[Early detection of methicillin resistance by real-time PCR in staphylococci isolated from blood cultures]. / Kan Kültürlerinde Üreyen Stafilokoklarda Metisilin Direncinin Gerçek Zamanli PCR ile Erken Tanisi

Bacteremia caused by methicillin-resistant Staphylococcus aureus (MRSA) has a significant morbidity and mortality. The aim of this study was to evaluate the efficiency of real-time polymerase chain reaction (Rt-PCR) targeting nuc and mec genes in the culture extracts of blood culture systems for the early diagnosis of MRSA. A total of 48 samples that gave positive growth signal in BACTEC 9000 MB (BD, USA) and stained as gram-positive cocci, were included in the study. The samples were collected between 2009 and 2010. VITEC 2 (bioMerieux, France) system was used for the identification and antibiotic susceptibility testing of the isolates. According to the culture results, 15 of the isolates were methicillin-resistant coagulase-negative staphylococci (MRCNS), four were MRSA, 14 were methicillin-susceptible coagulase- negative staphylococci (MSCNS) and 15 were methicillin-susceptible S.aureus (MSSA). However, Rt-PCR yielded 17 MRCNS, eight MRSA, 10 MSCNS and 13 MSSA results. Our findings indicated lack of concordance between blood culture and PCR technique. When the blood culture results were accepted as the gold standard for the determination of methicillin resistance, sensitivity, specificity, positive and negative predictive values of Rt-PCR were found as 73%, 62%, 56% and 78%, respectively. In conclusion, in contrast to the expectations, Rt-PCR was not considered as an appropriate method for the detection of MRSA in routine diagnosis.

Pseudo-outbreak of Serratia marcescens in a tertiary care hospital.

The aim of this study was to describe a pseudo-outbreak due to Serratia marcescens associated with laboratory contamination, and also the epidemiologic and laboratory methods used to determine the source of contamination. An apparent increase in positive culture results for Serratia marcescens was observed in the Clinical Microbiology Laboratory of Kocaeli University Hospital between September and November 2007. An outbreak investigation including retrospective and prospective studies using chart review, environmental sampling and random arbitrary polymorphic DNA-polymerase chain reaction (RAPD-PCR) of randomly selected isolates were performed by the Infection Control Committee. Nine out of 67 strains belonged to a real infection. S. marcescens was also isolated from saline solution in the specimen processing area of the laboratory. It was recognized that the technician has been using the same stock saline solution for processing specimens without changing the injector. RAPD patterns of the clinical isolates were identical to the pattern of saline strain. The contaminated saline solution was discarded, the technician was trained and no additional cases of suspected contamination have been observed. This pseudo-outbreak emphasize the importance of the observation of specimen processing procedures and the training of laboratory workers.

[Analysis of the results of NEQAS international external quality assessment programme for hepatitis B and C virus nucleic acid tests]. / Hepatit B ve C virus nükleik asit testlerinde NEQAS uluslararasi dis kalite kontrol degerlendirme programi sonuçlarinin analizi.

External quality assessment (EQA) has been playing an increasingly important role in the implementation of nucleic acid amplification techniques (PCR) for clinical diagnosis. In this study, the results of HBV-DNA quantification and HCV-RNA detection tests evaluated by United Kingdom National External Quality Assessment Scheme for Microbiology (NEQAS) were analysed and the performance of our laboratory was evaluated. Between April 2006-January 2008, in four different distribution panels including 16 freeze-dried serum and six plasma specimens for HBV-DNA and HCV-RNA testing, respectively, were received. Viral nucleic acids were extracted by magnetic particle technology (NucliSENS-easyMAG, bioMérieux, Boxtel, Holland). HBV-DNA and HCV-RNA tests were performed by Fluorion HBV quantitative v2.0 and Fluorion HCV quantitative v2.1 (lontek AS, Istanbul, Turkey) kits in real-time PCR (iCycler IQ v3.0a - BioRad Laboratories, Hercules, CA, USA) platform. The performance scores of all the quantification tests of HBV-DNA were 2 (completely correct result) and a strong correlation (r= 0.987) between the quantitative HBV data and the target values was found by linear regression analysis. The NEQAS scores of HCV-RNA testing, except for a false negative result (since the viral load in this specimen was very low--79 IU/ml--it was not scored by NEQAS), were 2 in all specimens. The evaluation of the data revealed 100% detection in HBV-DNA and 83.3% detection in HCV-RNA. In conclusion, the results of this study showed high accuracy of HBV quantification in the samples of HBV infected patients under antiviral therapy. However, the analytical sensitivity of HCV-RNA quantitative kit should be improved for the purpose of reliable HCV-RNA results. External quality control panels are important tools for monitoring the quality of diagnostic laboratory tests. Therefore, PCR laboratories should always have EQA in routine procedures.

