Fundamental niche prediction of the pathogenic yeasts cryptococcus neoformans and cryptococcus gattii in europe

Fundamental niche prediction of Cryptococcus neoformans and Cryptococcus gattii in Europe is an important tool to understand where these pathogenic yeasts have a high probability to survive in the environment and therefore to identify the areas with high risk of infection. In this study, occurrence data for C. neoformans and C. gattii were compared by MaxEnt software with several bioclimatic conditions as well as with soil characteristics and land use. The results showed that C. gattii distribution can be predicted with high probability along the Mediterranean coast. The analysis of variables showed that its distribution is limited by low temperatures during the coldest season, and by heavy precipitations in the driest season. C. neoformans var. grubii is able to colonize the same areas of C. gattii but is more tolerant to cold winter temperatures and summer precipitations. In contrast, the C. neoformans var. neoformans map was completely different. The best conditions for its survival were displayed in sub-continental areas and not along the Mediterranean coasts. In conclusion, we produced for the first time detailed prediction maps of the species and varieties of the C. neoformans and C. gattii species complex in Europe andMediterranean area

Evaluation of the significance of molecular methods in the diagnosis of invasive fungal infections: Comparison with conventional methods

Özellikle riskli hasta gruplarında gerçek ya da fırsatçı patojen mantarların neden olduğu enfeksiyonların tanısında direkt mikroskobik inceleme ve kültür, halen değerini koruyan standart konvansiyonel yöntemlerdir. Bu yöntemlerin uygulama ve değerlendirmelerinde karşılaşılan bazı sorunlar nedeniyle, günümüzde daha hızlı, kolay uygulanabilir, yüksek duyarlılık ve özgüllüğe sahip yeni tanı yöntemlerine gereksinim duyulmaktadır. Bu çalışmada, klinik örneklerde etken olan mantarların tespiti ve tanımlanmasında moleküler yöntemler ile alınan sonuçların, eş zamanlı olarak uygulanan konvansiyonel tanı yöntemleri ile karşılaştırılması amaçlanmıştır. Çalışmaya, immünsüpresif 50 hastaya ait klinik örnekler [24 bronkoalveoler lavaj (BAL); 14 kan; beş periton, dört plevra ve bir perikard sıvısı; bir beyin omurilik sıvısı (BOS), bir idrar] dahil edilmiştir. Tüm örnekler sabouraud dekstroz ve beyin kalp infüzyon agar besiyerlerine ekilerek 25-30°C ve 37°C’de 30 gün boyunca inkübe edilmiş; kan dışındaki örnekler ise %10-15 KOH + kalkoflor beyazı ile boyanarak direkt mikroskopi ile incelenmiştir. İzolatların konvansiyonel olarak tanımlanmasında temel morfolojik ve biyokimyasal özellikler esas alınmıştır. Polimeraz zincir reaksiyonu (PCR) için örneklerden mantar DNA’sının saflaştırılmasında, hem fenol-kloroform-izoamil alkol (9-10 saat süreli) hem de ticari DNA ekstraksiyon kiti (6-7 saat süreli) kullanılmıştır. PCR yöntemi, mantarların ITS1, ITS2, ITS3, ITS4, 5.8S rDNA ve 28S rDNA bölgelerinden seçilen genel ve türe özgül primerler (multipleks) kullanılarak uygulanmıştır. Genel primerler ile 550 baz çifti (bp) büyüklüğünde mantarlara özgül bantlar; türe özgül primerler ile de 273 bp’de Candida albicans, 320 bp’de Candida parapsilosis, 423 bp’de Candida glabrata, 357 bp’de Candida tropicalis, 385 bp’de Aspergillus fumigatus ve 136 bp’de Cryptococcus neoformans’a özgül bantlar değerlendirilmiştir. Çalışılan 50 örneğin 17 (%34)’sinin kültüründe üreme saptanmış (12 BAL, üç kan ve bir idrar örneğinde C.albicans, bir kan örneğinde C.parapsilosis), kan dışı 36 örneğin 7 (%19.4)’si direkt mikroskopi ile pozitif bulunmuş (tümü BAL örneği) ve 27 (%54) örnek PCR ile olumlu sonuç vermiştir. Kültürlerinde üreme olan 17 örneğin tümünde PCR ile de pozitif sonuç alınmış, kültürü negatif 23 örnek ise PCR ile de negatif bulunmuştur. Direkt misroskopi ve kültürü negatif bulunan beş BAL, dört periton sıvısı ve bir BOS olmak üzere toplam 10 örnekte PCR ile mantar DNA’ları saptanmış ve multipleks PCR ile bunların sekizi C.albicans, biri C.parapsilosis (periton sıvısı örneği) ve biri de C.neoformans (BOS örneği) olarak tanımlanmıştır. PCR negatif, kültür pozitif bir sonuç elde edilmemiştir. Kültür negatif, PCR pozitif bulunan bu 10 örneğin antifungal tedavi alan hastalara ait olduğu izlenmiş, kriptokokoz şüpheli bir hastanın BOS örneğinde sadece PCR ile C.neoformans DNA’sının saptanması, bu olguda hızla