[Evaluation of Antimicrobial Susceptibilities of Rapidly Growing Mycobacteria]. / Hizli Üreyen Mikobakterilerin Antimikrobiyal Ilaçlara Duyarliliklarinin Belirlenmesi.

Infections related to the rapidly growing mycobacteria (RGM), which are common in the environment, have clinical significance as they can affect both immunocompromised and immunocompetent patients. Treatment of RGM related infections is difficult, because they are resistant to many of the first-line tuberculosis agents, require a long-term multiple drug regimen, which is costly, and is associated with drugrelated toxicities. The aim of this study was to investigate the in vitro antimicrobial susceptibility profiles of RGM isolated in Dokuz Eylül University Hospital and also to reveal epidemiological data. A total of 58 isolates [(Mycobacterium fortuitum (n= 35), Mycobacterium abscessus (n= 19) and Mycobacterium chelonae (n= 4)], which were isolated in Dokuz Eylül University Hospital between 2013 and 2018, were subjected to in vitro testing for nine antimicrobial agents (amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, moxifloxacin and tobramycin) with the broth microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI). For M.abscessus; 73.68% of the isolates were found susceptible to amikacin; 73.68% of isolates were susceptible to clarithromycin at early reading and only 21.05% of them remained susceptible at late reading time. No resistance to imipenem were observed. M.abscessus isolates were highly resistant to tobramycin, doxycycline and fluoroquinolones. Antibiotic susceptibility testing of M.chelonae isolates demonstrated 100% susceptibility for amikacin, clarithromycin and tobramycin. No resistance to linezolid, imipenem and moxifloxacin were observed. None of the isolates were susceptible to cefoxitin. Ciprofloxacin and doxycycline also showed poor in vitro activity against M.chelonae isolates. For M.fortuitum clarithromycin susceptibility decreased from 32.35% to 2.94% after an additional incubation until 14 days. All tested isolates of the M.fortuitum were susceptible to amikacin, ciprofloxacin and moxifloxacin. None of the M.fortuitum isolates exhibited resistance to cefoxitin and imipenem. Most of the M.fortuitum isolates were resistant to tobramycin and doxycycline. When the results were evaluated together, RGM isolates in this study were highly susceptible to amikacin; and were highly resistant to doxycycline. In conclusion, this study supported that the status of antimicrobial susceptibilities were different between species and also showed the importance for hospitals to know susceptibility patterns of isolates in their region. It should be noted that accurate species determination is critical for treatment as well as susceptibility status of rapidly growing mycobacteria to the antimicrobials in use.

Evaluation of COVID-19 antibody response with using three different tests.

BACKGROUND AND OBJECTIVES: In this study, the performance of three different commercial antibody assays for COVID-19 was examined and parameters affecting the antibody response were investigated. The correlation of patients' chest CT results, procalcitonin, CRP, and D-dimer levels with the antibody response were retrospectively evaluated. MATERIALS AND METHODS: COVID-19 antibodies were detected by three commercially available assays in each patient. Two of the assays were rapid immunochromatographic tests and - one was an ELISA-based IgG assay. SARS-CoV-2 RNA was tested by "COVID-19 RT-qPCR Detection Kit" using nasopharyngeal swab samples. The results of antibody tests were compared with each other, RT-qPCR, Biochemical parameters and chest CT findings. RESULTS: RT-qPCR was positive in 46.6% (41/88) of the evaluated patients among which 77.3% (68/88) were healthcare workers. Seventeen (41.4%) of viral RNA positive patients had a positive antibody result with at least two assays. Both of the rapid immunochromatographic tests had identical sensitivity of 36.6% and specificity of 100%, compared to RT-qPCR assay; while the sensitivity of the ELISA based Euroimmune test was 43.9%, and the specificity was 95.7%. The sensitivity and specificity of the immunochromatographic tests were 75% and 100% respectively, compared to ELISA test result. There was a correlation between antibody positivity and old age and male gender. The presence of typical chest CT findings increased the antibody positivity 13.62 times. Antibody positivity was also increased with the decrease in Ct value of the PCR assay. There was no significant relationship between the biochemical parameters (CRP, D-dimer and procalcitonin values) and the antibody or RT-qPCR results. CONCLUSION: There was a correlation between antibody response and male gender, older age, presence of symptoms, typical chest CT findings and low PCR-Ct value.

