Arputharaj Kore

Development of a real-time quantitative PCR assay for detection of a stable genomic region of BK virus

BackgroundBK virus infections can have clinically significant consequences in immunocompromised individuals. Detection and monitoring of active BK virus infections in certain situations is recommended and therefore PCR assays for detection of BK virus have been developed. The performance of current BK PCR detection assays is limited by the existence of viral polymorphisms, unknown at the time of assay development, resulting in inconsistent detection of BK virus. The objective of this study was to identify a stable region of the BK viral genome for detection by PCR that would be minimally affected by polymorphisms as more sequence data for BK virus becomes available.ResultsEmploying a combination of techniques, including amino acid and DNA sequence alignment and interspecies analysis, a conserved, stable PCR target region of the BK viral genomic region was identified within the VP2 gene. A real-time quantitative PCR assay was then developed that is specific for BK virus, has an analytical sensitivity of 15 copies/reaction (450 copies/ml) and is highly reproducible (CV ≤ 5.0%).ConclusionIdentifying stable PCR target regions when limited DNA sequence data is available may be possible by combining multiple analysis techniques to elucidate potential functional constraints on genomic regions. Applying this approach to the development of a real-time quantitative PCR assay for BK virus resulted in an accurate method with potential clinical applications and advantages over existing BK assays.

The impact of intercurrent EBV infection on ATP levels in CD4+ T cells of pediatric kidney transplant recipients

Abstract:  ImmuKnow® measures ATP (ng/mL) in PHA‐activated CD4+ T cells from patient’s whole blood. According to published reports, median ImmuKnow® is 258 ng/mL in stable pediatric kidney transplant (PKT) recipients ≥12 yr, and 165 ng/mL in those <12 yr. However, data on the effect of infection or AR on ImmuKnow® are scarce. We studied the effect of Epstein‐Barr virus (EBV) viremia on ImmuKnow® in PKT with GD. Twenty‐eight PKT with GD were reviewed. Group 1 has 19 PKT ≥12 yr, and group 2 has nine PKT <12 yr. Mean follow‐up was 19.4 ± 12 months. All ImmuKnow® values discussed in this study were measured during GD ± fever. None had ImmuKnow® pretransplant. EBV DNA was isolated from patient blood by real‐time PCR. Group 1 has eight boys and 11 girls (mean age = 16.6 ± 2.4 yr). Group 2 has two boys and seven girls (mean age = 6 ± 3.1 yr). Median ImmuKnow® was 292 ng/mL in group 1, and 370 ng/mL in group 2. Nine children developed EBV viremia: two in group1 (median ImmuKnow® = 273 ng/mL), and seven in group 2 (median ImmuKnow® = 475 ng/mL). Overall mean ImmuKnow® in the nine EBV viremic patients was higher than that in the 19 non‐viremic ones (422 ± 176 ng/mL, and 302 ± 113 ng/mL, respectively, unequal variance t‐test, p = 0.08). Eight children developed AR (all in G1, median ImmuKnow® = 272 ng/mL). In group 1, one patient developed concurrent EBV viremia and rejection, while another patient developed EBV viremia six months following a rejection episode. In group 2, none developed simultaneous AR, CMV, or BK virus infection with EBV viremia. None developed post‐transplant lymphoproliferative disease. In summary, EBV viremia was paradoxically associated with high ImmuKnow® in PKT <12 yr. This suggests strong co‐stimulation of PHA‐activated CD4+ T cells by EBV‐transformed B cells.

Morbidity of 200 Consecutive Cases of Hand-Assisted Laparoscopic Living Donor Nephrectomies: A Single-Center Experience

Background. Recipients of laparoscopically procured kidneys have been reported to have delayed graft function, a slower creatinine nadir, and potential significant complications. As the technique has evolved laparoscopic donor nephrectomy technique is becoming the gold standard for living donation. Study Design. We retrospectively reviewed the data of the first 200 hand-assisted laparoscopic living donor nephrectomies performed between January 2003 and February 2009. The initial 41 donors and their recipients (Group 1) were compared to the next 159 donors and their recipients (Group 2). The estimated blood loss, serum creatinine at discharge and 6 months, and the incidence of delayed graft function and perioperative complications were analyzed. Results. The median donor serum creatinine at discharge and 6 months was 1.2 mg/dL in each group. None of the laparoscopic procedures required conversion to an open procedure, and none of the donors required perioperative blood transfusion. The median recipient serum creatinine at 6 months after transplant was 1.2 mg/dL for each group. No ischemic ureteral complications related to the laparoscopic technique were seen. Conclusions. HALDN with meticulous surgical technique allows kidney procurement with very low morbidity and no mortality. This improved safety and decreased invasiveness from laparoscopic approach may further decrease morbidity of the procedure and increase organ donation.

