Treatment of BK Polyomavirus-Associated Nephropathy in Paediatric Kidney Transplant Recipients: Leflunomide Versus Cidofovir.

OBJECTIVES: BK polyomavirus-associated nephropathy is a clinicopathological entity that negatively affects graft function in kidney transplant recipients. We compared the efficacy of leflunomide and cidofovir to treat BK polyomavirus-associated nephropathy in pediatric kidney transplant recipients. MATERIALS AND METHODS: Medical records of pediatric recipients with BK viremia for the period 2004 through 2019 were reviewed retrospectively, and patients diagnosed with BK polyomavirusassociated nephro-pathy were included in the study. A serum BK virus level above 104 copies/mL was accepted as BK viremia. We defined BK polyomavirusassociated nephropathy as detection of BK virus SV40 antigen on immunochemistry staining of renal graft tissue accompanied by signs of tubulointerstitial nephritis or elevated serum creatinine in addition to BK viremia. RESULTS: Of 304 kidney transplant recipients, 53 had persistent BK viremia; 36 of these patients (61.1% male) were included in the study with the diagnosis of BK polyomavirus-associated nephropathy. Twelve patients (33.3%) received cidofovir, and 14 (38.8%) received leflunomide. Results were similar between the cidofovir and leflunomide groups for serum creatinine level at last follow-up (0.91 ± 0.29 vs 0.94 ± 0.37 mg/dL, respectively; P = .843) and graft failure rate (8.3% vs 14.2%, respectively; P = .632). Graft failure was observed in 8.3% of patients with BK polyomavirus-associated nephropathy. CONCLUSIONS: Leflunomide and cidofovir showed similar efficacy for treatment of BK polyomavirus-associated nephropathy.

Predictive Factors of BK Virus Development in Kidney Transplant Recipients and the Effect of Low-Dose Tacrolimus Plus Everolimus on Clinical Outcomes.

OBJECTIVES: This study aimed to determine the predictive factors of BK virus viremia/nephropathy in kidney transplant recipients and to evaluate the effects of low-dose tacrolimus plus everolimus. MATERIALS AND METHODS: This study included 3654 kidney transplant recipients. The patients were divided into 2 groups: group 1 were BK virus negative (n = 3525, 96.5%) and group 2 were BK virus positive (n = 129, viremia 3.5%, nephropathy 1%). Predictive factors were determined by receiver operating characteristic curve analysis and logistic regression models.We also divided and analyzed patients with BK virus viremia/nephropathy into 2 groups according to immunosuppressive changes. Group 2a had been switched to low-dose tacrolimus plus everolimus (n = 54, 41.9%), and group 2b had been switched to other immunosuppressive protocols (n = 75, 58.1%). RESULTS: We found that use of anti-T-cell lymphocyte globulin and tacrolimus, deceased donor transplant, and rejection were predictive factors for BK virus viremia/nephropathy. In addition, patients who had low-dose calcineurin inhibitor plus mammalian target of rapamycin inhibitor regimens showed a low rate of BK virus development(only 6.2% of all cases). In Group 2a, both the BK polyomavirus-associated nephropathy rate (n = 23 [42.6%] vs n = 12 [16%] in group 2b; P = .001) and viral load (DNA > 104 copies/mL) (n = 49 [90.7%] vs n = 27 [36%] in group 2b; P = .001) were increased versus group 2b. Graft function, graft survival, viral clearance, and rejection rate were similar between the groups after protocol change. CONCLUSIONS: BK virus viremia/nephropathy rate was lower in patients who received low-dose calcineurin inhibitor plus mammalian target of rapamycin inhibitor protocols; the low-dose tacrolimus plus everolimus switch protocol after BK virus was more effective and safe than other protocols.

[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.

Follow-up of human adenovirus viral load in pediatric hematopoietic stem cell transplant recipients.

