Investigation of efflux pump genes in isoniazid resistant Mycobacterium tuberculosis isolates.

BACKGROUND: Tuberculosis (TB) is one of the most important infectious diseases worldwide. Resistance to antituberculosis drugs develops because of genetic mutations that render drug-activating enzymes inactive, changes in cell wall permeability, and increased expression of efflux pump genes and also combination therapy with efflux pump inhibitors may be more effective in drug-resistant TB patients. AIMS: To investigate the effect of verapamil (VR) on isonicotinic acid hydrazide (INH) resistance and the expression of 21 efflux pump genes in INH monoresistant MTBC clinical isolates. STUDY DESIGN: In vitro study. METHODS: In our mycobacteriology laboratory, 10 INH monoresistant and 10 primary anti-TB drug-susceptible MTBC clinical isolates were selected. Drug susceptibilities for INH and VR were studied by resazurin microtiter plate method and minimum inhibitory concentration (MIC) was determined. Additionally, mRNA gene expressions were investigated by quantitative Real Time Polymerase Chain Reaction for 21 efflux gene regions. RESULTS: While no change was observed in INH MICs of susceptible isolates under VR effect, 6 (60%) of the 10 INH-resistant isolates showed a decrease of less than one dilution in INH MIC under VR effect. VR significantly reduced resistance in resistant isolates (p â€‹< â€‹0.05). INH monoresistant MTBC isolates showed a 2.85-fold expression increase in the Rv1634 region of the Major Facilitator Superfamily efflux family under INH stress (p â€‹= â€‹0.029). No statistically significant change was observed in other efflux gene regions. Herein, increased expression was observed in the Rv1634 region, consistent with other studies in the literature, and this was associated with drug resistance. No significant change in expression was detected in other gene regions. CONCLUSION: The effect of efflux pump inhibitor VR on INH MIC levels is promising for the treatment of resistant TB. However, studies with more resistant strains are needed to evaluate the efficacy of efflux pump genes.

[Comparative Analysis of the Effect of Different Nucleic Acid Extraction Methods on SARS-CoV-2 Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction Results]. / Farkli Nükleik Asit Ekstraksiyon Yöntemlerinin SARS-CoV-2 Kantitatif Gerçek Zamanli Revers Transkripsiyon Polimeraz Zincir Reaksiyonu Sonuçlarina Etkisinin Karsilastirmali Analizi.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of Coronavirus diseases-2019 (COVID-19) disease. Rapid and accurate detection of the virus is vital to prevent transmission and effectively manage the pandemic. The gold standard diagnostic method for this agent is the real-time reverse transcription polymerase chain reaction (qrRT-PCR) test conducted on respiratory tract samples and one of the most critical steps affecting the sensitivity of this test is the nucleic acid extraction stage. However, restrictive factors such as reagent supply and storage conditions limit the testing capacity. Therefore, innovative and cost-effective alternatives are needed to speed up testing and minimize pre-processing steps. The aim of this study was to evaluate the impact and applicability of different methods to enhance the efficiency of the nucleic acid extraction stage in the SARS-CoV-2 qrRT-PCR test. As an alternative to the routinely used viral nucleic acid extraction buffer (vNAT), the modified vNAT method (MvNAT), which includes centrifugation, the R1-R2 kit and the heat treatment (HT) method, was applied to 118 respiratory tract samples. Samples determined with threshold cycle values of (Cq) of ≤ 35 (n= 10), > 35 (n= 42), indeterminate (n= 56) in routine results and negative controls (n= 10) were included in the study. The RNA quantities obtained after extraction for each method were measured and recorded using a spectrophotometric measurement device. All samples were processed using the SARS-CoV-2 qrRT-PCR kit targeting the RdRp region. The results were statistically analyzed using unpaired and paired t-tests and results with a p-value of < 0.05 were considered statistically significant. Excluding negative control samples, while the standard method yielded a Cq value of 48.1% (mean Cq value (Cqmean)= 39.5 ± 6.9) for a total of 108 samples, the MvNAT method produced a Cq value of 11.1% (Cqmean= 38.4 ± 5.2), the R1-R2 kit yielded 14.8% (Cqmean= 35.9 ± 7.1) and HT method resulted in 25% (Cqmean= 31.4 ± 6.3). When the variability in target gene Cq values was analyzed in all samples compared to the standard method, the HT method significantly provided lower Cq values (n= 16; p= 0.007; paired t-test) while the MvNAT method and R1-R2 kit yielded higher Cq values (n= 6; p= 0.025, n= 11; p= 0.004; paired t-test). Sensitivity rates were MvNAT= 31.6%, R1-R2= 57.9%, HT= 84.2%, with 100% specificity for all three methods. The HT method demonstrated a positive extraction efficiency because it is fast, easy and not dependent on reagents. Although this method provided lower Cq values than the standard method, especially in samples with a high viral load, it should be considered that it also has the potential to yield false-negative results in samples with Cq> 35. With this study, it was concluded that the extraction phase of the SARS-CoV-2 qrRT-PCR test can be carried out using various methods that do not require kits or reagents, such as the HT method. However, it is believed that multicenter studies involving a larger number of samples are necessary to standardize the test and assess the possibility of false negatives.

