated as 0.46. HaeIII restriction of genotype A, B and C isolates produced ten, one, and five restriction patterns (genotypes), respectively. By the addition of restriction enzyme analysis, the number of genotypes obtained was increased to 16, and the discriminatory power of the method to 0.79. Combining different genotyping methods increases the discriminatory power by increasing the number of genotypes obtained. However, there is also a risk to split certain strains in different genotypes by the different methods used and this makes the genotypic evaluation more difficult. On the other hand, combining 25S intron analysis with restriction enzyme analysis increases the discriminatory power without introducing a totally different method, and makes the method more suitable for epidemiological purposes and for genotyping clinical isolates. Different enzymes instead of HaeIII should be tested to evaluate the effect on the discriminatory power. In order to evaluate the relationship between the genotypes obtained by this method and parameters such as patient characteristics, clinical data, and antifungal susceptibilities, more sophisticated studies can be performed.

Two possible cases of Trichosporon infections in bone-marrow-transplanted children: the first case of T. japonicum isolated from clinical specimens.

Trichosporon spp. are emerging as opportunistic agents that cause systemic diseases in immunocompromised hosts. Trichosporonosis carries a poor prognosis in neutropenic patients. Trichosporon japonicum was isolated from the air and named by Sugita et al. Here we present the first case of T. japonicum isolated from a clinical specimen. Two cases of acute myeloid leukemia who had Trichosporon isolates are discussed because of their rarity and growing importance. T. asahii was isolated from the throat, feces and urine of the first patient. T. japonicum was isolated from the sputum of the second patient. Both cases produced high MICs to itraconazole, and low MICs to fluconazole and voriconazole. In virulance factor investigations there was (++) biofilm formation in T. japonicum but not in T. asahii. Conventional mycological studies were not adequate for the identification of the isolate at the species level. In our second case as in the first one, the isolate was identified as T. asahii with 99.9% accuracy by API 20C AUX. Although two T. asahii isolates from the same patient yielded identical typing profiles by arbitrary primed-PCR, the isolates of the two different patients showed different arbitrary primed-PCR typing profiles. However, the genetic identification of the other patient's strain gave the result of T. japonicum.

[Comparison of different primers used for the genotyping of Candida albicans clinical isolates by randomly amplified polymorphic DNA method]. / Candida albicans klinik izolatlarinin "randomly amplified polymorphic DNA" yöntemiyle genotiplendirmesinde kullanilan farkli primerlerin karsilastirilmasi.

Candida albicans causes severe infections with high mortality rates especially in immunocompromised patients. The aim of this study was to evaluate the efficiency of randomly amplified polymorphic DNA (RAPD) method and compare the discriminatory powers (DP) of different primers used for genotyping Candida albicans isolates. A total of 109 C. albicans strains recovered from throat, sputum, blood, feces, urine, vagina and wound cultures of 65 hospitalized paediatric patients with haematologic malignancy were evaluated by RAPD method using 10 different primers (OPE-03, OPE-04, OPE-12, OPE-18, OPE-19, OPE-20, OPF-10, OPF-12, P1 and P2) between June 1999-April 2003. Strains were separated into groups by analyzing band patterns derived from each primer and the DP was calculated. Reproducibility of the method was determined by evaluating randomly chosen 20 isolates with the same and different PCR devices under the same PCR conditions. C. albicans isolates generated 1-16 bands and were grouped in 41-80 genotypes depending on the primers used. DP of the RAPD method was calculated as > or = 0.90 for each primer (range between 0.90-0.99), which were accepted as reliable values. However, the strains clustered in the same group when studied with a primer could be dispersed into different groups by another primer. The reproducibility of the method was poor and the comparison of band patterns was difficult especially in isolates which generated many bands. In conclusion, for obtaining reliable results by RAPD method, using more than one primer and comparative analysis of these primers are appropriate. RAPD is an adequate method for studying small outbreaks in which a few number of isolates are evaluated, but it is laborious and unreliable for many number of isolates recovered in a long time period because of its poor reproducibility and difficulties in evaluating the strains generating many bands.

[The use of real-time polymerase chain reaction and enzyme immunoassay for the diagnosis of acute parvovirus B19 infections in immunosuppressed patients]. / Immünsüpresif hastalarda akut parvovirus B19 enfeksiyonunun tanisinda enzim immún yóntemi ile gerçek zamanli polimeraz zincir reaksiyonunun kullanimi.

Human parvovirus B19 (PV-B19) infection may lead to very serious clinical situations such as transient aplastic crisis in patients with hemolytic anemia, thrombocytopenia, neutropenia and transient arthritis accompanied with erythema infectiosum, especially in immunosuppressed patients. Early diagnosis of PV-B19 infection is of critical importance especially in immunosuppressed patients since the necessary precautions can be undertaken accordingly. In this study, PV-B19 IgM and IgG antibodies and viral DNA have been searched by enzyme immunoassay (ELISA) and real-time polymerase chain reaction (PCR), respectively, in 50 PV-B19 suspected immunosuppressed patients. Viral IgM, IgG and DNA positivities were detected in 7 (14%), 20 (40%) and 7 (14%) of the patients, respectively. During the first week three patients were found DNA and IgM positive but IgG negative, while four patients were found positive for the viral DNA, IgM and IgGs. The DNA copy numbers were high in all of the patients during the first week, with a gradual decrease during a seven-week follow-up period. IgM antibodies have disappeared in the sixth week in three of the patients and at the end of the seventh week in four of the patients. Although the IgG antibodies were negative in three patients in the first week, they became positive in the second week and the titers gradually increased during the following weeks. According to the results of this study, it can be concluded that, in high risk groups such as immunosuppressed patients, in addition to ELISA, real-time PCR method would be helpful for the early diagnosis of PV-B19 infections.