PCR Titers Research Articles

Sofosbuvir-Velpatasvir for Chronic Hepatitis C Virus-Infected Children and Young Adults: Efficacy and Safety

Background: Sofosbuvir-Velpatasvir has a high efficacy in adult patients with chronic hepatitis C infection .In this study, we reported treatment outcome with this drug in children and younger adults in Pakistani population.
 Objective: To assess the efficacy and safety of Sofosbuvir-velpatasvir in treating HCV-infected children and young adults. 
 Methods: This Interventional(Clinical trial) was done in Hepatitis clinic Jinnah Hospital, Lahore from April 10, 2021 to January 9, 2022.A total of 45 patients aged 6 to 18 years with detectable HCV RNA by PCR were registered. They were evaluated clinically. HCV genotyping was not done as Sofosbuvir – Velpatasvir is pangenotypic. All of the patients were given a single oral dose of Sofosbuvir-velpatasvir per day. Ribavirin was included for patients with compensated cirrhosis and for those who were treatment-experienced .Clinical assessment, CBC, LFT, RFT, and PT were performed monthly to determine side effects and safety. To evaluate treatment efficacy, HCV RNA was measured by PCR at 12 weeks (end of treatment) and 12 weeks post-treatment (sustained virological response). Treatment lasted for 24weeks for those with compensated cirrhosis. (Child Turcotte Pugh score 5 and 6; Child class A) or those who were treatment experienced. SPSS 24 was used for data analysis. 
 Results: Non cirrhotic patients had a mean age of 13.6±1.45 years, while those with compensated cirrhosis had 14.1±2.15 years. There were 63.6% males (28) and 36.4% female (17). 41 patients (91.11%) had undetectable HCV RNA by PCR ,12 weeks after treatment (Sustained virological response), indicating treatment effectiveness. Nine patients had HBV co-infection and took HBV medications as well during HCV treatment. At 12 weeks post-treatment, all HBV-coinfected patients had undetectable HCV RNA by PCR and lower HBV titers. Some of the patients experience minor side effects. However, none of the patients stopped their treatment due to these side effects Thus, this treatment was safe and effective. 
 Conclusion: Sofosbuvir/ velpatasvir has effectiveness and safety in treating HCV in children and young adults.

Significance of cytomegalovirus tests after three weeks of life in children with hearing loss

To determine the diagnostic role of viral markers for cytomegalovirus (CMV) when tested after the diagnostically critical period (postnatal 3 weeks) in children with sensorineural hearing loss (SNHL). A retrospective review of 104 subjects who underwent CMV diagnostic tests after the critical period of 3 postnatal three weeks but before 24 months of age. Infants included had not passed universal newborn hearing screening tests in at least one ear and thus underwent obligatory follow up audiology testing as well as either exome sequencing or magnetic resonance imaging in cases of SNHL. Our cohort was classified into four subgroups depending on the results from audiological and etiologic diagnostic tests (genetic and radiological tests): congenital CMV (cCMV)-related SNHL (Group 1, n=9), SNHL with another clear etiology (Group 2, n=34), and SNHL classified as neither Group 1 nor 2 (Group 3, n=18). We added age-matched, normal-hearing children (Group 4, n=43) as a control group. CMV related viral metrics were compared among these four groups. CMV PCR positivity, PCR titers, and culture positivity successfully differentiated Group 1 from Groups 2 and 4. Group 3 showed values of these parameters that were significantly different from Groups 2 and 4, while being more similar to those in Group 1, suggesting that a substantial portion of Group 3 truly had cCMV deafness. A hypothetical formula was developed to predict cCMV infections using logistic regression analysis. This is the first study to propose the clinical significance of CMV test results obtained after 3 weeks post-birth in children with SNHL and to suggest how we can utilize them.

Is There an Association between Concurrent Epstein–Barr Virus Infection and Sudden Hearing Loss?—A Case—Control Study in an East Asian Population

