Search

Due to limitations in commercial diagnostic methods, this study aimed to develop a reliable real-time polymerase chain reaction (Rt-PCR) assay for early diagnosis of brucellosis. Optimization of the Rt-PCR method was performed on serum samples spiked by Brucella melitensis with different densities ranging from 101 to 108 colony-forming units (cfu)/mL; each density was prepared in ten samples. The limit of detection was investigated by using Thermo DNA extraction kit with Maxima SYBR Green Rt-PCR and two TaqMan probe–based Rt-PCR protocols performed by QuantiTect and TEMPase multiplex PCR master mixes in two thermal cyclers, which were Rotor-Gene and Bio-Rad. The validation of the optimized protocol was carried on 20 brucellosis-negative samples and 20 samples spiked with B. melitensis by using a combination of Thermo DNA extraction kit with TEMPase PCR master mix. SYBR Green Rt-PCR yielded positive results on all samples having ≥ 104 cfu/mL of B. melitensis in both thermal cyclers. Its limit of detection was 112 DNA copies per reaction. The positivity of both probe-based Rt-PCR protocols was 100% and 80% on the samples having 103 cfu/mL and 102 cfu/mL of B. melitensis, respectively. The limit of detection of probe-based protocols was defined as 4 DNA copies per reaction. The optimized Rt-PCR protocol showed high-level accuracy, precision, specificity, and sensitivity, each having a rate of 100%. The current study indicated that the TaqMan probe–based Rt-PCR protocol optimized and validated with serum samples can be reliably used for early diagnosis of brucellosis.

Objective: Brucellosis is a zoonotic disease with various clinical presentations and early diagnosis is crucial to avoid severe complications. Due to limitations of conventional diagnostic methods, polymerase chain reaction (PCR) based approaches have gained importance in diagnosis.We aimed to evaluate diagnostic value of multiplex real time-PCR (mRT-PCR) in serum samples collected from brucellosis suspected patients by comparision sensitivity of mRT-PCR with those of conventional diagnostic methods. Material and Methods: A total of 249 serum samples collected from the suspected brucellosis patients admitted to the hospitals in three different provinces were analyzed by serological tests, culture and mRT-PCR. In laboratories of the participating hospital, serum samples were tested for the Brucella specific antibody by commercial serological kits including standart tube agglutination test (STAT), Coombs’ test, and immunocapture test (ICT). Blood culture was performed for 153 of the patients in the participating hospital. All serum samples were analyzed for the presence of Brucella DNA by mRT-PCR. Results: According to laboratory test results, 215 of the 249 suspected cases having comparible clinical data were identified as brucellosis cases. Of the 215 brucellosis cases, 36 were diagnosed as definitive cases, the remaning 179 patients were presumptive cases. Sensitivity of mRT-PCR in the samples that were positive by ICT, STAT, Coombs’ test, and blood culture was 70.2%, 77.3%, 83%, and 97.2%, respectively. By using mRT-PCR, additional 17 suspected patients were diagnosed as presumptive cases. Among the mRT-PCR positive serum samples, Brucella abortus was detected in 3 samples (1.9%), the remaining 156 samples (98.1%) had B. melitensis DNA. Conclusion: Our results indicate that mRT-PCR can be considered a useful diagnostic tool in patients who have negative serologic test results, and in detection of Brucella species. Keywords: Brucellosis, Multiplex real-time PCR, Serological diagnosis, Culture

