[Investigation of Ganciclovir Resistance in Cytomegalovirus Isolates by Phenotypic and Genotypic Methods]. / Sitomegalovirüs Izolatlarinda Gansiklovir Direncinin Fenotipik ve Genotipik Yöntemlerle Arastirilmasi.

Ganciclovir-resistant cytomegalovirus (CMV) strains are reported following long-term antiviral agent use, especially for immune-suppressive patients. In this study, it was aimed to investigate the mutations in the UL97 gene of CMV, which causes ganciclovir (GCV) resistance by genotypic and phenotypic methods in patients who developed CMV infection following hematopoietic cell (HCT) or solid organ transplantation (SOT). Thirty patients who had HCT or SOT in Mediterranean University Hospital and developed CMV infection during routine follow-up with a viral load of CMV over 1000 copies/mL were included in the study. CMV DNA was analyzed by an automated system (Cobas Ampliprep/COBAS TaqMan CMV Test, Roche Diagnostics, Germany) quantitatively. DNA sequence analysis of the regions including codons 420-664 in the UL97 gene region was done by the Sanger sequencing method to detect mutations causing antiviral resistance and compared with defined mutations. In order to investigate antiviral resistance by phenotypic methods, heparinized blood samples of the patients were collected, 'buffy coat (leukocyte layer)' was inoculated into MRC-5 cells by centrifugation method and CMV growth in these cells was controlled with monoclonal antibodies when growth was detected, virus titer was determined and plaque reduction test was applied as recommended. It was determined that 22 of the 30 patients were HCT recipients and eight were SOT (five kidney, three liver) recipients. When the CMV serology pattern of the patients was evaluated before transplantation, 29 (96.7%) patients were found to be seropositive and one (3.3%) patient was found to be seronegative. Totally, nine CMV UL97 mutations were detected in seven (23.3%) pediatric patients who had HCT, including six seropositive and one seronegative case. In addition, one mutation (D605E) not known to cause GCV resistance was detected in a seronegative recipient and three previously unidentified mutations were detected (1474T, F499S, V559A) in a seronegative recipient. Five of the mutations defined were UL97 mutations with a defined clinical resistance against GCV in each of the five recipients (C603W, C592G, H520Q, M460V, A594T). In the plaque reduction test using 3 µM, 12 µM, 48 µM and 96 µM concentrations of GCV in CMV strains, the IC50 value was determined to be ≥ 8 µM for the five CMV strains, and the phenotypic presence of GCV resistance was shown. Clinical resistance associated with CMV UL97 mutation was detected in five (22.7%) of 22 patients who had HCT. GCV resistance was also demonstrated in these patients by phenotypic methods. No UL97 mutation was detected in the patients who had SOT.

[P323L Mutation in a Case with Prolonged SARS-CoV-2 PCR Positivity]. / Uzamis SARS-CoV-2 PCR Pozitifligi Olan Olguda P323L Mutasyonu.

Coronavirus disease-2019 (COVID-19) emerged in the last months of 2019 and caused a pandemic effecting the whole world. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 has changed by various mutations since the day it was first identified, causing the pandemic to continue. Age, male gender, obesity, and comorbidity, which are general risk factors for COVID-19, can also cause prolonged PCR positivity. In this report, a case of 37-year-old male who is working in the hospital's COVID-19 molecular diagnostics laboratory was presented. He was vaccinated with three doses of inactivated vaccine, CoronaVac (Sinovac Biotech, Beijing-China), within the context of the vaccination program carried out in Türkiye. His first SARS-CoV-2 positivity was detected on 12.01.2021, four months after the last vaccination, and he continued to be detected positive for SARS-CoV-2 throughout a period of 39 days by quantitative reverse transcription polymerase chain reaction (qRT-PCR) tests performed with 2-3-day intervals. The patient has a 20-pack/year smoking history and his body mass index (BMI) was 29.8 kg/m2 at the time of his COVID-19. The case, which was clinically defined as mild COVID-19 with symptoms including back and headache, cough, fever (38.5°C), and loss of taste-smell, and without any additional complications or respiratory distress during the disease process. In the radiological examination, the lung was found within normal ranges. Prophylactic enoxaparin sodium anti-xa IU/0.6 ml was administered to the patient due to his cardiovascular risk, and no additional treatment was given. Whole genome sequencing was performed from nasopharyngeal swab samples of the patient at the beginning and 16th day of the infection to investigate the the specific genomic features and mutation pattern of the virus in the host over time, due to the prolonged SARS-CoV-2 PCR positivity. Library preparation for the whole next-generation sequencing (NGS) was performed by the SARS-CoV-2 Panel, Paragon CleanPlex kit (Paragon Genomics, USA), and indexing of the library was done by Clean-Plex Dual-Indexed PCR Primers for Illumina Set B kit (Paragon Genomics, USA). NGS analysis was performed on the Illumina Miniseq (Illumina, USA) platform. As a result of the bioinformatics evaluation, both samples were determined as SARS-CoV-2 Delta variant (Nextclade; 21J-Delta variant, Pango lineage; AY.43). Remarkably, the SARSCoV-2 sequences in the two samples taken 15 days apart; several identical mutations; such as D614G in the S gene, P323L in the ORF 1b gene region, and P1228L in the Nsp3 gene region, were detected. Besides that, when compared to the first sample, three additional mutations (P383L, P539S, L838I) were observed in the sequence of the second sample, which led to three amino acid changes, the clinical significance of which has not yet been determined in the literature. It is thought that; these mutations that change amino acid expression, as well as the other three mutations detected, may contribute to the improvement of the fitness of the virus and may be one of the factors responsible for the prolonged SARS-CoV-2 PCR positivity. Additional data to be obtained by further epidemiological sequencing studies will shed light on this issue.