[Elimination of PCR inhibitors in routine diagnostic real-time PCR assay and results of internal amplification control]. / Rutin gerçek zamanli PCR tani testlerinde PCR inhibitörlerinin eliminasyonu ve internal amplifikasyon kontrol sonuçlari.

Polymerase chain reaction (PCR) inhibitors which can be found in the clinical specimens lead to increased cost of these tests and also cause delay in the results particularly in the routine laboratories. Lysis/dilution methods are effective on the removal of PCR inhibitors and the performance of internal amplification control (IAC). This study was aimed to evaluate the effects of some methods, such as lysis (freezing and thawing) and dilution (1/10) of serum samples, used for the removal of PCR inhibitors, on IAC results. This evaluation was done by investigating the results of 1440 HCV-RNA and 2754 HBV-DNA (Fluorion HCV QNP and HBV QNP; Iontek, Turkey) tests that were performed in our laboratory during January 2005-October 2006 period. The nucleic acid isolation was done by "spin colon" (Qiagen, QIAamp DNA Mini Kit, Germany) and "magnetic particle" (Qiagen, BioRobot EZ1, Germany) technologies and PCR was performed by real-time PCR (iCycler IQ - BioRad Lab., USA). False negative IAC was detected in 211 samples during HCV-RNA tests and correct results were obtained in 66.4% of these when inhibitors were removed by lysis in 121 and by serum dilution in 19 of the samples. For HBV-DNA tests false negative IAC was detected in 15 samples and application of lysis method yielded correct results in 73.3% (11/15) of these. By the application of inhibitor removal methods the rate of false negative IAC decreased from 14.6% to 4.9% (71/1440) in HCV PCR and from 0.5% to 0.1% (4/2754) in HBV PCR. These data indicated that lysis/dilution methods were simple, economical and effective methods that could be used in routine PCR laboratories for the removal of PCR inhibitors and to achieve effective IAC.

[Entecavir resistance in entecavir naive lamivudine treated chronic hepatitis B patients]. / Lamivudin tedavisi uygulanmis ve entekavir naif kronik hepatit B'li hastalarda entekavir ilaç direnci.

Approved hepatitis B virus (HBV) therapies include interferon-alpha and nucleos(t)ide analogues. Lamivudine (LVD) is a nucleoside analogue and following long term LVD therapy, resistance emerges in a significant number of patients. Entecavir (ETV) is a novel deoxyguanosine analogue with potent activity against HBV in chronically infected patients. ETV is highly efficacious in treating nucleoside naive and LVD refractory patients. The aim of the present study was to determine the prevalence of ETV drug resistance in LVD treated/ETV naive (study group) and in untreated naive (control group) patients with chronic B hepatitis. DNA sequencing was applied to 80.-250. amino acid positions on HBV polymerase gene to investigate the ETV resistance and also HBV genotype and HBV polymerase gene overlapped S-gene segment mutations. Primary LVD and ETV drug resistance were detected in 37 (42.6%) and 4 (4.5%) of 87 patients, respectively in the study group. rtT184A, rtT1841 and rtT1845 mutations were found related to primary ETV resistance. In these patients also rtL180M and rtQ215S mutations were detected as compensatory mutations and YVDD and YIDD variants were observed at the 204. amino acid codon position. None of the patients in the control group had LVD or ETV resistance. Two of the patients in the study group had mutations at the positions sG145R and sC137G in the overlapped S-gene segment. However, mutations at the overlapped S-gene segment were not affected by the mutations associated with ETV resistance. All of the patients in the study and the control group were of HBV genotype D. The results obtained from this study may guide the treatment choices with ETV in chronic HBV patients treated with LVD and developed resistance to LVD.

