Diagnostic Utility of Cytomegalovirus (CMV) DNA Quantitation in Ulcerative Colitis.

Cytomegalovirus (CMV) colitis is a critical condition associated with severe complications in ulcerative colitis (UC). This study aimed to investigate the diagnostic value of the presence of CMV DNA in intestinal mucosa tissue and blood samples in patients with active UC. This study included 81 patients with exacerbated symptoms of UC. Patient data were obtained from the Hospital Information Management System. CMV DNA in colorectal tissue and plasma samples were analyzed using a real-time quantitative PCR assay. CMV markers were detected using immunohistochemistry and hematoxylin-eosin staining. Immunohistochemistry positivity was observed in tissue samples from eight (9.9%) patients. Only one (1.2%) patient showed CMV-specific intranuclear inclusion bodies. CMV DNA was detected in 63.0% of the tissues (median: 113 copies/mg) and in 58.5% of the plasma samples (median: 102 copies/mL). For tissues, sensitivity and the negative predictive value (NPV) for qPCR were excellent (100.0%), whereas specificity and the positive predictive value (PPV) were low (41.9% and 15.7%, respectively). For plasma, sensitivity and NPV were high (100.0%) for qPCR, whereas specificity and PPV were low (48.6% and 24.0%, respectively). CMV DNA ≥392 copies/mg in tissue samples (sensitivity 100.0% and specificity 83.6%) and ≥578 copies/mL (895 IU/mL) in plasma samples (sensitivity 66.7% and specificity 100.0%) provided an optimal diagnosis for this test. The qPCR method improved patient management through the early detection of CMV colitis in patients with UC. However, reliance on qPCR positivity alone can lead to overdiagnosis. Quantification of CMV DNA can improve diagnostic specificity, although standardization is warranted.

Comparison of the i-test COVID-19 Rapid Antigen Test with Real-Time Reverse Transcriptase PCR.

Objective: Improvements in SARS-CoV-2 diagnosis with easy, rapid and cost-effective approaches are required to control the COVID-19 pandemic. Antigen tests result in 5 to 30 minutes, providing an advantage over polymerase chain reaction (PCR) in duration. We tested the performance of the i-test COVID-19 rapid antigen test to real-time reverse transcriptase PCR in 200 symptomatic COVID-19 suspected patients. The cycle threshold (Ct) values of the patients were found to be between 21.6 and 34.4. The Ct value of 10 patients who tested positive in the PCR test was >30. We found that the sensitivity and specificity of the antigen test were 80.6 % and 93.7 %, respectively, for samples with a Ct value of <30, and overall agreement between antigen and PCR test was 91.6 % for these samples. i-test COVID-19 rapid antigen test can be used for screening in schools, factories, nursing homes, and everywhere where PCR test is unavailable.

Assessment of α9ß1 integrin as a new diagnostic and therapeutic target in Behcet's disease.

This study aimed to investigate the roles of α9ß1 integrin and its ligands in Behçet's disease (BD) by examining serum levels and gene expressions. 15 healthy controls and 30 BD patients (14 active and 16 inactive) were included in the study. Serum levels of ITGA9, ITGB1, TNC, OPN, VCAM-1, VEGF, TSP1, TGM2, Emilin-1, and vWF, were measured by ELISA. Gene expressions of α9ß1 (ITGA9 and ITGB1) and its ligands (TNC and SPP1) were evaluated by RT-PCR. Laboratory findings (CRP, ESR, HGB, WBC, RBC, neutrophil, lymphocyte, PLT, RDW, MPV, PCT, and HLA-B51) were obtained from the electronic database. Active BD patients had higher serum levels of α9ß1 integrin and its ligands than inactive patients and healthy controls. No significant difference was observed between healthy controls and inactive patients. Gene expressions of ITGB1 and SPP1 were increased in both patient groups compared to healthy controls. ITGA9 and TNC gene expression levels were lower in the active group than in the inactive group. No noticeable differences were found in ITGB1 and SPP1 gene expressions between the patient groups. BD patients exhibited elevated CRP, ESR, WBC, neutrophil, PLT, and PCT levels, while HGB, RBC, and RDW values were lower than healthy controls. Active patients had higher CRP, ESR, WBC, neutrophil, and PLT levels. Significant positive correlations were found between CRP, ESR, WBC, neutrophil, PLT, PCT and serum levels of α9ß1 integrin and its ligands. Increased release of α9ß1 integrin and its ligands is associated with BD, suggesting their potential as markers for disease severity.

