[Longitudinal Monitoring of Seroconversion Status in SARS-CoV-2 RT-PCR Positive Healthcare Workers]. / SARS-CoV-2 RT-PCR Pozitif Saglik Çalisanlarinda Serokonversiyon Durumunun Boylamsal Izlenmesi.

The impact of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome 2 (SARS-CoV-2) still continues. The duration of the immune response in individuals recovering from COVID-19 and its protection against future SARS-CoV-2 infection are not fully understood. This study aimed to longitudinally evaluate anti-SARS-CoV-2 seroconversion status in healthcare workers with positive SARS-CoV-2 Real-time reverse transcription polymerase chain reaction (rRT-PCR), test in Mersin University Hospital. A total of 68 healthcare workers with positive SARS-CoV-2 rRT-PCR test between 19 April and 27 November 2020 were included in the study. Blood samples were collected from healthcare workers for SARS-CoV-2 antibody testing in the 1st, 3rd and 5th months following PCR positivity. Healthcare workers were classified as symptomatic, asymptomatic and reinfected according to their clinical findings, and rRT-PCR cycle thresholds (Ct) were recorded. Elecsys Anti-SARS-CoV-2 (Roche Diagnostics, Germany) kit was used for antibody testing. Of the 68 healthcare workers; 46 were classified as symptomatic, 15 as asymptomatic, and seven as reinfected. Twenty-seven (39.7%) of the healthcare workers were male and 41 (60.3%) were female, and the mean age was 36.4 ± 9.04. Seroconversion was detected in 45 (66.2%) of 68 healthcare workers in the study, and only one person had sero-negative result at the end of the 5th month. While seroconversion was detected in 78.3% (n= 36/46) of symptomatic healthcare workers, it was observed in 26.7% (n= 4/15) of the asymptomatic healthcare workers. Seroconversion was detected in only one of the seven reinfected healthcare workers after primary infection. After reinfection, seroconversion was observed in five of seven reinfected healthcare workers. Antibody response was not detected in two of them after both infections. According to the rRT-PCR Ct values; the median of Ct value was found significantly lower in healthcare workers with seroconversion (23.26, IQR= 18.45-27.30), than the ones without seroconversion (36.20, IQR= 33.09-37.56) (p< 0.001). In those who had reinfection, the mean Ct value (31.77 ± 6.62) detected during the primary infection period was statistically higher than the Ct value (22.44 ± 5.54) detected during reinfection (p= 0.008). The most frequently recorded symptoms in healthcare workers were myalgia (57.3%), fatigue (51.5%), headache (51.5%) followed by sore throat (36.7%), fever (33.8%), cough (27.9%), diarrhea (23.5%) and dyspnea (16.2%). In addition, fever (52%) and fatigue (80.6%) were found to be significantly higher in seroconversion-positive healthcare workers than in those without seroconversion (p= 0.028; p= 0.005, respectively). As a result, a higher rate of antibody response was detected in healthcare workers who had symptomatic infection than those who were asymptomatic. It has been observed that patients with asymptomatic primary infection and without antibody response were more susceptible to reinfection. In addition, it was observed that the probability of immune response increased when the viral load increased (Ct value decreased) in symptomatic infections. Although these findings provide important information about the short-term seroconversion status of healthcare personnel; longer-term and larger-scale studies are needed to evaluate the long-term effectiveness of seroconversion and to better understand the effectiveness of the immune response developed after SARS-CoV-2 vaccine administrations.

[Comparison of Real Time PCR Cycle Threshold Values in Symptomatic and Asymptomatic COVID-19 Patients]. / Semptomatik ve Asemptomatik COVID-19 Hastalarinda Gerçek Zamanli PCR Döngü Esik Degerlerinin Karsilastirilmasi.