Urinary microbiota; Which non-invasive urine collection method should we use?

OBJECTIVE: The aim of this study is to establish the optimal non-invasive urine sample collection method for the microbiota studies. METHODOLOGY: Twelve men with bladder carcinoma underwent first voided and midstream urine collection. Urine samples were analysed using V3-V4 regions of bacterial 16s ribosomal RNAs. Bacterial groups with relative abundance above 1% were analysed in first voided urine and midstream urine samples at phylum, class, order and family level. At the genus level, all of the identified bacterial groups' relative abundances were analysed. The statistical significance (P < .05) of differences between first voided and midstream urine sample microbiota was evaluated using the Wilcoxon test. RESULTS: According to the analysis, 8 phyla, 14 class, 23 orders, 39 families and 29 different genera were identified in the first voided and the midstream urine samples. Statistical differences were not identified between first voided and midstream urine samples of all bacteria groups except the Clostridiales at order level (p:0.04) and Clostridia at class level (P: .04). CONCLUSIONS: Either first voided or midstream urine samples can be used in urinary microbiota studies as we determined that there is no statistically significant difference between them regarding the results of 16s ribosomal RNA analysis.

Hepatitis A, B and C seropositivity among first-year healthcare students in western Turkey: a seroprevalence study.

BACKGROUND: The risk of viral hepatitis among healthcare students (HCSs) is greater than that among the general population. Therefore, this study was conducted to investigate the seroprevalence of the hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV) among first-year HCSs at a university in Turkey and as a secondary objective, to determine the factors associated with HAV and HBV seropositivity. METHODS: This cross-sectional study was performed in first-year HCSs in Izmir, western Turkey. Data were collected using a self-administered questionnaire including items on sociodemographic characteristics, medical history, and hygiene. A total of 650 HCSs were tested for the HAV, HBV and HCV markers. Categorical variables were compared using the chi-square test. The association between independent variables and anti-HAV seropositivity and anti-HBs seropositivity was assessed by multinomial logistic regression analysis. RESULTS: The overall frequency of total anti-HAV seropositivity was 34.9%. HBsAg, total anti-HBc and anti-HBs seropositivity were found in 0.3, 1.2 and 93.7% of samples, respectively. All of the HCSs were negative for anti-HCV. Total anti-HAV seropositivity was found to be 1.73 times higher in those ≥21 years old, and it was 1.61 times higher in those who perceived their economic status to be average and 2.75 times higher in those who perceived their economic status to be low. Total anti-HAV seropositivity was found to be 4.37 times higher in those who lived in provinces with intermediate human development index levels. Total anti-HBs seropositivity was found to be 2.48 times higher in those ≤20 years old, and it was 2.13 times higher in those who perceived their economic status to be average. CONCLUSIONS: Approximately two out of three HCSs were susceptible to HAV infection. Since HCSs are at high risk for HAV infection, they should be vaccinated before medical clerkships begin. Our results indicate that there is a high prevalence of anti-HBs seropositivity among HCSs. This result may be largely attributed to the implementation of a successful vaccination program in Turkey since 1998.

Comparison of polymerase chain reaction-restriction enzyme analysis method and DNA sequence analysis results in the identification of non-tuberculous mycobacteria.

The typing of non-tuberculous mycobacteria (NTM) is important from a clinical and epidemiological perspective. The polymerase chain reaction-restriction enzyme analysis (PRA) method and DNA sequence analysis method were utilized to target a gene region that codes the 65-kDa heat-shock protein for typing 150 suspected NTM samples isolated from the respiratory tract. Mycobacterium abscessus, Mycobacterium xenopi, Mycobacterium fortuitum, and Mycobacterium peregrinum were most frequently found by both methods. Six isolates that could not be defined by the PRA method were defined as Nocardia cyriacigeorgica, Nocardia abscessus, and Mycobacterium intracellulare by DNA sequence analysis. Discordance between the results of the two methods was observed for only one isolate. The isolate that was defined as Mycobacterium gordonae type 6 by the PRA method was defined as Mycobacterium senegalense by sequence analysis. The PRA method is simple and gives rapid results. Compared with DNA sequence analysis, it gives consistent and reliable results up to a ratio of 90%. DNA sequence analysis is the gold standard method in which all strains can be defined. However, given our laboratory conditions, its disadvantage is that it takes longer to reach a diagnosis than through the PRA method.