BACKGROUND: The spectrum of human adenovirus (HAdV)-related disease is broad, and the virus acts on many organs and systems in hematopoietic stem cell transplantation (HSCT) recipients. We aimed to evaluate the effect of HAdV-DNA positivity with clinical and laboratory findings 4 months after HSCT. METHODS AND RESULTS: We retrospectively investigated HAdV-DNA in 153 HSCT recipients (≤18 years) by quantitative real-time polymerase chain reaction (RealStar; Altona Diagnostics). The results of samples from January 2014 to December 2017 are included. HAdV-DNA was positive for at least one sample type in 50 (32.67%) patients. HAdV-DNA positivity rate was 8.92% (N: 145/1625), 40.25% (N: 64/159), and 25% (N: 2/8) for plasma, stool, and urine samples, respectively. HAdV-DNA was positive in the plasma of 38 (24.83%) patients at a median 16 (range: 1-58 days) days after HSCT. The mortality rate was 23.68% and 6.95% in plasma HAdV-positive and HAdV-negative patients (p = .014). Moreover, HAdV-DNA positivity had an impact on overall survival for allogeneic-HSCT (p = .013), with the cumulative effect including graft-versus-host disease state in multivariate analysis (p = .014). CONCLUSIONS: Plasma HAdV-DNA positivity is a potential influencer that decreases survival in the early post-transplant period. Due to the high mortality rates, close monitoring is required of HAdV infections after HSCT with sensitive methods, especially at the early stage.

Incidence of BKV in the urine and blood samples of pediatric patients undergoing HSCT.

The aims were to investigate the incidence of BKV infection and the presence of HC in pediatric patients undergoing HSCT. Twenty-four children patients (M/F: 17/7) undergoing HSCT in a single center over a period of 1 year were included in the study. The presence of BKV DNA was determined by quantitative real-time PCR in plasma and urine samples at the following times: before transplantation, twice a week until engraftment time, and weekly for + 100 days. The mean age of the patients was 7.79 ± 5.03 years, the mean follow-up time was 95.6 ± 25.9 days, and the average number of samples per patient was 15.8 ± 3.2. BKV DNA was detected in at least one urine sample in 91.6% (n: 22) and at least one plasma sample in 75% (n:18) of the patients. The median time to the first BKV DNA positivity in urine and plasma samples was 11 (range: 1-80) and 32 days (range: 2-79), respectively. The median value of BKV DNA copies in urine and plasma were 1.7 × 106 (range: 2.8 × 101 -1.2 × 1014 ) and 1.9 × 103 copies/mL (range: 3-2.1 × 106 ), respectively. Thirteen patients (54.2%) had hematuria with BKV viruria; 8 (33.3%) patients had viremia. The median value of the BKV DNA copies in urine and plasma was 4.4 × 107 (range: 65-1 × 1011 ) and 2.9 × 103 (range: 7-7.8 × 104 ) copies/mL in these patients. Two (15.4%) of the 13 patients with BKV viruria and hematuria were diagnosed with BKV-related HC. BKV DNA viral load monitoring of urine and plasma in pediatric HSCT patients with a high risk for viral infections is valuable for understanding the development of BKV-related HC.

Investigation of Torque Teno Virus (TTV) DNA as an immunological and virological marker in pediatric hematopoietic stem cell transplantation (HSCT) patients.

BACKGROUND: High viral loads are observed in Torque Teno Virus (TTV) infection after hematopoietic stem cell transplantation (HSCT). We aimed to analyze the kinetics of plasma TTV-DNA load in pediatric patients who received immunosuppressive therapy and developed infection complications in the first 100 days after HSCT. METHODS: As a patient group; 113 plasma samples taken from 33 pediatric HSCT recipients at a time interval after transplantation and as a control group; 38 plasma samples from 38 children without known chronic disease were included in the study. Viral nucleic acid isolation was performed by using the NucliSENS easyMAG (bioMerieux, France) system. A laboratory designed quantitative polymerase chain reaction process was performed on 7300 Real-Time PCR system (Applied Biosystems, CA, USA) with the amplification mixture containing primer and probe sequences for the UTR gene region. RESULTS: TTV-DNA was detected in all patient's samples and the median viral load was calculated as 7.67 Log10 copies/mL (range: 2.84-9.59). In the control group, the TTV-DNA median viral load was calculated as 5.51 Log10 copies/mL (range: 2.50-7.04), except for one negative sample. A significant difference was observed between the control group and the patient group in terms of TTV viral load levels. In nine patients, a median 2.15 Log10 copies/mL viral load increase was observed at 31-60 days post-transplant compared to the pre-transplant period. CONCLUSION: TTV-DNA levels should be closely monitored to understand the immune status of the first 100 days after transplantation and the effects of treatment regimens on patients with HSCT.

