Effect of climate change on nature and human health with a special focus on infectious diseases in the Mediterranean region.

INTRODUCTION: In this short review, the effect of climate change on nature and human health with a special focus on infectious diseases in the Mediterranean region is discussed. This research is a part of the Mediterranean Convention of Human Rights project, which is an organizational work on human rights issues that was established in cooperation with civil society and the national authorities of the Mediterranean Region. METHODOLOGY: Previously published data were collected by retrieving published literature from PubMed, Google Scholar, and Web of Science using "climate change", "the Mediterranean region", "infections in Mediterranean Region", "infectious diseases", "biodiversity", and "the Mediterranean Sea" as keywords. The collected data were then evaluated and reviewed. The recommendations and guidelines were analysed by the preferred reporting items for systematic reviews and meta-analyses (PRISMA). CONCLUSIONS: The Mediterranean region presents a typical example witnessing a dramatic change in climate events and their adverse impact on biodiversity, ecosystems and public health are multiple. This negative impact is in part due to the geographical particularities, and sociocultural and geopolitical conflicts that are progressively worsening the burden of climate change. While most of these changes cannot be totally avoided, many of the health risks related to climate change could be monitored. This can be done by establishing health systems with policies to reduce and prevent the risks of infectious diseases and to recover and support the affected areas, which may identify priority and management of high-risk events.

Molecular epidemiological investigation of carbapenem resistant Klebsiella pneumoniae isolated from intensive care unit patients of six geographical regions of Turkey.

INTRODUCTION: Klebsiella pneumonia causes serious infections in hospitalized patients. In recent years, carbapenem-resistant infections increased in the world. The molecular epidemiological investigation of carbapenem-resistant K. pneumoniae isolates was aimed in this study. METHODOLOGY: Fifty carbapenem-resistant K. pneumoniae isolates from six geographical regions of Turkey between September 2019-2020 were included in the study. The disk diffusion method was used for the antibiotic susceptibility testing. The microdilution confirmed colistin susceptibility. Genetic diversity was investigated by MLST (Multi-Locus Sequence Typing). RESULTS: The resistance rates were as follows: 49 (98%) for meropenem, 47 (94%) imipenem, 50 (100%) ertapenem, 30 (60%) colistin and amoxicillin-clavulanate, 49 (98%) ceftriaxone, 48 (96%) cefepime, 50 (100%) piperacillin-tazobactam, 47 (94%) ciprofloxacin, 40 (80%) amikacin, 37 (74%) gentamicin. An isolate resistant to colistin by disk diffusion was found as susceptible to microdilution. ST 2096 was the most common (n:16) sequence type by MLST. ST 101 (n:7), ST14 (n:6), ST 147 and ST 15 (n:4), ST391 (n:3), ST 377 and ST16 (n:2), ST22, ST 307, ST 985, ST 336, ST 345, and ST 3681 (n:1) were classified in other isolates. In Istanbul and Ankara ST2096 was common. Among Turkey isolates, the most common clonal complexes (CC) were CC14 (n:26) and CC11 (n = 7). CONCLUSIONS: In Turkey, a polyclonal population of CC14 throughout the country and inter-hospital spread were indicated. The use of molecular typing tools will highlight understanding the transmission dynamics.

COVID-19 vaccine hesitancy in Turkey: A systematic review and meta-analysis.

This systematic review and meta-analysis aims to estimate the prevalence of coronavirus disease 2019 (COVID-19) vaccine hesitancy in Turkey, which can aid future health policies and strategies. A comprehensive search was conducted on various databases using keywords related to COVID-19 vaccine hesitancy in Turkey. Quality assessment was performed using Joanna Briggs Institute (JBI) checklist for prevalence studies. Data extraction was conducted. The random effect model (DerSimonian and Laird method) was used in pooled prevalence data analysis (95% confidence interval [CI]). A total of 1,072 articles were identified. After removing duplicates and excluding articles, 61 articles remained for bias assessment. Among these, 19 articles with low risk of bias were included in the review and meta-analysis. Total population included in the analysis was 15,164, vaccine hesitancy was 30.5% (95% Cl: 24.3-36.8%). Prevalence of the vaccine hesitancy was found to be 39.8% (95% Cl: 31.4-48.2%) in studies conducted before the initiation of vaccination, while in studies conducted after the commencement of vaccination, hesitancy was 20.4% (95% Cl: 12.9-28%). We suggest conducting high-quality studies in different populations to understand the level of vaccine hesitancy, as many of the previous studies have mainly focused on healthcare workers and students, and rest were community-based studies, which have generally shown high bias. Also, we suggest that early vaccination can reduce vaccine hesitancy.