Evaluation of the BD Phoenix CPO Detect Test for the detection of carbapenemase-producing Enterobacterales.

Aims: This study aimed to evaluate the performance of the BD Phoenix CPO Detect Test (BD Diagnostic Systems) for the detection and classification of carbapenemase-mediated carbapenem resistance. Methods: A total of 447 Enterobacterales strains were included in the study. All strains were tested with the BD Phoenix CPO Detect Test and the modified carbapenem inactivation method. Results: Carbapenemase production was detected in 157 of 159 carbapenemase producers, including 95.7% of class B and 99.2% of class D isolates using the BD Phoenix CPO Detect Test. BD Phoenix CPO Detect has a sensitivity of 98.7% and a specificity of 95.5% in detecting carbapenemase production. Conclusion: The classification of OXA-48 and class B carbapenemases, the most common carbapenemases circulating in Turkey, was highly accurate.

Enterobacterales are a type of bacteria that usually live harmlessly in the gut of humans. However, if the bacteria get access to the bladder or bloodstream, they can cause infection. Carbapenemase-producing Enterobacterales (CPE) are a type of bacteria that can cause carbapenem antibiotic-resistant infections, a group of powerful antibiotics. The rapid spread of CPE will pose an increasing threat to public health and medical treatment practices; therefore, rapid detection of CPE is crucial. This study assessed the performance of the BD Phoenix CPO Detect Test for the detection of carbapenemase-producing Enterobacterales. The BD Phoenix CPO Detect Test offers both the detection of carbapenemase production and antimicrobial susceptibility testing simultaneously and can be clinically useful for determining possible treatment options.

One-Year Post-Vaccination Longitudinal Follow-Up of Quantitative SARS-CoV-2 Anti-Spike Total Antibodies in Health Care Professionals and Evaluation of Correlation with Surrogate Neutralization Test.

Numerous vaccines have been generated to decrease the morbidity and mortality of COVID-19. This study aims to evaluate the immunogenicity of the heterologous boosts by BioNTech against homologous boosts by CoronaVac at three-month intervals in two health care worker (HCW) cohorts, with or without prior COVID-19, for one year post-vaccination. This is a prospective cohort study in which the humoral responses of 386 HCWs were followed-up longitudinally in six main groups according to their previous COVID-19 exposure and vaccination status. Anti-SARS-CoV-2 spike-RBD total antibody levels were measured and SARS-CoV-2 neutralization antibody (NAbs) responses against the ancestral Wuhan and the Omicron variant were evaluated comparatively using international standard serum for Wuhan and Omicron, as well as with the aid of a conversion tool. The anti-SARS-CoV-2 spike-RBD total Ab and Nab difference between with and without prior COVID-19, three months after two-dose primary vaccination with CoronaVac, was statistically significant (p = 0.001). In the subsequent follow-ups, this difference was not observed between the groups. Those previously infected (PI) and non-previously infected (NPI) groups receiving BioNTech as the third dose had higher anti-SARS-CoV-2 spike total Ab levels (14.2-fold and 17.4-fold, respectively, p = 0.001) and Nab responses (against Wuhan and Omicron) than those receiving CoronaVac. Ab responses after booster vaccination decreased significantly in all groups at the ninth-month follow-up (p < 0.05); however, Abs were still higher in all booster received groups than that in the primary vaccination. Abs were above the protective level at the twelfth-month measurement in the entire of the second BioNTech received group as the fourth dose of vaccination. In the one-year follow-up period, the increased incidence of COVID-19 in the groups vaccinated with two or three doses of CoronaVac compared with the groups vaccinated with BioNTech as a booster suggested that continuing the heterologous CoronaVac/BioNTech vaccination, revised according to current SARS-CoV-2 variants and with at least a six-month interval booster would be an effective and safe strategy for protection against COVID-19, particularly in health care workers.

Association of SARS-CoV-2 IgG Antibody Levels with Clinical/Laboratory Characteristics in COVID-19 Patients: Data from a Turkish Study.