[Evaluation of the Two Different Real Time Polymerase Chain Reaction Methods Used for BK Virus (BKV) Quantification and BKV Genotype Assignment]. / BK Virüs (BKV) Kantitasyonunda Kullanilan Iki Farkli Gerçek Zamanli Polimeraz Zincir Reaksiyonu Yönteminin Karsilastirmasi ve BKV Genotip Tayini.

BK virus (BKV) viral load quantification has a distinct role in the clinical control of BKV nephropathy and organ rejection among renal transplant recipients. In this study, it was aimed to compare BKV DNA measurement values performed with two different real-time polymerase chain reaction (PCR) methods and to determine BKV genotypes in renal transplant recipients. Totally, 150 clinical samples tested previously in two different laboratories (Lab-1 and Lab-2) from adult and pediatric renal transplantation patients were included in the study. Fifty plasma samples of 50 different patients from Lab-1, 50 plasma and 50 urine samples of 58 different patients from Lab-2 were included in the study. Viral nucleic acid extraction was performed with automatized systems in Lab-1 and Lab-2 (EZ1, Qiagen, Germany and MagNA Pure 96, Roche Diagnostics, Germany; respectively;). Real-time PCR procedure was carried out in Lab-1 with an amplification mixture of primer, probe sequences targeting VP-1 gene region using RotorGene (Qiagen, Germany) and in Lab-2 with an amplification mixture of primer, probe sequences targeting VP-2 gene region using ABI Prism 7500 (Applied Biosystems, USA). BKV genotyping was performed with multiplex PCR using primer, probe sequences for BKV genotypes I-IV. In both of the laboratories, 82 (54.6%) of the samples were found as positive, 37(24.6%) samples were found as negative and a moderate agreement was found between qualitative results of two real-time PCR methods (ƙ= 0.56, p<0.001). Median viral load values were 4.1 x 104 copies/ml (321-6 x 109) in Lab-1 and 3.3 x 105 copies/ml (224-8.3 x 1010) in Lab-2 for positive samples. According to the lineer regression analysis of quantitative results, moderate (R2= 0.52, p<0.001) and high (R2= 0.88, p<0.001) correlation was found for plasma (n= 52) and urine (n= 30) samples, respectively. Bland-Altman analysis yielded a mean difference of -0.58 log10 for all samples. For plasma samples mean difference was -0.29 log10, while it was -1.1 log10 for urine samples. In all samples, Lab-1 measurements were lower than Lab-2 measurements. A mean difference of -1.1 log10 indicated that the measurement values of Lab-2 were more higher than Lab-1 measurments with an average of 1.1 log10. Supporting this result, 71.9% of the samples had a measurement difference more than 0.5 log 10 and 29.2% of the samples had a measurement difference more than 1 log10. Only 28.1% of the samples were measured within clinically acceptable log difference range (less than 0.5 log10). BKV genotyping was performed only for 74 different patient samples with sufficient copy numbers and genotype I (81.7%), IV (15.5%), II (1.4%), I+IV (1.4%) were detected. When the results were compared; 66.6% (n= 12) of the genotype IV samples had more than 1 log10 and 83.3% of them had more than 0.5 log10 viral load measurement difference. Correlation and linear regression analyzes were insufficient for the comparison ofthe results of the two different tests. It will be appropriate for each center to monitor patients with the same test until the international BKV standard developed by the World Health Organization is optimized. The clinical correlation of the tests is limited to the currently used test. The result of incorrect BKV quantification affects the clinical decision. Measurements less than the actual value will lead to the development of BKV nephropathy, and higher measurements will lead to unnecessary allograft biopsy and unnecessary reduction of immunosuppression.

Evaluation of Samples with DFS Staining Pattern Detected by Indirect Immunofluorescence Assay.