Frequency of azole resistance in clinical and environmental strains of Aspergillus fumigatus in Turkey: a multicentre study.

OBJECTIVES: Aspergillus fumigatus causes several diseases in humans and azole resistance in A. fumigatus strains is an important issue. The aim of this multicentre epidemiological study was to investigate the prevalence of azole resistance in clinical and environmental A. fumigatus isolates in Turkey. METHODS: Twenty-one centres participated in this study from 1 May 2018 to 1 October 2019. One participant from each centre was asked to collect environmental and clinical A. fumigatus isolates. Azole resistance was screened for using EUCAST agar screening methodology (EUCAST E.DEF 10.1) and was confirmed by the EUCAST E.DEF 9.3 reference microdilution method. Isolates with a phenotypic resistance pattern were sequenced for the cyp51A gene and microsatellite genotyping was used to determine the genetic relationships between the resistant strains. RESULTS: In total, resistance was found in 1.3% of the strains that were isolated from environmental samples and 3.3% of the strains that were isolated from clinical samples. Mutations in the cyp51A gene were detected in 9 (47.4%) of the 19 azole-resistant isolates, all of which were found to be TR34/L98H mutations. Microsatellite genotyping clearly differentiated the strains with the TR34/L98H mutation in the cyp51A gene from the strains with no mutation in this gene. CONCLUSIONS: The rate of observed azole resistance of A. fumigatus isolates was low in this study, but the fact that more than half of the examined strains had the wild-type cyp51A gene supports the idea that other mechanisms of resistance are gradually increasing.

Association of SARS-CoV-2 cycle threshold (Ct) values with clinical course and serum biomarkers in COVID-19 patients.

INTRODUCTION: Our knowledge has gaps regarding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication levels and its association to severity of Coronavirus disease 2019 (COVID-19). The aim of this study was to investigate the association of SARS-CoV-2 viral load with disease severity and serum biomarkers in COVID-19 patients. METHODOLOGY: Viral load was determined via cycle threshold (Ct) values of SARS-CoV-2 real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in 214 adult patients. Ct values were compared with clinical severity, biochemical and hematological biomarkers. RESULTS: Clinical course of the disease was mild (49.1%), moderate (40.2%), and severe (10.7%). Median Ct value was 28.2 (IQR: 22.2-33.8) during the first week of the disease. Ct values were lower within five days after symptom onset [lowest Ct value on the third day (median: 24, IQR: 20.6-32.3)], but they increased significantly during the second and third weeks. No association was detected between admission Ct values and disease severity. Gender, age, co-morbidity, and mortality did not differ significantly in patients with low (≤ 25) and high (> 25) Ct values. White blood cell, neutrophil, platelet, and especially lymphocyte counts, were significantly lower in patients with low Ct values. CONCLUSIONS: No definitive/clear correlation between SARS-CoV-2 viral load and severity and mortality was found in the studied COVID-19 patients. However, neutrophil, platelet, and especially lymphocyte count were significantly lower in patients with a high viral load.

Changing epidemiology of influenza and other respiratory viruses in the first year of COVID-19 pandemic.