Purpose In this study we aimed to propose an algorithm for initial anti HCV EIA reactive blood donations in Turkey where nucleic acid amplification tests are not yet obligatory for donor screening. Methods A total of 416 anti HCV screening test reactive donor samples collected from 13 blood centers from three cities in Turkey were tested in duplicate by Ortho HCV Ab Version 3.0 and Radim HCV Ab. All the repeat reactive samples were tested by INNO-LIA HCV Ab 3.0 or Chiron RIBA HCV 3.0 and Abbott Real Time HCV. Intra-assay correlations were calculated with Pearson r test. ROC analysis was used to study the relationship between EIA tests and the confirmatory tests. Results The number of repeat reactive results with Ortho EIA were 221 (53.1%) whereas that of microEIA, 62 (14.9%). Confirmed positivity rate was 14.6% (33/226) by RIBA and 10.6% (24/226) by NAT. Reactive PCR results were predicted with 100% sensitivity and 95% specificity with S/CO levels of 8.1 with Ortho EIA and 3.4 with microEIA. Conclusions Repeat reactivity rates declined with a second HCV antibody assay. Samples repeat reactive with one HCV antibody test and negative with the other were all NAT negative. All the NAT reactive samples were RIBA positive. None of the RIBA indeterminate or negative samples were NAT reactive. Considering the threshold values for EIA kits determined by ROC analysis NAT was decided to be performed for the samples above the threshold value and a validated supplemental HCV antibody test for the samples below.

Summary Introduction Human papillomavirus infection (HPV) is one of the most common sexually transmitted infections and can cause penile and anal cancer in men, and invasive cervical cancer in women. Objective To evaulate the colonization of 32 HPV subtypes in the foreskin of boys. Study design A prospective analysis was made of the data of 62 healthy boys who had undergone standard circumcision. Deoxyribonucleic acid (DNA) was isolated from the foreskin tissues, and the integrity of DNA was tested. The DNA of each patient was further assessed with real-time polymerase chain reaction (PCR) and the presence of 32 subtypes of HPV was explored. To confirm the results, melting curve analysis and agarose gel electrophoresis (AGE) were performed for all samples. Further analysis was made using LCD-array on six randomly selected samples to confirm the results together with negative and positive controls. Results The mean age of the boys was 6.8 ± 2 years at the time of surgery. All positive controls and samples were positive, all negative controls were negative in the first HPV amplification assay. All positive controls had typical melting curve peaks, whereas all sample amplifications had non-specific, atypical melting curves not fitting with those of the positive controls. Two bands of expected sizes (124 and 405 bp) were only observed in positive controls, but not in negative controls or samples on AGE. The same results were observed on the 6 randomly selected samples using LCD-array. Consequently, all the foreskin samples were evaluated as negative for the 32 HPV types investigated in the study. Discussion Literature shows a high prevalence of genital HPV in newborns, in early infancy, late adolescence and adulthood. However there is a lack of data in literature on the prevalence in early and late childhood. The negative results of HPV colonization on the foreskin in the current study may be attributed to the conservative and mostly monogamous nature of most family structures in Turkey. Conclusion The results of the present study have shown that foreskin tissue is not a natural reservoir for HPV and subclinical HPV infection is not likely in the absence of suspected sexual contact.

Background/aim: This study aimed to evaluate the risk factors of patients colonized with carbapenem-resistant Enterobacteriaceae (CRE).Materials and methods: The study was conducted between January 2010 and March 2016. The colonized group consisted of patients who had a CRE strain in their rectal swab cultures, whereas patients with negative rectal surveillance cultures for CRE who were concurrently hospitalized in the same units with the colonized group patients were included in the control group.Results: The number of patients in the colonized and the control group was 71 and 120, respectively. Both groups were evaluated for demographic and healthcare-associated characteristics. Isolated microorganisms in rectal surveillance cultures for CRE were Klebsiella pneumoniae (75.5%), Escherichia coli (15.5%), Enterobacter cloacae (4.2%), Klebsiella oxytoca (1.4%), and Klebsiella terrigena (1.4%). The isolates were resistant to imipenem, meropenem, and ertapenem (52.1%, 73.2%, and 100%, respectively). In multivariate analysis, presence of decubitus, colistin usage, glycopeptide usage, and fluoroquinolone usage were found to be independent risk factors for CRE colonization. There was no significant difference between the two groups with regards to mortality (P = 0.070).Conclusion: These results are in agreement with the current literature. The findings of this study could be useful for improvement of infection control strategies related to CRE.