BACKGROUND: In this study, we aimed to determine the presence of anti DFS70 antibody by a specific IB method in samples showing the DFS pattern and to determine the distribution of DFS pattern in different patient groups. METHODS: 2,401 serum samples, which were received for ANA screening, were tested by IIF method at Akdeniz University Hospital Diagnostic Laboratory. Out of 139 samples with DFS pattern, 75 samples were tested for the presence of anti DFS70 antibody by IB and were included in the study. Patients' clinical diagnoses were obtained retrospectively from medical records. RESULTS: 63 (84%) of 75 samples, which showed DFS patern by IIF, were found to have anti DFS70 antibody by IB. Five of these patients were diagnosed with SARD while the rest (58) had diseases other than SARD. CONCLUSIONS: DFS pattern detected by IIF and isolated anti DFS70 antibody positivity detected by IB show high concordance. However IIF results should be confirmed because of the patterns that can be misidentified as DFS pattern. The presence of anti-DFS70 antibodies, which help to exclude SARD, prevent further unnecessary referral demands.

[Determination of cytomegalovirus glycoprotein B genotypes in different geographical regions and different patient groups in Turkey]. / Türkiye'de farkli cografi bölgelerde ve farkli hasta gruplarindaki sitomegalovirus izolatlarinin glikoprotein B genotiplerinin belirlenmesi.

Cytomegalovirus (CMV), a common virus found all around the world, usually causes asymptomatic infections in immunocompetent hosts, however it may lead to serious complications in immunodeficient patients and in the fetus. CMV is divided into four genotypes according to the polymorphisms in UL55 gene that encodes for envelope glycoprotein B. Nucleotide polymorphisms of CMV gB gene can affect the cell tropism of the virus and host immune response and believed to have important changes in the pathogenesis of CMV. The aim of this study was to determine the gB genotypes of CMV isolates from different patient groups selected from different regions of Turkey. A total of 136 clinical specimens from patients (66 female, 70 male; age range: 0-65 years, mean age: 24.03 ± 17.17) who were diagnosed to have CMV infection by polymerase chain reaction (PCR) and/or antigenemia tests, between 2001-2014, in the medical school hospitals of Akdeniz, Ege, Istanbul Cerrahpasa and Erciyes Universities (located at Mediterranean, Aegean, northwest and central Anatolia regions, respectively), were included in the study. The patient group consisted of 80 renal transplant (RT) recipients, 35 stem cell transplant (SCT) recipients, 13 newborns, seven heart transplant (HT) recipients and one pregnant woman. CMV gB genotypes were determined by PCR-RFLP (restriction fragment length polymorphism) method, and DNA sequencing and phylogenetic analysis were performed for the randomly selected 15 isolates with different genotypes. Among 136 (135 plasma, 1 amnion fluid) samples, the most frequent genotype was gB1 (n= 44, 32.4%), followed by gB2 (n= 39, 28.6%), gB3 (n= 36, 26.5%) and gB4 (n= 8, 5.9%); however nine (6.6%) samples could not be genotyped. When analysis were interpreted according to the patient groups, it was determined that the genotypes in RT recipients were gB1 32.3%, gB2 28.7%, gB3 26.5% and gB4 5.9%; in SCT recipients gB1 34.3%, gB2 28.6%, gB3 22.9% and gB4 5.7%; in HT recipients gB3 57.1%, gB1 14.3% and gB2 14.3%; in newborns gB1 38.4%, gB3 30.8%, gB2 15.4% and gB4 7.7%, and gB2 genotype in the pregnant woman. As our study was a descriptive study to determine the genotypes of CMV gB, the relationship between the genotypes and the variants such as viral load, symptomatic disease and prognosis were not analyzed. As a result, the isolation of different gB genotypes in various case groups from four distinctive provinces, underlines the diversity of CMV gB genotypes in Turkey.

Quantitative analysis of BKV-specific CD4+ T cells before and after kidney transplantation.