Multicenter evaluation of crystal violet decolorization assay (CVDA) for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis.

The aim of this multicenter study was to evaluate the performance of the crystal violet decolorization assay (CVDA) for detection of multidrug resistant tuberculosis (MDR-TB). This study was performed in 11 centers in two phases. A total of 156 isolates were tested for INH and RIF resistance. In the phase I, 106 clinical isolates were tested in the Center 1-7. In the phase 2, 156 clinical isolates were tested in the center 1-6, center 8-11. Eighty six of 156 tested isolates were the same in phase I. Agreements were 96.2-96.8% for INH and 98.1-98.7% for RIF in the phase I-II, respectively. Mean time to obtain the results in the phase I was 14.3 ± 5.4 days. In the phase II, mean time to obtain the results was 11.6 ± 3.5 days. Test results were obtained within 14days for 62.3% (66/106) of isolates in the phase I and 81.4% (127/156) of isolates in the phase II. In conclusion, CVDA is rapid, reliable, inexpensive, and easy to perform for rapid detection of MDR-TB isolates. In addition, it could be adapted for drug susceptibility testing with all drugs both in developed and developing countries.

[Multicenter evaluation of the indirect nitrate reductase assay for the rapid detection of multidrug-resistant tuberculosis]. / Çok ilaca dirençli tüberkülozun hizli tespiti için dolayli nitrat redüktaz testinin çok merkezli degerlendirilmesi.

Multidrug-resistant tuberculosis (MDR-TB) is defined as resistance to at least isoniazid (INH) and rifampicin (RIF), and it complicates the implementation of tuberculosis control programmes. The rapid detection of MDR-TB is crucial to reduce the transmission of disease. The nitrate reductase assay (NRA) is one of the colorimetric susceptibility test methods for rapid detection of MDR-TB and based on the ability of reduction of nitrate to nitrite by Mycobacterium tuberculosis. The aim of this study was to evaluate the performance of the NRA for the rapid detection of MDR-TB. A total of 237 M.tuberculosis complex (MTC) isolates that were identified by the same method (BD MGIT(TM) TBc Identification Test, USA) from nine different medical centers in Turkey were included in the study. The susceptibility results of the isolates against INH and RIF obtained by reference test (Bactec MGIT(TM) 960, BD, USA) were then compared with NRA. In order to ensure consistency between centers, Löwenstein-Jensen (LJ) medium with antibiotics and without antibiotics (growth control) and Griess reagent solution were prepared in a single center (Ondokuz Mayis University School of Medicine, Medical Microbiology Department) and sent to all participant centers with the standardized test procedure. After the inoculation of bacteria into the test tubes, the tubes were incubated at 37°C, and after seven days of incubation, 500 µl Griess reagent was added to the LJ medium without antibiotics. If a color change was observed, an equal volume of Griess reagent was added to test LJ media with antibiotics. When a color change was observed in LJ media with antibiotics, it was considered that the isolate was resistant to tested antibiotics. Among 237 MTC isolates, 16 were resistant only to INH and nine were resistant only to RIF; 93 isolates (39.2%) were resistant (MDR) and 119 isolates (50.2%) were susceptible to both of the drugs determined with the reference susceptibility test. In the study, five INH-resistant isolates determined with reference method were found susceptible with NRT and eight INH-susceptible isolates determined with reference method were found resistant with NRT. In contrast, one RIF-resistant isolate determined with reference method was found susceptible with NRT and three RIF-susceptible determined isolates were found resistant with NRT. Accordingly, the concordance rate between the reference method and NRA were estimated as 94.5% for INH and 98.3% for RIF. The sensitivity, specificity, positive and negative predictive values of NRA were detected as 95.4%, 93.7%, 92.8% and 96% for INH, and 99%, 97.8%, 97.1% and 99.2% for RIF, respectively. The results of the 111 isolates were obtained on the seventh day, while the rest of the results were obtained between 10-14 days. In conclusion, the data of this multicenter study showed that NRA is a reliable, relatively inexpensive and practical method to perform for the rapid detection of MDR-TB.