First year real life experience with tenofovir alafenamide fumarate: The pythagorean cohort.

Background and Aim: In chronic hepatitis B infection, antiviral therapy significantly reduces the incidence of complications. This study aimed to present real-life 12-month effectiveness and safety data for TAF. Materials and Methods: This Pythagoras Retrospective Cohort Study included patients from 14 centers in Turkiye. The study presents 12-month results of 480 patients treated with TAF as initial therapy or after switching from another antiviral drug. Results: The study shows treatment of about 78.1% patients with at least one antiviral agent (90.6% tenofovir disoproxil [TDF]). The rate of undetectable HBV DNA increased in both treatment-experienced and naive patients. In TDF-experienced patients, the rate of alanine transaminase (ALT) normalization increased slightly (1.6%) within 12 months, but the change was not statistically significant (p=0.766). Younger age, low albumin, and high body mass index and cholesterol were identified as risk factors for abnormal ALT after 12 months, but no linear relationship was detected. In TDF-experienced patients, renal and bone function indicators showed significant improvement three months after the transition to TAF and remained stable for 12 months. Conclusion: Real-life data demonstrated effective virological and biochemical responses with TAF therapy. After switching to TAF treatment, gains in kidney and bone functions were achieved in the early period.

Culture and culture-independent diagnostic tests in Campylobacter enteritis.

INTRODUCTION: Campylobacter infections are among the most common causes of bacterial enteritis. This study aims to determine the sensitivity, specificity and positive predictive values (PPV) of culture and culture-independent tests for the diagnosis of Campylobacter enteritis. METHODOLOGY: A total of 400 stool samples were included in the study. BD MAX enteric bacterial panel (BD Diagnostics, Franklin Lakes, NJ, USA) and EntericBio Gastro Panel II (Serosep, Limerick, Ireland) were used as commercial molecular tests. RIDA®QUICK Campylobacter (R-Biopharm, Darmstadt,, Germany) and CerTest (Biotec, Zaragoza, Spain) were used to detect Campylobacter antigens. Samples were cultured in CCDA media and subjected to bacterial identification by mass spectrometry. RESULTS: Among the 400 specimens, 41 (10.2%) were evaluated as Campylobacter positive; 21 were culture-positive and 20 were detected as positive by both PCR methods. Of the 21 isolates grown in culture, 16 (76.2%) were identified as C. jejuni and 5 (23.8%) as C. coli. While all 21 culture-positive specimens were detected as positive by both molecular tests, 18 of the specimens were found positive by RidaQuick, and 16 by Certest ICA. Of the 20 culture-negative Campylobacter cases, 18 were positive by RidaQuick and 12 by Certest ICA. Sensitivities of culture, ICA-RidaQuick and ICA-CerTest were 51.2%, 87.8 and 68.3, respectively. The specificities of all tests were in the range of 90-100 %. PPV of molecular tests, ICA-RidaQuick and ICA-CerTest were > 95%, 72 % and 48.3 %, respectively. CONCLUSIONS: Molecular tests were superior to culture and ICA in terms of sensitivity, specificity, and positive predictive value.

Occult hepatitis B infection in Turkish HIV-infected patients: A multicentre, retrospective, cross-sectional study, Schindler study.

OBJECTIVE: Occult hepatitis B infection (OHBI) appears to have a higher prevalence in populations at high risk for hepatitis B virus (HBV) infection with concomitant liver disease. The aim was to assess the prevalence of OHBI in a sample of human immunodeficiency virus -1 positive and HBV surface antigen-negative (HIV-1+/HBsAg-) Turkish patients. METHODS: Ten centres in Turkey were included in the study. Patients were selected on the basis of a power calculation with a known population size of HIV-positive patients and a reported prevalence of OHBI. Gender, age, occupation, place of residence, treatment and clinical status, and laboratory results, including immunodeficiency panel, antibody tests, hemogram, biochemistry, and coagulation studies were evaluated retrospectively. RESULTS: The number of HIV-infected patients followed in these centres was 3172 and the sample population numbered 278. All 278 were HBsAg negative. The mean age of the sample was 37.2 ± 13.1 years and 235 (84.5%) were male. All but one patient (99.6%) had been treated with antiretroviral therapy. Of the 278 patients, 169 (60.6%) were positive for Anti-HBs and 125 (44.8%) were positive for Anti-HBc IgG. HIV RNA was detected in 203/278 (73%) of the patients. Four HBV DNA (1.4%) were diagnosed with OHBI. There was no significant difference in hemogram, hemoglobin or bilirubin concentrations in those with OHBI compared with the other patients. CONCLUSION: In a representative sample of HIV+ patients from 10 Turkish centres, the prevalence of OHBI was found to be 1.4%. In HIV positive patients, it is important to identify those with OHBI for optimal clinical management and prognosis.

