[Evaluation of the Methods Used for the Detection of Entamoeba histolytica in Stool Samples of Patients with Diarrhea]. / Ishalli Hastalarin Diski Örneklerinde Entamoeba histolytica'nin Saptanmasinda Kullanilan Yöntemlerin Degerlendirilmesi.

Amoebic dysentery (amebiasis) is a parasitic infection caused by Entamoeba histolytica. The diagnosis of invasive amebiasis has traditionally been based on direct and stained microscopic examination of stool samples. Stool microscopy exhibits low sensitivity and it is difficult to distinguish E.histolytica cysts and trophozoites from cells such as leukocytes, macrophages and non-pathogenic Entamoeba species in the stool by microscopy. Therefore more sensitive and specific diagnostic methods such as enzyme linked immunosorbent assay (ELISA) tests which investigate the presence of E.histolytica-specific antigen in stool, and polymerase chain reaction (PCR) are being widely used. In this study it was aimed to study stool samples of the patients who applied with the clinical signs of amebiasis by using direct and permanent stained microscopy, E.histolytica adhesin antigen ELISA test and real-time PCR-based BD Max Enteric Parasite Panel (BD Max EPP) test and to evaluate the diagnostic values of these tests. A total of 546 faecal samples with blood and/or mucus were analyzed in the study. In these samples, the presence of E.histolytica was investigated by direct and permanent stained microscopy, E.histolytica adhesin antigen ELISA and BD Max EPP PCR. Of the samples 36.3% were suspected to contain E.histolytica/dispar/moshkovskii cyst and/or trophozoite by direct microscopic examination. Trichrome staining was performed on these samples and 49 samples were found suspicious for the presence of E.histolytica/dispar/moshkovskii cysts and/or trophozoites. The presence of E.histolytica and other Entamoeba species was not confirmed in 75.2% of the samples. BD Max EPP PCR and E.histolytica adhesin antigen ELISA tests were studied in 49 faecal samples that were suspected by trichrome staining. None of these samples were positive by ELISA. Forty-four samples were negative by PCR and invalid test results were obtained in five samples. In this study, E.histolytica was not detected in the patient population. The results of this study showed that microscopic examination alone is not sufficient for the detection of E.histolytica. It is concluded that it is necessary to use a more sensitive and specific also rapid diagnostic test such as E.histolytica-specific antigen detection test or PCR in the diagnosis of amebiasis to avoid misdiagnosis and unnecessary treatment of patients.

Evaluation of an Automated Yeasts Identification System for Identification of Yeast Isolates.

BACKGROUND: Invasive candidiasis is the most important health-care-associated fungal infection worldwide. In the last two decades, the cause of the increase of fungal infections is immunosuppression or serious underlying diseases. Additionally, Rhodotorula species, Blastoschizomyces capitatus, and Trichosporon species are emerging as important human pathogens in immunocompromised patients with hematological malignancy. METHODS: Between January 2012 and January 2018, a total of 603 fungal organisms were isolated from blood culture samples and included in the study. All of the isolates were identified by using standard mycological methods, MALDI TOF MS system, and the Phoenix system. Sequence analysis was performed on yeasts that could not be definitively identified by using SMM and incompatible according to the results with Phoenix and MALDI-TOF MS analysis. RESULTS: 603 fungal isolates including 594 Candida spp. and 9 other yeasts like species were analyzed. C. albicans was the most frequently isolated species. The results of identification by conventional methods and MALDI TOF MS were compared to the results of the Phoenix system. The observed concordance was 99.2%. The compatibility with other systems of the Phoenix system was 100%, 100%, 97.3%, 100%, and 96.9% for C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, and C. krusei, respectively. CONCLUSIONS: The BD Phoenix system was found to be a simple, reliable, and effective method to identify the main species of the genus Candida in our study.