INTRODUCTION: We aimed to determine the epidemiological change in influenza and other respiratory tract viruses isolated from patients with nasopharyngeal swab samples in our hospital during the COVID-19 period. METHODS: We investigated nasopharyngeal swabs for respiratory viruses between March 2020 and February 2021 during the first year of pandemic in Turkey. We used QIAStat Dx Respiratory panel (Qiagen, Germany) in QIAStat Dx (Qiagen, Germany) for detection of respiratory viruses between March 2020 and February 2021. Respiratory panel kit included influenza A, B, influenza A H1N1, rhinovirus/enterovirus, parainfluenza (PIV) 1,2,3,4, coronaviruses (CoVs) NL 63, 229E, OC43 and HKU1, human metapneumovirus (MPV) A/B, bocavirus, respiratory syncytial virus (RSV) A/B and adenovirus. RESULTS: We retrospectively analyzed the results of 319 nasopharyngeal swab samples. The average age of 199 (62.4%) male and 120 (37.6%) female patients between the ages of 0-92 was 16 years. We found that 101 (31.7%) samples were positive for viruses. Rhino/enteroviruses were the most common viruses in all age groups. Influenza positivity rate during the first year of pandemic declined to 2.3% from 17.3% among the previous year. MPV infection activity did not change during the pandemic. DISCUSSION: According to our findings we argue that epidemiology of respiratory viruses has changed during the pandemic period. Despite the current clinical focus on the COVID-19 pandemic, clinicians should keep in mind that rhino/enterovirus and MPV infections may mimic COVID-19 and respiratory infections should be differentially diagnosed with rapid multiplex kits containing SARS-CoV-2, rhino/enterovirus and MPV.

[Comparison of Clinical Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution methods for determining the susceptibilities of Candida isolates]. / Candida izolatlarinin antifungal duyarliliginin belirlenmesinde Klinik Laboratuvar Standartlari Enstitüsü (CLSI) ve Avrupa Antimikrobiyal Duyarlilik Testleri Komitesi (EUCAST) sivi mikrodilüsyon yöntemlerinin karsilastirilmasi.

Candida species are among the top 10 pathogens causing bloodstream infections associated with high morbidity, mortality. In spite of the development of new antifungal drugs, epidemiological studies have shown that resistance to antifungal drugs among Candida isolates is becoming a serious problem. The aim of this study was to compare the antifungal broth microdilution methods of the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) for amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole and anidulafungin susceptibility of Candida blood isolates. The study consisted of 74 Candida albicans, 67 Candida parapsilosis, 30 Candida glabrata, and 18 Candida tropicalis isolates. The minimum inhibitory concentrations were determined after 24 and 48 hour of incubation with CLSI method and only 24 hour of incubation with EUCAST method except anidulofungin. The MIC values obtained by both methods were considered to be compatible within ± 2 dilution limits. The categorical agreement between methods for each antifungal agent was assessed using clinical break points and epidemiological cut-off values. The agreement (± 2 dilution) between the methods was found to be species, drug, and incubation time dependent. After 24 hour incubation, good agreement category (> 90%) was detected between amphotericin B, itraconazole, posaconazole and anidulofungin, but was lower category (< 85%) was determined with fluconazole and voriconazole especially for relatively slow growing C.glabrata and C.parapsilosis isolates. Excellent categorical agreement (100%) was observed for amfoterisin B/C.parapsilosis, C.glabrata, C.tropicalis and anidulofungin/C.albicans, C.glabrata, C.tropicalis but least category was determined for posaconazole and C.albicans (71.6% at 24 hour; 73% at 48 hour). In vitro resistance of therapeutically used fluconazole and anidulafungin determined by both methods was rare among C.albicans (1.3%, 2.7% respectively), C.glabrata (0%, 3.3% respectively) and C.tropicalis (0%, 5.6% respectively) isolates but, an increase of non-susceptible isolates were observed among C.parapsilosis (11.9% at 24 hour of incubation; 17.9% at 48 hour of incubation) for fluconazole. There was also a cross resistance between fluconazole and voriconazole for three C.parapsilosis isolates and one multidrug resistant (fluconazole, itraconazole, posaconazole and anidulofungin) C.albicans isolate (fluconazole, itraconazole, posaconazole and anidulofungin). As a result in this study, it was determined thatboth methods were similar and can be used according to preference of laboratories. The CLSI antifungal susceptibility test results can be assessed at the end of 24 hour incubation, but sometimes it is important that the evaluation should be performed as a result of 48 hour incubation in slow growing species such as C.glabrata.