A Healthcare-Associated Outbreak of Urinary Tract Infections Due to Myroides odoratimimus.

Myroides spp. are low-grade opportunistic pathogens. Outbreaks due to Myroides spp. have rarely been described in the literature to date. We report a healthcare-associated outbreak of urinary tract infections (UTIs), caused by Myroides odoratimimus, in a Turkish hospital. As of March 2019 until May 2019, 6 strains of M. odoratimimus were isolated from the urine samples of patients, all of whom were hospitalized in intensive care units. After identification and antibiotic susceptibility testing using the VITEK 2 system, MALDI-TOF-MS and 16S rRNA-based sequencing methods were performed for confirmation and species-level identification. Pulsed-field gel electrophoresis (PFGE) was performed in order to investigate the clonal relatedness of the isolates. All the patients were immunocompromised and underwent urinary catheterization. None of the patients had urinary neoplasm, surgery, or calculi. VITEK 2 and MALDI-TOF-MS systems revealed that the isolates belonged to the Myroides genus; however, the aforementioned systems neglected to identify the isolates at the species level. The isolates were all successfully identified as M. odoratimimus through 16S rRNA-based sequencing. The isolates were resistant to every antibiotic tested. All isolates had an indistinguishable PFGE pattern, thus indicating cross-transmission between cases. Although M. odoratimimus is rarely isolated from human specimens, clinicians should be aware of its ability to cause UTIs and infectious outbreaks.

Convalescent plasma therapy in patients with COVID-19.

There are currently no licensed vaccines or therapeutics for COVID-19. Anti-SARS CoV-2 antibody-containing plasmas, obtained from the recovered individuals who had confirmed COVID-19, have been started to be collected using apheresis devices and stored in blood banks in some countries in order to administer to the patients with COVID-19 for reducing the need of intensive care and the mortality rates. Therefore, in this review, we aim to point out some important issues related to convalescent plasma (CP) and its use in COVID-19. CP may be an adjunctive treatment option to the anti-viral therapy. The protective effect of CP may continue for weeks and months. After the assessment of the donor, 200-600 mL plasma can be collected with apheresis devices. The donation interval may vary between countries. Even though limited published studies are not prospective or randomized, until the development of vaccines or therapeutics, CP seems to be a safe and probably effective treatment for critically ill patients with COVID-19. It could also be used for prophylactic purposes but the safety and effectiveness of this approach should be tested in randomized prospective clinical trials.

A healthcare-associated outbreak of urinary tract infections due to Myroides odoratimimus.

Myroides spp. are low-grade opportunistic pathogens. There were only a few outbreaks due to Myroides spp. described in the literature to date. We report a healthcare-associated outbreak of urinary tract infections caused by Myroides odoratimimus in a Turkish hospital. From March to May 2019, six strains of M. odoratimimus were isolated from the urine samples of patients hospitalized in the intensive care units (ICUs). After identification and antibiotic susceptibility testing with VITEK 2 system, MALDI-TOF-MS and 16S rRNA based sequencing methods were performed for confirmation and species level identification. Pulsed-field gel electrophoresis (PFGE) was used to investigate clonal relatedness of the isolates. All the patients were immunocompromised and underwent urinary catheterization. None of them had urinary neoplasm, surgery or calculi. VITEK 2 and MALDI-TOF-MS systems revealed that the isolates belong to the Myroides genus but lacked to identify the isolates at the species level. 16S rRNA based sequencing successfully identified all the isolates as M. odoratimimus. The isolates were resistant to all antibiotics tested. All isolates had indistinguishable PFGE pattern indicating cross-transmission between cases. Although M. odoratimimus is rarely isolated from human specimens, clinicians should be aware of its ability to cause UTIs and outbreaks.

How CRISPR-Cas System Could Be Used to Combat Antimicrobial Resistance.