Viral infection serves as the crucial etiology for the development of sudden sensorineural hearing loss (SSNHL). We aimed to investigate whether there is an association between concurrent Epstein-Barr virus (EBV) infection and SSNHL in an East Asian population. Patients who were older than 18 years of age and met the criteria of sudden hearing loss without an identifiable etiology were enrolled from July 2021 until June 2022, followed by the serological testing of IgA antibody responses against EBV-specific early antigen (EA) and viral capsid antigen (VCA) with an indirect hemagglutination assay (IHA) and real-time quantitative polymerase chain reaction (qPCR) of EBV DNA in serum before the treatment was initiated. After the treatment for SSNHL, post-treatment audiometry was performed to record the treatment response and degree of recovery. Among the 29 patients included during enrollment, 3 (10.3%) had a positive qPCR result for EBV. In addition, a trend of poor recovery of hearing thresholds was noted for those patients with a higher viral PCR titer. This is the first study to use real-time PCR to detect possible concurrent EBV infection in SSNHL. Our study demonstrated that approximately one-tenth of the enrolled SSNHL patients had evidence of concurrent EBV infection, as reflected by the positive qPCR test results, and a negative trend between hearing gain and the viral DNA PCR level was found within the affected cohort after steroid therapy. These findings indicate a possible role for EBV infection in East Asian patients with SSNHL. Further larger-scale research is needed to better understand the potential role and underlying mechanism of viral infection in the etiology of SSNHL.

Codivir suppresses SARS-Cov-2 viral replication and stabilizes clinical outcome: In vitro and Phase I clinical trial results.

Treatment of severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) remains a significant challenge in the face of increased worldwide morbidity and mortality. The acute illness caused by SARS-CoV-2 is initiated by a viral phase, followed by an inflammatory phase. Numerous anti-inflammatory and anti-viral therapies, with a relatively minor clinical effect, have been applied. Developing a safe and efficient direct anti-viral treatment is essential as it can block disease progression before significant complications ensue and potentially prevent transmission. The present phase 1 study aimed to determine the safety of Codivir, a newly developed anti-viral agent, and to preliminarily assess its anti-viral activity in patients infected by COVID-19. In vitro studies were conducted to determine the direct anti-viral effect of Codivir using an immunofluorescence-based assay and to assess its cytotoxic effect by tetrazolium assay (MTT). In a phase I clinical trial, Codivir was administered for ten days in 12 patients who were followed for its safety. Patients were followed for clinical manifestations during administration. Sequential nasal viral PCR titers (Cycle Threshold, CT) were determined preceding and during treatment. In vitro, Codivir showed activity against SARS-CoV-2 with 90% viral replication suppression and minimal cytotoxicity. The anti-viral activity was demonstrated at the early stages of infection, post-entry of the virus in the cell. Codivir was safe in all 12 patients in phase I clinical trial and significantly suppressed viral replication in 5/7 fully assessed patients, with an anti-viral effect noted as early as three days. The present study's data support the safety of Codivir administration in humans and suggest its significant anti-COVID-19 effect. These results support the testing of the drug in more extensive controlled trials in patients with SARS-CoV-2.

Curcumin inhibits Singapore grouper iridovirus infection through multiple antiviral mechanisms

Singapore grouper iridovirus (SGIV) is one of the main pathogens that causes high mortality and substantial economic losses in the grouper aquaculture industry. Curcumin is a polyphenolic compound extracted from the traditional Chinese medicinal material, turmeric. In this study, we investigated the antiviral properties of curcumin against SGIV based on quantitative real-time PCR (qRT-PCR), western blotting, and viral titer assessment with in vitro models, and found that different concentrations of curcumin inhibited SGIV infection. Furthermore, curcumin significantly impaired attachment of the virion. Moreover, the qRT-PCR results showed that cellular immune and inflammatory responses related to the NF-κB signaling pathway were regulated by curcumin. Examinations of the expression of antioxidant enzymes indicated that the antioxidant capacity played a critical role in preventing SGIV infection, which may depend on the Nrf2/Keap1 signaling pathway. Importantly, curcumin also had a strong antiviral activity in vivo according to the increased survival rates of groupers and inhibition of SGIV gene expression. This study demonstrated that curcumin could inhibit SGIV infection both in vitro through multiple antiviral mechanisms and reduce the mortality of SGIV-infected groupers in vivo. Overall, our findings show that SGIV is sensitive to curcumin and its antiviral activity makes it a potential candidate drug for treating SGIV infection.

The Cost-Effectiveness of Requesting a Complete Blood Count (CBC) in the Management of COVID-19 in Saudi Arabia.