Diffuse Hepatic Epithelioid Hemangioendothelioma Developed in a Patient with Hepatitis C Cirrhosis

Hepatic epithelioid hemangioendothelioma (HEHE) is an infrequent vascular tumor of endothelial origin that primarily occurs in women in the mid-fifth decade of life without underlying chronic liver disease or cirrhosis. Liver transplant should be the first-line of therapy in patients with large or diffuse unresectable tumors even in the presence of metastatic disease due to the favorable long-term outcome. We report the case of a 48-year-old female who complained of abdominal pain and weight loss. She has a history of cirrhosis secondary to chronic hepatitis C (HCV) and was treated with interferon and ribavirin with sustained virological response. Her work-up revealed multiple confluent infiltrating bilobar liver masses diagnosed as HEHE. She underwent a successful liver transplant without evidence of recurrent HCV infection. She developed cervical spine (C4-C6) HEHE metastases 4 years after transplant. She underwent surgical resection and local radiotherapy after resection with good clinical response. To the best of our knowledge, this is the first report of HEHE that developed in a patient with HCV cirrhosis successfully treated with antiviral therapy before transplant and liver transplant with good allograft function without evidence of recurrent liver tumor or HCV infection but developed metastases to the cervical spine 4 years after transplant.

Post-Transplant Diabetes Mellitus After Kidney Transplant in Hispanics and Caucasians Treated with Tacrolimus-Based Immunosuppression

Background Development of post-transplant diabetes mellitus after kidney transplant (PTDM) significantly increases kidney graft loss and mortality. Several risk factors for PTDM have been reported, including Hispanic ethnicity and the use of calcineurin inhibitors and corticosteroids. The incidence and impact of PTDM in the Hispanic kidney transplant population is unknown. Material/Methods We retrospectively reviewed the medical records of 155 Hispanic and 124 Caucasian patients, who were not diabetics and underwent kidney transplant between January 2006 and December 2011. We analyzed their clinical outcomes at 12 months post-transplant, including the incidence of PTDM, acute rejection rates, and patient and graft survival. Results Hispanics who developed PTDM (n=22) were more than 10 years older and had higher body mass index (BMI) than Hispanics without PTDM (p<0.001 and p=0.001, respectively). Caucasians with PTDM (n=13) were non-significantly older (2.5 years) and had higher BMI than Caucasians without PTDM (p=0.526, p=0.043, respectively). The incidence of PTDM was not significantly different between Hispanics and Caucasians treated with tacrolimus-based immunosuppression (14.2% and 10.5%, respectively). Conclusions PTDM did not cause significant difference in short-term outcomes after kidney transplant in Hispanics or Caucasians. Larger multicenter prospective and long-term clinical trials are needed to validate these findings.

Comparison of outcomes of hand-assisted laparoscopic to open donor nephrectomy for pediatric recipients.

The purpose of this study is to compare the outcome of pediatric recipients of kidneys procured using a hand‐assisted laparoscopic (HALDN group) to an open technique (ODN group). Twenty‐eight patients ≤18 yr old (HALDN group) were compared with 17 patients (ODN group). The serum creatinine for HALDN and ODN groups at discharge were 0.93 ± 0.48 and 0.94 ± 0.54 mg/dL (p = 0.917), respectively. The serum creatinine for HALDN and ODN groups at six and 12 months was 1.01 ± 0.44 and 1.11 ± 0.55, and 1.04 ± 0.52 and 1.14 ± 0.46 mg/dL (p = 0.516, p = 0.554), respectively. The eGFR for HALDN and ODN groups at discharge was 108.66 ± 37.23 and 106.1 ± 50.55 mL/min/1.73 m2 (p = 0.845), respectively. The eGFR for HALDN and ODN groups at six and 12 months was 97.77 ± 28.25 and 81.73 ± 27.46, and 94.56 ± 28.3 and 85.74 ± 30.1 mL/min/1.73 m2 (p = 0.085, p = 0.344), respectively. The patient and graft survival for both groups were 100% at 12 months post‐transplant. In conclusion, the short‐term outcome of recipients of kidneys procured via HALDN is comparable to that of kidneys procured via ODN in pediatric patients.

HIGH PREVALENCE AND CLINICAL SIGNIFICANCE OF HYPERTENSIVE RESPONSE TO DOBUTAMINE STRESS IN END STAGE RENAL DISEASE PATIENTS UNDERGOING EVALUATION FOR RENAL TRANSPLANTATION

Patients with end stage renal disease (ESRD) have a cardiovascular mortality about 15-30 times the general population. Dobutamine stress echocardiography (DSE) is commonly performed in their preoperative cardiac evaluation before renal transplantation. Hypertensive response during DSE defined as