Rapid emergence of antibiotic-resistant bacteria has made it harder for us to combat infectious diseases and to develop new antibiotics. The clustered regularly interspaced short palindromic repeats - CRISPR-associated (CRISPR-Cas) system, as a bacterial adaptive immune system, is recognized as one of the new strategies for controlling antibiotic-resistant strains. The programmable Cas nuclease of this system used against bacterial genomic sequences could be lethal or could help reduce resistance of bacteria to antibiotics. Therefore, this study aims to review using the CRISPR-Cas system to promote sensitizing bacteria to antibiotics. We envision that CRISPR-Cas approaches may open novel ways for the development of smart antibiotics, which could eliminate multidrug-resistant (MDR) pathogens and differentiate between beneficial and pathogenic microorganisms. These systems can be exploited to quantitatively and selectively eliminate individual bacterial strains based on a sequence-specific manner, creating opportunities in the treatment of MDR infections, the study of microbial consortia, and the control of industrial fermentation.

Clinical manifestation, diagnosis, prevention and control of SARS-CoV-2 (COVID-19) during the outbreak period.

The novel coronavirus SARS-CoV-2 (Covid-19), spreading from Wuhan, China, is one of the causes of respiratory infections that can spread to other people through respiratory particles, and can cause symptoms such as fever, dry cough, shortness of breath, anorexia, fatigue and sore throat in infected patients. This review summarizes current strategies on the diagnosis. Additionally, treatments, infection prevention and control of the SARS-CoV-2 are addressed. In addition to the respiratory system, this virus can infect the digestive system, the urinary system and the haematological system, which causes to observe the virus in the stool, urine and blood samples in addition to throat sample. The SARS-CoV-2 causes changes in blood cells and factors and makes lung abnormalities in patients, which can be detected by serological, molecular, and radiological techniques by detecting these changes and injuries. Radiological and serological methods are the most preferred among the other methods and the radiological method is the most preferred one which can diagnose the infection quickly and accurately with fewer false-negatives, that can be effective in protecting the patient's life by initiating treatment and preventing the transmission of infection to other people.

Optimization and validation of a real-time polymerase chain reaction protocol for the diagnosis of human brucellosis.

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.

Antisense peptide nucleic acids againstftsZ andefaA genes inhibit growth and biofilm formation of Enterococcusfaecalis.

Enterococcus faecalis is one of the important causes of nosocomial infections. Nowadays, increasing prevalence of antibiotic-resistant bacteria and slow progress in recognizing new antimicrobial agents has limited the efficiency of conventional antibiotics, which cause to find novel strategies to overcome bacteria. Therefore, in this study, we aimed to assess the role of efaA gene in the biofilm formation and the role of ftsZ gene in the controlling of bacterial growth by the anti-sense PNAs(Peptide Nucleic Acid).E. faecalis ATCC® 29212™was used for the study of PNAs designed to targeting the start codon section of the ftsZ andefaA genes. PNA attachment to RNA was confirmed by blotting. Electroporation technique was used for the intracellular transfer of anti-ftsZ PNAs. The spot-plating method was used to the assessment of alteration in bacterial growth. Biofilm formation assay and real-time PCR were used for detection of biofilm inhibitory effect of cell penetrating peptide (CPP) conjugated to anti-efaA PNAs.ByftsZ PNAs treatment, no growth was seen from the strain in agar by a spot plating method and the inhibition zone of anti-ftsZ PNAs was not seen. PNAs against the efaA gene decreased by 95% the expression of the efaA gene and biofilm formation. In addition, the(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) MTT assay showed no toxicity on MCF7 cells for both of anti-ftsZand anti-efaA PNAs.This study used new genetic and molecular tools to inhibit pathogenicity and infection by E. faecalis. In this study, we suggested that efaA gene plays a critical role in the biofilm formation and anti-efaA PNAs could decrease the formation of biofilm, as well as, anti-ftsZ PNAs could eliminate bacterial growth.

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.

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.

Antimicrobial Activity of Garlic Derivatives on Common Causative Microorganisms of the External Ear Canal and Chronic Middle Ear Infections.