uygun tedaviye başlanmasına olanak sağlamıştır. İstatistiksel değerlendirmede, kültür ile PCR arasındaki uyum önemli düzeyde (k= 0.61; p< 0.001), mikroskopi ile PCR arasındaki uyum ise minimal düzeyde (k= 0.24; p< 0.001) saptanmış; kültür referans yöntem olarak alındığında, PCR yönteminin duyarlılığı %100, özgüllüğü ise %69.7 olarak belirlenmiştir. Çalışmamızda, kültürde üretilen ve konvansiyonel yöntemlerle tanımlanan tüm mantar türlerinin, uygulanan multipleks PCR ile de aynı şekilde tanımlandığı gözlenmiştir. Sonuç olarak, konvansiyonel yöntemler göz ardı edilmeksizin, hastanın klinik durumu ve laboratuvar koşulları dikkate alınarak, invazif mantar enfeksiyonlarının tanısında duyarlılığı yüksek bulunan PCR temelli testlerin kullanılmasının uygun olacağı düşünülmüştür.Direct microscopy and culture methods are still valuable standard conventional methods for the diagnosis of infections caused by true or opportunistic fungal pathogens, especially in high risk patients. However, some of the problems concerning the application and interpretation of those methods, indicate a need for more rapid, practical and reliable tests with high sensitivity and specificity. This study was conducted to compare the results obtained by molecular methods with the results of conventional methods performed simultaneously for the detection and identification of causative fungi in clinical samples. Clinical samples [24 bronchoalveolar lavage (BAL); 14 blood; 5 peritoneal, 4 pleural and 1 pericardial fluids; 1 cerebrospinal fluid (CSF), 1 urine] from 50 immunosuppressed patients were included in the study. All of the samples were cultivated on Sabouraud dextrose and brain-heart infusion agar media and incubated at 30&deg;C and 37&deg;C for 30 days. Samples other than blood were stained with 10-15% KOH + calcofluor white and examined by direct microscopy. Conventional identification of the isolates were performed by using basic morphological and biochemical characteristics. The isolation of fungal DNAs for polymerase chain reaction (PCR) was achieved by classical phenol-chloroform-isoamylalcohol procedure (9-10 hours) and commercial DNA extraction kit (6-7 hours) and general and species-specific primers (multiplex) from ITS1, ITS2, ITS3, ITS4, 5.8S rDNA and 28S rDNA regions were choosen for amplification. In PCR results, 550 base-paired (bp) bands obtained with universal primers were evaluated as fungal DNA positivity, and 273 bp, 320 bp, 423 bp, 357 bp, 136 bp and 385 bp bands with species-specific primers were evaluated as Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Cryptococcus neoformans and Aspergillus fumigatus positivities, respectively. Seventeen (34%) of the 50 samples yielded fungal growth on culture (C.albicans in 12 BAL, 3 blood, 1 urine sample, and C.parapsilosis in 1 urine), while seven BAL out of 36 (19.4%) non-blood samples gave positive result by direct microscopy. Of the samples 27 (54%) were found positive by PCR. All of the 17 culture positive samples were also found PCR positive, and all of the 23 culture negative samples were also found PCR negative. However, fungal DNAs were detected by PCR in 10 of the samples (5 BAL, 4 peritoneal fluids, 1 CSF) which were negative by direct microscopy and culture methods. These fungi were identified as C.albicans (n= 8), C.parapsilosis (n= 1, from peritonal fluid) and C.neoformans (n= 1, from CSF) by multiplex PCR. No samples yielded PCR negative, culture positive result. All of those 10 PCR positive, culture negative samples belonged to patients who were under antifungal treatment. The detection of C.neoformans DNA from CSF sample of a patient with suspected cryptococcosis only with PCR provided the chance for rapid therapy. In statistical evaluation, the concordance between culture and PCR methods were found significantly high (k= 0.61; p&lt; 0.001), whereas it was minimal (k= 0.24; p&lt; 0.001) between direct microscopy and PCR. When considering culture as the reference method, the sensitivity and specificity of PCR were estimated as 100% and 69.7%, respectively. In addition, multiplex PCR was as successful as culture and conventional identification methods in the identification of all fungal species. As a result, without disregarding conventional methods, use of PCR might be recommended for the identification of fungal species on the basis of clinical status of the patient and conditions of the laboratory