COVID-19-associated pulmonary aspergillosis (CAPA): identification of Aspergillus species and determination of antifungal susceptibility profiles.

Among the co-infectious agents in COVID-19 patients, Aspergillus species cause invasive pulmonary aspergillosis (IPA). IPA is difficult to diagnose and is associated with high morbidity and mortality. This study is aimed at identifying Aspergillus spp. from sputum and tracheal aspirate (TA) samples of COVID-19 patients and at determining their antifungal susceptibility profiles. A total of 50 patients with COVID-19 hospitalized in their intensive care units (ICU) were included in the study. Identification of Aspergillus isolates was performed by phenotypic and molecular methods. ECMM/ISHAM consensus criteria were used for IPA case definitions. The antifungal susceptibility profiles of isolates were determined by the microdilution method. Aspergillus spp. was detected in 35 (70%) of the clinical samples. Among the Aspergillus spp., 20 (57.1%) A. fumigatus, six (17.1%) A. flavus, four (11.4%) A. niger, three (8.6%) A. terreus, and two (5.7%) A. welwitschiae were identified. In general, Aspergillus isolates were susceptible to the tested antifungal agents. In the study, nine patients were diagnosed with possible IPA, 11 patients were diagnosed with probable IPA, and 15 patients were diagnosed with Aspergillus colonization according to the used algorithms. Serum galactomannan antigen positivity was found in 11 of the patients diagnosed with IPA. Our results provide data on the incidence of IPA, identification of Aspergillus spp., and its susceptibility profiles in critically ill COVID-19 patients. Prospective studies are needed for a faster diagnosis or antifungal prophylaxis to manage the poor prognosis of IPA and reduce the risk of mortality.

Evaluation of amino acid profile in serum of patients with Covid-19 for providing a new treatment strategy.

Background: Amino acids have an important role in metabolism and may affect COVID-19-related outcomes. In our study, the amino acid serum level of hospitalized COVID19 patients was evaluated to determine a new treatment strategy. Methods: The amino acid profile covering 43 amino acids in 68 subjects, comprising 30 (14 men and 16 women) controls and 38 (16 men and 22 women) COVID-19 patients, were examined. The amino acid profiles of the participants were screened by LC-MS/MS. Results: Compared with the control group, serum levels of 27 amino acids increased in the patient group. Alpha-aminopimelic acid, sarcosine, and hydroxyproline amino acids were considerably higher in the control group than in the patient group (p<0.0001). There was no notable difference among control group and the case group for 13 amino acids (p≥0.05). A significant positive correlation was seen among the control and the patient groups in the mean amino acid values (r=0.937; p<0.0001). Conclusions: These results postulated a clear picture on the serum levels of amino acid in the COVID-19 patients. Serum amino acids measured in hospitalized COVID-19 patients can explain the patient's metabolic status during the disease.

[Comparison of Candida Polymerase Chain Reaction Method and Blood Culture in Patients with High Risk of Candidemia in Intensive Care Unit]. / Yogun Bakim Ünitesinde Takip Edilen Kandidemi Riski Yüksek Hastalarda Candida Polimeraz Zincir Reaksiyonu Yöntemi ve Kan Kültürünün Karsilastirilmasi.