Evaluation of clinical, laboratory, and therapeutic features of 145 tularemia cases: the role of quinolones in oropharyngeal tularemia.

Tularemia outbreaks have occurred in various regions of Turkey in recent years. In this study, clinical (145 patients) and laboratory (97 patients) features of patients with oropharyngeal tularemia were evaluated during the tularemia outbreak in the district of Gölcük in Kocaeli, Turkey. We analyzed the risk factors for therapeutic failure and prolonged recovery time, and compared the efficacy of three antibiotic groups, namely aminoglycoside, tetracycline and quinolone. The most common physical sign and laboratory findings in patients were lymphadenopathy (LAP) and increased erythrocyte sedimentation rate, respectively. Treatment failure was observed in 55 of the 145 (38%) patients during one-year follow-up and the most successful results were obtained in the quinolone group. It was determined that antimicrobial therapy initiated 14 days after onset of symptoms was a statistically significiant risk factor, reducing the success rate (p=0.0001, OR=13.10, 95% CI=5.69-30.15) and prolonging the recovery period (p=0.001, OR=3.23, 95% CI=1.63-6.40) in oropharyngeal tularemia cases. These results suggest that antimicrobial treatment should be started early, and quinolones such as moxifloxacin and ciprofloxacin seem to be new alternatives in the treatment of oropharyngeal tularemia.

Emergence and spread of carbapenem-resistant Acinetobacter baumannii in a tertiary care hospital in Turkey.

An intensive care unit (ICU)-based OXA-23-producing multiple-drug resistant Acinetobacter baumannii (MDRAB) outbreak was detected between October 2005 and October 2006. A total of 47 patients were infected/colonized with the outbreak strain. Clinical data were available from 37 patients. The all-cause mortality rate among the patients exposed to the epidemic strain was 35% (13/37). The outbreak strain and the resistance determinants were characterized both by microbiological methods and by molecular techniques. Cloning and sequencing experiments identified ISAbaI-associated bla(oxa-23) on the chromosome. Screening of imipenem-resistant Acinetobacter isolated from the ICU during the outbreak period with PCR identified 97 isolates as positive for the ISAbaI-bla(oxa-23) structure. Pulsed-field gel electrophoresis and plasmid analyses with selected nonrepetitive isolates revealed the clonality. Disk diffusion on cloxacillin-supplemented agar media and the real-time PCR experiments showed that outbreak isolates are overexpressing the ampC enzyme. This study highlights the occurrence of OXA-23-producing and ampC-overexpressing MDRAB in ICUs.

[A small water-borne tularemia outbreak]. / Su kaynakli küçük bir tularemi salgini.

The aim of this study was to investigate a small tularemia outbreak in a village of Karamürsel county of Kocaeli province (located in North-west part of Turkey), between 22 January - 8 March 2005 and to present the anti-epidemic measures implemented. Following diagnosis of oropharyngeal tularemia in two patients living in the same village, a field investigation was performed at this region. All patients have undergone physical examination. Blood samples and if possible throat swabs and lymph node aspirates were taken from the patients and water samples from the natural spring water suspected to be the source of the infection, were also taken. Cultures were performed from the clinical samples and filtrated water samples. Francisella tularensis antibodies were screened by microagglutination (MA) test in the serum samples of the patients. F. tularensis DNA was investigated in the filtrated water samples by real-time PCR assay. A total of 17 patients were diagnosed as tularemia with their clinical features and MA test results (> or = 1/80). All the patients had oropharyngeal tularemia except one who had ulceroglandular form. The age range of the patients was 27-80 years (mean age: 48 +/- 17 years), and 10 (59%) were female. Weakness (100%), swelling on the neck (94%) and sore throat (88%) were the most common symptoms, whereas cervical lymphadenopathy (94%) was the most frequently seen clinical finding. F. tularensis could not be grown in the cultures, however F. tularensis DNA was detected in the samples of the natural spring water by real time PCR. The patients were treated with streptomycin, ciprofloxacin, or doxycycline, and all the patients have recovered. The outbreak was taken under control after cleaning the spring water tank and chlorination of the water.