[Comparison of Two Commercial Quantitative Cytomegalovirus (CMV) Polymerase Chain Reaction Tests Calibrated by World Health Organization International CMV Standard]. / Dünya Saglik Örgütü Uluslararasi Sitomegalovirüs (CMV) Standardi ile Kalibre Edilmis Iki Ticari Kantitatif CMV Polimeraz Zincir Reaksiyonu Testinin Karsilastirilmasi.

Cytomegalovirus (CMV) viral load quantitation is important in diagnosis, follow-up, and monitoring of antiviral therapy in transplanted patients. In this study, it was aimed to compare the results of the two commercial World Health Organization (WHO) International CMV standard calibrated polymerase chain reaction tests, CMV Cobas Ampliprep/Cobas Taqman (CMV-CAP/CTM) (Roche, Germany) and Artus CMV QIASymphony-Rotorgene (CMV-QS-RGQ) (Qiagen, Germany). Both tests were performed simultaneously on 244 plasma samples. The results were measured in copies/ml and converted to IU/ml by multiplying with 0.91 for CMV-CAP/CTM and 1.64 for CMV-QS-RGQ, as specified by the manufacturers. CMV DNA was detected in 174 (71.3%) and was not detected in 52 (21.3%) of the samples and eighteen (7.4%) samples had discordant results by both of the tests. In 16 out of 18 samples with discordance, the viral load was below the dynamic measuring ranges of both tests. In one sample, CMV DNA could not be detected by CMV-CAP/CTM but detected by CMV-QS-RGQ with 497 copies/ml, and 334 copies/ml CMV DNA was detected by CMV-CAP/CTM in another sample where it could not be detected by CMV-QS-RGQ. A high degree of agreement was found between the qualitative results of the both tests (kappa= 0.80, p< 0.001). For quantitative results in the dynamic measuring range of both assays (n= 129), the median viral load values measured by CMV-CAP/CTM and CMV-QS-RGQ were 1140 copies/ml (range: 151-254000) and 1826 copies/ml (range: 189-551521). When the results were converted to IU/ ml, the median viral load values measured by CMV-CAP/CTM and CMV-QS-RGQ were 1037 IU/ml (range: 137-231140) and 2993 IU/ml (range: 310-904133), respectively. There was a very strong correlation (r= 0.94, p< 0.001; r= 0.94, p< 0.001, respectively) between the log10 values of the quantitative results in the dynamic measuring ranges (n= 129) as copies/ml and IU/ml of both tests. CMV-QS-RGQ values corresponding to 150, 1000, 3000 copies/ml in CMV-CAP/CTM were as 94.5, 1571, 323.5 copies/ml and CMV-QS-RGQ values corresponding to 137, 910, 2730 IU/ml in CMV CAP/CTM were as 154, 2557.6, 6965.9 IU/ml, respectively. A variation of 0.45 log10 was determined between these values. In a total of 131 samples; 129 of them with the result of both tests in the dynamic measuring range and two of them which CMV DNA was not detected in one of the tests; it was found that 112 (85.5%) results for copy/ ml, 73 (56%) results for IU/ml were within the measurement difference of ± 0.5 log10 and 19 (14.5%) results for copy/ml and 58 (44%) results for IU/ml were greater than ± 0.5 log10. Bland-Altman analysis showed that CMV-CAP/CTM test made lower measurements than CMV-QS-RGQ and the average difference for copy/ml and IU/ml results were 0.22 log10 copies/ml and 0.47 log10 IU/ml. In conclusion; when the results were converted to IU/ml, the number of samples with an acceptable measurement difference between the two test results (≤ 0.5 log10) decreased and the number of samples with a measurement difference > 0.5 log10 increased and the difference was found as statistically significant (p< 0.001). Calibrating the Roche CMV CAP/CTM and Artus CMV-QS-RGQ tests with the WHO international CMV standard did not increase comparability between quantitative results in plasma samples, on the contrary, it was found that when the results were converted to IU/ml, a measurement difference indicating biologically significant viral replication was detected between the two test results.

