Evaluation of Viral and Bacterial Pathogens in the Central Nervous System Infections with Multiplex PCR.

OBJECTIVE: To evaluate the bacterial and viral causes of central nervous system (CNS) infection by multiplex PCR. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Department of Medical Microbiology, Pamukkale University Faculty of Medicine, Turkey, from March 2016 to December 2021. METHODOLOGY: Cerebrospinal fluid (CSF) samples of patients prediagnosed with CNS infection were included in the study. Viral pathogens were detected with the Multiplex real-time PCR panel (FTD Neuro9, Fast Track Diagnostics, Luxembourg) and bacterial pathogens with the multiplex real-time PCR panel (FTD Bacterial Meningitis, Fast Track Diagnostics, Luxembourg). The identification of bacteria growing in samples was done by conventional methods and with the Phoenix™ (Becton Dickinson Diagnostics, USA) automated system. RESULTS: CSF samples of 440 patients were evaluated using multiplex PCR panel. The viral factors included adenovirus (14.2%), human herpes virus 7 (1.5%), varicella zoster virus (1.3%), herpes simplex virus 1 (1.3%), cytomegalovirus (1.3%), Epstein-Barr virus (0.8%), human herpes virus (0.8%), herpes simplex virus 2 (0.3%), varicella zoster virus (0.3%), and parvovirus B19 (0.3%); and bacterial factors included Streptococcus pneumoniae (7.0%) and Neisseria meningitidis (0.9%). The bacterial growth was detected in the CSF culture was 4.9%. Among the growing bacteria, there were six different types that were not found on the multiplex PCR panel. CONCLUSION: The use of a comprehensive bacterial multiplex PCR panel containing common pathogens will be more effective in pathogen detection. Care should be taken, especially when interpreting the viral Multiplex PCR. KEY WORDS: Viral multiplex PCR, Bacterial multiplex PCR, Bacteria culture.

The Presence of Protozoan Parasites in Drinking Waters and Environmental Waters in Denizli City Center

Objective: The presence of protozoan parasites in agricultural irrigation and drinking water resources at Denizli city center was investigated in detail for the first time. Methods: The research was carried out between October 2017 and October 2018 and 84 water samples were taken from 7 different stations identified from the Denizli city center. After examining the samples by direct visualization (Native-Lugol), they were stained with quinton's acid fast, giemsa and trichrome dyes. The preparations were evaluated parasitologically under a light microscope. Results: Cryptosporidium spp. was detected in 21 (25%) of 36 agricultural irrigation water samples collected during the study, Cyclospora cayetanensis in 5 samples (5.95%) and Giardia spp. in 12 samples (14.28%). No parasite findings were found in any of the 48 drinking water samples collected. Conclusion: The widespread use of animal husbandry and agriculture as grazing land in the sampling stations, the mixing of domestic wastewater into these waters without any treatment, and seasonal conditions cause the protozoan parasites to be seen more in certain periods. It is thought that waterborne protozoan infections that may occur in the future can be significantly prevented by taking the necessary precautions in terms of public health and environmental animal husbandry.

Rotavirus and adenovirus prevalence in patients with acute viral gastroenteritis in Denizli, Turkey, 2017-2021.

This study aims to determine retrospectively the prevalence of rotavirus and enteric adenovirus in patients with gastroenteritis symptoms and the distribution of pathogens by gender, age, seasons, and years. The stool samples sent to Pamukkale University Healthcare Research and Practice Hospital's Medical Microbiology laboratory between January 2017 and December 2021 were evaluated for rotavirus/adenovirus antigen positivity. Rotavirus and adenovirus antigens were studied with the Rotavirus-Adenovirus Combo Rapid Cassette Test (Acro Biotech) kit. Rotavirus was detected in 683 (8.2%) of the 8315 stool samples evaluated, and 180 (2.2%) samples were positive for adenovirus. Coinfection was detected in 21 (0.25%) samples. Rotavirus was found at the highest rate in 2019 (p = 0.001). The adenovirus was detected in 2020 at a lower rate than in other years (p = 0.0001). The rotavirus was observed at a higher rate in 0-<3, 3-<6, and 6-<13 age groups and adenovirus was detected at a higher rate in 3-<6 and 6-<13 age groups compared to other age groups (p = 0.001, p = 0.003, respectively). The highest rate of incidence of the rotavirus was found in spring and adenovirus in winter. In the etiology of gastroenteritis, especially in children, adenovirus and rotavirus should not be ignored in winter and spring. The prevalence of rotavirus was observed to have decreased in 2020 and onwards, and the prevalence of adenovirus decreased in 2020.