Comparison of galactomannan, beta-D-glucan, and Aspergillus DNA in sera of high-risk adult patients with hematological malignancies for the diagnosis of invasive aspergillosis.

BACKGROUND/AIM: Invasive aspergillosis (IA) is a fatal infection that is difficult to diagnose in immunocompromised patients. In this study, Aspergillus-specific DNA was searched using real-time PCR (RT-PCR) in serum samples. Galactomannan (GM) and/or beta-D-glucan (BDG) tests were previously performed on these samples for 70 neutropenic patients with hematological malignancy. MATERIALS AND METHODS: The patients were categorized according to the criteria of the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG). Among the patient serum samples, the first positive GM or BDG test sample and the median sample of GM or BDG test for negative patients were used to detect DNA levels by RT-PCR method (Light Cycler 480, Roche Molecular Biochemicals, Meylan, France) using a commercial kit (Way2Gene Fungi; Genmar, Izmir, Turkey). RESULTS: When the proven and probable IA group were considered as real patients, sensitivity of Aspergillus-specific DNA test was 90%, specificity was 73.3%, positive predictive value was 81.8%, and negative predictive value was 84.6%. CONCLUSION: This study found that searching for specific DNA by RT-PCR method has a sensitivity as high as the GM test. Although specificity was rather low, it was concluded that it can be used jointly with GM and BDG tests after decreasing contamination by severe laboratory applications.

First determination of azole resistance in Aspergillus fumigatus strains carrying the TR34/L98H mutations in Turkey.

Aspergillus fumigatus is the most important etiological agent of invasive aspergillosis. Recently, an increasing number of azole-resistant A. fumigatus isolates have been described in various countries. The prevalence of azole resistance was investigated in this study using our culture collection of A. fumigatus isolates collected between 1999 and 2012 from clinical specimens. Seven hundred and forty-six A. fumigatus isolates, collected from 419 patients, were investigated. First, all isolates were screened for resistance to itraconazole by subculturing on Sabouraud dextrose agar that contained 4 mg/L itraconazole. For isolates that grew on the itraconazole containing agar, the in vitro activities of amphotericin B, itraconazole, voriconazole and posaconazole were determined using the Clinical and Laboratory Standards Institute (CLSI) M38-A reference method. After PCR amplification, the full sequence of the cyp51A gene and its promoter region was determined for all in vitro azole-resistant isolates. Itraconazole resistance was found in 10.2% of the A. fumigatus isolates. From 2000 onwards, patients were observed annually with an itraconazole-resistant isolate. According to in vitro susceptibility tests, amphotericin B exhibited good activity against all isolates whereas the azoles were resistant. Sequence analysis of the promoter region and CYP51A gene indicated the presence of TR34/L98H in 86.8% (n = 66) of isolates. This initial analysis of the resistance mechanism of A. fumigatus from Turkey revealed a common TR34/L98H mutation in the cyp51A gene.

[Development of a real-time polymerase chain reaction method for the identification of Candida species]. / Candida türlerini tanimlayan bir gerçek zamanli polimeraz zincir reaksiyonu yönteminin gelistirilmesi.