Background: Since the beginning of the COVID-19 pandemic, studies have attempted to determine the value of the different laboratory investigations. This study aims to assess the cost-effectiveness of requesting a complete blood count (CBC) for COVID-19 patients, as opposed to ordering a COVID-19 antibody titer in Saudi Arabia. Methods: The prices of a CBC, COVID-19 PCR, and antibody titer were obtained from 40 healthcare establishments in Saudi Arabia, between January and February 2022. Results: Requesting a CBC is significantly cheaper than requesting a COVID-19 antibody titer, which was available in almost all of the establishments, as compared to COVID-19 PCR and antibody titer testing. The investigation prices did not differ significantly between hospitals and private laboratories, nor across cities in Saudi Arabia. Conclusions: CBC, which provides valuable information on the patient’s condition and prognosis, is a cost-effective and widely available tool for managing COVID-19. The price and availability of CBC warrant it to be included in the COVID-19 management protocols.

Effect of CMV Viremia on HeartCare

<h3>Purpose</h3> CMV infection post-heart transplantation presents complex challenges affecting changes in immunosuppression and antiviral therapy that have both short- and long-term consequences. AlloMap gene expression profiling (AM), a non-invasive method to detect acute cellular rejection was affected by the presence of CMV infection. The aim of this study was to evaluate the impact of CMV infection on donor derived cell free DNA (AS) in patients post heart transplantation (HT). <h3>Methods</h3> The Surveillance Using HeartCare Outcomes Registry (SHORE) is a multicenter study for post-heart transplant patients followed with AM/AS for 5 years following heart transplantation. Patients enrolled in the SHORE registry were analyzed based on CMV infection. AM/AS were evaluated before, at the time, and following the viremic episodes. Both individual trends and the differences between the median AS and AS levels were studied. Nonparametric tests were used to assess categorical and longitudinal variables. <h3>Results</h3> 1702 patients were included, 1245 (73%) male, median age 57, 1156 (68%) white. CMV infection was diagnosed in 360 (21%) patients occurring at a mean time of 229 days after HT. CMV(-) patients had similar demographics to those patients CMV(+ve), but CMV(+) were older (p=0.025). CMV(+ve) patients had an increase in both AS (0.07% v. 0.05%, p<0.001) and AM (34 v. 30, p<0.001) compared to those without CMV [Figure 1a]. Levels returned to baseline within 5 weeks following the infection [Figure 1b]. The threshold to discriminate CMV infection with a NPV of 95% with GEP is 34 and dd-cfDNA is 0.12% . <h3>Conclusion</h3> AS does not seem to be confounded by CMV, but the presence of CMV infection may be associated with an elevated AS if there is allograft injury. Recovery from CMV infection was associated with normalization of AS and AM. This study points to the need to evaluate CMV in the presence of an increased AS, with further work to compare PCR titers and AS levels planned.

Outpatient Clinical Trial in Dogs With Leptospirosis Treated With Enrofloxacin Hydrochloride-Dihydrate (ENRO-C).

Pharmacokinetics of enrofloxacin HCl-2H2O (enro-C) in dogs and Monte-Carlo simulations against Leptospira spp. prompted a clinical study to treat the clinically apparent phase of this disease. Leptospirosis was diagnosed by real-time PCR from blood, micro-agglutination titers (MAT), clinical signs and blood parameters of the liver and kidney. In order to determine the clinical ability of the participants to diagnose leptospirosis on the first exam and establish an early treatment to avoid excessive organ damage, patients were clinically classified as: high-risk or medium-risk. Forty-five dogs were included in this trial (from 2017 to early 2019). The treatment consisted of IM injections of a 5% aqueous enro-C suspension (10 mg/kg/day) for 10 days, and subsequently enro-C was administered orally for another 7 days in gelatin capsules. Thirty-four high-risk and 11 medium-risk dogs were treated, including 6 puppies (4 high-risk with ages between 6 to 10 months and 2 medium-risk dogs with an average age of 6 and 7 months). Other ages ranged from 1 to 5 years. Fifteen cases had a history of having received prior treatment with other antibiotics, including all puppies. The clinical diagnostic error was 13.5% (7/52 cases), and only one of the misdiagnosed dogs had been classified as a high-risk patient. Three to 5 days after finishing treatment with enro-C, 82.2% of the dogs were negative to real-time PCR from urine samples and 100% negativity was observed on day 30 after treatment, when antibody titrations dropped to 1:100–1:200. Based on the absence of clinical signs, real-time PCR, and MAT titers, all treated dogs were considered as successful treatments. Within 6–24 months of clinical follow-up, no relapses were recorded. Adverse effects were inconsequential. This study represents the first report of a successful treatment of canine leptospirosis using a fluoroquinolone, and due to its efficacy, it is suggested that enro-C be considered as a viable option for the treatment of this disease.