[Evaluation of a Commercial Multiplex Tandem Polymerase Chain Reaction Method for the Identification of the Yeasts Isolated from Blood Cultures]. / Kan Kültürlerinden Izole Edilen Mayalarin Tanimlanmasinda Ticari Bir Multipleks Tandem Polimeraz Zincir Reaksiyonu Yönteminin Degerlendirilmesi.

Candidemia is one of the most important health care-associated infections worldwide. Candida species have species-specific antifungal susceptibility profiles and it has been shown that the identification of the Candida species is necessary for the appropriate treatment of the patients with candidemia. Various methods are used to shorten the identification time for the determination of the causative species. Fungal ID multiplex tandem polymerase chain reaction (MT-PCR) (AusDiagnostics, Australia) is a test developed to identify yeasts and molds isolated from clinical specimens. In this study, we aimed to evaluate the Fungal ID MT-PCR test (AusDiagnostics, Australia) for the identification of the yeasts from positive blood cultures in Akdeniz University Hospital Central Laboratory. Between December 2016 and December 2017, blood culture samples from 92 consecutive patients with yeast cells detected in Gram stained smears were tested by Fungal ID MT-PCR and the reference method. After the subculture of the positive signaling blood culture bottles to Sabouraud dextroz agar (SDA), the identification of the yeasts were performed by morphological identification methods (Germ tube test, Corn Meal Tween® 80 agar media, etc.), BD Phoenix Yeast ID Panel (Becton Dickinson, Sparks, MD) and Bruker Biotyper matrix-assisted laser desorption ionization-time of mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Germany) systems. Identification with MALDI-TOF MS have been accepted as the reference method. Thirty-five of the isolates were identified as Candida albicans, 17 were Candida glabrata, 13 were Candida parapsilosis, 12 were Candida tropicalis, seven were Candida krusei , two were Candida guilliermondii, two were Candida dubliniensis, two were Candida inconspicua, one was Candida kefyr and one was Saprochaete capitata by the reference method. In our study, no blood culture sample yielded more than one yeast species. 94.6% of the strains were presumptively identified by the morphological identification methods. Discordant results were not detected between the BD Phoenix Yeast ID Panel and the reference method. Thirty-three of the isolates were identified as C.albicans, 15 were C.glabrata, 13 were C.parapsilosis, 11 were C.tropicalis, five were C.krusei , two were C.guilliermondii, one was C.dubliniensis, one was C.kefyr and 10 were Candida spp. by Fungal ID MT-PCR assay. Since C.inconspicua and S.capitata were not included in the test panel, C.inconspicua was identified as Candida spp. in two samples, while S.capitata could not be identified in one sample. Concordance between Fungal ID MT-PCR and the reference method were found to be 88% at the species level and 98.9% at the genus level. The sensitivity of the Fungal ID MT-PCR test in in the detection of C.krusei and C.glabrata was 71.4% and 88.2%, respectively. Fungal ID MT-PCR test has shown a high performance in the identification at the genus level, but the identification at the species level, which is important for the treatment management, was moderate. Fungal ID MT-PCR can be used as an adjunct test to the traditional identification methods for the early identification of the Candida species.

[Evaluation of Microscopic Examination, Culture and Polymerase Chain Reaction Tests in the Diagnosis of Trichomonas vaginalis Infection]. / Trichomonas vaginalis Enfeksiyonu Tanisinda Mikroskobik Inceleme, Kültür ve Polimeraz Zincir Reaksiyonu Testlerinin Degerlendirilmesi.