BACKGROUND: COVID-19 causes clinical manifestations ranging from asymptomatic infection to multi-organ failure. It is reported that those with severe disease have higher anti-SARS-CoV-2 antibody titers compared to asymptomatic or mild cases. We evaluated the correlation of antibody responses with laboratory and clinical indicators in COVID-19 patients. METHODS: Seventy-nine male and 66 female patients (mean age: 39) with at least one positive SARS-CoV-2 RT-PCR test and SARS-CoV-2 IgG antibody result after acute infection were included. RESULTS: Seventy-six (52%), 45 (31%), and 24 (17%) patients had mild, moderate, and severe clinical findings, respectively. Patients with high body mass index and advanced age had significantly more severe disease (p < 0.001). A significant correlation was found between the increase in lymphopenia, C-reactive protein, ferritin, D-dimer, and lactate dehydrogenase and the severity of clinical findings (p = 0.0001). SARS-CoV-2 IgG antibody test was positive in 128 (88.3%) patients. A significant correlation was found between disease severity and antibody levels in the comparison of all groups (p < 0.001). CONCLUSIONS: Long-term monitoring of immune responses will be required to determine the appropriate time for the administration of new vaccines.

Can the Prognosis of COVID-19 Disease Be Determined by Fecal Markers and Cytokines?

The coronavirus disease 2019 (COVID-19) pandemic has affected the entire world, and has a variety of clinical presentations. The aim of this study is to determine the relationships of fecal cytokines and markers with the symptoms and prognosis of children with COVID-19 infection, and to identify noninvasive markers during follow-up. In a cohort of 40 COVID-19-positive children and 40 healthy controls, fecal cytokines and markers were examined in stool samples. A binary logistic model was used to assess the potential of cytokines as risk factors for hospitalization. Odds ratios (ORs) with 95% confidence intervals (CIs) were reported. A P-value <0.05 was accepted as statistically significant. Levels of fecal lysozyme, myeloperoxidase, hemoglobin, and interleukin-5 (IL-5) (P < 0.05) were significantly higher among the patients than controls. In a logistic regression analysis, fecal IL-2 (OR = 3.83; 95% CI: 1.44-15.92), IL-4 (OR = 2.96; 95% CI: 1.09-12.93), IL-5 (OR = 4.56; 95% CI: 1.18-27.88), IL-10 (OR = 2.71 95% CI: 1.19-7.94), interferon-gamma (IFN-γ) (OR = 4.03; 95% CI: 1.44-15.73), IFN-α (OR = 3.02; 95% CI: 1.08-11.65), calcium-binding protein B S100 (S100 B) (OR = 4.78; 95% CI: 1.31-27.82), neutrophil elastase (NE) 2 (OR = 4.07; 95% CI: 1.17-19.69), and matrix metalloproteinase 1 (MMP-1) (OR = 3.67; 95% CI: 1.1-18.82) levels were significantly higher in hospitalized patients with COVID-19 infection than outpatients. We demonstrated that various fecal cytokines and markers were increased in patients who had COVID-19. Fecal IL-2, IL-4, IL-5, IL-10, IFN-γ, IFN-α, S100 B, NE, and MMP-1 levels were significantly elevated in hospitalized patients. We suggest that the fecal and serum levels of cytokines could be used to predict the prognosis of COVID-19 disease, although more studies are needed to confirm this.

Comparison of the Clinical and Laboratory Findings and Outcomes of Hospitalized COVID-19 Patients Who Were Either Fully Vaccinated with Coronavac or Not: An Analytical, Cross Sectional Study.

COVID-19 vaccines are highly protective against severe disease; however, vaccine breakthrough infections resulting in hospitalization may still occur in a small percentage of vaccinated individuals. We investigated whether the clinical and microbiological features and outcomes were different between hospitalized COVID-19 patients who were either fully vaccinated with Coronovac or not. All hospitalized COVID-19 patients who had at least one dose of Coronavac were included in the study. The oldest unvaccinated patients with comorbidities, who were hospitalized during the same period, were chosen as controls. All epidemiologic, clinical and laboratory data of the patients were recorded and compared between the fully vaccinated and unvaccinated individuals. There were 69 and 217 patients who had been either fully vaccinated with Coronavac or not, respectively. All breakthrough infections occurred in the first 3 months of vaccination. Fully vaccinated patients were older and had more comorbidities than unvaccinated patients. There were minor differences between the groups in symptoms, physical and laboratory findings, anti-spike IgG positivity rate and level, the severity of COVID-19, complications, and clinical improvement rate. The mortality rate of fully vaccinated patients was higher than the mortality rate in unvaccinated patients in univariate analysis, which was attributed to the fact that vaccinated patients were older and had more comorbidities. The severity and clinical outcomes of hospitalized patients with breakthrough COVID-19 after Coronavac vaccination were similar to those of unvaccinated patients. Our findings suggest that the immune response elicited by Coronovac could be insufficient to prevent COVID-19-related severe disease and death within 3 months of vaccination among elderly people with comorbidities.

Carbapenem-resistant Klebsiella pneumoniae outbreak in a COVID-19 intensive care unit; a case-control study.