Candida species are one of the major causes of nosocomial infections and are the fourth most common agent involved in bloodstream infections. The impact of non-albicans Candida species is increasing, however C.albicans is still the most common species. Since the antifungal susceptibility pattern among Candida spp. may be different, rapid diagnosis and identification of non-albicans Candida spp. are important for the determination of antifungal agents that will be used for treatment. The aim of the study was to describe a real-time polymerase chain reaction (Rt-PCR) assay that rapidly detects, identifies and quantitates Candida species from blood culture samples. A total of 50 consecutive positive blood culture bottles (BACTEC, Beckton Dickinson, USA) identified at our laboratory between June-November 2013, were included in the study. Reference strains of Candida spp. (C.albicans ATCC 10231, C.glabrata ATCC 90030, C.tropicalis ATCC 1021, C.krusei ATCC 6258, C.parapsilosis ATCC 22019 and C. dubliniensis CD36) grown on Sabouraud dextrose agar were used for quality control. BACTEC bottles that were positive for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were also studied to search the cross-reactivity. A commercial kit (Zymo Research, USA) was used for DNA extraction. Real-time PCR was performed on LightCycler 480 (Roche, Germany) with primers and probes specific for 18S rRNA of Candida species. Twenty microlitres of the reaction mix contained 2 µl of extracted DNA, 2 µl of LightCycler Fast Start DNA Master Probe (Roche Diagnostics, Germany), 2 µl of MgCl(2) (5 mmol), 2 µl of 10x PCR buffer (Roche Diagnostics, Germany), 0.5 µl of each primer (0.01 nmol/µl) and 1 µl of each probe (0.1 µmol/µl) (TibMolBiol, Germany). Amplification was performed using the following conditions; 95°C for 10 mins and 50 cycles of denaturation at 95°C for 10 secs, annealing at 62°C for 10 secs and polymerisation at 72°C for 20 secs. A melting curve was created by cooling the producs at 50°C for 30 secs and then heating to 80°C at a rate of 0.1°C/sec measuring of the fluorescence simultaneously. For the quantitation of fungal DNA according to the standard curve, serial dilutions of C.albicans ATCC 10231 DNA from 3 x 10(5) to 3 x 10(2) ng/µl were used. All of the strains were also identified by conventional methods and sequence analysis in order to compare the results obtained by Rt-PCR. In our study, all patient and standard samples could be amplified, identified and quantitated by this developed Rt-PCR method. A total of 50 strains, of them 26 were C.parapsilosis, 15 were C.glabrata, 6 were C.albicans, and 3 were C.tropicalis have been detected and identified among patient samples. The results were completely concordant with the sequencing and conventional methods, so the sensitivity and specificity of this method were estimated as 100 percent. In conclusion, it was novel Rt-PCR developed and evaluated in this study is considered as a rapid, accurate, reproducible, sensitive and specific method for the detection, identification and quantitation of commonly observed Candida spp. strains.

Investigation of methicillin resistant Staphylococcus aureus in neonatal intensive care unit.

Methicillin resistant Staphylococcus aureus (MRSA) strains lead to severe infections in immunosupressive patients, geriatric population and premature infants. 27 MRSA strains isolated in the Neonatal Intensive Care Unit was considered as an outbreak and it was aimed to investigate the genetic and epidemiologic relation of the MRSA outbreak. MecA gene was investigated in the S. aureus strains and pulsed field gel electrophoresis (PFGE) was used to investigate the genetic relation between outbreak strains. MecA gene was showed in all isolates. PFGE revealed that there were two different strains and most of the isolates (25/27) were owing to same clone. One of the samples were found closely related with the common strain and the other sample was found genetically unrelated. To terminate the outbreak; liquid baby food was gained to the baby food kitchen, no more new patient was imported to the neonatal unit and none of the patients were exported from neonatal unit to other clinics during outbreak, education about infection control precautions was given to all the staff and nursing bottle dishwasher was obtained. To manage and terminate the outbreak, besides the infection control precautions, tests to determine the genetic relation between outbreak strains which are done in the microbiology laboratory are needed. Molecular analysis of outbreak strains will contribute to prove the epidemiologic and evolution of outbreaks.

[Comparison of Phoenix™ Yeast ID Panel and API® ID 32C commercial systems for the identification of Candida species isolated from clinical samples]. / Klinik örneklerden izole edilen Candida türlerinin tanimlanmasinda Phoenix™ Yeast ID Panel ile API(®) ID 32C ticari sistemlerinin karsilastirilmasi