[Can HBsAg levels guide to differentiate inactive HBsAg carriers from HBeAg negative chronic B hepatitis?]. / Inaktif HBsAg tasiyiciligi ile HBeAg negatif kronik B hepatiti ayiriminda HBsAg düzeyleri yol gösterici olabilir mi?

It is valuable to differentiate the inactive HBsAg carrier state from HBeAg negative chronic B hepatitis (CBH) which develops due to precore or core promoter region mutations in hepatitis B virus (HBV). The aim of this study was to investigate the role of HBsAg S/N (sample rate/index calibrator mean rate) levels in the differentiation of inactive HBsAg carriers from HBeAg negative CBH cases. A total of 134 HBsAg positive patients followed-up in Kocaeli University Medical Faculty hospital between June 2004-September 2005, were included to the study. The patients were classified into four groups according to their serological patterns (Group 1: HBeAg and HBV-DNA negative 34 cases with normal ALT levels; Group 2: HBeAg negative, anti-HBe positive, HBV-DNA >10(5) copies/ml, 36 cases with increased ALT levels; Group 3: HBeAg negative, anti-HBe positive, HBV-DNA 102-10(5) copies/ml, 32 cases with normal ALT levels; Group 4: HBeAg positive, HBV-DNA >10(5) copies/ml, 32 cases with increased ALT levels). The age and gender distributions of the groups were similar. HBV markers have been detected by microparticle enzyme immunoassay (AxSYM System, v3.0, Abbott Laboratories, USA), and viral load were investigated by real-time polymerase chain reaction (iCycler IQ, v3.0a, Bio Rad Laboratories, USA). As a result, the mean HBsAg S/N level in group 2 who were HBeAg negative with a viral load of >10(5) copies/ml, was found significantly higher than group 1 who were inactive HBsAg carriers (285.9+/-78.8 and 214.4+/-108.6, respectively; p<0.05). In contrast there was no statistically significant difference between group 1 (HBV-DNA negative) and group 3 (HBV-DNA <10(5) copies/mL) by means of mean HBsAg S/N levels (214.4+/-108.6 and 216.3+/-57.2, respectively; p>0.05). Although HBsAg levels seem to guide the differentiation of inactive HBsAg carriers from HBeAg negative CBH cases with high viral loads (>10(5) copies/ml), advanced studies are needed to clarify this relationship with the use of quantitative HBsAg measurements (IU/ml) in large patient groups and by performing mutation analysis.

A sporadic case of visceral leishmaniasis from Kocaeli, Turkey.

Visceral and cutaneous leishmaniasis are endemic in the western and southeastern parts of Turkey. We report a sporadic case of visceral leishmaniasis from Kocaeli, which is not an endemic area. The patient, a 10-month-old male infant, had since birth never been outside the city. He was referred to our hospital with a one-month history of fever. Antibiotics were administered but fever persisted. There were Leishman bodies in the bone marrow aspirate, both in macrophages and in clusters among other cells. Immunofluorescence antibody test (IFAT) detected no antibodies in the mother. Liposomal amphotericin B was administered. Visceral leishmaniasis should be considered in the differential diagnosis of patients with persistant fever, hepatosplenomegaly and cytopenia, even in nonendemic areas.

[Low positive anti-HCV microparticle enzyme immunoassay results: do they predict hepatitis C virus infection?]. / Mikropartikül enzim immunoassay ile düsük titrede saptanan anti-HCV pozitifligi hepatit C virus enfeksiyonunun tanisinda kullanilabilir mi?