We analysed a carbapenem-resistant Klebsiella pneumoniae (CRKP) outbreak in the coronavirus disease (COVID) ICU. We retrospectively collected data from ICU records. We identified 25 cases between 12 November 2020 and 19 December 2020, and compared them to 42 controls present in the ICU during the same period. The presence of a femoral haemodialysis catheter was strongly associated with invasive CRKP infections (cases, 9 [36%]; controls, 0 [0%]; odds ratio [OR] 95% confidence intervals [CIs], 21 (5; 89)). We found a significant association between old age and CRKP infection with adverse outcomes. Sequence analysis revealed three distinct carbapenemase genes: blaNDM-1, blaOXA-48 and blaKPC-2. We launched rectal swab sampling upon admission to the ICU, cohorted colonized patients and cases and conducted an intensive training programme for newly employed staff. This study revealed that the emergence and dissemination of CRKP in COVID ICUs were associated with increased adverse outcomes. The presence of a femoral haemodialysis catheter was a significant risk factor for CRKP infections.

Anti-SARS-CoV-2 IgG and Neutralizing Antibody Levels in Patients with Past COVID-19 Infection: A Longitudinal Study

Background: Monitoring the longevity of immunoglobulin G (IgG) responses following severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections is vital to understanding the role of antibodies in preventing infection. Aims: To determine the quantitative IgG responses specific to the Spike-S1 (S1) receptor-binding domain (S1/RBD) region of the virus in serum samples taken between 4 weeks and 7 months after polymerase chain reaction (PCR) positivity in patients who are diagnosed with coronavirus disease-2019 (COVID-19). Study Design: A longitudinal study. Methods: This study included 113 patients with a clinical and molecular diagnosis of COVID-19. The first and second serum samples were taken 1 and 7 months, respectively, after the PCR positivity. S1/RBD-specific IgG antibody response was assayed using anti-SARS-CoV- 2 QuantiVac ELISA (IgG) kit (Euroimmun, Lübeck, Germany). The neutralizing antibodies were investigated in 57 patients whose IgG test results were above the cut-off value. Results: In 57 patients with SARS-CoV-2 IgG, the anti-SARS-CoV-2 IgG quantitative antibody levels significantly decreased after 7 months (Z = −2.197, p = 0.028). A correlation was detected between the anti-SARS-CoV-2 IgG and nAb percent inhibition (IH%) levels detected in 1 month (rs = 0.496, p < 0.001), but without significant correlation in serum samples taken on 7 months. The nAb IH% levels of the first and second were compared for COVID-19 severity and revealed no statistical difference (p = 0.256). In the second serum sample, the nAb IH%s of patients with moderate COVID-19 showed a statistically significant difference from patients with mild COVID-19 (p = 0.018), but without significant differences between severe and moderate or mild COVID-19. Conclusion: SARS-CoV-2 quantitative IgG antibody titers are significantly reduced at long-term follow-up (> 6 months). Due to the limited information on seroconversion, comprehensive studies should be conducted for long-term follow-up of the immune response against SARS-CoV-2.

Effect of BTN162b2 and CoronaVac boosters on humoral and cellular immunity of individuals previously fully vaccinated with CoronaVac against SARS-CoV-2: A longitudinal study.

BACKGROUND: It is essential to know about immune response levels after booster doses of the two different types of vaccines, mRNA, and the inactivated, currently used against COVID-19. For this purpose, we aimed to determine the effects of BNT162b2 (BNT) and CoronaVac (CV) boosters on the humoral and cellular immunity of individuals who had two doses of CV vaccination. METHODS: The study was conducted in three centers (Koc University Hospital, Istanbul University Cerrahpasa Hospital, and Istanbul University, Istanbul Medical School Hospital) in Istanbul, Turkey. Individuals who had been previously immunized with two doses of CV and no history of COVID-19 were included. The baseline blood samples were collected 3-5 months after the second dose of CV. Follow-up blood samples were taken 1 and 3 months after administration of third doses of CV, or one dose of BNT boosters. Neutralizing antibody titers were measured by plaque reduction assay. The CD4+ T cell, CD8+ T cell, effector CD4+CD38+CD69+ T cell, and effector CD8+CD38+CD69+ T cell ratios were determined by flow cytometry. The intracellular IFN-γ and IL-2 responses were measured by ELISpot assay. RESULTS: We found a 3.38-fold increase in neutralizing antibody geometric mean titers (NA GMT, 78.69) 1 month after BNT booster and maintained at the third month (NA GMT, 80). Nevertheless, in the CV booster group, significantly lower NA GMT than BNT after 1 month and 3 months were observed (21.44 and 28.44, respectively) (p < .001). In the ELISpot assay, IL-2 levels after BNT were higher than baseline and CV booster (p < .001) while IFN-γ levels were significantly higher than baseline (p < .001). The CD8+CD38+CD69+ and CD4+CD38+CD69+ T cells were stimulated predominantly in the third month of the BNT boosters. CONCLUSION: The neutralizing antibody levels after 3 months of the BNT booster were higher than the antibody levels after CV in fully vaccinated individuals. On the contrary, ratio of the effector T cells increased along with greater IFN-γ activation after BNT booster. By considering the waning immunity, we suggest a new booster dose with BNT for the countries that already had two doses of primary CV regimens.