The diagnosis of human brucellosis requires culture or serological tests for the conformation of the clinical findings. Isolation of the bacteria is used as a gold standard, however it is time consuming and processing of positive cultures has a potential risk for laboratory acquired human brucellosis. Polymerase chain reaction (PCR) based methods have offered new approaches for early diagnosis of brucellosis and reduce the risk of laboratory acquired human brucellosis. A major limitation of the PCR method is the difficulty to remove the inhibitors in specimens. The aim of this study was to determine the performance of two DNA extraction kits by using two separate PCR master mixes and to determine appropriate "extraction kit - PCR master mix" combination for the diagnosis of Brucella from whole blood samples and blood culture bottles. Two commercial DNA extraction kits, NORGEN Blood DNA isolation kit (Norgen) and Thermo Scientific GeneJet Whole blood genomic DNA purification kit (Thermo Fisher Scientific, USA) and two PCR master mixes, QuantiTect multiplex PCR (QuentiTect, Qiager, Almanya) and Ampliqon Multiplex TEMPase (Amliqon, Denmark) were assessed on 30 simulated blood samples with known concentrations (102-104 cfu/ml) of Brucella melitensis ATCC 23456 strain and 10 blood culture bottles that gave positive signal. By using different combinations of extraction kits and PCR master mixes, a total of 160 different multiplex real-time PCR (Rt-PCR) trials were performed with probes and primers specific to Brucella spp., B.melitensis, and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All the 120 Rt-PCR trials performed on the DNA samples extracted from blood samples gave positive results with GAPDH probe/primers. The rate of positive PCR results for Brucella spp. was 96.7% for the combination of Norgen-QuantiTect, 93.3% for Thermo-Ampliqon, 93.3% for Thermo-QuantiTect, and 86.7% for Norgen-Ampliqon. The frequency of positive B.melitensis results for these combinations were 96.7%, 93.3%, 56.7% and 90%, respectively. In the samples with the bacterial density of 102 cfu/ml, Brucella spp. detection rates were 80% for Thermo-Ampliqon and Norgen-Ampliqon, and 90% for Thermo-QuantiTect and Norgen-QuantiTect; and for B.melitensis positivite rates were 90%, 70%, 20%, and 90%, respectively. Rt-PCR assays with the DNA samples extracted from blood culture bottles using Norgen isolation kit yielded 80% positivite result. However, the frequency of PCR positivite results was only 20% in the DNA samples extracted by Thermo DNA extraction kit. PCR result for GAPDH gene was also negative in 80% of the samples extracted by Thermo kit. Our results revealed that for the removal of inhibitors and detection of even low number of Brucella spp./B.melitensis in blood samples and blood culture bottles, NORGEN Blood DNA isolation kit can be used with a combination of QuantiTect multiplex PCR or Ampliqon Multiplex TEMPase.

This study aimed to determine the presence of vancomycin resistance (vanA and vanB) and virulence genes (esp, asa1, gelE, ace, hyl, cylA, cpd and ebpA) in vancomycin-resistant Enterococcus faecium (VREfm) strains and to analyse the clonal relationships among the strains. E. faecium strains were identified from rectal and clinical specimens by biochemical tests and the API-20 Strep kit. Susceptibility testing was performed using disc-diffusion and broth-dilution methods. PFGE was used for molecular typing of the VREfm strains. The vancomycin resistance and virulence genes were amplified by two-step multiplex PCR. All 55 VREfm isolates were resistant to penicillin G, ampicillin and high-level gentamicin but were susceptible to quinupristin/dalfopristin and linezolid. Multiplex PCR analysis indicated that all isolates harboured vanA and that 41 (75 %) were positive for virulence genes. The esp gene was the most common virulence factor and was detected in nine (41 %) invasive and 32 (96.7 %) non-invasive isolates. Multiple virulence genes were observed only in two non-invasive isolates; one harboured esp and ebpA and the other harboured esp, ebpA, asa1, gelE and cpd. PFGE typing yielded 16 different types, seven of which were clusters with two to 14 strains each. The clustering rates of the rectal swab, blood and urine isolates were 72.7 %, 61.5 % and 87.5 %, respectively. The genetic similarity observed among the VREfm isolates indicated cross-transmission in the hospital. Further studies on the virulence factors present in the strains might provide insight into the acquisition of these traits and their contribution to increased prevalence of VREfm.