Sexually transmitted infections (STIs) are important as a public health problem all over the world. There are some difficulties in prevention and control programs of STIs due to clinical and laboratory diagnostic problems.The most common STIs are Chlamydia trachomatis infections, trichomoniasis and gonorrhea. The study aimed to investigate the direct microscopic examination, culture and polymerase chain reaction (PCR) tests in the diagnosis of Trichomonas vaginalis infection; to determine other microbiological agents that may cause vaginal discharge and to evaluate the various social variables in women with vaginal discharge admitted to the outpatient clinic of Obstetrics and Gynecology in Akdeniz University Hospital. Two hundred and fifteen patients were enrolled in the study. The socio-demographic features of the patients were recorded. Vaginal/endocervical swab specimens taken from patients were evaluated by microscopic examination. Swab specimens were inoculated into blood agar, MacConkey agar and chocolate agar for bacterial culture. Modified Trichosel broth with 5% horse blood (Becton Dickinson, USA) was used for Trichomonas spp. culture. The presence of C.trachomatis, Neisseria gonorrhoeae, and T.vaginalis in swab samples were investigated by multiplex PCR assay (BD Max CT/GC/TV, Becton Dickinson, USA). At least one pathogen was detected among 65 (30.3%) samples. T.vaginalis was detected by microscopic examination and PCR in four of 215 (1.9%) patients. Existence of yeast morphology was observed in 21 (9.8%) specimens by microscopic examination. Twenty four (11.2%) patients were diagnosed as bacterial vaginosis microscopically according to Nugent score system. Candida species grew in 32 (14.9%) and Streptococcus agalactiae grew in 2 (0.9%) of the specimens. C.trachomatis was detected in 2 (0.9%) samples and N.gonorrhoeae in 1 (0.5%) sample by PCR. In this study, 95.3% of the patients were married and 96.7% had only one sexual partner in the mean time. The rate of detection of pathogens were statistically higher in women who have had two or more pregnancies (p<0.05). In our study, T.vaginalis together with N.gonorrhoeae and C.trachomatis were investigated by PCR method in women with vaginal discharge. The use of multiplex PCR test allowed simultaneous investigation of multiple pathogens in the patient samples.

Evaluation of a nucleic acid amplification assay for the diagnosis of Clostridioides difficile infection.

Clostridioides difficile is the leading cause of healthcare-associated diarrhea and the laboratory diagnosis of Clostridioides difficile infection (CDI) continues to be challenging. Accurate and rapid identification of C. difficile will reduce unnecessary antibiotic use and ensure contact isolation to control the spread of CDI. In this study, diagnostic performance of BD MAX Cdiff assay (Becton Dickinson, USA) was evaluated for the detection of C. difficile in 2502 fresh stool samples from hospitalized children and adult patients and the results were compared to toxigenic culture. The frequency of CDI in adults and pediatric patients were found as 3.3% and 6.2%, respectively. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of BD MAX Cdiff assay were found as; 100%, 99.7%, 93%, and 100% for all patients; 100%, 99.7%, 96.2%, and 100% for pediatric patients; and 100%, 99.6%, 90.2%, and 100% for adult patients, respectively. We concluded that BD MAX Cdiff assay with high sensitivity, specificity, and PPV is useful for the diagnosis of CDI. With a high NPV of 100%, BD MAX Cdiff assay is also suitable for the exclusion of CDI.

[Evaluation of the BD Phoenix100 System and Colistin Broth Disk Elution Method for Antimicrobial Susceptibility Testing of Colistin Against Gram-negative Bacteria]. / Kolistinin Gram-negatif Bakterilere In Vitro Etkinliginin Belirlenmesinde BD Phoenix100 Sistemi ve Kolistin Sivi Disk Elüsyon Yöntemlerinin Degerlendirilmesi.