CMV reactivation in allogeneic hematopoietic stem cell transplant patients receiving post-transplant cyclophosphamide.

e18533 Background: Post transplant cyclophosphamide (PTCY) has been shown to be an effective treatment for prevention of graft versus host disease (GVHD). However, this increased immune suppression rates may increase the risk of CMV reactivation. There is limited published data addressing CMV reactivation in this patient population. Additionally there is no data on the efficacy of prophylactic letermovir in the patients who have received PTCY. In this study we analyzed the incidence of CMV reactivation in patients treated with PTCY and those not treated with PTCY, as well as the efficacy of letermovir in preventing CMV reactivation in the PTCY population. Methods: We conducted a retrospective review of MUD, MRD, and haploidentical stem cell transplant patients at our institution from 1/1/2014 until 12/10/2018. We analyzed the incidence of CMV reactivation (PCR &gt; 137 DNA IU/ml), peak of CMV PCR titer and time to reactivation within the first 100 days post-transplant. Results: There were 150 patients with at least 60 days of follow-up that were included in this study. These patients were split into three groups: No post-transplant cyclophosphamide (NPTCY) (N = 64), received post-transplant cyclophosphamide (PTCY) (N = 70), and received PTCY and letermovir prophylaxis. (L-PTCY) (N = 15). The incidence of CMV reactivation was increased in the PTCY patients when compared to the NPTCY (44% vs 29%). In the NPTCY patients the donor (D) serostatus increased the risk of CMV reactivation (Recipient (R)+ D+ 73% vs R+D-36%) conversely in the PTCY group the donor CMV status did not influence reactivation rates (R+D+ 52% vs R+D- 81%). The CMV reactivation rate in the L-PTCY patients was lower when compared to the PTCY patients (21% vs 44%), additionally the L-PTCY patients had much lower peak CMV titers compared to PTCY group (445 vs 2112 IU/ml). Conclusions: This study demonstrates that there is an increased incidence of CMV reactivation in patients who receive PTCY. Additionally, the donor CMV serostatus does not appear to influence the incidence of CMV reactivation in patients receiving post-transplant CY. Although the number of patients in the L-PTCY group is small, it does appear to be an effective prophylactic treatment in patients receiving PTCY.

Early Cytomegalovirus Reactivation and Donor Constellation Reduce Early and Late Relapse Incidence of Acute Myeloid Leukemia in a Long-Term Study

IntroductionAfter allogeneic cell transplantation (HCT) for acute myeloid leukemia (AML) relapse remains the main cause of morbidity and mortality. Relapse rates directly translate into patients' overall survival. The connection between early human cytomegalovirus (CMV) replication and reduced leukemic recurrence has first been established by this group (Elmaagacli et. al. Blood 2011), using a pp65-antigenemia assay and confirmed by Green et al. (Blood 2013), Takenaka et al. (BBMT 2015) and others. In a more recent study by Teira et al. (Blood 2016) using PCR to detect reactivation, this effect has been questioned and the controversy remains probably due to methodological differences between studies.MethodsPatients with AML received allogeneic HCT at the Department of Bone Marrow Transplantation of the University Hospital Essen between 10/1997 and 10/2017. All patient-, donor-, HCT- and virology data was prospectively documented and retrospectively analyzed. The immunosuppressive regimen consisted of cyclosporine A and methotrexate. The conditioning was according to physician's choice and included only non-anti-thymocyte globuline (ATG) conditioning regimens. Early supportive and follow-up care was identical for all patients. Patients were followed for up to 60 months. Surviving patients were censored at last follow-up date. Competing risk analysis was performed for calculating cause-specific relapse-incidences with non-relapse mortality as competing risk. CMV titers were measured at the Department of Virology using quantitative PCR (qPCR) and a pp65 antigenemia assay. CMV reactivation was defined as a replication of >500 CMV copies per ml EDTA blood or as >25 pp65 antigen expressing cells per 5×105 white blood cells. If several CMV reactivation episodes occurred, only the interval to the first episode after HCT was analyzed.ResultsA total of 420 consecutive adult patients (median age, 48 years; range, 16-73 years) with AML and HCT were included for analysis. Over 60 months of follow-up, cumulative incidence of CMV reactivation was detected in 28% of patients and AML relapsed in 34%. In this cohort, the sensitivity of qPCR or pp65 detection methods did not differ (p=0.35) with regard to the incidence of CMV reactivation. CMV reactivation associated with significantly reduced AML relapse risk. The relapse incidence (RI) for patients with CMV reactivation was 0.18 (95% Confidence interval (CI), 0.12-0.25) compared to 0.41 (95% CI, 0.35-0.46) for patients without CMV reactivation (p<0.0001). This significant difference was reproduced for prognostic AML subgroups in first complete remission (RI, 0.14 with CMV reactivation versus 0.35 without; p=0.02) and for higher risk AML (RI, 0.20 versus 0.45; p<0.0001). Notably, donor-recipient serostatus constellation significantly (p=0.005) associated with AML relapse reduction, independently of CMV reactivation. CMV positive donors had a RI of 0.40 (95% CI, 0.33-0.46) compared to 0.28 in CMV negative donors (95% CI, 0.22-0.34).DiscussionThis new PCR dataset confirms previous reports (Elmaagacli et al. 2011; Takenaka et al. 2015) of an independent reduction of AML relapse risk after CMV reactivation, using qPCR detection in a homogeneous population of AML patients. While the association between CMV reactivation and early relapse (< day+100) has also been confirmed by Green et al. (Blood 2013) and relapse (>day +100) by Takenaka et al. (BBMT 2015), our data shows that late relapse (>24 months) may occur in patients without CMV reactivation. While the aforementioned studies exclusively used pp65 antigenemia titers as a determinant of CMV reactivation, Teira et al. (Blood 2016) analyzed PCR samples. The sensitivity of CMV reactivation using PCR or pp65 antigen titers was identical in our dataset. With regard to the on-going controversy on the role of CMV reactivation and AML relapse, this data shows a reduction of relapse in the quantitative PCR age. [Display omitted] DisclosuresTurki:Neovii Biotech: Other: subsidies for the costs of travel. Beelen:Medac: Consultancy, Other: Travel Support.