The identification of Candida species isolated from clinical specimens of immunocompromised patients with PCR and determination of antifungal resistance genes with RFLP and sequencing analysis

Amaç: Bu çalışmanın amacı immun sistemi baskılı hastalardan elde edilen klinik örneklerden izole edilen Candida türlerinde PCR tekniğinin ve RFLP ve sekans analizi ile antifungal direnç genlerinin araştırılmasıdır. Gereç ve yöntem: Çalışmada immün sistemi baskılı 200 hastadan alınan klinik örnekler (96 bronkoalveoler lavaj, 56 biyopsi-apse, 8 kan, 15 periton diyaliz sıvısı, 15 plevra sıvısı, 5 beyin omurilik ve 5 perikard sıvısı) geleneksel ve moleküler yöntemler ile incelenmiş ve sonuçlar değerlendirilmiştir. Örneklerden mantar DNA’sı saptandıktan sonra elde edilen ürünün, multipleks PCR yardımı ile tür düzeyinde tanısı yapılmış, antifungal direnci E-test, direnç genleri ise RFLP analizi ile saptanmıştır. Bulgular: İkiyüz örneğin 30’unda (%15) kültür yöntemleri ile pozitif sonuç alınmış [20 Candida albicans (%67), beş Candida parapsilosis (%17), beş Candida tropicalis (%17)], 170 (%85) örnekte üreme olmamıştır. Genel primerler ile yapılan PCR testi sonucunda kültürleri olumlu bulunan 30 örneğin tümünün mantar DNA’sı içerdiği belirlenmiştir. İzole edilen suşların biri E-test yöntemiyle flukanozole dirençli, ikisi flukanozole doza bağlı duyarlı olarak saptanırken 27’si duyarlı olarak bulunmuştur. Seçilen uygun primerler ile BamHI ve SalI enzimleri kullanılarak RFLP uygulanmış, yapılan analizler sonucunda dirençli bir C.albicans suşunda 600 bp’lik bant görülmüştür. Candida dubliniensis (950 bp) ve Candida krusei (360 bp) ile yapılan multipleks PCR optimizasyonunda başarı sağlanmıştır. Suşların ERG genine uygun primer çiftleri ile PCR’ları yapıldıktan sonra yapılan sekans analizi sonucunda dirençli C.albicans suşu referans gen ile karşılaştırıldığında D132E, E216D mutasyonları belirlenmiştir. Sonuç: Moleküler test yöntemleri özellikle immün sistemi baskılanmış hasta populasyonunun kısa sürede doğru tedavisine olanak sağladıkları için yaşamsal açıdan önemlidir.Objectives: The aim of this study is to investigate PCR technique and antifungal resistance genes with RFLP and sequencing analysis in Candida species isolated from clinical specimens of immune-compromised patients. Materials and methods: Clinical samples (96 bronchoalveolar lavages, 56 biopsy-abscess, 8 blood specimens, 15 peritoneal fluid specimens, 15 pleural fluid, 5 cerebrospinal fluid and 5 pericard fluid specimens) from 200 immunosuppressed patients were studied by conventional and molecular methods. Antifungal susceptibility testing was performed by the E-test method. Firstly, fungal DNA was isolated from specimens, and then the resultant products are defined with multiplex PCR. Antifungal resistance and resistance genes were established by E-test and RFLP analysis. Results: Thirty of 200 samples (15%) were culture positive [20 Candida albicans (67%), five Candida parapsilosis (17%), five Candida tropicalis (17%)], and 170 of samples were found culture negative (85%). PCR with the universal primers detected fungal DNA in all 30 culture positive samples. One strain was determined as resistant; 2 strains were dose dependent susceptible and 27 strains were sensitive to fluconazole by E-test. The resistance gene (ERG11) was detected by BamHI and SalI enzymes revealed fluconazole resistance in one of C.albicans strains. The identification was successful in Candida dubliniensis (950 bp) and Candida krusei (360 bp) with multiplex PCR. D132E and E216D mutations were detected in sequencing of ERG 11 gene of this isolate and compared with reference gene in GenBank by clustal analysis. Conclusion: The molecular test methods supplies correct therapy rather early in immunosuppressive patients therefore it is important for the survival