Performance of tuberculin skin test and interferon gamma assay for the diagnosis of latent tuberculosis infection in juvenile idiopathic arthritis.

The objective of this prospective cross-sectional study was to compare a Mycobacterium tuberculosis-specific interferon gamma (IFN-γ) enzyme linked immunosorbent assay [QuantiFERON-TB Gold In-Tube (QFT-GIT)] test with tuberculin skin test (TST) for detection of latent tuberculosis infection (LTBI) in patients with juvenile idiopathic arthritis (JIA). To our knowledge, this is the first study evaluating the performance of QFT-GIT in comparison with TST in JIA. A cross-sectional study of 39 children with JIA and 40 healthy controls was conducted in Izmir, Turkey. Blood was for drawn for the QFT-GIT assay prior to administration of the TST using 5 tubercullin units (TU) of purified protein derivative (PPD-S). A positive TST was defined as ≥10 mm for JIA and ≥15 mm for controls. Statistical analysis was performed using SPSS version 16.0 for Windows. There were no significant differences between JIA patients and controls for age, sex, and Bacillus Calmette-Guérin (BCG) vaccination. Of patients, 70% had active JIA disease. The median TST induration was 5.8 mm (±5.7 mm) for JIA and 10.7 mm (±4.5 mm) for the control group, which was statistically significant (p = 0.000). The rate of patients who showed no reaction to TST was 38%, of which 93% had active disease. There were two patients who had positive IFN-γ results but negative TST, who had systemic and polyarticular type JIA, respectively. Overall agreement between TST and QFT-GIT was low both in JIA and control group (κ value =0.06 and 0.10, respectively). TST may be inadequate to diagnose LTBI in JIA patients. The IFN-γ assay may be useful to identify false negative TST response in cases with latent M. tuberculosis infection. The combination of IF QFT-GIT method with TST would provide successful diagnostic screening for LTBI in JIA, particularly prior to anti-tumor necrosis factor treatment. Long-term prospective studies are still necessary to appreciate the advantages and the applicability of these tests in pediatrics.

[Three step MIRU-VNTR for routine mycobacteriology laboratory practice]. / Rutin mikobakteriyoloji laboratuvarlari için üç adimda MIRU-VNTR.

Several methods are available for the molecular typing of Mycobacterium tuberculosis complex isolates. The results of the recent research demonstrated that Mycobacterial Interspersed Repetitive Unit (MIRU)-Variable Number Tandem Repeats (VNTR) method has high discriminatory power and reproducibility, is easy to perform, and available for multi-center studies and automation. However, there is insufficient data about the MIRU-VNTR profiles in Turkey. The aim of this study was to determine the most appropriate MIRU-VNTR combinations to distinguish cross contaminations and nosocomial infections in routine mycobacteriology laboratory practice. Following molecular typing of 152 clinical isolates which were consecutively isolated from different patients in two years period (August 2004-July 2006) in our laboratory, a retrospective analysis of MIRU-VNTR data of 12 loci primers was performed by an "in-house" computer based programme. The programme was prepared by using Microsoft QuickBASIC programming language and all of the data were calculated by the help of this programme. The best combinations to differentiate the clusters and to identify the unique isolates were determined out of 4095 possible results of 12 different primer pairs. According to our 152 MIRU-VNTR results, to determine cross contaminations and nosocomial infections in routine mycobacteriology laboratory practice, we recommend to use primers 26, 40, 16, 10 and 23 in the first step; primers 31, 27, 20 and 2 in the second step, and primers 4, 24 and 39 in the third step. The created software is user friendly, fast and meets the requirements of routine clinical mycobacteriology laboratories. Besides its discriminatory power, the speed and cost-effectiveness of a typing method is also considerable. According to the results of this study it was suggested that for more rapid and economic molecular typing of M.tuberculosis and related epidemiological investigations, MIRU-VNTR should be performed in a stepwise manner.