Inactive SARS-CoV-2 vaccine generates high antibody responses in healthcare workers with and without prior infection.

BACKGROUND AND OBJECTIVES: Healthcare workers (HCWs) were among the first groups to be vaccinated in Turkey. The data to be obtained by the vaccination of HCWs would guide wide spread vaccination programs. MATERIALS AND METHODS: The study included 330 HCWs working at Istanbul University-Cerrahpasa, Cerrahpasa Medical Faculty Hospital and vaccinated with inactive CoronaVac (Sinovac Life Sciences, China) SARS-CoV-2 vaccine in two doses (28 days apart). Anti-Spike /RBD IgG levels were measured 14 days after the first dose and 28 days after the second dose. Chemiluminescent microparticle immunoassay (CMIA) (ARCHITECT IgG II Quant test, Abbott, USA), which is 100% compatible with plaque reduction neutralization test (PRNT), was used. RESULTS: Of the participants, 211 (63.9%) were female, 119 (36.1%) were male, and mean age was 39.6 ± 7.7 years. In those without prior COVID-19 history; (n = 255) antibody positivity was detected as 48.2% (95% CI: 42.1-54.3) 14 days after the first dose of vaccine, and 99.2% (95% CI: 98.1-100) at day 28 after the second dose. Antibody titers were significantly lower in patients with hypertension (p = 0.011). In those with prior history of COVID-19 (n = 75); both the antibody positivity rates after the first vaccine (48.2% vs 100%, p = 0.000) and the anti-spike/RBD antibody levels after the second vaccine (with a ≥ 1050 AU/mL titer equivalent to PRNT 1/80 dilution) was significant than infection-naive group (25.9% vs. 54.7%, p = 0.000). Antibody positivity after two doses of vaccination for all study group was 99.4% (95% CI: 98.6-100). CONCLUSIONS: Two doses CoronaVac produce effective humoral immunity in HCWs. Antibody response is significantly higher in those with prior history of COVID-19 than infection-naive group. Given no significant benefit of the second dose, a single shot of vaccination may be sufficient for those with prior history of COVID-19. Monitoring humoral and cellular immune responses, considering new variants, is required to validate this approach.

Detection of SARS-CoV-2 RNA in Upper Respiratory Swap Samples by Pooling Method

Background: Widespread and effective use of molecular diagnostic tests is indispensable for protecting public health and containing the severe respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. More than 1 year into the pandemic, as resources have reached a point of depletion, grouping samples in pools of certain sizes appears to be a reasonable method to reduce both the costs and the processing time without necessitating additional training, equipment, or materials. Aims: To assess whether the pooling strategy that was used in past outbreaks and is used in blood tests prior to transfusion for screening large populations can also be used in SARS CoV-2 tests. Study Design: Diagnostic accuracy study. Methods: This prospective study was conducted with 2815 samples, sent to the coronavirus disease 2019 (COVID-19) Laboratory of our hospital between February 12 and 21, 2021, to be tested for the presence of SARS-CoV-2. The samples were examined individually and in pools of five 100 µl taken from each sequential sample, using 3 different SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) kits, the Allplex™ 2019-nCoV Assay kit (Seegene, Republic of Korea), the GeneMAP™ 2019-nCoV detection V.3 kit (GenMark, Türkiye), and the Bio-Speedy™ SARS-CoV-2 Double Gene™ RT-qPCR kit (Bioeksen, Türkiye) on the BioRAD CFX96™ Touch (Bio-Rad Laboratories Inc., Hercules, CA, USA) platform available in our laboratory. Results: Following the extraction of serial dilutions prepared from the SARS-CoV-2 RNA positive (cycle of threshold: 20) sample, the standard curves of RT-PCR were analyzed. By evaluating the efficiency (E) values, all 3 kits showed high sensitivity and similar results; while the highest level was detected with the Allplex™ 2019-nCoV Assay kit in the nucleocapsid (N) gene (E: 124%), the lowest was detected with the Double Gene™ RT-qPCR kit in the N and ORF 1ab genes (E: 90%). Of the samples included in the study, only 1 positive sample with low viral load was found to be negative when studied by pooling. The total number of kits to be used in pooled tests and then to individually retest the 5 samples in positive pools was calculated as 827 and the savings rate as 69.91% (1968/2815). Conclusion: The pooling strategy is an effective approach to extend the impact of limited testing resources and reagents available in certain periods of the COVID-19 pandemic. Testing by pooling samples requires improvement of RNA extraction methods and careful monitoring of RT-PCR test sensitivity to avoid missing low-positive entities. Therefore, based on the prevalence of COVID-19 in their regions, laboratories should conduct their own validation of pooling studies for RNA extraction and amplification methods they use.