A Case of Acute Hepatitis With Co—Infections: Co—Existent Hepatitis E With a Positive Serology for Double Mono

Epstein—Barr virus (EBV), Cytomegalovirus (CMV) and Hepatitis E virus (HEV) are all known to cause mild and usually self—limiting acute viral hepatitis. Acute hepatitis caused by simultaneous presence of two viruses has been reported in literature. However, patients with positive serology for three acute viral infections causing acute hepatitis is very rare. A 23—year—old Caucasian female with an unremarkable past medical history presented with one—week duration of nausea, vomiting, generalized abdominal pain, fever and one day duration of scleral icterus and jaundice. The patient had stable vital signs but elevated liver enzymes on admission with AST 385 IU/L, ALT 290 IU/L, Alkaline phosphatase 461 IU/L, Total bilirubin 9 mg/dL and Direct bilirubin of 5.1 mg/dL. White blood cell (WBC) count was 13500/uL. Work up was unremarkable for acute hepatitis A, B & C viruses, Influenza A & B and infectious mono—spot test along with a negative pregnancy test. When Ultrasound and Computed tomography scan of the abdomen was unrevealing a magnetic resonance cholangiopancreatography was obtained and was unremarkable. Anti—mitochondrial antibody, anti—smooth muscle antibody, anti—nuclear antibody, Alpha—1 antitrypsin level and ceruloplasmin level were all un—remarkable. Infectious disease was consulted when the patient became febrile to 102.0 F despite negative work up. Serologic work up for acute EBV, CMV and HEV infections turned out to be positive; with positive PCR for EBV (Table. 1). The patient was subsequently managed conservatively per infectious disease recommendations and improved. She was discharged on her fourth hospital day with a 6 week follow up showing normalization of liver enzymes. The above patient was diagnosed with Infectious mononucleosis with associated acute hepatitis due to EBV infection. Positive CMV serology was thought to be false positive as the patient's CMV PCR titers were low. HEV PCR wasn't obtained but given the significantly elevated bilirubin in combination with positive serology, it was thought to be a true infection. The patient improved only with conservative management. This lead to two important conclusions: (1) If the patient has positive serology concerning for co—infection of EBV, CMV and/or HEV; it should be confirmed via PCR and (2) if the patient truly is co—infected supportive treatment is one management option in immunocompetent patients.3047.tif Figure 1: No Caption available.

Impact of Vital Dyes on Cell Viability and Transduction Efficiency of AAV Vectors Used in Retinal Gene Therapy Surgery: An In Vitro and In Vivo Analysis.