BACKGROUND: BK virus (BKV) is the main infectious cause of renal allograft dysfunction. Although recent studies showed an inverse correlation between BKV-specific T-cell responses and viral load after transplantation, the importance of pre-transplant response in the process of virus reactivation has only been studied once. In this study, we aimed to determine whether pre-transplant CD4+ T-cell response can be used for prediction of BKV reactivation and BKV nephropathy (BKVN), by a method that can practically be used in routine patient monitoring. METHODS: BKV-specific CD4+ T-cell responses of 31 kidney recipients (all from live donors) were measured by an IFN-γ-enzyme-linked-immunospot (ELISPOT) method using mixture of peptides, at day 0 and +1, +3, and +6 months posttransplant. Additionally, seven other reactivation patients as another group were also analyzed. BKV viral loads in plasma were measured by real-time polymerase chain reaction (PCR). Responses of 10 healthy people were also included as controls in the analysis. RESULTS: All but one patient and all of the controls had detectable CD4+ T-cell responses. Reactivation occurred in 8 out of 31 patients. There was no significant association between pretransplant BKV-specific CD4+ T-cell responses and BKV reactivation and between BKV DNA levels and CD4+ T-cell responses. In the additional group consisting of reactivation patients, four patients who had BKVN showed negative correlation between BKV-DNA levels and BKV-specific CD4+ T-cell responses (p<0.05). One patient who developed BKVN, however, was not able to mount a similar CD4+ T-cell response to viral reactivation despite immunosuppressive reduction. CONCLUSION: Even though our cohort is small, our results may suggest that pre-transplant measurement of BKV specific CD4+ T-cell response may not be necessary, and that post-transplant monitoring, particularly during reactivation, may be more helpful in the management of the infection.

Epstein-Barr virus infection in children with renal transplantation: 17 years experience at a single center.

OBJECTIVES: The aim of this study was to detect the frequency, time of occurrence, management and outcome of Epstein-Barr virus (EBV) infection and related complications in pediatric renal transplant recipients. METHODS: Pediatric renal allograft recipients transplanted between August 1994 and December 2011 at our hospital was evaluated retrospectively. The patients were divided into two groups; Groups 1 and 2 were composed of patients transplanted before and after November 2007, respectively, when plasma EBV DNA levels were periodically measured. RESULTS: The study included 166 children, 89 (53.6%) boys, with a mean age of 12.2 ± 3.8 years. Prior to transplantation, 144 patients (86.7%) were EBV seropositive. Within a median follow-up period of 36 months, 11 of 22 seronegative children (50%) developed primary EBV infection. EBV reactivation was observed in 23 of 144 children (15.9%). Two patients with primary infection developed post-transplant lymphoproliferative disorder, one of whom died. Elevated serum creatinine levels or graft loss were not observed in any patient with EBV reactivation. CONCLUSIONS: EBV DNA monitoring by PCR in high-risk pediatric renal transplant recipients will provide early diagnosis and treatment of EBV infections.

Analysis of human herpes virus 6 infections with a quantitative, standardized, commercial kit in pediatric stem cell transplant recipients after transplantation.

BACKGROUND AND OBJECTIVES: The aim of the study was to assess the incidence and clinical relevance of active Human Herpes Virus 6 (HHV6) infections in pediatric patients after allogeneic stem cell transplantation. DESIGN AND SETTINGS: Retrospective analysis of samples prospectively collected at Akdeniz University Medical Faculty Hospital, Antalya, Turkey, between May 2006 and July 2007 from 15 pediatric patients with allogeneic hematopoietic stem cell transplantation (HSCT). SUBJECTS AND METHODS: A commercial quantitative real-time polymerase chain reaction kit was used to analyze plasma samples collected from 15 pediatric allogeneic HSCT recipients. RESULTS: HHV6 DNA was found positive in 8 (53%) patients. HHV6 DNA levels above 1000 copies/mL were found only in 2 patients and they were also consecutively positive for HHV6 DNA. Age at transplantation, use of ATG, and receiving grafts other than HLA identical siblings increased the risk, with a statistically significant difference, of having HHV6 reactivation with levels exceeding 1000 copies/mL (P values, respectively, P=.03, .001, .025). Active HHV6 infections with HHV6 viremia levels higher than 1000 copies/mL were associated with subsequent delayed platelet engraftment (P=.001), acute graft versus host disease (P=.001), skin rash, and fever of unknown origin. CONCLUSION: More than half of pediatric allogeneic HSCT patients develop active HHV6 infection, and especially in patients with high viremic loads, the infection can result in serious clinical situations. A clinically significant cutoff value for viremia seems to be necessary to predict serious clinical complications.