Patients infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) show different clinical courses ranging from asymptomatic to severe infection requiring intensive care treatment and death. Real-time reverse transcription polymerase chain reaction (rRT-PCR), used in the diagnosis, screening and surveillance of coronavirus-2019 (COVID-19), provides the viral load as a cycle threshold (Ct) value. It has been reported that the Ct value may be related to the course of the infection and the clinical condition of the patient. In this study, it was aimed to compare the Ct and C reactive-protein (CRP) results of symptomatic and asymptomatic patients who were found to be positive with rRT-PCR. Between 14 April and 29 August 2020, a total of 355 patients aged 18 years and older with positive SARS-CoV-2 rRT-PCR test were included in the study. The COVID-19 rRT-PCR test was performed with Bio-speedy SARS-CoV-2 rRT-PCR kit (Bioeksen, Turkey) versions, the kit targeting the RdRp gene region, and the dual gene kit versions targeting the N and ORF1ab gene regions were used. Patients were classified as symptomatic and asymptomatic according to their clinical findings. Ct and CRP results of the patients were analyzed statistically. Of the 355 patients included in the study, 237 (66.7%) were symptomatic and 118 (33.2%) were asymptomatic patients. The mean age of symptomatic patients (46.68 ± 18.03) was observed significantly higher than asymptomatic patients (38.27 ± 13.82) (p<0.001). When the patients are evaluated according to the age groups, the rate of asymptomatic patients was significantly higher in the 21-39 age group, while the rate of symptomatic patients was significantly higher in 65 years and older group (p<0.05). The rate of comorbidity was significantly higher in symptomatic patients (n= 69, 29.1%) than in asymptomatic patients (n= 11, 9.3%) (p<0.001). Hypertension (12.2%), diabetes mellitus (9.7%), chronic respiratory disease (9.3%) and cardiovascular diseases (5.5%) were the most common diseases in symptomatic patients. However, among these, hypertension and chronic respiratory disease were found significantly higher in symptomatic patients (p<0.05). Increased CRP rate in symptomatic patients (64.6%) was found significantly higher than asymptomatic patients (27.3%) (p<0.001). The median of Ct value was found significantly higher in asymptomatic patients (26.34, IQR= 19.78-35.48), than in symptomatic patients (21.77, IQR= 17.81-26.51) (p<0.001). Regarding the medians of Ct values obtained from target genes; RdRp gene Ct value was found significantly higher in asymptomatic patients than in symptomatic patients (p<0.001). However, no statistical difference was found between symptomatic and asymptomatic patients in the ORF1ab and N genes Ct value medians (p> 0.05). As a result, it was observed that SARS-CoV-2 PCR positive patients were symptomatic in the presence of advanced age and comorbidity. Increased CRP value at the time of admission to the hospital was found significantly higher in symptomatic patients. Ct value has been shown to be lower in symptomatic patients, as expected. Although Ct and CRP values are thought to be useful in monitoring the clinical course and prognosis of patients with COVID-19, more detailed studies are needed to prove their clinical value.

[Dihydropteroate Synthase (DHPS) Gene Mutations and Mitochondrial Large Subunit (mtLSU) rRNA Genotype Variations in Pneumocystis jirovecii Strains]. / Pneumocystis jirovecii Izolatlarinin Dihidropteroat Sentaz (DHPS) Gen Mutasyonlari ve Mitokondriyal Büyük Alt Birim (mtLSU) rRNA Genotip Varyasyonlarinin Belirlenmesi.

Pneumocystis jirovecii is an atypical fungus that causes Pneumocystis pneumonia (PCP) in HIV/AIDS and immunocompromised patients. Antibiotics containing sulfa and sulfone groups are widely used in PCP prophylaxis and treatment. Especially, long-term use of trimethoprim sulfamethoxazole (TMP-SMX) is known to cause certain point mutations associated with drug resistance in the P.jirovecii dihydropteroate synthase (DHPS) gene. In addition, DHPS and mitochondrial large subunit (mtLSU) rRNA genotype characterization provides important data on the epidemiology of P.jirovecii. In this study, it was aimed to investigate the DHPS and mtLSU rRNA gene polymorphisms of P.jirovecii strains isolated from immunocompromised patients in Mersin University Hospital. In this study, 16 P.jirovecii positive samples, which isolated from 96 patients samples, between August 2016 and February 2018, were included. P.jirovecii mtLSU rRNA genotypes were determined by sequence analysis according to polymorphisms at the 85th and 248th nucleotide positions. Nested PCR and RFLP method was applied for mutation analysis of DHPS locus, 165th and 171st nucleotide positions. In the DHPS mutation analysis, 12/16 (75%) wild type (W165/W171) and 4/16 (25%) mutant type (M165/W171) were detected. Two mutant types belonged to HIV/AIDS positive patients with PCP and had a history of prophylaxis; the other 2 mutant types belonged to patients with colonization. In the study, a history of prophylaxis in 3 (19%) of the 16 patients were recorded, and mutant type was detected in these 2 of 3 patients. According to mtLSU-rRNA analysis, 3 different genotypes were obtained from 16 P.jirovecii isolates. In our region, genotype 2 (43.75%; n= 7) was the most common genotype, genotype 1 (37.5%; n= 6) was the second common and genotype 3 (18.75%; n= 3) was the least one. Genotype 4 was not detected in our region. When DHPS and mtLSU-rRNA were evaluated as multilocus, five different genotypes were observed. As a result, these findings provided important data on P.jirovecii epidemiology in our region and potential drug-resistant strains showed a risk of transmission in immunosuppressive patients. Multicenter studies involving more P.jirovecii isolates are needed to better define the epidemiology of P.jirovecii in our region and in our country.

[Investigation of Pneumocystis jirovecii Infection and Colonization in Immunocompromised Patients with Pneumonia]. / mmün Sistemi Baskilanmis Pnömonili Hastalarda Pneumocystis jirovecii Enfeksiyon ve Kolonizasyonlarinin Arastirilmasi.