Comparative Evaluation of Re 529-Bp Sequence and B1 Gene in the Detection of Toxoplasma gondii Through PCR in Water Samples of Denizli, Turkey.

PURPOSE: While Toxoplasma gondii (T. gondii) infection is asymptomatic in immunocompetent individuals, it is a life-threatening protozoan in immunocompromised individuals. Its water-borne transmission to humans poses a serious public health concern. Polymerase Chain Reaction (PCR) has a considerable potential for the sensitive and specific detection of T. gondii oocysts in waters. METHODS: Comparative evaluation of RE 529-bp sequence and B1 gene to detect T. gondii tachyzoites and oocysts via PCR in agricultural irrigation water taken from downtown Denizli, Turkey and water samples collected from neighborhood fountains was performed for the first time in Turkish context. RESULTS: Based on real-time PCR targeting the B1 genetic markers and RE 529-bp sequence, T. gondii DNA was identified in 6 (16.7%) out of 48 samples collected from agricultural irrigation water. Besides, our PCR analysis did not establish any presence of T. gondii in drinking water samples. CONCLUSION: T. gondii showed lower sensitivity in B1-based PCR than in PCR targeting RE 529-bp sequence.

[Flow Cytometry Optimization of Three Different BODIPY Molecules for Staining Lipid Droplets of Cryptococcus neoformans]. / Cryptococcus neoformans'in Lipit Damlaciklarinin Boyanmasinda Üç Farkli BODIPY Bilesiginin Akan Hücre Ölçer için Optimizasyonu.

The pathogenic Cryptococcus neoformans causes life-threatening disease in immunocompromised patients. Although there are hypotheses about the role of lipid droplets in C.neoformans pathogenesis, there is still not extensively data determined on the subject yet. Lipid droplets are dynamic cytoplasmic energy storage bodies in yeasts. Diazo dyes, Nile red, LD540 and borradiazaindasen (BODIPY) molecules are frequently used in the lipid droplets studies. BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyestuffs are among the brightest green light emitting fluorophores. These neutral molecules have high lipophilicity and can easily pass through the cell wall and membrane. In this study, for the future studies on lipid droplets of C.neoformans, we aimed to optimize three different BODIPY (BODIPY480/525, BODIPY480/530, BODIPY480/535) molecules for fluorescence microscopy and flow cytometry system. Ten molecularly confirmed environmental C.neoformans strains were grown on Sabouraud dextrose agar with and without oleic acid. BODIPY staining protocols at different concentrations were used for C.neoformans lipid droplets fixed with paraformaldehyde. The visualization (by fluorescence microscopy) and detection (by flow cytometer) of the lipid droplet structures of C.neoformans strains were evaluated. Forwardscatter and side-scatter analysis were performed to evaluate the number of lipid droplets determined in the cytoplasmic region quadrant in flow cytometry. The staining of the lipid droplets of C.neoformans of all three BODIPY molecules used in the study was observed by fluorescence microscope with creating distinct brightness and sharp contrast with the background. All BODIPY molecules could be examined by fluorescence microscopy without loss of brightness in more than one minute. The optimal dye concentration of BODIPY compounds were found as 2 µM. Incubation at room temperature for five minutes was sufficient for fluorochrome staining. They were also shown to be able to stain lipid droplets in heatinactivated C.neoformans strains in all three compounds. The synthesized BODIPY480/525, BODIPY480/530 and BODIPY480/535 molecules were evaluated in accordance with the staining of lipid droplets, which were claimed to play a role in the pathogenesis of C.neoformans, and analysis by fluorescence microscopy and analysis with flow cytometer. BODIPY molecules may exhibit different properties in staining lipid droplets. These molecules should be tested for demonstrating the presence of lipid droplets in different yeast species and their suitability for pathogenesis studies.

Association Between S100b Levels and COVID-19 Pneumonia: A Case Control Study.