Candida species are responsible for 70-90% of invasive fungal infections in the intensive care unit. Early diagnosis and treatment is important in candidemia. Improper diagnosis and treatment increases mortality and morbidity significantly. Because of the late results of blood cultures and low sensitivity of the serological tests when used alone, molecular methods should be investigated in this field. In this study, the results of the Candida real-time polymerase chain reaction (Rt-PCR) test, which was studied from blood culture and whole blood, were compared in patients with high candidemia risk who were followed in the General Surgery Intensive Care and Anesthesiology and Reanimation Unit of Cukurova University Faculty of Medicine. It was aimed to investigate the practical utility of Candida RT-PCR test, which is a rapid diagnosis method in patients with suspected candidemia. In our study, 90 patients with high risk of candidemia according to the criteria determined according to the guidelines were evaluated prospectively. Urine, perineum, axilla, tracheal aspirate culture and two sets of blood cultures were obtained from the patients. Blood sample was also drawn into an ethylenediaminetetraacetic acid (EDTA) tube and stored at -40°C for later Candida Rt-PCR study. In Candida Rt- PCR, species-specific primers were used to distinguish species. Candida score (CS) of the patients was calculated. Forty one (45.5%) of the patients were female and 49 (55.5%) were male. The median age of the patients was 61.5 years. Candida was positive in blood culture in three (3.3%) of the patients included in the study, while Candida Rt-PCR was positive in 17 (18.9%). Candida species detected in the blood culture and Rt-PCR test were compatible with each other. Rt-PCR was significantly more positive (p= 0.006). Candida Rt-PCR positivity was significantly higher in patients receiving total parenteral nutrition (p= 0.028), malignancy (p= 0.021), and history of surgery in the last three months (p=0.003). The difference in CS between patients with PCR positive and PCR negative was statistically significant (p= 0.015). Our study was conducted in a high-risk population for candidemia and the results of Candida Rt-PCR was found to be more positive than blood culture. Rt-PCR positivity and blood culture positivity were associated with high CS. In the light of these data, it was thought that it would be appropriate to use molecular methods in the diagnosis and support them with CS, especially in patients with high risk of candidemia. Considering that blood culture, which is the gold standard for the diagnosis of candidemia, gives late results and is 50% positive, using faster diagnostic methods for candidemia is important to reduce mortality and morbidity. The fast and good results of Candida PCR method have shown that it can be used in diagnosis. However, lack of standardization of primers used in PCR tests may cause false positives. Additional studies are needed in this respect.

Evaluation of rapid antibody test and chest computed tomography results of COVID-19 patients: A retrospective study.

The purpose of this study was to evaluate the SARS-CoV-2 immunoglobulin M/immunoglobulin G (IgM/IgG) rapid antibody test results in symptomatic patients with COVID-19 and their chest computed tomography (CT) data. A total of 320 patients admitted to our hospital for different durations due to COVID-19 were included in the study. Serum samples were obtained within 0-7 days from COVID-19 patients confirmed by reverse-transcription polymerase chain reaction (RT-PCR) and chest CT scan. According to the SARS-CoV-2 RT-PCR results, the patients included in the study were divided into two groups: PCR positive group (n = 46) and PCR negative group (n = 274). The relationship between chest CT and rapid antibody test results were compared statistically. Of the 320 COVID-19 serum samples, IgM, IgG, and IgM/IgG were detected in 8.4%, 0.3%, and 11.6% within 1 week, respectively. IgG/IgM antibodies were not detected in 79.7% of the patients. In the study, 249 (77.8%) of 320 patients had positive chest CT scans. Four (5.6%) of 71 patients with negative chest CT scans had IgM and two (2.8%) were both IgM/IgG positive. IgM was detected in 23 (9.2%), IgG in one (0.4%), and IgM/IgG in 35 (14%) of chest CT scan positive patients. The rate of CT findings in patients with antibody positivity was found to be significantly higher than those with antibody negativity. The results of the present study show the accurate and equivalent performance of serological antibody assays and chest CT in detecting SARS-CoV-2 within 0-7 days from the onset of COVID19 symptoms. When RT-PCR is not available, we believe that the combination of immunochromatographic test and chest CT scan can increase diagnostic sensitivity for COVID-19.