The role of Bifidobacterium lactis B94 plus inulin in the treatment of acute infectious diarrhea in children.

BACKGROUND/AIMS: In contrast to many other studies of probiotic species, the number of publications evaluating Bifidobacterium lactis and its combinations with prebiotics as treatments for acute infectious diarrhea is limited. We investigated the synbiotic effects of B. lactis B94 plus inulin on acute infectious diarrhea. MATERIALS AND METHODS: The study was conducted on children with acute diarrhea between the ages of 2 and 60 months. The patients were administered 5×1010 colony-forming units (CFU) of B. lactis B94 plus 900 mg inulin or placebo, once a day for five days. Stools were examined for Rotavirus, Adenovirus, Entamoeba histolytica, Salmonella, Shigella, Campylobacter, Clostridium difficile, Cryptosporidium, and parasites. RESULTS: We examined 79 patients in the synbiotic group and 77 patients in the placebo group. The duration of diarrhea was significantly reduced in the synbiotic group in comparison with the placebo group (3.9±1.2 days vs. 5.2±1.3 days, respectively; p<0.001). Moreover, the number of diarrheal stools on the third day was significantly lower in the synbiotic group than in the placebo group (5.5±2.9 vs. 8.3±3.01, respectively; p<0.001). Diarrhea in the synbiotic-group patients with rotavirus infection was of a significantly shorter duration (3.2±1.3 days vs. 5.2±1.3 days, respectively; p=0.001). Duration of diarrhea in patients who started the synbiotic treatment within the first 24 h was shorter than that in the patients who started the treatment later (3.9±1.1 days vs. 4.8±1.8 days, respectively; p=0.002). CONCLUSION: Treatment with 5 × 1010 CFU of B. lactis B94 plus 900 mg inulin shortened the duration of acute watery diarrhea by an average of 31 h. This decrease was most pronounced in cases of Rotavirus diarrhea.

Evaluation of vancomycin resistance 3 multiplexed PCR assay for detection of vancomycin-resistant enterococci from rectal swabs.

BACKGROUND: Active screening for vancomycin-resistant enterococci (VRE) using rectal specimens is recommended to limit the spread of antimicrobial resistance within certain high-risk populations. We evaluated the diagnostic performance of Vancomycin Resistance 3 Multiplexed Tandem PCR assay (AusDiagnostics, Australia), a rapid multiplex real-time PCR assay that detects vanA and/or vanB. METHODS: Two-hundred-and-eleven rectal swabs from Hematology and Oncology unit were submitted for VRE surveillance via direct detection of vanA and/or vanB by culture and by using Vancomycin Resistance 3 Multiplexed Tandem PCR assay. Enterococci were identified to the species level by using standard biochemical tests and BD Phoenix Automated Microbiology System (BD Diagnostic Systems, USA). Vancomycin susceptibility of enterococci was determined using Etest (BioMerieux, France). RESULTS: Compared to the culture method, Vancomycin Resistance 3 Multiplexed Tandem PCR assay had a sensitivity of 84.0%, specificity of 98.8%, positive predictive value (PPV) of 91.3%, and negative predictive value (NPV) of 97.6%. The assay failed to detect 18 (8.5%) specimens because of the presence of PCR inhibitors; of the remaining 193 specimens, 25 (12.9%) were positive, 23 for vanA, and 2 for vanB. Although both sensitivity and specificity for vanA VRE was 100% compared to the culture method, all vanB-positive specimens tested negative by VRE culture. CONCLUSIONS: Vancomycin Resistance 3 Multiplexed Tandem PCR assay is a rapid and laborsaving option for VRE surveillance for direct use on rectal swabs. However, the high rate of PCR failure owing to the inhibitors in the specimens and the low specificity for vanB should be considered when interpreting the results.