Evaluation of the Significance of Molecular Methods in the Diagnosis of Invasive Fungal Infections: Comparison with Conventional Methods

Direct microscopy and culture methods are still valuable standard conventional methods for the diagnosis of infections caused by true or opportunistic fungal pathogens, especially in high risk patients. However, some of the problems concerning the application and interpretation of those methods, indicate a need for more rapid, practical and reliable tests with high sensitivity and specificity. This study was conducted to compare the results obtained by molecular methods with the results of conventional methods performed simultaneously for the detection and identification of causative fungi in clinical samples. Clinical samples [24 bronchoalveolar lavage (BAL); 14 blood; 5 peritoneal, 4 pleural and 1 pericardial fluids; 1 cerebrospinal fluid (CSF), 1 urine] from 50 immunosuppressed patients were included in the study. All of the samples were cultivated on Sabouraud dextrose and brain-heart infusion agar media and incubated at 30 C and 37 C for 30 days. Samples other than blood were stained with 10-15% KOH + calcofluor white and examined by direct microscopy. Conventional identification of the isolates were performed by using basic morphological and biochemical characteristics. The isolation of fungal DNAs for polymerase chain reaction (PCR) was achieved by classical phenol-chloroform-isoamylalcohol procedure (9-10 hours) and commercial DNA extraction kit (6-7 hours) and general and species-specific primers (multiplex) from ITS1, ITS2, ITS3, ITS4, 5.85 rDNA and 28S rDNA regions were choosen for amplification. In PCR results, 550 base-paired (bp) bands obtained with universal primers were evaluated as fungal DNA positivity, and 273 bp, 320 bp, 423 bp, 357 bp, 136 bp and 385 bp bands with species-specific primers were evaluated as Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Cryptococcus neoformans and Aspergillus fumigatus positivities, respectively. Seventeen (34%) of the 50 samples yielded fungal growth on culture (C.albicans in 12 SAL, 3 blood, 1 urine sample, and C.parapsilosis in 1 urine), while seven SAL out of 36(19.4%) non-blood samples gave positive result by direct microscopy. Of the samples 27 (54%) were found positive by PCR. All of the 17 culture positive samples were also found PCR positive, and all of the 23 culture negative samples were also found PCR negative. However, fungal DNAs were detected by PCR in 10 of the samples (5 BAL, 4 peritoneal fluids, 1 CSF) which were negative by direct microscopy and culture methods. These fungi were identified as C.albicans (n = 8), C.parapsilosis (n = 1, from peritonal fluid) and C.neoformans (n = 1, from CSF) by multiplex PCR. No samples yielded PCR negative, culture positive result. All of those 10 PCR positive, culture negative samples belonged to patients who were under antifungal treatment. The detection of C.neoformans DNA from CSF sample of a patient with suspected cryptococcosis only with PCR provided the chance for rapid therapy. In statistical evaluation, the concordance between culture and PCR methods were found significantly high (k = 0.61; p < 0.001), whereas it was minimal (k = 0.24; p < 0.001) between direct microscopy and PCR. When considering culture as the reference method, the sensitivity and specificity of PCR were estimated as 100% and 69.7%, respectively. In addition, multiplex PCR was as successful as culture and conventional identification methods in the identification of all fungal species

Identifying the knowledge of hygiene and sanitation levels of kitchen personnel working in the Turkish Republic of Northern Cyprus hospitals

European Biotechnology Congress -- MAY 25-27, 2017 -- Dubrovnik, CROATIAWOS: 000413585400282

The place of molecular methods in the identification of dermatophytes and the determination of their feasibility