Infections with multidrug resistant gram-negative bacteria is a growing problem especially in health care settings. Colistin is one of the last resort antibiotics for such infections in which treatment options are limited. Increasing resistance to colistin is a global problem. Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) study groups have recommended the ISO-standard broth microdilution method (20776-1) as the reference method for the determination of colistin susceptibility. Since the broth microdilution method is not a practical method, it is rarely used in routine clinical microbiology laboratories, yet simple and accurate phenotypic detection methods for the determination of colistin resistance in routine microbiology laboratories are not precisely defined. The aim of this study was to evaluate BD Phoenix100 (Becton Dickinson, USA) system and colistin broth disk elution method for the detection of in vitro activity of colistin against gram-negative bacteria. A total of 419 gram-negative bacteria, including 199 Klebsiella pneumoniae, 163 Acinetobacter baumannii, 34 Escherichia coli, 20 Enterobacter spp., and three Citrobacter spp. isolates which were isolated from various clinical samples in our hospital between 2016-2018 were tested. The broth microdilution method was used as the reference method applying ISO-standard broth microdilution methods (20776-1) and CLSI/EUCAST recommendations. For colistin broth disk elution method, final concentrations of 0 (growth control), 1, 2 and 4 µg/ml were obtained by adding 10 µg colistin disks to four tubes containing 10 ml cation-adjusted Mueller Hinton broth per isolate. After incubation at room temperature for 30 minutes, 50 µl of standardized inoculum suspensions were added to the tubes. Colistin minimum inhibitor concentration (MIC) values were read visually after 16-20 hours of incubation at 35°C in ambient air. Manufacturer's recommendations were followed for BD Phoenix100 system. The categorical agreement between the reference broth microdilution method and the colistin broth disk elution method was 99.3%, very major error and major error rates were 0.2% and 0.5%, respectively. For BD Phoenix100 system, the categorical agreement was 95%, with a very major error rate of 5%. Our results showed that colistin broth disc elution method worked well compared to the reference broth microdilution method. The BD Phoenix100 system, with a high very major error rate, does not reliably distinguish colistin-resistant and colistin-susceptible strains.

Nitrate Reductase Assay for Rapid Determination of Methicillin-Resistant Staphylococcus aureus Clinical Isolates.

OBJECTIVE: To evaluate the performance of nitrate reductase assay (NRA), a rapid, colorimetric method for the determination of methicillin resistance in Staphylococcus aureus isolates obtained from the culture collection of the Akdeniz University Hospital Central Laboratory, Antalya, Türkiye. MATERIALS AND METHODS: Identification for all 290 S aureus isolates at the species level was performed via matrix-assisted laser desorption/ionization-time of flight. Isolates were tested with NRA for methicillin resistance. The cefoxitin broth microdilution (BMD) method recommended by the Clinical and Laboratory Standards Institute was used as the reference method in the study. S aureus ATCC 29213 and S aureus ATCC 43300 strains were used for quality control. RESULTS: According to Food and Drug Administration criteria, the category agreement between NRA and BMD was found to be 100%. The essential agreement between both methods was determined to be 96.20%. There is no minor, major, or extremely major discrepancy between both methods. CONCLUSION: The results show that NRA is a rapid, practical, and reliable colorimetric method for detecting MRSA.

Quercetin: Synergistic Interaction with Antibiotics against Colistin-Resistant Acinetobacter baumannii.

Infections caused by resistant strains of Acinetobacter baumannii are now a global problem that requires the immediate development of new antimicrobial drugs. Combination therapy is one of the strategies used to solve this problem. Based on this information, the purpose of this study was to determine whether quercetin (QUE), in combination with three antibiotics, is effective against colistin-resistant A. baumannii strains (ColR-Ab). The effects of the combination of QUE with colistin (COL), amikacin (AMK), and meropenem (MEM) were evaluated according to the checkerboard synergy test. The combinations of QUE + COL and QUE + AMK showed synergistic activity on ColR-Ab strains with FICI values in the range of 0.1875-0.5 and 0.1875-0.2825, respectively. A 4- to 16-fold decrease in COL MIC and a 16- to 64-fold decrease in AMK MIC values were detected. Synergistic activity was confirmed by the time-kill test, and these combinations were found to be bactericidal at the end of 24 h. According to spectrophotometric measurements, the combinations of QUE + COL and QUE + AMK induced membrane damage, leading to the leakage of nucleic acids. Cell lysis and cell death were confirmed with SEM observations. The detected synergy offers an opportunity for the future development of treatment strategies for potential infections caused by ColR-Ab strains.

Resazurin microplate test method for rapid determination of colistin resistance in carbapenem-resistant Acinetobacter baumanii isolates.