Antibody screening tests for hepatitis C virus (anti-HCV) can yield false positive results among populations such as Turkey, with a low prevalence of HCV infection. This may lead to the unnecessary use of expensive tests like HCV RNA for confirmation. However, HCV RNA is not always positive in samples with low S/Co (signal to cutoff) ratios. This retrospective study was conducted to determine the frequency of HCV RNA positivity in the samples with low anti-HCV levels. A total of 655 patients who were positive for anti-HCV by microparticle enzyme immunoassay (MEIA; v 3.0, AxSYM System, Abbott Laboratories, USA) and positive for HCV RNA by real-time polymerase chain reaction (PCR; iCycler IQ, v 3.0a, Bio Rad Laboratory, USA) between June, 2002 and November, 2005 were evaluated. Anti-HCV positivity was found in 368 (56%) patients and HCV RNA was not detected in samples with S/Co ratios lower than 3.8. As a result, for the confirmation of the samples yielding low and/or indeterminate grey zone anti-HCV levels, immunoblot methods, retesting of the same sample with another enzyme immunassay or testing of a new sample, would be appropriate and economical prior to use of HCV RNA tests.

Intensive care unit-acquired infections: incidence, risk factors and associated mortality in a Turkish university hospital.

In this prospective study, 93 intensive care unit (ICU)-acquired infections seen in 131 ICU patients were evaluated. Infection rates were found to be 70.9 in 100 patients and 56.2 in 1,000 patient-days. Pneumonia (35.4%) and bloodstream infections (18.2%) were the most common infections; Staphylococcus aureus (30.9%) and Acinetobacter spp. (26.8%) were the most frequently isolated microorganisms. The results of multivariate logistic regression analyses estimating the risk factors for ICU-acquired infections were as follows: length of stay in ICU (>7 days) (odds ratio [OR]: 7.02; 95% confidence interval [CI]: 2.80-17.56), respiratory failure as a primary cause of admission (OR: 3.7; 95% Cl: 1.41-9.70), sedative medication (OR: 3.34; 95% CI: 1.27-8.79) and operation (before or after admission to ICU) (OR: 2.56; 95% CI: 1.06-6.18). In logistic regression analyses, age (>60 years) (OR: 3.65; 95% CI: 1.48-9.0), APACHE II score >15 (OR: 4.67; 95% CI: 1.92-11.31), intubation (OR: 3.60; 95% CI: 1.05-12.39) and central venous catheterization (OR: 7.85; 95% CI: 1.61-38.32) were found to be significant risk factors for mortality. The difference in mortality rates between patients with ICU-acquired infection and uninfected patients was not statistically significant (mortality rates: 42.3 and 45.6%, respectively). A high incidence of nosocomial infections was found, and the risk factors for ICU-acquired infections and mortality were determined.

Surgical site infection rates in 16 cities in Turkey: findings of the International Nosocomial Infection Control Consortium (INICC).

BACKGROUND: Surgical site infections (SSIs) are a threat to patient safety; however, there were no available data on SSI rates stratified by surgical procedure (SP) in Turkey. METHODS: Between January 2005 and December 2011, a cohort prospective surveillance study on SSIs was conducted by the International Nosocomial Infection Control Consortium (INICC) in 20 hospitals in 16 Turkish cities. Data from hospitalized patients were registered using the Centers for Disease Control and Prevention (CDC) National Healthcare Safety Network (NHSN) methods and definitions for SSIs. Surgical procedures (SPs) were classified into 22 types according to International Classification of Diseases, Ninth Revision criteria. RESULTS: We recorded 1879 SSIs, associated with 41,563 SPs (4.3%; 95% confidence interval, 4.3-4.7). Among the results, the SSI rate per type of SP compared with rates reported by the INICC and CDC NHSN were 11.9% for ventricular shunt (vs 12.9% vs 5.6%); 5.3% for craniotomy (vs 4.4% vs 2.6%); 4.9% for coronary bypass with chest and donor incision (vs 4.5 vs 2.9); 3.5% for hip prosthesis (vs 2.6% vs 1.3%), and 3.0% for cesarean section (vs 0.7% vs 1.8%). CONCLUSIONS: In most of the 22 types of SP analyzed, our SSI rates were higher than the CDC NHSN rates and similar to the INICC rates. This study advances the knowledge of SSI epidemiology in Turkey, allowing the implementation of targeted interventions.

[Seroprevalence of hepatitis B and C virus, human immunodeficiency virus and syphilis in the blood donors]. / Kan donörlerinde hepatit B ve C virusu, insan immün yetmezlik virusu ve sifiliz seroprevalansi.