Amaç: Dermatofitler geleneksel izolasyon besiyerlerinde fırsatçı mantarlar gibi birkaç günde izole edilemezler. Uygun ortamda üreme süreleri yaklaşık olarak iki haftayı kapsar ve identifikasyonunda tipik makroskopik, mikroskopik özellikler ve biyokimyasal testler gibi geleneksel yöntemlerden yararlanılır. Ancak fenotipik özellikler ile her zaman başarılı sonuçların alınamayışı, bu nedenle tanı ve tedavide oluşabilecek gecikme ve sorunlar, nükleik asit amplifikasyon temeline dayalı yöntemlerden yararlanmayı gerekli kılmıştır. Bu çalışmada geleneksel yöntemler ile identifikasyonu yapılan 56 dermatofit suşunun moleküler yöntemlerle de identifiye edilerek her iki yöntemin birbirleriyle uyum derecelerinin araştırılması ve moleküler yöntemlerin rutin laboratuvarlarda kullanılabilirliklerinin belirlenmesi amaçlanmıştır. Gereç ve Yöntemler: Dermatofitoz ön tanılı 270 hastanın çeşitli klinik örnekleri (saç+saçlı deri, deri ve tırnak kazıntısı) öncelikle geleneksel yöntemlerle incelenmiş; Sabouraud dekstroz agar, mısır unlu agar ve patates dekstroz agar besiyerleri izolasyon amacı ile kullanılmıştır. Gerektiğinde üreyi hidrolize etme, Trichophyton agar besiyerlerindeki çeşitli vitaminleri kullanabilme özellikleri araştırılmıştır. Moleküler tanı amacı ile polimeraz zincir reaksiyonu (PCR) ve sekans analizi yapılmıştır. Bulgular: Geleneksel yöntemler ile suşların 37 (%66,1)’sinin Trichophyton (T) rubrum, dördünün (%7,1) T. mentagrophytes, dördünün (%7,1) T. tonsurans, birinin (%1,8) T. violaceum, sekizinin (%14,3) Trichophyton cinsinden, birinin (%1,8) Microsporum(M) canis, birinin (%1,8) Microsporum cinsinden olduğu saptanmıştır.Moleküler (T1 PCR, 25 GA PCR, ITS PCR-RFLP ve sekans analizi) identifikasyon sonuçlarına göre ise 41 suş (%73,2) T. rubrum, 10 suş (%17,8) T. interdigitale, bir suş (%1,8) T. violaceum, iki suş (%3,6) M. canis, bir suş (%1,8) Peacilomyces lilacinus, bir suş (%1,8) Aspergillus fumigatus olarak belirlenmiştir. Sonuç: Çalışmanın sonuçları identifikasyonlarında zorluk çekilen dermatofitlerin cins ve tür düzeyinde tanımlanmasında moleküler yöntemler ile hızlı ve güvenilir sonuçlar alındığını göstermiştir.Background and Design: Unlike opportunistic fungi, dermatophytes cannot be isolated on the conventional culture media in a few days. Their growing periods cover approximately two weeks in a suitable media and identification are made with conventional methods as typical macroscopic and microscopic appearance. However, successful results are not always obtained with the phenotypic features, and thus, diagnostic problems and delay in diagnosis and treatment may arise. For this reason, the methods based on nucleic acid amplification have been necessary. In this study, we aimed to identify 56 dermatophytes strains, which were identified by conventional methods, by molecular methods and to investigate the correlation between the two methods and to determine the usability of molecular methods in routine laboratories. Materials and Methods: Several clinical samples of 270 patients with suspected dermatophytoses (hair+scalp, skin and nail scrapings) were examined by conventional methods; Sabouraud dextrose agar, corn meal agar and potato dextrose agar were used for isolation. In case of necessity to hydrolyze urea, to be used different vitamins in Trichophyton agar media were investigated. Polymerase chain reaction (PCR) and sequence analyses were done for the molecular diagnosis. Results: Using conventional methods, 37 strains (66,1%) were identified as Trichophyton(T) rubrum, four (7.1%) - T.mentagrophytes, four (7.1%) - T.tonsurans, one (1.8%) - T.violaceum, eight (14.3%) - Trichophyton spp., one (1.8%) - Microsporum(M) canis, and one (1.8%) - Microsporum spp. According to the molecular and sequence analyses results (T1PCR, 25GAPCR, ITSPCR-RFLP and sequence analyses), 41 (73.8%) strains were identified as T.rubrum, 10 (17.8%) - T.interdigitale, one (1.8%) - T. violaceum, two (3.6%) - M. canis, one (1.8%) - Peacilomyces lilacinus, and one (1,8%) - Aspergillus fumigatus. Discussion: This study suggests that, molecular methods offer fast and reliable results in identification of dermatophytes