WOS: 000384907900008, PubMed ID: 27236394, BACKGROUND: Knowing risk factors for colistin resistance is important since colistin is the only remaining choice for the treatment of infections caused by multi-drug resistant microorganisms. OBJECTIVE: Evaluate risk factors associated with infection by colistin-resistant microorganisms. DESIGN: Retrospective study. SETTINGS: Tertiary healthcare centers. PATIENTS AND METHODS: An e-mail including the title and purpose of the study was sent to 1500 infectious disease specialists via a scientific and social web portal named "Infeksiyon Dunyasi (Infection World)". Demographic and clinical data was requested from respondents. MAIN OUTCOME MEASURE(S): Colistin-resistance. RESULTS: Eighteen infectious disease specialists from twelve tertiary care centers responded to the invitation. Data was collected on 165 patients, 56 cases (39.9%) and 109 (66.0%) age-and sex-matched controls. The colistin-resistant microorganisms isolated from cases were 29 Acinetobacter baumannii (51.8%), 18 Pseudomonas aeruginosa (32.1%) and 9 Klebsiella spp. Colistin, carbapenem, and quinolone use in the last three months were risk factors for colistin resistance in the univariate analysis. Previous quinolone use in the last three months (P=.003; RR: 3.2; 95% CI: 1.5-6,7) and previous colistin use in the last three months (P=.001; RR: 3.6; 95% CI: 1.63-7.99) were significant risk factors in the multivariate analysis. CONCLUSION: Clinicians should limit the use of quinolones and remain aware of the possibility of resistance developing during colistin use. LIMITATIONS: The lack of a heteroresistance analysis on the isolates. No data on use of a loading dose or the use of colistin in combination.

Introduction: Colistin use has increased over the last ten years because of multidrug-resistant microorganisms. The aim of this study was to compare the clinical and microbiological efficacy of colistin alone or in combination with sulbactam or carbapenem in the treatment of ventilator-associated pneumonia (VAP) due to multidrug-resistant (MDR) and extremely drug-resistant (XDR) A. baumannii. Methodology: Cases treated for VAP because of MDR and XDR A. baumannii between January 2011 and January 2013 were included in the study. The primary and secondary outcome for colistin alone, colistin with sulbactam, and colistin with carbapenems were evaluated. The primary outcomes were clinical efficacy and microbiological efficacy; the secondary outcomes were nephrotoxicity, length of hospitalization, and mortality. Results: A total of 70 VAP patients were evaluated. A total of 17 patients (24.3%) were administered colistin alone, 20 patients (28.6%) were administered colistin and sulbactam, and 33 patients (47.1%) were administered colistin and carbapenem. Clinical and microbiological response rates were higher in the carbapenem combination group (63.6% and 63.6% in both) than in the sulbactam combination group, which registered 55.0% and 60.0%, respectively. However, this did not represent a significant difference statistically (p > 0.05). There was also no significant difference between colistin alone and the combination groups regarding clinical and microbiological efficacy and mortality. Conclusions: Neither the administration of colistin alone nor colistin combined with either sulbactam or carbapenem had any noticeable advantage in the treatment of VAP in terms of clinical response, microbiological response, nephrotoxicity, length of hospitalization, and mortality.

Macrolide resistance in 156 consecutive group B streptococcal isolates was investigated. Thirty-five isolates (22.4%) had inducible (80%) or constitutive (20%) erythromycin resistance. The genes responsible were erm (B), erm (A) subclass erm (TR), and erm (B) plus erm (TR) in 62.9, 2.9, and 8.6% of isolates, respectively. Nine isolates (25.7%) harbored neither mef nor detectable erm genes.