Real-life experience: sensitivity and specificity of nasal and saliva samples for COVID-19 diagnosis.

BACKGROUND: COVID-19 (coronavirus disease 2019) outbreak has spread rapidly around the world, continues to show its effect, and it is not clear how long it will continue. For the diagnosis of COVID-19, it is important to ensure the comfort of the patients and to protect the healthcare workers (HCWs) by reducing the use of protective equipment. AIMS: To evaluate or assess whether the samples taken by the patient for COVID-19 testing during this pandemic period can be used in real-life experience. METHODS: Three different samples (nasopharyngeal taken by the healthcare worker, nasopharyngeal, and saliva taken by the patient) from 132 patients were evaluated for the diagnosis of COVID-19. The sensitivity and specificity of the samples in the diagnosis of COVID-19 were compared with real-life experience. RESULTS: Paired analyzes were performed by comparing each sample taken by the healthcare worker with the sample taken by the patient. The sensitivity of the three samples (nasopharyngeal taken by the healthcare worker, nasopharyngeal, and saliva taken by the patient) in the diagnosis of the COVID-19 was (100%, 98.7%, and 96.1%, respectively) accepted to be accurate. CONCLUSIONS: The sample taken by the paramedic was compatible compared to the real-life experience for the samples taken by the patient in the COVID-19 pandemic period. During the pandemic that is unknown when it will end, this study demonstrated that taking the sample of the patient alone for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test is a beneficial approach to the protection of the healthcare worker, reducing the need for protective equipment, increasing the patient's comfort and rapid sampling.

Novel SARS-CoV-2 Omicron variants in Istanbul; Rapid Preponderance of BA.2 and BA.5.

Objective: In Turkey, the fourth wave of SARS-CoV-2 started in December 2021 and peaked in mid-January 2022. Afterward, peaks were seen in the number of COVID-19 cases because of Omicron BA.2 and BA.5 variants. Our study aimed to observe the prevalence and viral load-related transmissibility rates of the Omicron BA.2 and BA.5 variant infections in our region between January 21 and July 01, 2022, using an easy and cost-effective PCR screening method. Methods: The frequency of BA.2 and BA.5 were determined by the two-stage allele-specific and drop-out RT-PCR method targeting NSP6 105-107del, spike 69-70del, and spike L452R mutation-specific primers. Transmissibility of the Omicron variants was assessed using cycle threshold (Ct) values (a proxy for SARS-CoV-2 viral load and infectivity). Also, using the next generation sequencing (NGS) method, existing mutations were analyzed by generating full-length sequences of the representative, randomly selected samples from the Omicron variants determined by PCR screening test. Results: We defined the first case of BA.2 on January 19, 2022, in Istanbul University-Cerrahpasa School of Medicine COVID-19 Molecular Diagnosis Laboratory. Following this, it was observed that BA.1 lost its dominance due to the increased transmissibility of BA.2. On May 5, we defined the first case of BA.5, and as of July this Omicron variant rapidly became preponderant, with a frequency of more than 85%. Compared with BA.1, BA.2 and BA.5 were associated with 2.82 (95% CI: 2.33-4.12) and 2.49 (95% CI: 2.16-3.55) fewer cycles, respectively, meaning higher transmissibility. As confirmed by the NGS results, it was concluded that screening with NSP6 105-107del, spike 69-70del and spike L452R mutation targeted PCR method, which is used uniquely in our hospital in Turkey, can be an easy and cost-effective method in the follow-up of Omicron variants. Conclusion: The higher viral load detection in infections with BA.2 and BA.5 reflects a prolonged disease period, and increased transmissibility, so rapid expansion of these Omicron variants in Turkey is inevitable. Even though the prevalence of the Omicron variants in the population can be monitored in near real-time by the PCR screening method, more sequencing studies are needed for the early identification of new mutations that will emerge.

Tocilizumab in COVID-19: The Cerrahpasa-PREDICT score.

BACKGROUND: Cytokine release syndrome (CRS), characterized by overproduction of proinflammatory cytokines in the course of severe coronavirus disease 2019 (COVID-19), has been suggested as the major cause of mortality. Tocilizumab, a recombinant humanized monoclonal antibody against human IL-6 receptor, poses a therapeutic option for the treatment of CRS leading to severe acute respiratory syndrome in coronavirus-2 (SARS-CoV-2) infection. METHODS: We performed a single-center retrospective study to reveal the outcome of COVID-19 patients on tocilizumab and proposed "the Cerrahpasa-PREDICT score", a new clinical scoring system using clinical and laboratory parameters that would help predicting the 28-day mortality of COVID-19 patients receiving tocilizumab. RESULTS: Eighty-seven patients (median age: 59 years) were included of whom 75.8% were male. Tocilizumab use significantly improved clinical and laboratory parameters. The 28-day mortality rate on tocilizumab was 16.1%. The Cerrahpasa-PREDICT score, consisting of platelet counts, procalcitonin, D-dimer levels, SO2R and the time from symptom onset to tocilizumab administration had a positive predictive value of 94.5% and negative predictive value of 92.9% for anticipating 28-day mortality. CONCLUSIONS: Severe COVID-19 should closely be monitored for the signs of hyperinflammation. We showed that administration of tocilizumab early in the course of the disease (prior to ICU admission) resulted in a favorable outcome. Close monitoring usually aids identifying patients who would benefit from tocilizumab. In this regard, the Cerrahpasa-PREDICT score might serve as a practical tool for estimating the 28-day mortality in COVID-19 patients who received tocilizumab and would facilitate timely recognition of fatal cases to be evaluated for other therapeutic options.