Molecular characterization of Mycobacterium tuberculosis isolates from Izmir, Turkey.

In recent years, molecular typing methods have been used in epidemiologic studies of Mycobacterium tuberculosis isolates in various areas of the world. However, there have been few data on this issue in Turkey. We describe the molecular characterization of 56 Mycobacterium tuberculosis isolates recovered from individual patients in Izmir and the surrounding area by three different molecular methods. Isolated M. tuberculosis strains were characterized by IS6110 RFLP, spoligotyping and major genetic group designation. In total, 51 RFLP and 35 spoligopatterns were identified. Fourteen (25%) isolates were indicated as low copy number. Based on three genotypic characterization methods together, five clusters with two isolates each were identified. Most of the isolates (98.2%) were assigned as genetic groups 2 or 3. Only one isolate was identified as Beijing family strain (principal genetic group 1). The shared international clades were found to be Beijing-family, var T1 (ST 37), LAM (Latin-American-Mediterranean) 7 (ST 41), LAM 9 (ST 42), Haarlem 1 (ST 47), Haarlem 3 (ST 50) and T1 (ST 53). In this study, IS6110 RFLP, spoligotyping and major genetic group designation were found to be useful methods for molecular epidemiologic studies.

Is it valuable to examine more than one sputum smear per patient for the diagnosis of pulmonary tuberculosis?

The simplest, cheapest, and fastest diagnostic method for tuberculosis (TB) is the detection of acid-fast bacilli (AFB) by microscopy. The algorithm advised for the diagnosis of TB recommends examination of three consecutive sputum specimens from TB suspects for the presence of AFB. In the present study, we evaluated the contribution of each specimen to the final detection of TB suspect patients with culture-proven disease. The collection and analysis of retrospective data on patients with culture-proven pulmonary TB, from June 2002 to August 2006 at Dokuz Eylul University Hospital, Turkey, have enabled us to assess the value of examining two sputum specimens in diagnosing this disease. AFB were detected from one or more sputum specimens with direct microscopy in 42% of the cases. An analysis of results of smear examination showed that 97% of AFB were detected from the first specimen and only 3% were obtained from the second smear. The third specimen did not have any additional diagnostic value for the detection of AFB by microscopy. As a conclusion the present study shows that examining two sputum smears is sufficient for the early detection of AFB in our laboratory.

The role of calcium utilization of intestinal flora in urinary calcium excretion.

OBJECTIVE: We aimed to evaluate whether Ca(+2) utilization of intestinal flora (IF) has an effect on urinary excretion of Ca(+2) (UCaE) levels. MATERIALS AND METHODS: Fecal samples (0.1 g/mL) of children who underwent UCaE examination in the past year were implanted in broths. Labeled (45)Ca (5 muL) was added to the samples and incubated. From these samples, a 200-muL quantity was filtrated with a 0.45-micrometer membrane and was rinsed in 200 muL pure water. (45)Ca activity in the membrane was measured and defined as percent activity per bacteria ((45)Ca(act) %/CFU). Levels of aerobic and anaerobic (45)Ca(act) %/CFU and their correlations with UCaE were compared between hypercalciuric (Group I) and normocalciuric (Group II) patients. RESULTS: Levels of (45)Ca %/CFU were similar between groups (P > .05). Aerobic and anaerobic (45)Ca(act)%/CFU levels were not significantly correlated to UCaE, either in normocalciuric (P = .079, r = -0.503; P = .260, r = -0.420, stray mart respectively) or in hypercalciuric children (P = .509, r = 0.223; P = .623, r = -0.257, respectively). CONCLUSION: Similar (45)Ca(act)%/CFU levels in the 2 groups imply that calcium utilization of IF does not have a distinct effect on UCaE.

Ultrastructural stages of biofilm development of Escherichia coli on urethral catheters and effects of antibiotics on biofilm formation.