Macrolide-lincosamide-streptogramin B (MLSB) resistance determinants of group A streptococci (GAS) in Ankara, Turkey, were defined for the first time. ISOLATES AND METHODS: A total of 1355 GAS isolates, collected from three different regions of Ankara, were screened for erythromycin resistance. Resistance phenotypes were determined by a triple-disc test, and the gene determinants responsible were determined by PCR. MICs of erythromycin, clindamycin and spiramycin were measured for the resistant isolates, and susceptibility rates to some further antibiotics were determined.Thirty-six isolates (2.6%) were resistant to erythromycin. Of these, 17 (47.2%) expressed macrolide-restricted resistance (M phenotype), while the remainder expressed inducible (16 isolates, 44.4%) or constitutive (three isolates, 8.3%) MLSB resistance. All isolates of the M phenotype harboured the mef(A) gene. Of non-M isolates, 14 harboured erm(A) subclass erm(TR) and five had erm(B) genes. There was a significant relationship between tetracycline resistance and the inducible phenotype (P0.05). Macrolide resistance was significantly higher in adults (P0.05), and increased more than two-fold in 2002 compared with 2001 (P0.05).The prevalence of macrolide resistance in GAS is low in Ankara; therefore, routine antimicrobial susceptibility testing against these agents seems unwarranted.

To investigate about serum PCT, IL-6 and IL-8 levels and how they are affected by the treatment in diabetic foot patients.Fifty patients' blood samples were taken to study ESR and CRP, IL-6, IL-8 and PCT before and at the 14th day of the treatment.The pretreatment results of the 50 patients showed positive correlations between PCT and either ESH (r=0.49, p0.001), or CRP (r=0.56, p0.001). Similarly, there was a positive correlation between IL-6 and ESH (r=0.46, p=0.001), just like as it was between IL-6 and CRP (r=0.54, p0.001). At the 14th day, the levels of ESR (70 ± 30.2 and 58.4 ± 26.2, p=0.02), CRP (63.8 ± 73.1 and 18.1 ± 19.7, p0.001) and PCT (0.6 ± 2.1 and 0.05 ± 0.02, p=0.007) were significantly decreased while IL-6 was decreased at a close range to statistical significancy at healing patients (97.5 ± 147.2 and 47.1 ± 77.6; p=0.05), but they did not at nonhealing patients. IL-8 levels were not changed anyhow.PCT was significantly decreased such as ESR and CRP were in the early phase of healing; IL-6 and IL-8 levels were also decreased by the treatment, but not statistically significantly. IL-6 and PCT were affected in correlation with the other inflammatory parameters in the beginning, but IL-8 was not. PCT and IL-6 may be useful like CRP and ESR in the diagnosis and follow up of diabetic foot infection, but IL-8 is not. Further investigation is needed.

In this study, 153 clinical isolates of Shigella were screened for extended-spectrum beta-lactamase (ESBL) production by double-disk synergy test. After being confirmed by the combined disk-test and inhibitor-combined E-test strips, all positive isolates were tested for the bla gene by PCR and the enzymes by isoelectric focusing. DNA sequencing of PCR products revealed that five Shigella sonnei isolates had CTX-M-3 type ESBL enzymes. All of them were resistant to ampicillin, sulfamethoxazole and cefotaxime but susceptible to ofloxacin and ceftazidime. All isolates displayed identical patterns in pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR. This study reports multidrug-resistant (MDR) S. sonnei isolates producing CTX-M-3 type ESBL from successive pediatric bacillary dysentery patients, indicating widespread and rapid spread of CTX-M type ESBL in Shigella spp. To counter this emerging threat to public health, the surveillance of CTX-M type beta-lactamases should be considered, together with measures designed to prevent outbreaks of MDR Shigella in the community.