Opportunistic fungal pathogens are one of the important causes of nosocomial infections, and several different types of yeasts, especially Candida species are increasingly recovered from immunocompromised patients. Since many of the yeasts are resistant to the commonly used antifungal agents, the introduction of appropriate therapy depends on rapid and accurate identification. The aims of this study were to compare the commercial identification systems namely API® ID 32C (bioMerieux, France) and Phoenix™ Yeast ID Panel (Becton Dickinson Diagnostics, USA) for the identification of Candida species and to evaluate the effect of morphological findings in the identification process. A total of 211 yeast strains isolated from different clinical samples (111 urine, 34 blood/vascular catheter, 27 upper/lower respiratory tract, 16 abscess/pus, 13 throat/vagina swabs and 10 sterile body fluids) of 137 patients hospitalized in Uludag University Health and Research Center between October 2013 to January 2014, were included in the study. Samples were cultured on blood agar, chromogenic agar (CHROMagar Candida, BD, USA) and Saboraud's dextrose agar (SDA), and isolated yeast colonies were evaluated with germ tube test and morphological examination by microscopy on cornmeal/Tween-80 agar. The isolates were identified as well by two commercial systems according to the manufacturers' recommendations. Discrepant results between the systems were tried to be resolved by using morphological characteristics of the yeasts. Of the isolates 159 were identified identical by both of the systems, and the concordance between those systems were estimated as 75.4%. According to the concordant identification, the most frequently isolated species was C.albicans (44.1%) followed by C.tropicalis (9.9%), C.glabrata (9.5%), C.parapsilosis (8.5%) and C.kefyr (8.1%). The concordance rate was 81.7% in identification of frequently isolated species (C.albicans, C.tropicalis, C.parapsilosis, C.glabrata, C.kefyr), however it was 38.7% for the rarely isolated ones (C.krusei, C.lusitaniae, C.inconspicua/C.norvagensis, C.catenulata), representing statistical significance (p= 0.034; x2 test). Although not significant (p= 0.31; x2 test), the rate of concordance was increased (88.1%), when adding the morphological findings to the identification process. Of 211 isolates 37 (17.5%), 50 (23.7%) and 124 (58.8%) were identified according to their growth characteristics on chromogenic agar, blood agar and SDA, respectively, indicating no statistically significant difference between the media (p> 0.05). Although genotypic identification is essential, phenotypic methods are more commonly used in routine laboratories for the identification of yeast species. However, since genotypic identification could not be performed in this study, none of the systems were accepted as the standard method and therefore the sensitivity and specificity of the systems were not calculated. On the other hand, our data indicated that the two identification systems were comparable and careful observation of yeast morphology could add confidence to the identification. In conclusion, since the Phoenix™ Yeast ID system was found more practical with easier interpretation, and the results were obtained earlier than those of the API® ID 32C system (16 hours versus 48 hours), it was thought that Phoenix™ Yeast ID system may be used reliably in the routine laboratories. However, as none of the methods evaluated was completely reliable as a stand-alone, careful evaluation is necessary for species identification.

Comparative evaluation of galactomannan optical density indices and culture results in bronchoscopic specimens obtained from neutropenic and non-neutropenic patients.

Aspergillus infections are major causes of morbidity and mortality among immunocompromised patients. This study was designed to investigate the galactomannan assay optical density (OD) indices relative to the culture results in bronchoscopic samples obtained from neutropenic and non-neutropenic patients. Galactomannan OD indices from 1427 samples from 2005 to 2012, which were sent from 839 patients and were composed of bronchial lavage (BL = 727) and bronchoalveolar lavage fluids (BAL = 700), were retrospectively analysed. The recovery rates of Aspergillus species from these specimens were 9.4% from the combined patient group and 13.3% from the neutropenic group. Aspergillus fumigatus complex was the most frequently isolated species. The mean and median OD indices of the positive and negative culture samples are approximately 5 and 1, respectively, and 91% of all culture-positive samples have ≥1 OD index value. The receiver-operating characteristics curve analysis demonstrated that the feasibility of the Aspergillus galactomannan assay and Aspergillus galactomannan test has superior accuracy in BAL compared to BL fluids, and the test is not affected by the immune status of the patient. We suggest that the Aspergillus galactomannan test, which uses bronchoscopic material, leads to an earlier diagnosis and if the OD index is found ≥1, fungal growth can be expected.

Investigation of vancomycin resistant Enterococcus faecium outbreak in neonatal intensive care unit.