BACKGROUND: Colistin-resistant Acinetobacter baumannii isolates are extremely important pathogens for hospital-acquired infections. OBJECTIVE: To investigate the effectiveness of the resazurin microplate assay (REMA) for the rapid determination of colistin resistance. METHODS: Susceptibility for colistin was investigated in vitro by the broth microdilution method (BMD) and the resazurin microplate assay (REMA) on 106 carbapenem-resistant Acinetobacter baumannii isolates. RESULTS: The results of both test methods were compared, and the categorical agreement between them was found to be 100%. No minor, major, or very major discrepancy was observed between the 2 methods. CONCLUSIONS: The most important advantages of REMA are that the results are obtained within 6 hours compared to the reference method, that it is easy to evaluate because it is colorimetric, and that the susceptibility result can be reported to the clinician on the same day as bacterial identification.

Fungal keratitis caused by Scedosporium apiospermum: a case report.

BACKGROUND: We present a case of fungal keratitis caused by Scedosporium apiospermum, which is a rare agent. Case description A 34-year-old Caucasian male patient was admitted to our clinic with complaints of pain and blurred vision in the left eye. The patient had a history of wearing contact lenses for 3 years. According to the Snellen chart, the patient's visual acuity was 20/20 and counting fingers at 30 cm, for right and left eyes, respectively. A 3 × 3 mm corneal abscess at the center of the cornea with hypopyon in the patient's left eye was observed. After the patient was hospitalized, fortified gentamicin and fortified cefazolin drops were started 24 times per day. Intravenous fluconazole 1 × 800 mg loading, 1 × 400 mg maintenance dose, intravenous vancomycin 4 × 500 mg and intravenous cefoperazone + sulbactam 2 × 2 g treatments were started. We observed S. apiospermum in the corneal scraping sample, which the identification was performed by combined phenotypic characteristics and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry on the sixth day of treatment. The drops were revised as fortified vancomycin, ceftazidime, and voriconazole drops 24 times per day. Intravenous voriconazole 2 × 6 mg/kg loading and 2 × 4 mg/kg maintenance dose treatments were started. Three weeks later, left eye visual acuity increased to 20/40, and the corneal abscess regressed. On second-year follow-up, his visual acuity increased to 20/25 for the left eye and the cornea was transparent. CONCLUSION: Scedosporium group is an opportunistic filamentous fungus that is very rarely seen and causes severe keratitis infections. In the literature, to the best of our knowledge, three cases of keratitis due to S. apiospermum after contact lenses were reported, and all were treated with penetrating keratoplasty. In this case, unlike the others, only medical treatment was applied. In cases with suspected fungal keratitis, medical treatment should be started without waiting for the culture result, the findings should be followed and penetrating keratoplasty should be performed in the case of no response to treatment.

Evaluation of surface characteristic and bacterial adhesion of low-shrinkage resin composites.

This study aimed to examine the surface characteristics of low shrinkage composites and adhesion of Streptococcus mutans and Streptococcus mitis to these materials. Control material (glass) and three low shrinkage composites (Charisma Diamond, Kalore GC, Beatiful II LS) were used. After polishing procedure was applied to composite specimens, surface roughness (SR), surface free energy (SFE), and contact angle measurements were performed. Surfaces of composite were analyzed using scanning electron microscope and energy-dispersive X-ray spectroscopy. After pellicle formation with artificial saliva, S. mutans and S. mitis biofilms were incubated in 5% CO2 for 24 h at 37°C and were analyzed using confocal laser scanning microscopy. The lowest SR and highest SFE values were found in the control group. While the contact angle of control was statistically lower than composites, statistically difference was not found between composite groups. S. mutans adhesion of composites was significantly lower than control group, but there was no significant difference between composites. S. mitis adhesion of all groups was statistically similar. SR did not affect the S. mutans and S. mitis adhesion. Less adherence of S. mutans to low shrinkage composites was associated with low SFE and high contact angle values. Even though the highest SR was observed in the Charisma Diamond, no difference was found between the composites in terms of bacterial adhesion.