[Viral Respiratory Tract Infection Agents Detected Between Years 2019-2021, COVID-19, and Co-Infections]. / 2019-2021 Yillari Arasinda Saptanan Viral Solunum Yolu Enfeksiyonu Etkenleri, COVID-19 ve Ko-Enfeksiyonlar.

Respiratory tract infections are a major cause of morbidity and mortality at all ages and are seen as a very important public health problem all over the world. In this study, we aimed to evaluate the effect of the pandemic on the epidemiological and seasonal characteristics of the agents by analyzing the respiratory viral infection agents, viral co-infections and associations with Coronavirus diseases-2019 (COVID-19) studied by multiplex polymerase chain reaction (PCR) test in the molecular microbiology laboratory in a three-year period, including the one-year period before the pandemic. Between March 2019 and December 2021, 8825 respiratory tract specimens accepted to the molecular microbiology laboratory with respiratory tract multiplex PCR test requests were included in the study. In addition, severe acute respiratory syndrome (SARS-CoV-2) PCR test results of the patients with positive results with respiratory tract multiplex PCR test, which were studied within ± 3 days, were evaluated retrospectively. Respiratory viral pathogens were detected using FTD Respiratory Pathogens 21 kit (Fast Tract Diagnostics, Siemens Healthineers Company). Two different kits based on real-time reverse transcription PCR were used for SARS-CoV-2 RNA detection in different periods. According to our results, at least one viral agent was detected in 2156 (24.4%) of a total of 8825 samples and a single agent was detected in 1843 (85.5%) of these. The distribution of viruses in the samples with a single agent was determined as RV, RSV A/B, HCoVs, AdV, flu A virus, MPV A/B, PIV 1-4, flu B virus, EV, BoV and PeV, in order of frequency. Multiple agents were found in 313 (14.5%) of these 2156 samples. They were found to be two agents in 291 samples, three in 21 samples and four in one sample. When the SARS-CoV-2 PCR test results of the patients who had positive results with respiratory tract multiplex PCR and who were studied within ± 3 days were evaluated retrospectively, SARS-CoV-2 RNA was detected in 45 (3.5%) of 1277 samples in which at least one agent was detected. In four of these patients, SARS-CoV-2 was found together with multiple agents. Consequently, there was a sharp decrease in the prevalence of all viral agents during the pandemic period. It was evaluated that besides the COVID-19 infection, the restrictions applied during the pandemic period were also effective in this situation.

[Comparison of two commercial DNA extraction kits and PCR master mixes for the detection of Brucella from blood samples and blood culture bottles]. / Kandan ve kan kültür siselerinden Brucella saptanmasinda iki farkli ticari DNA ekstraksiyon kiti ve PCR master kiksinin karsilastirilmasi.

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.

Investigation of carbapenemase and mcr-1 genes in carbapenem-resistant Klebsiella pneumoniae isolates.

INTRODUCTION: Carbapenem-resistant Klebsiella pneumoniae are a major problem. We aimed to investigate carbapenemase-encoding genes and transferable mcr-1 genes among 57 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates from hospitalized patients. METHODOLOGY: Antibiotic susceptibility tests were performed by Phoenix (BD). Results for ertapenem and colistin were confirmed by gradient diffusion and microdilution methods. Carbapenemase and mcr-1 genes were investigated by Polymerase Chain Reaction (PCR). RESULTS: Thirty-two (56.14%) isolates were from intensive care units (ICU). Antibiotic resistance rates by Phoenix: 52.63% for amikacin; 73.69% trimethoprim sulfamethoxazole; 91.23% cefepime; 82.46% tigecycline; 59.65% colistin. Carbapenemases positivity: 82.45% (47) for blaOXA-48, 40.35% (23) blaOXA-55, 3.50% (2) blaOXA-51, 1.75% (1) blaOXA-23, 1.75% (1) blaOXA-24, 1.75% (1) blaIMP. blaOXA-58, blaKPC, blaNDM-1, and blaVIM were not detected. Twenty (35.08%) isolates had both blaOXA-48 and blaOXA-55. Three isolates were mcr-1 (+) and blaOXA-48 (+). One mcr-1 (+) isolates was blaOXA-51 (+). One colistin sensitive isolate determined by Phoenix, was found colistin resistant by microdilution. CONCLUSION: OXA-48 and OXA-55 co-harboring isolates and mcr-1 gene (+) isolates were spreading. Automated colistin susceptibility results should be confirmed by microdilution method. Resistance mechanisms in Enterobacteriaceae should be determined and the isolates should be monitored by molecular epidemiological methods. Effective infection control measures will contribute to reduce risk of antibiotic resistant bacterial infections and dissemination of antibiotic resistance.

Analysis of the effect of smoking on the buccal microbiome using next-generation sequencing technology.

PURPOSE: This study aimed to investigate the effect of smoking on the buccal microbiome and to analyse the descriptive ability of each of the seven hypervariable regions in their 16S rRNA genes. METHODOLOGY: Microbiome compositions of 40 buccal swab samples collected from smokers (n =20) and non-smokers (n =20) were determined using 16S rRNA sequencing. Seven different 16S rRNA hypervariable regions (V2, V3, V4, V6-7, V8 and V9) in each sample were amplified using the Ion Torrent 16S Metagenomics kit and were sequenced on the Ion S5 instrument. RESULTS: Seven hypervariable regions in the 16S rRNA gene were successfully sequenced for all samples tested. The data obtained with the V2 region was found to be informative but the consensus data generated according to a number of operational taxonomic unit reads gathered from all seven hypervariable regions gave the most accurate result. At the phylum level, no statistically significant difference was found between smokers and non-smokers whereas relative abundances of Veillonella atypica, Streptococcus australis, Prevotella melaninogenica, Prevotella salivae and Rothia mucilaginosa showed significant increases in the smoker group (P-adj=0.05). Alpha diversity results did not show a significant difference between the two groups; however, beta diversity analysis indicated that samples of smoker and non-smoker groups had a tendency to be clustered within themselves. CONCLUSION: The results of the current study indicate that smoking is a factor influencing buccal microbiome composition. In addition, sequencing of all seven hypervariable regions yielded more accurate results than those with any of the single variable regions.