PurposeTreatment of inherited retinal degenerations using adeno-associated viral (AAV) vectors involves delivery by subretinal injection. In the latter stages, alteration of normal anatomy may cause difficulty in visualizing the retinotomy, retinal detachment extension, and vector diffusion. Vital dyes may be useful surgical adjuncts, but their safety and impact on AAV transduction are largely unknown.MethodsThe effects of Sodium Fluorescein (SF), Membrane Blue (MB), and Membrane Blue Dual (DB) at a range of dilutions were assessed on human embryonic kidney cells in vitro using an AAV2-green fluorescent protein (GFP) reporter at different multiplicities of infection. Flow cytometry analysis was performed to assess both cell viability and transduction efficiency. The effect on quantitative (q)PCR titer was determined. Balanced salt solution (BSS) or dilute DB (1:5 in BSS) were delivered subretinally into left/right eyes of C57BL/6J mice (n = 12). Retinal structure and function were analyzed by optical coherence tomography, autofluorescence, dark-and light-adapted full-field electroretinography.ResultsDB and MB were not toxic at any concentration tested, SF only when undiluted. The presence of dyes did not adversely affect the genomic titer. DB even increased the values, due to presence of surfactant in the formulation. AAV2-GFP transduction efficiency was not reduced by the dyes. No structural and functional toxic effects were observed following subretinal delivery of DB.ConclusionsOnly undiluted SF affected cell viability. No effects on qPCR titer and transduction efficiency were observed. DB does not appear toxic when delivered subretinally and improves titer accuracy. DB may therefore be a safe and helpful adjunct during gene therapy surgery.Translational RelevanceThis paper might be of interest to the retinal gene therapy community: it is a “bench to bedside” research paper about the potential use of dyes as a surgical adjunct during the gene therapy surgery. We have tested the potential toxicity and impact on transduction efficiency in an in vitro and in vivo model.

Evaluation and Monitoring of Mycobacterium leprae Transmission in Household Contacts of Patients with Hansen's Disease in Colombia.

Leprosy in Colombia is in a stage of post elimination—since 1997, prevalence of the disease is less than 1/10000. However, the incidence of leprosy has remained stable, with 400–500 new cases reported annually, with MB leprosy representing 70% of these case and 10% having grade 2 disability. Thus, leprosy transmission is still occurring, and household contacts (HHCs) of leprosy patients are a population at high risk of contracting and suffering from the effects of the disease during their lifetime. We performed a cross-sectional study with the aim of evaluating leprosy transmission within Family Groups (FGs) from four Colombian departments: Antioquia, Bolívar, Córdoba and Sucre. This study included 159 FGs formed by 543 HHCs; 45 FGs were monitored twice, first in 2003 and again in 2012. Migration, forced displacement by violence, loss of contact with the health center and the lack of an agreement to participate in the second monitoring were the primary reasons not all FGs were tested a second time. In each HHC, a clinical examination was performed, epidemiological data recorded, the bacillary index determined, DNA was isolated for M. leprae detection by nested PCR and IgM anti-phenolic glycolipid-I (PGL-I) titers were inspected. Further, DNA from M. leprae isolates were typed and compared among FGs. Twenty-two (4.1%) of the 543 HHCs had IgM anti-PGL-I positive antibody titers, indicating infection. Nasal swabs (NS) taken from 113 HHCs were tested by RLEP PCR; 18 (16%) were positive for M. leprae DNA and two new leprosy cases were detected among the HHCs. Of the confirmed HHCs with leprosy, it was possible to genotype the bacterial strains from both the index case and their HHCs. We found that the genotype of these two strains agreed at 9 markers, showing the individuals to be infected by the same strain, indicating familiar transmission. HHCs of leprosy patients not only are a high-risk population for M. leprae infection, they can act as M. leprae carriers and therefore serve as sources for transmission and infection. Our results confirm familiar leprosy transmission and suggest that follow-up of HHCs is a good strategy for early diagnosis of leprosy and to monitor its transmission.