[Evaluation of the Diagnostic Performance of Different Principles of SARS-CoV-2 Commercial Antibody Tests in COVID-19 Patients]. / SARS-CoV-2 ile Ilgili Farkli Prensipli Ticari Antikor Testlerinin COVID-19 Hastalarindaki Tanisal Performanslarinin Degerlendirilmesi.

Following the emergence of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) and using only PCR for diagnosis, antibody tests have been rapidly developed by various commercial companies. There are differences between the sensitivity and specificity of these tests due to the usage of different viral target proteins and antibody subclasses. In order to evaluate the diagnostic use of these tests, we aimed to examine the diagnostic performance, especially sensitivity and specificity, of SARS-CoV-2 IgM, IgA and IgG tests of various companies (Abbott, Roche, Euroimmun, Dia.Pro, Anshlabs, Vircell, UnScience and RedCell), which have different principles (ECLIA/CLIA, EIA, LFA). Current (n= 180) and past (n= 180) COVID-19 patients with clinical and molecular diagnosis of COVID-19 admitted to Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine Hospital, Pandemic Polyclinic with suspected COVID-19 infection, were included in our study. The patients admitted within the first 3 weeks after the onset of symptoms were included in the current patient group, and those admitted at the third and after the third week were included in the past patient group. Serum samples (n= 180) obtained from Istanbul Sisli Hamidiye Etfal Training and Research Hospital, Blood Center between April and June 2018 before the COVID-19 pandemic were included in the study as a control group. All the tests included in our study were studied with the recommendations of the manufacturer companies. Between the IgG detection tests with different principles in patients with past COVID-19, the sensitivity and specificity values of the most effective tests were; 86.7%/99.4% (Abbott), 86.1%/98.9% (Dia.Pro), 91.3%/95% (RedCell). Between the IgM detection tests with different principles in current COVID-19 patients, the sensitivity and specificity values were; 67.8%/99.4% (Abbott), 68.9%/98.6% (Vircell), 50%/97.5% (RedCell). Abbott IgM with a kappa coefficient of 0.67 and Vircell IgM + IgA test with a kappa coefficient of 0.65 showed the best fit in patients with current COVID-19 infection. In patients with past COVID-19, Abbott IgG with 0.86 kappa coefficient and Dia.Pro IgG test with 0.85 kappa coefficient showed the best match. Due to the low sensitivity of IgM detection antibody tests, they should not be preferred instead of real-time reverse transcriptase polymerase chain reaction in routine diagnosis. IgG detection tests may be preferred to detect the antibody response and the titers in people who have had COVID-19 for population seroprevalence and especially therapeutic immune plasma production. However, it is thought that the combined use of both ECLIA/CLIA-based and EIA/ELISA-based tests together may be more effective in routine use for SARS-CoV-2 IgG tests.

The role of typical and atypical pathogens in acute exacerbations of chronic obstructive pulmonary disease.

OBJECTIVES: The exact role of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila in the development of chronic obstructive pulmonary disease exacerbations remains to be elucidated. This study was conducted to identify nonspecific and atypical pathogens associated with acute exacerbations of COPD. MATERIALS AND METHODS: Between February 2013 and February 2015, 107 patients were analyzed. Sixty-nine comprised the inpatient and 38 comprised the outpatient treatment group. RESULTS: When nonspecific culture results were taken into consideration only a causative organism could be detected in 46.7% of the patients. The detection rate increased to 85.1% with the additional use of polymerase chain reaction (PCR), direct fluorescent antibody (DFA) test, and culture methods. More than one causative agent was responsible for COPD exacerbation in 53.3% of patients: two agents in 37.3%, three agents in 15%, and four agents in 0.9%. H. influenzae was detected in 63 (58.9%) patients, S. pneumoniae in 57 (53.2%), P. aeruginosa in 15 (14.0%), and L. pneumophila in 11 (10%). L. pneumophila was the more commonly isolated agent in the inpatient group (P = 0.002). Patients receiving continuous oxygen therapy and noninvasive mechanical ventilation were more likely to have an exacerbation associated with P. aeruginosa (P = 0.008 and P = 0.009, respectively). CONCLUSION: The additional use of DFA for Legionella and multiplex PCR in combination with nonspecific microbiological culturing methods greatly improves the ability to identify infectious agents in acute exacerbations of COPD. There should be a high index of suspicion for P.aeruginosa as a causative organism, particularly in subjects receiving continuous oxygen therapy and/or using NIV and L. pneumophila should certainly be taken into consideration in severe COPD exacerbations.