Pneumocystis jirovecii is an atypical fungus that causes P.jirovecii pneumonia (PCP) in immunocompromised patients. Currently, while the incidence of AIDS-related PCP is decreasing, PCP has become more common in HIV-negative immunosuppressive patients as a result of increased diseases requiring immunosuppressive therapy. In this study, it was aimed to investigate PCP and colonizations by microscopy, polymerase chain reaction (PCR) and Krebs von den Lungen-6 (KL-6) tests in symptomatic immunosuppressive inpatients with the sign of radiologically atypical pneumonia in Mersin University Hospital. A total of 96 patients, between August 2016 and February 2018 were included in the study. Seventy two (75%) of the 96 patients were under immunosuppressive therapy. P.jirovecii was investigated in the respiratory tract samples [sputum (n= 88), tracheal aspirate (n= 6) and bronchoalveolar lavage (n= 2)] by mtLSUrRNA nested PCR and microscopic staining methods [immunofluorescence assay (IFA), Toluidine Blue O (TBO)], and KL-6 levels were tested in serum samples. P.jirovecii was detected in 16 (16.7%) samples by PCR, in five (5.2%) samples by IFA, in three (3.1%) samples by TBO stain method. When IFA was taken as a reference test, sensitivity and specificity of TBO and PCR were calculated as 60% and 100%; 100% and 87.9%, respectively. In P.jirovecii PCR positive patients, the distribution of underlying diseases; cancer (n= 6), hematological malignancy (n= 3), HIV/AIDS (n= 3), COPD (n= 2), and interstitial lung disease (n= 2) were found as 11 (68.75%) of the 16 positive patients, received immunosuppressive therapy (HIV positive non-Hodgkin lymphoma); of the 3 (18.75%) patients of were immunocompetent, and only 2 (12.5%) were HIV/AIDS. Five of the 16 PCR positive the patients that have positive microscopic examination were definited PCP [HIV/AIDS (n= 3), lung cancer (n= 1), interstitial lung disease (n= 1)]; three patients were PCR positive and microscopy negative probable PCP [multiple myeloma (n= 1), interstitial lung disease (n= 1), cholangiocellular carcinoma (n= 1)] and eight other patients were identified as colonized. In the study, when the frequency of the detection of P.jirovecii was evaluated according to the underlying diseases, it was found statistically significantly higher only in HIV/AIDS patients (p= 0.012). When KL-6 was evaluated among the patients defined as PCP/possible PCP and colonization, sensitivity and specificity were determined as 62.5% and 75%, respectively. As a result, nested PCR method was found as sensitive and successful for the detection of P.jirovecii from sputum samples. KL-6 test was not found sufficient for the differentiation of colonization and the infection in PCR positive patients. The results obtained in the study showed that PCP should be on the differential diagnosis list according to the immune status and the clinical features of the inpatients. More researchs are required with more patients to achieve for detailed reliable results in these groups. In addition, molecular epidemiological studies related to genotyping and resistance against anti-PCP drugs are needed to understand P.jirovecii infections in our region and country.

[Investigation of the clonal relationship between nosocomial Acinetobacter baumannii isolates by Rep-PCR]. / Hastane enfeksiyonu etkeni olan Acinetobacter baumannii izolatlari arasindaki klonal iliskinin Rep-PCR ile arastirilmasi

Acinetobacter spp. are opportunistic bacterial pathogens primarily associated with hospital-acquired infections and the spread of multidrug resistant Acinetobacter strains is a growing problem in terms of infection control. The aim of this study was to determine the clonal relationship between strains of nosocomial Acinetobacter baumannii by using rep-PCR method. A total of 75 Acinetobacter strains isolated from various clinical samples of the hospitalized patients between October 2011-May 2012 were included in the study. Antibiotic susceptibilities of Acinetobacter isolates were investigated by Kirby-Bauer disk diffusion method according to CLSI guidelines. According to disk diffusion test, the resistance rates for piperacillin, piperacillin-tazobactam, cefepime, ceftazidime, imipenem, meropenem, gentamicin, amikacin, tetracycline, levofloxacin, ciprofloxacin and trimetoprim-sulfamethoxazole were 96%, 96%, 97.3%, 89.3%, 96%, 94.6%, 66.7%, 85.3%, 68%, 82.7%, 97.3% and 89.3% respectively. In this study, 73 (97%) strains were found resistant to three or more than three antibiotics (multidrug resistant). Rep-PCR analysis have shown the presence of eight clones, including two major clones [A (7subtypes), B (3 subtypes)] and six unique clones (C-H). Clone A was found to be the dominant type. Fifty-four (72%) of the 75 Acinetobacter strains belonged to clone A, 13 (17.3%) to clone B, two strains to clone C, D, and one of each to the other clones (E, F, G, H). Clone A was isolated from 71% (20/28), 70% (7/10) and 100% (6/6) of the samples sent from reanimation intensive care unit, surgery ward and internal diseases intensive care units, respectively. The time interval between the first and last strain was eight months. The results of this study indicated an increase in the resistance rates of Acinetobacter strains in our hospital and this increase was attributed to the clonal dissemination of the strains. Strains of the clone A were found to be dominant at the intensive care and other clinics of our hospital. It is contemplated that Acinetobacter strains were scattered as a result of cross transmission and patient transfer among clinics. The rep-PCR method which was used in this study was evaluated as a rapid, easily applicable and successful procedure for epidemiological studies. Clonal distribution of resistant strains in the hospital environment emphasizes the significance of infection control measures.