OBJECTIVE: Today, antibiotic resistance is increasing and evolving into an important health problem. Therefore, it is important to research on alternative therapies to antibiotics. This study aimed to investigate the inhibitory effect of four garlic derivatives on microorganisms commonly isolated in ear infections. METHODS: The antimicrobial activities of allicin, s-allyl cysteine (SAC), diallyl disulfide (DADS), and s-allyl mercaptocysteine (SAMC) were investigated on standard strains of commonly isolated microorganisms using the broth microdilution method. The test strains were selected among the microorganisms responsible for chronic suppurative otitis media and otitis externa. These microorganisms were Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, Candida albicans, and Candida tropicalis. RESULTS: Minimum inhibitory concentration (MIC) values of allicin and SAC ranged from 0.125 to 20 µg/mL for fermentative bacteria (E. coli and K. pneumoniae), 20 to 80 µg/mL for non-fermentative bacteria (P. aeruginosa and A. baumannii), 5 to 10 µg/mL for gram-positive cocci (S. aureus and E. faecium), and 40 to 80 µg/mL for yeasts (C. albicans and C. tropicalis). MIC values of DADS ranged from 40 to 80 µg/mL for fermentative bacteria, 40 to 160 µg/mL for non-fermentative bacteria, 40 to 80 µg/mL for gram-positive cocci, and 20 to 40 µg/mL for yeasts. The MICs of SAMC were >640 µg/mL for the tested bacteria and yeasts. CONCLUSION: Both allicin and SAC showed antimicrobial activity against the tested microorganisms, even at low concentrations. These two derivatives may be used to treat infections in the future.

Comparison of multiplex real-time polymerase chain reaction with serological tests and culture for diagnosing human 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.

[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.

Role of efflux pump and OprD porin expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates.

INTRODUCTION: In recent years, the prevalence of multidrug-resistant P. aeruginosa has remarkably increased. Thus, we wanted to investigate the carbapenem resistance mechanisms and clonal relationship among 80 carbapenem-resistant P. aeruginosa strains. METHODOLOGY: Carbapenemase production was detected using the Modified Hodge Test (MHT), EDTA combined disc method (ECD), and PCR. Expression levels of efflux and porin genes were mesured by real-time reverse transcription PCR. Clonal relationship of the isolates was investigated by pulsed-field gel electrophoresis (PFGE). RESULTS: Carbapenemase production was detected in 7.5% of the isolates with MHT/ECD tests and in 11.3% of the isolates with PCR. Although the specificity of MHT/ECD was high, the sensitivitivity was low. oprD downregulation and mexB, mexY, and mexD overexpression were demonstrated in 55%, 16.3%, 2.5%, and 2.5% of the isolates, respectively. Multiple carbapenem resistance mechanisms were found in nearly a quarter of the isolates. PFGE typing of the 80 P. aeruginosa isolates yielded 61 different patterns. A total of 29 isolates (36.3%) were classified in 10 clusters, containing 2 to 7 strains. We could not find a strict relationship between PFGE profile and carbapenem resistance mechanisms. CONCLUSIONS: Although oprD downregulation and MexAB-OprM overexpression were the most common mechanisms, carbapenem resistance was associated with multiple mechanisms in the study. MHT/ECD tests should not be used alone for investigation of carbapenemase production in P. aeruginosa. Rapid tests with high sensitivity and specificity should be developed for the detection of carbapenemase production in P. aeruginosa.

Assessment of the underlying causes of the immune thrombocytopenia: Ten years experience.

OBJECTIVE: Immune thrombocytopenia (ITP) is an immune haematologic disorder causing platelet destruction mediated by anti-platelet antibodies. In this study we aimed to evaluate the clinical and laboratory variables of ITP patients in southeast of Turkey. METHODS: In this retrospective study 167 ITP patients between 2005 and 2015 were evaluated. All patients were screened for immunological parameters including ANA (antinuclear antibodies), anti dsDNA (anti-double-stranded-DNA), ACA(anti-cardiolipin) IgM and IgG, LA (lupus anticoagulants). All patients were screened for Helicobacter pylori, HBsAg (Hepatitis B surface antigen), anti-HCV (hepatitis C virus antibody), and anti-HIV ½ (HIV antibody) and brucellosis.. RESULTS: Among the patients, 50 (29.9%) patients were male, 117 (70.1%) were female. The age range of patients was 18-86 (mean 38.16±14). In 56 patients (33.5%) splenectomy was performed. 36 patients (21.6%) were positive for ANA, 5 (3%) were positive for anti dsDNA, 14 (8.4%) for ACA Ig G, and 14 (8.4%) patients for ACA IgM. LA was tested in 165 patients and 30 (18%) patients were positive for LA. Microbiologic evaluation was as follows: 16 patients (9.6%) were positive for HbsAg, 109 (65.3%) positive for Anti-HBs, 5 positive for anti-HCV (3%), 56 (33.5%) patients were positive for Helicobacter pylori antigen, 5 (2.9%) for Brucella and one patient was positive for anti-HIV ½. CONCLUSIONS: Immune thrombocytopenia patients have to be evaluated according to their demographic characteristics and laboratory results. Secondary causes of ITP were HIV, HCV, Helicobacter pylori, brucellosis, tuberculosis, and autoimmune diseases in our region. Management of ITP patients can change in different regions.