OBJECTIVES: Biofilm formation on biomaterials by various kinds of bacteria renders the infection chronic, and the bacteria can become resistant to the immune system and antibiotics. Developmental biofilm stages of Escherichia coli on urethral catheters have not yet been thoroughly demonstrated. We aimed to show biofilm formation of E. coli on urethral catheters, and the effects of various antibacterial agents on this formation using scanning electron microscopy. METHODS: Using urine infected with uropathogenic E. coli type O4 (10(5) to 10(6) colony forming units/mL), biofilm was formed on latex/silicone balloon catheters in a modified Robbin's device. The study included an infected-only group and four antibiotic study groups (ciprofloxacin, cefuroxime, gentamicin, and trimethoprim). The catheters were infused with the antibiotic solutions once before placement in the modified Robbin's devices. Ten 5-mm catheter samples were taken for all groups on the first, fourth, and seventh days. The 4 and 12-hour and 2-day samplings were also taken from the infected-only group. The catheter samples were evaluated by scanning electron microscopy and given scores according to the level of formation. RESULTS: The biofilm layers emerged between 4 and 12 hours after infection in the infected-only group and had developed completely between 12 and 24 hours. The antibiotics, especially cefuroxime, significantly delayed this process for up to 4 days. However, the biofilm had developed completely in almost all catheter samples after 4 to 7 days. CONCLUSIONS: Biofilm of E. coli on urethral catheters had completed their maturation at 12 to 24 hours. For short-term urethral catheterization, a single dose of antibiotic can delay the development of biofilm for up to 4 days but eventually cannot prevent it.

[Microbiological results of bronchoalveolar lavage that was performed for opportunistic pulmonary infections]. / Firsatçi pulmoner infeksiyon ön tanisiyla yapilan bronkoalveoler lavajin mikrobiyolojik sonuçlari.

Between 2001-2002; in 62 cases, 33 (53%) male, 29 (47%) female, mean age 51.4 +/- 18.1 years) bronchoalveolar lavage (BAL) was performed for diagnosis of opportunistic pulmonary infection and specimens were evaluated for results of microbiological examinations. There was hematological malignancy in 18 (29%) and solid organ malignancy in 13 (21%) cases. Thirty-one (50%) cases were immunocompromised for reasons other than malignancy. By endoscopic evaluation endobronchial lesion was seen in 2 (3%) cases, indirect tumor signs were seen in 2 (3%) cases and signs of infection were seen in 11 (18%) cases. Forty-even (76%) cases were endoscopically normal. Acid-fast bacilli (AFB) direct examination was positive in 3 (5%) cases. In 4 (6%) cases mycobacterial culture was positive, Mycobacterium tuberculosis-polymerase chain reaction (PCR) was also positive in these four cases. Examination of gram-stained smears for bacteria was associated with infection in 14 (23%) cases. Bacteriologic cultures were positive for single potential pathogen in 10 (16%) cases, and for mixed pathogens in 7 (11%) cases for a total number of 17 (27%). Fungal cultures were positive in 3 (5%) cases all of which had hematological malignancy. As a result in 24 (39%) cases microbiological agent of infection is determined: in four mycobacteria, in 17 bacteria other than mycobacteria and in three fungi.

Evaluation of Cobas Amplicor MTB test to detect Mycobacterium tuberculosis in pulmonary and extrapulmonary specimens.

Tuberculosis (TB) is one of the major public health problems in the world. Effective control of TB depends on rapid and correct diagnosis and appropriate treatment. The aim of this study was to evaluate the performance of Cobas Amplicor MTB (CA-MTB) test for pulmonary and extrapulmonary specimens isolated in our laboratory. A total of 424 specimens obtained from the suspected TB patients from January 2003 to August 2004 were included in this study. All specimens (173 pulmonary and 251 extrapulmonary specimens) were processed, stained, cultured and assayed using the CA-MTB test for identification of Mycobacterium tuberculosis. The CA-MTB test results were compared to culture and acid-fast staining as gold standard. The sensitivity, specificity, positive and negative predictive values of CA-MTB were determined as 73%, 100%, 100%, and 97% for pulmonary specimens, and 45%, 100%, 100% and 96% for extrapulmonary specimens respectively. The sensitivity of the test for acid-fast bacilli (AFB) smear positive pulmonary and extrapulmonary specimens was 92% and 75%. These results indicate that the CA-MTB is a rapid test for detection of tuberculosis in pulmonary specimens, but does not perform well enough in extrapulmonary specimens.