[BK virus infections in pediatric kidney transplant recipients]. / Pediatrik renal transplant alicilarinda BK virus enfeksiyonlari

Primary BK virus (BKV) infections acquired mainly during childhood are usually asymptomatic. Several studies revealed its seroprevalence in adult population as high as 90% worldwide. Following primary infection, virus persists as latent infection in the urogenital tract. In renal transplant recipients, primary infection and reactivations affect 10% of patients and without treatment, more than half of these patients lose their grafts. The only way of preventing graft loss due to BKV nephropathy (BKVN), seems to monitor BKV infection after transplantation and to diagnose patients developing BKVN during the early period and treat them accordingly. In this study, we analyzed BKV presence in plasma and urine samples with real-time PCR method and evaluated the renal biopsies of pediatric renal transplant recipients after transplantation, retrospectively. A total of 142 children (63 female, 79 male; mean age: 11.7 ± 3.9 years) who had renal transplantation in Akdeniz University Medical Faculty, Antalya, Turkey, between February 2006 and April 2011 were enrolled in the study. After transplantation, peripheral blood and urine samples were collected bi-weekly for the first three months, monthly till the sixth month and every three months thereafter. BKV DNA was additionally screened in patients with unexplained rise in serum creatinine or in patients receiving anti-rejection therapy. In any plasma positivity or during the BKVN therapy, BKV DNA analysis was done bi-weekly. After DNA extraction by automated system, an 83 base pair fragment in VP1 region was amplified. Signal detection for the target region was performed with a TaqMan probe dual-labelled at the 5' end with 6-carboxyfluorescein (FAM) and the 3' end with 6-carboxytetramethylrhodamine (TAMRA). Histopathological examinations of renal biopsies were done with routine histological stains and immunohistochemical staining with monoclonal antibodies directed to SV40 antigen. From 2171 plasma and 1995 urine samples without PCR inhibitors, 442 (20%) (range: 300-4.5 x 10(7) copies/ml; mean: 2.0 x 10(5) ± 2.2 x 10(6) copies/ml) and 800 (40.1%) (range: 300-3 x 10(12) copies/ml; mean: 5.9 x 10(9) ± 1.1 x 10(11) copies/ml) were found positive for BKV DNA, respectively. For 114 (80.3%) patients, at least one urine sample was positive and more than half of those patients (68/114, 59.6%) had viremia. Of the patients, 19.7% (28/142) had viral DNA above 10(4) copies/ml, which was choosen as a cut-off value for its high positive predictive value for BKVN. For all these 28 patients, prior to renal biopsy, immunosupressive treatment was decreased. Cidofovir and/or leflunomid were initiated to nine patients who did not respond to lowered immunosupressive therapy and eight of them had renal biopsy for the confirmation of BKVN. All renal biopsy results were compatible with BKVN. From these nine patients who were receiving cidofovir and/or leflunomid, two lost their grafts because of BKVN. Since viruria is frequently encountered and the viral load is usually in low quantities and transient, it is more appropriate to use blood samples for screening programmes after renal transplantation. The efficacy of antiviral treatment in BKVN could not be evaluated since it was only applied in patients non-responding to lowered immunosuppressive therapy and had decreased renal functions. Multicenter prospective studies are required to enlighten this important issue. Early diagnosis with close monitoring of renal function and viremia, seems to be the most effective way for controlling BKVN.

[Investigation of adenoviruses in children with lower respiratory tract infections]. / Alt solunum yolu enfeksiyonu olan çocuklarda adenoviruslarin arastirilmasi