Two children aged 12 and 11 years with a similar history of abdominal pain, nausea, vomiting and fever with abdominal tenderness, and muscle guarding at the right lower quadrant for few days were admitted to our hospital. They subsequently developed diarrhea but without clinical relief. Just before the decision of laparotomy, both patients were diagnosed as having Blastocystis hominis infection with light microscopic examination of the stools and were treated uneventfully with the appropriate antibiotics.

Published reports dealing with rotavirus infections in Turkey are very scarce. This study included 1099 consecutive paediatric patients with diarrhoea, who sought care at 3 hospitals in Ankara, Turkey between 1999 and 2002 and were investigated for the presence of rotavirus antigen in faeces. Rotavirus antigen was detected by an immunochromatographic test, Simple Rotavirus (Operon, Spain). Other clinical and laboratory data were extracted from patient journals. A total of 404 (36.8%) patients were positive for rotavirus antigen. Rotavirus antigen was more frequently detected in boys than girls (40.8 vs 31.8%) and in children younger than 2 y (62.7%). The proportion of rotavirus-positive children was higher in the winter season (49.6%; November to April) and the highest proportion was observed in December (55.4%). Rotavirus-associated diarrhoea had a more severe clinical presentation than non-rotaviral diarrhoea; 55.3% of all patients who required hospitalization were rotavirus-positive. The seasonal and epidemiological characteristics of rotavirus diarrhoea in Ankara were similar to those in the USA and Europe. For reliable nationwide information about the epidemiology of rotavirus-associated disease in Turkey, more individual studies and reliable official statistics of gastroenteritis cases are needed.

Brucellosis is a zoonotic disease caused by a Gram-negative coccobacillus from the Brucella genus. The disease has a broad spectrum of clinical manifestations. The musculoskeletal system involvement is frequent and, rarely, arthritis can be the only clinical feature of the disease. We report a case of monoarthritis caused by Brucella melitensis.

Production of chlamydospores is one of the phenotypic features used to differentiate Candida albicans and Candida dubliniensis. C. albicans produces few chlamydospores on only cornmeal/rice-Tween agar at room temperature, whereas C. dubliniensis produces abundant chlamydospores at this temperature both on cornmeal agar and some other commonly used media. We tried to determine whether the room temperature is the main factor that induces chlamydospore production of C. dubliniensis, regardless of the medium used. For this purpose, 100 C. albicans and 24 C. dubliniensis isolates were tested for chlamydospore production at room temperature and at 37 degrees C on some routinely used media, including eosin-methylene blue agar (EMB), nutrient agar (NA), nutrient broth (NB), and also on an investigational medium, phenol red agar (PR). At 37 degrees C, none of the isolates produced chlamydospores on any of the tested media. At 26 degrees C, all C. dubliniensis isolates produced abundant chlamydospores and pseudohyphae after 24-48 h on all tested media. At this incubation temperature, all C. albicans isolates failed to produce chlamydospores and pseudohyphae on EMB, NA, and NB, whereas 2 of the C. albicans isolates produced a few chlamydospores on PR. We also observed that all C. dubliniensis isolates tested on EMB and PR produced rough colonies with a hyphal fringe around the colonies, whereas none of the C. albicans isolates showed this property. In conclusion, incubation at 26 degrees C may play the key role for production of abundant chlamydospores and pseudohyphae by C. dubliniensis. Comprehensive molecular studies are needed to clarify the genetic basis of this observation. Using EMB and PR may be an inexpensive, a time-saving, and a simple way of presumptive identification of C. dubliniensis based on chlamydospore formation and colony morphology.

A Shigella sonnei strain resistant to cefotaxime and aztreonam (but not ceftazidime) was isolated from the stool sample of a 7-y-old outpatient. Double disk synergy test, isoelectric focusing, polymerase chain reaction and sequence analysis confirmed that the isolate produced CTX-M-3, an extended-spectrum beta-lactamase (ESBL). This is the first report from Turkey of Shigella spp. producing an ESBL, and of CTX-M type enzyme.