BACKGROUND/AIM: Extracellular S100b effects are mediated by the receptor for advanced glycation end products (RAGE), which is the S100b membrane receptor. RAGE belongs to the immunoglobulin superfamily of cell surface molecules and serves as a multiligand receptor and is expressed in high abundance by alveolar type I (AT-I) cells in adult pulmonary tissue. This study aimed to provide an insight into the association between the severity of COVID-19 disease and serum S100b levels during admission to the emergency department (ED). PATIENTS AND METHODS: A total of 64 patients (34 mild cases; 30 severe cases) were diagnosed with COVID-19 pneumonia and 30 healthy volunteers were admitted to study. Serum S100b levels were measured by using enzymle linked immunoassay method from blood serum samples. RESULTS: Serum S100b levels showed a significantly higher mean value in mild and severe disease cohorts than in healthy controls (p=0.036 and p=0.028 respectively). Receiver operating characteristic (ROC) analysis indicated greater area under the curve (AUC) for serum S100b levels of the COVID-19 patients (AUC=0.663, 95% CI=0.541-0.785; p=0.014). In addition, serum S100b concentration was measured as 151.7 ng/ml at 79.3% sensitivity and 51.7% specificity (p=0.014). Serum S100b protein levels can serve as a valuable clinical marker in establishing diagnosis of patients. Though not useful in identifying different stages of COVID-19 infection, serum S100b concentration along with other known markers can be utilized to reliably predict clinical severity along with other clinical parameters.

Photodynamic inactivation of Staphylococcus aureus using tetraethylene glycol-substituted Zn(II) phthalocyanine.

Methicillin resistant Staphylococcus aureus infections are increasing, especially in intensive care units. A new method for photodynamic inactivation (PDI) generates reactive oxygen species by photosensitization to kill bacteria. We investigated the PDI effect of tetraethylene glycol-substituted Zn(II) phthalocyanine (TEG-P) on S. aureus strains including two standards (ATCC 25923 and ATCC 43400) and 20 clinically isolated methicillin sensitive and 20 methicillin resistance strains. We also investigated three treated groups: 650 nm laser only, TEG-P only and TEG-P + laser, plus one control group. Treatments included 0.5, 1, 2, 4, 8, 16, 32 µg/ml concentrations of TEG-P. No suppression of bacterial growth was observed in the control, laser only and TEG-P only groups whether or not S. aureus was methicillin resistant. Bacterial growth was suppressed by 85% using 8 µg/ml TEG-P and completely suppressed by 32 µg/ml TEG-P in the TEG-P + laser group. A combination of TEG-P + laser treatment may be an alternative to conventional antibiotics for routine treatment of S. aureus infections, although further investigation of the effect at the tissue level is required.

[Prevalence and genotype distribution of human papillomavirus in patients attending to gynecology polyclinics]. / Jinekoloji poliklinigine basvuran hastalarda insan papillomavirüs prevalansi ve genotip dagilimi.

Human papillomavirus (HPV) infection is the most significant risk factor of the development of cervical cancer. The distribution of HPV prevalence and genotype varies widely between regions. In this study, it was aimed to investigate the prevalence and genotype distribution of HPV, retrospectively. One thousand one hundred and seventy patients who applied to the department of obstetrics and gynecology were included in this study. Samples were collected from patients for cervical HPV DNA and Pap smear. The Pap samples taken for Pap smear were fixed with alcohol and analyzed according to the modified Bethesda system. HPV identification and typing were performed using the "Linear Array HPV Genotyping Test (Roche Molecular System, USA)". Patients were divided into 5 groups due to their age. Total HPV ratio was most frequently found among the patients who were between 17-30 years old, while HR-HPV was most frequently found between 51-60 years. Nine hundred seventy-eight of 1170 (83.6%) patients had normal cytologic findings whereas 192 (16.4%) had abnormal cytologic findings. HPV was detected in 37.2% of the total patients. high-risk HPV (HR-HPV) rate was 21.2%, probable high risk (PR-HPV) rate was 6.4% and low risk HPV (LR-HPV) rate was 9.5%. When the relationship between cytologic findings and HPV was examined, normal cytology/HPV negative 67.8%; abnormal cytology/HPV negative 37.5%, normal cytology/HPV positive 32.2%, abnormal cytology/HPV positive 62.5% were detected. The highest prevalence of HPV was 8.9% with HPV 16, followed by 6, 53 and 52/53/35/58. A total of 354 patients were biopsied, 177 of whom were normal, 111 of whom were cervical intraepithelial neoplasia (CIN) 1, 66 of whom were CIN 2 and over. In the group with normal pathological findings, HR-HPV ratio was found as 15.8%, while in CIN 1 44.1% in CIN 2-3 63.6%. Sensitivity, specificity, positive predictive value, and negative predictive value of screening tests were examined in CIN 2 and more lesions. Sensitivity and specificity for HR-HPV were 63.6% and 73.3%, respectively, the same rates were 81.8% and 58.7% for HPV. The highest sensitivity was found in combination of HRHPV and Pap smear, the highest specificity in HPV. In conclusion, the HPV prevalence and genotype distribution in our study are similar to those reported in the world, but higher than previous studies in our country. These results may be due to our methodology and hospital based nature of our study group. We conclude that only smear or HR-HPV testing are not sufficient as a single pronged screening test, and that the participation of other genotypes of HPV in screening increases the sensitivity.

