ONİKOMİKOZLU OLGULARIN DİREKT KAZINTI ÖRNEKLERİNDEN DERMATOFİTLERE AİT DNA İZOLASYONU VE TRICHOPHYTON RUBRUM TANIMLANMASI

Bu çalısmada, onikomikozlu hastalardan alınan tırnak örneklerinde en sık görülen etken olan Trichophyton rubrum tanısı için gerçek zamanlı polimeraz zincir reaksiyonu (PZR)’nun katkısı degerlendirilmistir. Ayrıca dermatofitozların rutin tanısında kullanılan direk mikroskobi, kültür ile PZR sonuçları karsılastırılmıstır. Bu amaçla saglıklı kontrol grubu, direk mikroskobisi negatif onikomikoz süpheli ve direk mikroskobisi pozitif onikomikoz ön tanılı örnekler çalısılmıstır. Saglıklı kontrol grubunda tüm testler negatif bulunmustur. Direk mikroskobisi pozitif 72 hastanın 20’sinde (%27.8) kültürde T. rubrum üremistir. Bu grupta 67 hastada (%93) ise PZR pozitif bulunmustur. Direk mikroskobisi negatif 18 hastanın hiçbirinin kültüründe üreme olmamıstır, sekiz hastada ise (%44.4) PZR pozitif bulunmustur. Direk mikroskobik inceleme standart yöntem olarak alındıgında PZR’nin duyarlılıgı % 93, özgüllügü %56, pozitif prediktif degeri %89, negatif prediktif degeri % 67 olarak bulunmustur. Gerçek zamanlı PZR ile yapılan incelemenin tanı açısından çok daha etkin ve hızlı oldugu görülmüstür. Fiyat karsılastırması yapıldıgında da PZR pahalı gibi görünse de alt yapısı hazır merkezlerde kültürle arasında maliyet açısından anlamlı bir fark olmadıgı saptanmıstır. Sonuç olarak dermatofitlerin tanısında moleküler bir yöntem kullanılarak tırnak kazıntısı gibi zor bir klinik örnekten DNA elde edilebilmis, rutin uygulamada kullanılabilecek bir protokol olusturulmus, hızlı ve güvenilir tür tanımı yapılabilmistir.In this study the value of real time polymerase chain reaction (PCR), for the identification of T.rubrum from nail samples, taken from the patients with onychomycosis, was evaluated. Direct microscopy, culture and PCR results were compared, for the routine identification of dermatophytosis. Healty control group, was compared with two onychomycosis prediagnosed group, which were direct microscopy positive or negative. All diagnostic tests were found to be negative in the healty control group. A total of 20 T. rubrum colonies (%27.8) were isolated from 72 patients who were direct microscopy positive. In this microscopy positive group, 67 patients (%93) were found to be positive for PCR assay. No fungal growth was seen in the samples of 18 patients who were direct microscopy negative, where as 8 patients were found to be positive for PCR ( %44.4). In the case of direct microscopy was evaluated as a standart method for the diagnosis of dermatomycosis, sensitivity, spesifity, positive predictive value and negative predictive value of PCR assay were calculated as %93, %56, %89 and %67, respectively. Investigation using PCR was evaluated as a rapid and effective method. Economically, PCR was found to be more expensive than culture assay. But for the centres which were ready technologically for the molecüler methods, no important difference was seen. As a conclusion, DNA isolated from a difficult sample, as nail scraping. Routine protocole was organized and rapid and reliable species identification was performed

DNA Extraction and Identification of Trichophyton rubrum by Real-Time Polymerase Chain Reaction from Direct Nail Scraping Specimens of Patients with Onycomycosis

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 (Light Cycler-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

Interdigital foot infections: Corynebacterium minutissimum and agents of superficial mycoses

Interdigital foot infections are mostly caused initially by dermatophytes, yeasts and less frequently by bacteria. Erythrasma caused by Corynebacterium minutissimum can be confused with superficial mycoses. The aim of the study was to determine the prevalence of the etiologic agents of superficial mycoses and the frequency of Corynebacterium minutissimum in interdigital foot infections. All the samples obtained from the 121 patients with interdigital foot infections were examined directly with the use of 20% potassium hydroxide mounts and Gram stain under the microscope and cultured on Sabouraud's dextrose agar plates. In identification of superficial mycoses, the rate was found to be 14% with the cultural method and 14% with direct microscopic examination. Using a combination of direct microscopic examination and culture, a 33.8% ratio was achieved. In the culture of these samples, the most isolated factor was Trichophyton rubrum (33.7%). In 24 of the patients (19.8%) Corynebacterium minutissimum was detected by Gram staining, in 6 of these patients Trichophyton rubrum was found, Trichophyton mentagrophytes was found in 2 and Trichosporon spp. was found in 1. The examination of interdigital foot lesions in the laboratory, the coexistence of erythrasma with dermatophytes and yeast should be considered

Moxifloxacin-associated neutropenia

A 32-y-old woman presented with pneumonia. Treatment was started with moxifloxacin. On day 2 of moxifloxacin treatment the patient developed neutropenia. After discontinuing the moxifloxacin, neutrophil counts were normal on day 4. Clinicians should be aware of the possibility of this adverse effect in patients treated with moxifloxacin.A 32-y-old woman presented with pneumonia. Treatment was started with moxifloxacin. On day 2 of moxifloxacin treatment the patient developed neutropenia. After discontinuing the moxifloxacin, neutrophil counts were normal on day 4. Clinicians should be aware of the possibility of this adverse effect in patients treated with moxifloxacin.</p