Transfusion-transmitted infections are the most commonly encountered complications in transfusion practice. Serological markers for hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV) and Treponema pallidum were screened in blood banks routinely. These tests are obligatory for transfusion safety and may give an idea about the seropositivity rates of a specific region. In this study, the results of 29,049 volunteer blood donors who were admitted to Blood Center of Kocaeli University Hospital between January 1999 to January 2004, have been retrospectively evaluated. HBsAg, anti-HCV and anti-HIV-1/2 were screened by microparticle enzyme immunoassay (MEIA), and syphilis antibodies were screened by rapid plasma reagin (RPR) test in the Blood Center Laboratory. The seropositivity rates for HBsAg, anti-HCV and RPR were found as 2.3%, 0.37% and 0.02%, respectively. Eight donors were found seropositive for anti-HIV-1/2, but they were all accepted as false positive results, since the western blot confirmation tests were all negative.

[Dermatophyte species isolated from patients prediagnosed as onychomycosis and the value of fungal culture]. / Onikomikoz ön tanili olgulardan izole edilen dermatofit türleri ve mantar kültürünün tanisal degeri.

In this study, the clinical specimens taken from 390 patients who were prediagnosed as onychomycosis have been investigated by direct microscopy and culture methods, in the Mycology Unit of Central Laboratory of Kocaeli University Medical Faculty. Twenty-one (41.2%) of the 51 microscopically positive samples revealed positive cultures for dermatophytes, whereas 32 (9.4%) samples were culture positive among the 399 samples which were microscopically negative. The most frequently isolated dermatophyte was found to be Trichophyton rubrum (73.6%), followed by Trichophyton mentagrophytes (24.5%) and Microsporum canis (1.9%).

Erysipelothrix rhusiopathiae pneumonia in an immunocompetent patient.

Erysipelothrix rhusiopathiae is a Gram-positive bacillus that causes infections primarily in animals. In humans, this bacterium usually causes localized cutaneous infections called erysipeloid. Here we report a case of pneumonia with isolation of E. rhusiopathiae from bronchoalveolar lavage and sputum. To our knowledge, this is the first report of a pneumonia case caused by E. rhusiopathiae confirmed by culture.

Epidemiology and risk factors of intensive care unit-acquired infections: a prospective multicentre cohort study in a middle-income country.

INTRODUCTION: This study aimed to determine the incidence and risk factors of infections among patients admitted to intensive care units (ICUs) in tertiary care hospitals in Turkey. METHODS: Adult patients who were admitted to the ICUs of five tertiary care hospitals for over 48 hours between June and December 2007 were monitored daily. Potential risk factors such as age, gender, comorbidities, diagnosis at admission, severity of disease (Acute Physiology and Chronic Health Evaluation II scores), exposure to antibiotics, history of invasive procedures and significant medical interventions were evaluated. A multivariate analysis of these risk factors was carried out using Cox regression. RESULTS: A total of 313 patients with a median ICU stay of 12 days were selected for the study. 236 infectious episodes (33.8/1,000 ICU-days) were diagnosed among 134 patients (42.8/100 patients) in this group. Multivariate analysis revealed that exposure to a cephalosporin antibiotic (hazard ratio [95% confidence interval] 1.55 [1.10-2.19]) was an independent risk factor, whereas having a tracheostomy cannula (0.53 [0.36-0.81]) or nasogastric tube (0.48 [0.33-0.70]) was protective. Patients admitted to the ICUs from surgical wards were significantly more exposed to cephalosporins. CONCLUSION: ICU-associated infections, which are quite high in Turkey, are largely due to inadequate infrastructure and facilities and understaffing. Abuse of antibiotics, particularly in patients who have undergone surgery, and prolonged ICU stays are significant risk factors for such infections.

A case of pneumonia caused by Bacillus anthracis secondary to gastrointestinal anthrax.

We present herein an unusual case of anthrax pneumonia secondary to gastrointestinal infection. In this case, severe abdominal pain occurred during the course of a stent placement procedure. The patient had undergone surgery with the prediagnosis of intestinal ischemia. On the second postoperative day, pneumonia developed and B. anthracis grew as the etiologic agent. Pathological examination of small-bowel sections revealed findings in accordance with anthrax.