[Evaluation of virulence factors in enterococcus species]. / Enterokok türlerinin virulans faktörlerinin arastirilmasi.

Enterococci have recently become important due to their increased isolation rates in community-based and nosocomial infections and resistance to many antibiotics, including glycopeptides. The aim of this study was to evaluate the antimicrobial susceptible patterns and virulence factors of various clinical specimens; urine (n= 149), blood (n= 38), wound (n= 17), stool (n= 13), and other (n= 12) with a total of 229 enterococci including 138 E.faecalis and 91 E.faecium isolates. Aggregation factor (AF), enterococcus surface protein (esp), cytolysins and gelatinase encoding genes (asa1, esp, cylM, cylBcyl A, cylll, cylls, gelE, respectively) were investigated by molecular methods. Haemolysin production and gelatinase were studied phenotypically. A total of 30 isolates, 29 of E.faecium and one of E.faecalis isolates were resistant to vancomycin. High-level gentamicin and high-level streptomycin resistance in E.faecalis were 40.7% and 63.7% however, they were 47.1% and 55.8% in E.faecalis isolates. All strains were susceptible to linezolid. Ampicillin, penicillin and vancomycin resistance in E.faecium isolates were found to be higher than E.faecalis isolates (p= 0.001, p= 0.008 and p< 0.001). Asa1 (p< 0.001), cylll (p= 0.002) and cylls (p< 0.001) as well as gelatinase activity in isolates of E.faecalis were significantly higher than the isolates of E.faecium (p< 0.001). The most common virulence genes in our study were asa1 gene (45%), cyLs gene (33.2%) and esp gene (32.3%). Ciprofloxacin resistance in cylLL and cyLs gene positive isolates of E.faecalis were significantly higher compared to isolates that do not contain these genes (p= 0.035 and p= 0.047). Likewise, haemolysin producing E.faecium isolates were significantly more resistant to vancomycin compared to isolates that do not produce hemolysin (p< 0.001). When the virulence factors of vancomycin resistant and susceptible isolates were compared, the esp gene level in VRE E.faecium isolates was found to be 24.1%, while no esp gene was found in VRE E.faecalis isolates. The existence of asa1was negative in both VRE E.faecium and VRE E.faecalis isolates. The activity of hemolysin was found 42.3% for E.faecalis and 19.3% for E.faecium. In vancomycin-sensitive enterococcus (VSE) species, esp gene activity was 35.1% for E.faecalis, 29.4% for E.faecium, asa1 gene activity was 60.8% for E.faecalis and 47.1% for E.faecium, hemolysin activity was 52.8% for E.faecalis and 23.5% for E.faecium. In our study, it was found that VSE isolates have more virulence genes than VRE isolates. It should be kept in mind that VRE can causeinfections which are difficult-to-treat especially in hospitalized patients and VSE have significant virulence factors that can cause severe infections.

[Investigation of the effect of ibuprofen on wound healing in experimental Staphylococcus aureus soft tissue infections]. / Deneysel Staphylococcus aureus yumusak doku enfeksiyonlarinda ibuprofenin yara iyilesmesi üzerindeki etkisinin arastirilmasi.