Adenoviruses which are one of the causative agents of acute respiratory tract infections at all age groups worldwide, can lead to epidemic, endemic or sporadic infections year-round. Adenovirus infections in lower respiratory tract can be presented as bronchitis, bronchiolitis and pneumonia. The aim of this study was to investigate the presence of adenoviruses as the etiologic agent of lower respiratory tract infections (LRTIs) in children by cell culture, polymerase chain reaction (PCR) and direct fluorescence antibody (DFA) test. The results of the laboratory tests were evaluated in the light of patients' clinical findings. The study consisted of 206 patients aged between 0-5 years old and who were admitted to the hospital with the complaints of LRTI between January 2011 and April 2012. The clinical, radiological and laboratory findings of the patients were recorded. Nasopharyngeal specimens were taken with flocked swab from all patients and adenoviruses were investigated by shell-vial cell culture, real-time PCR and DFA test, simultaneously. Of all the samples 89.3% were taken in January, February and March and 38% of the patients have one or more chronic underlying diseases as chromosomal abnormalities, congenital heart disease, heart failure, asthma, cystic fibrosis, leukemia, kidney failure and prematurity. Adenovirus was detected in 12 (5.8%) of the samples by PCR, seven (3.4%) of the samples by cell culture method. While seven samples were found positive with both PCR and cell culture, 194 samples yielded negative results in both tests. Five samples, which were found positive by PCR, were not grown in cell culture method. Twelve of the 153 samples examined with DFA test, could not be evaluated due to insufficient amount of cells, however 2.8% (4/141) of the samples were found positive for adenovirus antigens by DFA method. Those samples were also positive ones in the other two methods. Compared with cell culture, the sensitivity, specificity, positive and negative predictive values of PCR were 100%, 97.5%, 58.3% and 100%, respectively; those values were 57%,100%,100% and 97.7%, respectively for DFA testing. Compared to PCR the sensitivity of cell culture is very low (16.6%) after three days of incubation, however, it increased to 58.3% after five days' of incubation. There was no significant relationship between adenovirus positivity and the presence of chronic diseases, the radiological findings and the laboratory findings. Of all adenovirus positive samples 83.3% were obtained in January, February and March. Our data indicated that the etiological agent was adenovirus in approximately 6% of children with LRTI. The most important step for the isolation of adenovirus from respiratory tract, is high quality and sufficient amounts of sample. The flexible flocked swabs made it easy to take nasopharyngeal swab from children. Although cell culture is still the gold standard for the diagnosis of adenovirus infections, PCR which is a fast method with high sensitivity and specificity can also be used. However, specific care should be taken during the DNA extraction stage, since the amount of the nucleic acid in the sample is critical for the best results. Even though the low sensitivity of DFA restricts its use in routine diagnosis of adenovirus infections, it should always be kept in mind that the quality of the clinical samples is most reliably evaluated by this method.

Preliminary results of the first human uterus transplantation from a multiorgan donor.

OBJECTIVE: To describe the first-year results of the first human uterus transplantation case from a multiorgan donor. DESIGN: Case study. SETTING: University hospital. PATIENT(S): A 21-year-old woman with complete müllerian agenesis who had been previously operated on for vaginal reconstruction. INTERVENTION(S): Uterus transplantation procedure consisting of orthotopic replacement and fixation of the retrieved uterus, revascularization, end to site anastomoses of bilateral hypogastric arteries and veins to bilateral external iliac arteries and veins was performed. MAIN OUTCOME MEASURE(S): Resumption of menstrual cycles. RESULT(S): The patient had menarche 20 days after transplant surgery. She has had 12 menstrual cycles since the operation. CONCLUSION(S): We have described the longest-lived transplanted human uterus to date with acquirement of menstrual cycles.

Bilateral tonsillar and esophageal Kaposi sarcoma in an HIV-negative patient.

Tonsillar involvement in Kaposi sarcoma is extremely rare, as only a few such cases have been reported; all but 1 of these previously reported cases occurred in patients with human immunodeficiency virus (HIV) infection. We describe what to the best of our knowledge is the first reported case of concurrent bilateral tonsillar and esophageal Kaposi sarcoma in an HIV-negative patient. A 68-year-old man presented with sore throat and dysphagia. Clinical examination revealed the presence of bilateral and asymmetrical tonsillar masses, as well as generalized lymphadenopathy in the cervical chain. The masses were resected, and findings on histopathologic analysis were consistent with Kaposi sarcoma. In addition, human herpesvirus 8 was demonstrated on a tonsil specimen by polymerase chain reaction, and microinvasive squamous cell carcinoma was also detected. Later, another Kaposi sarcoma lesion was detected in the lower third of the esophagus. We recommend that clinicians not discount the possibility of oral classic Kaposi sarcoma in the workup of an immunocompetent patient with oral vascular lesions.