A cross-sectional study of real life data of HCV from Turkey south region.

INTRODUCTION: This study investigated demographic characteristics and the prevalence of viremia among anti-HCV-positive patients. METHODOLOGY: Hospital records of adult patients with anti-HCV positivity between June 2016 and October 2018 were screened retrospectively. Demographic characteristics, genotype distribution, history of injection drug use (IDU), treatment data of HCV RNA-positive patients were investigated. RESULTS: The rate of anti-HCV seropositivity was 1.7% and 54.5% of these were viremic. 69.5% of the 869 viremic patients were male. The mean age was 62 ± 15 (18-95) years for women and 42 ± 19 (18-90) years for men (p < 0.0001). 42.7% of these patients had IDU history. Regarding age, patients with IDU history accounted for 95% of the 18-29 age group. The most common genotype in patients younger than 40 was genotype 3, and genotype 1b in those older than 40. Only 52% of viremic patients had received DAA therapy. Also, 62.2% of patients aged < 40 and 36% of patients > 40 did not receive treatment (p < 0.0001). The SVR12 rate in patients receiving DAA treatment and follow-up was 100%; SVR24 was 99.5%. CONCLUSIONS: A shift in the demographic structure of HCV-infected patients due to the changing trends of the HCV transmission mode was observed in this study. On the other hand, the proportion of patients who received DAA therapy was low. A substantial proportion of untreated patients were young with a history of IDU. This indicates that without strategies targeting the patients, the patient load due to HCV-related cirrhosis and hepatocellular carcinoma may persist in the future.

[Detection and Molecular Analysis of Moxifloxacin MIC Values of Mycobacterium tuberculosis Strains Isolated from Clinical Specimens]. / Klinik Örneklerden Elde Edilen Mycobacterium tuberculosis Izolatlarinin Moksifloksasin MIK Degerlerinin Tespiti ve Moleküler Analizi.

Tuberculosis (TB) is a chronic, granulomatous and necrotizing disease caused by microorganisms belonging to the Mycobacterium tuberculosis complex group. In 2017, 6.4 million new TB cases have been reported according to the World Health Organization 2018 Global Tuberculosis Report. TB remains among the major health problems of our time due to the increasing drug resistance problem and the difficulties in definitive diagnosis in recent years. It is stated by clinicians that intensive use of quinolone group drugs with oral form in simple indications such as respiratory or urinary tract infections may lead to resistance and this may result in treatment failures. The aim of this study was to determine the moxifloxacin susceptibility of M.tuberculosis isolates obtained from clinical specimens by phenotypical methods, to determine the resistance rates of moxifloxacin and to investigate the relationship between phenotypical resistance and mutations in the gyrA gene. A hundred (n= 100) consecutive non-multidrug resistant and 37 non-consecutive multidrug resistant M.tuberculosis strains isolated from the clinical specimens of patients with pulmonary tuberculosis were included in the study. The moxifloxacin susceptibility of the isolates was determined by using Löwenstein-Jensen medium and their epidemiological properties were investigated and also mutations detected by gyrA region were compared with drug susceptibility rates. Of the 137 isolates tested for phenotypical susceptibility, 25 (18.2%) were found to be resistant to moxifloxacin. Resistance rate among non-multidrug resistant and multidrug resistant isolates were determined as 17% and 21.6%, respectively. According to the results of the sequencing analysis, of the gyrA regions of all the isolates included in the study, a single base mutation was found in a total of six samples. The location positions of the mutations were determined as D94Y, D94G, A90V, G88A and among two strains as D89N. Two of the isolates with mutations were found to be phenotypically susceptible to moxifloxacin. In our study, it was found that moxifloxacin resistance in M.tuberculosis isolates was higher than similar studies and it was found that different mechanisms may be responsible for the existing resistance other than the mutations in the gyrA gene. It was concluded that the data obtained from the study should be shared with all clinicians in the country due to the possibility of resistance development to this group of drugs in a short time and considering this drug will have an important role in the treatment of TB, it should be used more limited in non-specific indications. Further studies using larger case groups and isolates are needed for the continuation of the research.