Soft tissue infections (STIs) occur as a result of the colonization of pathogenic bacteria upon the destruction of normal skin microbial flora and the skin integrity. Streptococci and staphylococci are the most frequent causes of bacterial STIs. Non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen are often used in STIs because of their analgesic and antipyretic effects. However, evidence suggests that these drugs might delay both epithelization and angiogenesis in the early phases of wound healing because of an antiproliferative effect. The aim of this study was to investigate the effect of ibuprofen on the wound healing in STIs caused by Staphylococcus aureus in immunosuppressed mice. A total of 120 female Balb/c mice were used in the study and the mice were assigned to four test groups and two control groups. The test groups were defined as follows; B (Bacteria group, n= 23), BI (Bacteria + Ibuprofen group, n= 23), BA (Bacteria + Ampicillin group, n= 23), BIA (Bacteria + Ampicillin + Ibuprofen group, n= 21); and the control groups were defined as follows; S1B2 (only immunosuppressed controls, n= 15) and S2B2 (Sham group). Immunosupression was induced with cyclophosphamide and the experimental infection was generated by subcutaneous inoculation of bacterial suspension (2 x 10(8) cfu/ml) of methicillin-sensitive S.aureus ATCC 25923 to the right hind leg. Ibuprofen was given to the mice by gastric gavage (50 mg/kg/day), and ampicillin (100 mg/kg/day) by intramuscular injection. Wound sizes that appear in the animals were measured on a daily basis. Serum and tissue (epithelial tissue, connective tissue, sebaceous glands, sweat glands) samples were obtained on the first, third and seventh days. The tissue samples were examined histopathologically by hematoxylin-eosin (HE) staining method and IL-1, IL-6, TNF-α and VEGF (Vascular Endothelial Growth Factor) levels were determined in serum samples by ELISA method. The tissue cytokine reactions were also evaluated by immunohistochemical (immunoperoxidase staining) method in tissue samples. In our study, no significant change was detected in the wound sizes of B and BI groups from the second day to the end of study period (p> 0.05). On the other hand the wound dimensions of BA and BIA groups gradually decreased and remained superficial. The average serum levels of TNF-α and IL-1 was detected low in all groups. The mean value of serum IL-6 on the first day in group B was determined to be higher compared to other groups, and when this difference was compared to groups BI and BA, and the control group, it was found statistically significant (p< 0.05). In addition, the VEGF levels which were detected low in all groups in the third day of infection increased significantly at the seventh day. The results of histopathologic and immunohistochemical studies have supported the results of ELISA and yielded similar results with serum cytokine patterns. In conclusion, our data indicated that ibuprofen has no negative effect on the wound healing in soft-tissue infections caused by S.aureus.

[Evaluation of airborne molds in Laodikeia's recreation work environment during an excavation period]. / Bir kazi döneminde laodikeia rekreasyon ortaminda hava küflerinin degerlendirilmesi.

Microclimatic environments including hot and damp variations are convenient backgrounds to fungi-related diseases for archeology workers. In this study, indoor air viable mold flora of Laodikeia's antique recreation warehouse and their effect on archeology workers were investigated during an annual excavation period. Work environment and workers' symptoms have been detailed by a questionnaire. Air sampling of viable molds were periodically collected within the excavation period in 2005. The mean indoor viable mold concentrations were 528, 578 and 1023 cfu/m3 while outdoor mold concentrations were 352, 409 and 553 cfu/m3 during July, August and September, respectively. The prevalent culturable indoor fungal type was Cladosporium spp. (57.7%), Aspergillus spp. (17.8%), Alternaria spp. (15.8%) and Penicillium spp. (3.4%). In outdoor environment, Cladosporium spp. (60.4%) was the predominant type followed by Alternaria spp. (16.8%), Aspergillus spp. (11.8%) and Penicillium spp. (3%). Aspergillus spp. were found in higher concentrations for indoor air than outdoor samples in late-summer period owing to higher humidity and lower temperature levels (p < 0.05). The rate of Aspergillus niger isolation was higher than the other Aspergillus species (p < 0.05). Among the 41 workers hay fever, bronchial asthma and eczama were detected in 8 (19.2%) of them and three workers stated increase in their complaints during their work in the recreation warehouse. According to the questionnaire results, moisture and irritable particle problems were more prevalant among archeology workers in recreation warehouse. As in Laodikeia experience, re-building of warehouses or restriction of recreation period between early and mid summer may be recommended in order to avoid to be confronted with xerophilic fungi. Climate monitorization and questionnaire results may help to decide on the screening procedures to enlighten the ventilation problems related to toxigenic molds and also to demonstrate the presence of xerophilic fungi in the moist environment.