Enterococci are one of the major agents of community-acquired and nosocomial infections. In this study we aimed to analyze the clonal relation of the vancomycin-resistant Enterococci outbreak seen at the Neonate Intensive Care Unit (NICU) of Uludag University Hospital. Vancomycin resistance gene was investigated in the Enterococcus faecium strains and pulsed field gel electrophoresis (PFGE) was used to investigate the genetic relation between outbreak strains. Enterococci grown in all patient samples were identified as Enterococcus faecium by BD Phoenix 100 (Becton Dickinson, USA). We found vanA resistance gene in all of the swab samples by Xpert VanA/B test on Cepheid (Cepheid, USA). PFGE band patterns revealed two different strains, of which the majority of them (22/24) had the same clonal origin. The common clonal origin was also isolated from rectal probes. Perianal swab culture positivity was evaluated as colonization but culture growth in two blood cultures, two urine cultures and one wound culture was evaluated as infection and treated with linezolid. All of the patients survived the outbreak. Besides the infection control precautions determining the genetic relation between outbreak strains which can be done in the microbiology laboratory is necessary to control an outbreak. PFGE is a reliable method in the microbiologic analysis of outbreaks. Molecular microbiologic analysis of outbreak strains will contribute to prove the epidemiologic and evolution of outbreaks.

[A real-time PCR assay for the quantification of hepatitis B virus DNA and concurrent detection of YMDD motif mutations]. / Hepatit B Virus DNA Düzeyi Tayini ve Es Zamanli Olarak YMDD Motif Mutasyonu Analizi Yapan Gerçek Zamanli PCR Testi.

Monitoring therapy in chronic hepatitis B patients receiving lamivudine therapy, is done by two different assays; determination of viral load and genotypic resistance. These methods are labor intensive and time consuming. It was aimed to develop an assay to quantitate hepatitis B virus (HBV) DNA in serum and detect YMDD (thyrosine, methionine, aspartate, aspartate) motif mutations in the same run. The assay was based on real-time polymerase chain reaction (Rt-PCR) with YMDD-specific hybridization probes. Determination of YMDD motif was done by melting temperature analysis. External standard curve was used for quantifying viral DNA, which was generated by standard sera (VQC S2220) including HBV-DNA between concentrations of 1000 to 3 million copies/ml. The assay was compared with commercial quantitative kit (Artus HBV RG PCR; Qiagen, Germany), commercial line prob assay (INNO-LiPA HBV DR v1.0; Innogenetics, Belgium) and direct DNA sequencing method. Thirty-eight serum samples obtained from 20 chronic hepatitis B patients (7 female, 13 male; age range: 27-70 years) treated with only lamivudine and were negative for HIV and HCV antigen and antibodies were tested in the study. The analytical sensitivity of the assay was found as 200 copies/ml, with a dynamic range of 1 x 103 to 3 x 107 copies/ml. PCR efficiency of the in-house assay was found to be 1.98. Comparison of log10 HBV-DNA concentrations determined by the in-house and commercial quantitative kits showed a significant correlation (r= 0.681). Melting temperature (Tm) analysis was used for the YMDD motif determination and found to be 59.86°C for YMDD, 56.34°C for YVDD and 55.10°C for YIDD. The results of the in-house assay, DNA sequencing and LiPA were concordant in samples with homogeneous virus population, and in-house assay could also detect the major type of YMDD motif in mixed viral populations The Rt-PCR method which was developed in this study is a rapid, accurate and reproducible method for quantifying HBV-DNA and detecting the predominant YMDD motif in the same run in two hours duration. It was concluded that this method may be a convenient tool for monitoring HBV-infected patients receiving lamivudine treatment.

A new hepatitis B virus vaccine escape mutation in a renal transplant recipient.

Surface antigen mutations of hepatitis B virus (HBV) may lead to immune escape and cause failure of immunization. In this report, the development of a chronic HBV infection in a vaccinated renal transplant recipient with pre-existing anti-HBs antibody is documented. The sequencing data showed that the HBV strain carried five amino acid substitutions in the major hydrophilic region of the S protein, one (sS143L) located at the "a" determinant. A commercial HBsAg assay failed to detect the mutant antigen.