[Investigation of Ganciclovir Resistance in Cytomegalovirus Strains Obtained from Immunocompromised Patients]. / Immün Yetmezlikli Hastalardan Elde Edilen Sitomegalovirüs Izolatlarindaki Gansiklovir Direncinin Arastirilmasi.

CMV is a virus that is asymptomatic in healthy individuals but can cause serious mortality and morbidity in transplant patients and patients with acquired immunodeficiency syndrome (AIDS). Ganciclovir (GCV) is a nucleoside analog that significantly reduces morbidity and mortality in CMV-related infections and is used as the first choice in treatment. It is the first drug shown to be effective in the treatment of CMV disease in humans, and is also homologous to acyclovir. Long-term antiviral therapy is required to prevent or treat CMV disease, but this can cause antiviral resistance which was reported to be 8-14% in CMV. In CMV strains, GCV resistance is most common in the UL97 kinase gene region. The aim of this study was to investigate GCV resistance in CMV strains obtained from the patients with immune deficiency. A total of 49 patients, including 20 children, 29 adults, who were followed in the department of hematology were included in the study. Fifty-three samples from 49 patients with CMV DNA viral load ≥ 103 copies/ml were examined for GCV resistance. In the study, DNA sequences were determined by Sanger sequence analysis method 3500 Abi Prism Genetic Analyser (Applied Biosystems, Thermo Fisher Scientific, USA) in the 674 bp part of the UL97 gene region. The next generation sequencing (NGS) method was applied to the samples that could not be evaluated with this method. GCV resistance was not detected in 35 (66%) of 53 samples with the Sanger method. C592G, C607S and M460I GCV resistance mutation was detected in three patients. Since the sequences were mixed, resistance analysis could not be evaluated with Sanger in 15 patient samples and the resistance was not detected in these samples studied with NGS. Antiviral resistance mutation was detected in three of 49 patients (6.1%). In 20 patients included in the study, three variant sequences (A442G, C592F, A427V) reported in the literature and determined to be sensitive to drugs by phenotypic tests and 78 variant sequences that were not reported in the literature were detected. As a result, the detection of antiviral resistance is important in the follow-up of the patients and guides the clinician in planning of the treatment. It was concluded that the samples that could not be evaluated with the Sanger method should be studied with NGS and further studies are needed to determine the role of the variant sequences detected for the first time in drug resistance.

Epstein-Barr virus, cytomegalovirus and BK polyomavirus burden in juvenile systemic lupus erythematosus: correlation with clinical and laboratory indices of disease activity.

OBJECTIVES: Clinical and laboratory investigations have revealed that Epstein-Barr virus (EBV) is involved in altered immunological response of systemic lupus erythematosus (SLE). Higher seroprevalence rates of anti-EBV antibodies and increased viral load are demonstrated in adult SLE patients. The prevalence of BK polyomavirus (BKV) reactivation is also suggested to be higher in SLE. Herein, we aimed to evaluate the immune response of children with SLE to EBV antigens in addition to EBV and BKV DNA. We also tried to evaluate whether these serological results differ from another connective tissue disease - juvenile systemic sclerosis (jSS) - and healthy individuals. METHODS: Serum levels of EBV early antigen diffuse (EA-D) IgG, EBV nuclear antigen-1 IgG, EBV viral capsid antigen (VCA), cytomegalovirus (CMV) IgG, EBV DNA, CMV DNA and urinary BKV DNA were evaluated in healthy controls and in patients with a diagnosis of juvenile SLE (jSLE) and jSS. RESULTS: A total of 70 jSLE patients, 14 jSS patients and 44 sex-matched healthy individuals were involved in the study. EBV VCA was positive in 84.2% of jSLE patients, 85.7% of jSS patients and 36.3% of healthy controls. EBV EA-D IgG positivity was significantly higher in jSLE patients compared to jSS patients and healthy controls (20% vs. 7.1% and 0%, p = 0.005). EBV VCA positivity was associated with malar rash and immunological disorder, but there was no statistical significance in other antibody positivity in terms of clinical and haemogram findings and autoantibody positivity. CMV DNA positivity was present in only 2.8% of jSLE patients. None of the jSS patients or the healthy controls had CMV DNA positivity. EBV DNA and BKV DNA were also negative in all three groups. CONCLUSION: The results of our study assume a relationship between SLE and EBV, but we could not demonstrate an association between CMV and BKV. The negative DNA results in contrast to serological positivity can be interpreted as an altered and impaired immune system and increased viral susceptibility. These results suggest that EBV contributes to disease continuity, even if it does not directly cause development.