[Does pre-incubation period affect detection of Pseudomonas aeruginosa and Acinetobacter baumannii species with automatized BACTEC 9120 blood culture system?]. / Pre-inkübasyon süresi Pseudomonas aeruginosa ve Acinetobacter baumannii türlerinin otomatize BACTEC 9120 kan kültür sisteminde saptanmasini etkiliyor mu?

In this study, the effect of the pre-incubation period on detection of non-fermentative species in blood culture system was investigated. Different concentrations of Pseudomonas aeruginosa and Acinetobacter strains were inoculated into BACTEC-9120 plus+Aerobic/F bottles and incubated for 0, 4, 8, 16 hours at 36 dgerees C and then were loaded to the system. Both P. aeruginosa and A.baumannii strains yielded positive signals within 24 hours after loading. In all preincubation periods, as the concentration of P. aeruginosa strains decreased, the detection time was increased. The higher the concentration of A.baumannii strains, is the longer the signalling time as the pre-incubation period is increased, whereas the lower the concentration of A. baumannii strains, is the shorter the signalling time as the preincubation period is increased. Our study indicated that the BACTEC-9120 blood culture system determined non-fermentative bacteria after a pre-incubation time of 16 hours.

[Species distribution and antibiotic resistance of enterococci isolated from cattle farmers and cattles]. / Besici ve sigirlardan izole edilen enterokok türlerinin dagilimi ve antibiyotik duyarliliklari.

The aim of this study was to detect the species distribution and antibiotic resistance of enterococci with a special view for vancomycin resistance, isolated from stool and rectal swab samples of cattle farmers and cattles in Denizli region, Turkey. All the specimens were inoculated onto Enterococcosel agar plate and into Enterococcosel broth supplemented with and without vancomycin (6 microg/ml). The agar plates were incubated at 37 degrees C for 24 hours, while broth cultures were incubated at 37 degrees C for 48 hours. Enterococcus spp. were isolated from 96 (86.4%) of 111 farmers' and 65 (90.2%) of 72 cattles' specimens. The most commonly isolated species from both humans and animals were E. faecalis (39.6% and %32.3%, respectively), E. faecium (28.1% and 24.6%, respectively) and E. raffinosus (8.3% and 13.8%, respectively). None of the isolates were found to be resistant to vancomycin and teicoplanin. One high-level streptomycin (300 microg) resistant E. faecium, and six (2 E. faecalis, 2 E. durans, 2 E. hirae) high-level gentamicin (120 microg) resistant strains have been isolated from the specimens of cattle farmers and cattles, respectively. There was no significant difference between the rectal swabs and stool specimens for the isolation of Enterococcus spp. (p>0.05). Enterococcosel broth was found to be more sensitive than Enterococcosel agar for the isolation of Enterococcus spp. (p=0.001). It was concluded that the lack of vancomycin resistant enterococci may be related to the prohibition of avoparcin use in our country.

[Investigation of the inter-observer agreement rates for morphometric evaluation of the growth of Microsporum canis colonies]. / Microsporum canis kolonilerinin morfometrik degerlendirmesinde arastirici uyumu.

Morphometry is a newly applied method for investigation of the in-vitro growth dynamics of dermatophyte colonies. This study was undertaken to evaluate the correlation and agreement rates between the morphometric data obtained by different observers. For this purpose, five different Microsporum canis growth data were evaluated by six observers. The results of the study suggested that the agreement rates among the data obtained by different observers were low (R1 = -5.3509), while the results obtained by a single observer at different reading time points were consistent and correlated with the estimated growth rate. We thus recommend the evaluation of morphometric test results by a single observer for optimal standardization and consistency.