PP13.2 – 3042: Infantile spasms in an infant with anti-NMDA receptor encephalitis secondary to HSV-1 encephalitis

Objective The recent identification of NMDAR antibodies causing choreoatethosis post-herpes simplex encephalitis (HSE) has provided a proof of principle that relapsing symptoms post-HSE are immune mediated. We report the first patient with anti-NMDAR encephalitis post-HSE and infantile spasms. Methods Review of patient's clinical information. Immunological studies as previously reported. Results An 11-month-old boy was admitted to our Pediatric Intensive Care Unit for status epilepticus and fever. The CSF showed 25 WBC/uL and the PCR was positive for HSV1. Intravenous acyclovir was started and over the next days 3 days he developed simple partial seizures that were initially well controlled with valproate (VPA). On day 19th after HSE onset he was readmitted for irritability, impaired interaction, involuntary movements (head shaking and facial twitching), and spasms with EEG hypsarrhythmia. The CSF revealed mild pleocytosis, negative HSV-1 PCR and high titers of NMDAR antibodies. Repeated MRI showed progression of T2/FLAIR abnormalities and intense contrast enhancement that was not present in previous studies. He was treated with midazolam, VPA, vigabatrin and immunotherapy (iv methylprednisolone, IVIg, rituximab, cyclophosphamide) without symptom improvement. Spasms and EEG paroxysmal activity progressively worsened and evolved towards a super-refractory status epilepticus, which was eventually controlled with barbiturate coma, lacosamide and ketogenic diet (KD). Seventy days after HSE onset he underwent plasma exchange for persisting coma and generalized choreoathetosis with partial clinical improvement of symptoms. KD was discontinued after 3 months of seizure control and EEG activity improvement. Conclusion Early onset infantile spasms post-HSE can be caused by anti-NMDAR encephalitis. Recent advances in the genetics of West syndrome (GRIN2B mutations – encoding the NR2B subunit of the NMDA receptor) and the animal model for West syndrome (induced by NMDA) support this hypothesis. Plasma exchange and KD were the most effective treatments in our patient.

Genomics of BK Viremia in Kidney Transplant Recipients

This study aimed to investigate global gene expression profiles of BK viremia and nephropathy (BKVN) samples using microarrays to investigate the immunologic response to BK virus. Patients were monitored for BK viremia in the blood monthly for 6 months, then at 9 and 12 months after kidney transplantation. BKVN and normal transplant kidney biopsy samples, and whole blood samples of patients with and without BK viremia were analyzed by Affymetrix Human Gene 1.0 ST Arrays. During a mean follow-up of 917±325 days, 61 of the 289 patients (21%) developed BK viremia at a median 149 (27, 1,113) days after transplantation with a median peak PCR titers of 35,900 (1,000, 2,677,000). The only significant risk factor for development of BK viremia was induction with anti-thymocyte globulin (P=0.03). Only four patients developed BKVN (1.3%). Pathogenesis-based transcript analysis revealed a significant increased expression of interferon-gamma and rejection induced (GRIT), quantitative cytotoxic T-cell (QCAT), quantitative constitutive and alternate macrophage, B-cell and natural killer cell-associated transcripts (NKAT), indicating an active inflammatory immune response in BKVN biopsies (n=3) compared to normal transplant kidney biopsies with (n=3) and without BK viremia (n=11). The whole blood gene expression profiles of 19 BK viremia patients revealed significant increased expression of GRIT, QCAT, and NKAT compared to 14 patients without viremia. The results showed increased activity of cytotoxic T cells and natural killer cells in BKVN and viremia samples resembling acute rejection and suggested the involvement of both adaptive and innate immunity.

Concurrent Epstein Barr Virus Viremia and Hodgkin Lymphoma In Two Patients With Chronic Lymphocytic Leukemia

IntroductionChronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder (LPD) derived from mature B-cells with heterogeneous outcomes. High-grade lymphoma can arise from CLL in 3-10% of cases, a process known as Richter's transformation (RT). The majority of RT results in high grade B-cell lymphomas, though rarely transformation to Hodgkin lymphoma (HL) has been reported. Epstein Barr virus (EBV) viremia can arise in patients with CLL secondary to immunosuppression and can lead to an EBV driven LPD. In patients with CLL, this has been seen with fludarabine based treatments but has been reported with alemtuzumab. HL is also been associated with EBV virus in tumor cells but cases of HL and EBV viremia have not been reported. We report the first two cases in the English literature of CLL with EBV viremia and HL. CasesCase 1 A 66 year old male was diagnosed with a RAI stage I, del13q14, Zap-70 (-), IgvH mutated, CD38 (+) CLL in 2004. Worsening thrombocytopenia resulted in the initiation of treatment in April 2012 using Ofatumumab and high dose methylprednisolone followed by lenalidomide maintenance therapy (HiLOG clinical trial). The patient responded to induction therapy and was continued on lenalidomide maintenance for 12 cycles. In May of 2013 the patient developed progressive fevers, chills, cough, weakness, weight loss, and hypercalcemia. Evaluation revealed an EBV viremia with whole blood PCR titers of 484,550 copies/mL. Bone marrow biopsy was consistent with CLL and full body PET CT scan showed diffuse lymphadenopathy with max SUV of 30.7 and splenomegaly. Biopsy of a left external iliac node showed large atypical lymphoid cells that were surrounded by small lymphocytes. The large atypical lymphoid cells were CD30 (+), EBV (+), Pax-5 (+), CD 20 (-), CD15 (-), CD5 (-), and CD3 (-) confirming HL. The patient has currently received 4 weekly treatments with rituximab for EBV driven LPD and one cycle of Adriamycin, bleomycin, vinblastine, and dexamethasone (ABVD) for HL with resolution of symptoms and a whole blood EBV PCR to <500 copies/mL. Case 2A 68 year old male was diagnosed in England with an unknown stage del13q and IgVH unmutated CLL in 2002. He was started on treatment with 5 cycles of fludarabine, cyclophosphamide, rituximab in 2011 and achieved a complete response. His post therapy course was complicated with infection resulting in septic shock with renal failure resulting in dialysis. He stabilized but continued to have hypogammaglobulenemia and required intravenous immunoglobulin (IVIG) replacement. In April of 2013 he developed fevers to 104F, night sweats, weight loss and, fatigue. PET CT scan showed diffuse adenopathy with max SUV of 22.5, and bone marrow biopsy showed 60% CLL and a component of large cells that were CD 30 (+), CD15 (+), Mum1 (+), and EBER-ISH (+). The patient's whole blood EBV PCR was 276,600 copies/mL. Based on the clinical picture the patient was diagnosed with an EBV positive HL, EBV viremia, and chronic CLL. He has completed 4 weekly treatments with rituximab for EBV driven LPD and cycle 1 of ABVD for HL with resolution of symptoms and a whole blood EBV PCR of <500 copies/mL. DiscussionAlthough EBV viremia has been reported in CLL and EBV staining can be positive in HL, these two cases are the first to report CLL with EBV viremia and EBV positive HL. The high EBV viral load likely provided a base for an EBV driven LPD, in these cases HL. In patients with constitutional symptoms who have stable CLL disease burden, the clinician should screen for RT and EBV viremia. When a concurrent process is found, as in our cases, treatment against both the EBV driven LPD and HL should be initiated. Further research is needed to establish a biological relationship between EBV viremia and HL in patients with CLL. Disclosures:No relevant conflicts of interest to declare.

Perforation of the Nasal Septum as the First Sign of Histoplasmosis Associated with AIDS and Review of Published Literature

Disseminated histoplasmosis in South America is associated with AIDS in 70-90 % of cases. It is visceral and cutaneous, compromising the oral, pharynx, and laryngeal mucous membranes. The involvement of the nasal mucosa is unusual. Two patients with perforation of the nasal septum as the only sign of their disease were clinically and histopathologically diagnosed as leishmaniasis. The revision of the biopsies and the culture of nasal discharge secretions showed that the pathogens seen were not amastigotes but Histoplasma capsulatum. Other mycotic lesions were not detected, nor there was history of cutaneous leishmaniasis. The leishmanin skin test, available only for the male patient, was negative. The PCR and immunofluorescence antibody titers for Leishmania were negative in both patients. They were HIV positive; in the male, his CD4+ T cell count was 60/mm(3) and in the female 133/mm(3). The nasal ulcer was the only manifestation of histoplasmosis and the first of AIDS in both patients. The male patient recovered with amphotericin B and itraconazole treatment. The female has improved with itraconazole. Both patients received antiretroviral treatment. Nasal mucous membrane ulcers should include histoplasmosis among the differential diagnosis. In conclusion, two patients had perforation of their nasal septum as the only manifestation of histoplasmosis, a diagnosis confirmed by nasal mucosa biopsy and by culture of H. capsulatum, findings which demonstrated that both patients had AIDS.

Abstract 4159: Progastrin inhibits Helicobacter-associated gastric corpus carcinogenesis in mice

Abstract Background &amp; Aims: We have previously reported that overexpression of amidated gastrin in transgenic (INS-GAS) mice with Helicobacter felis (H. felis) infection accelerated gastric corpus carcinogenesis, while overexpression of non-amidated glycine-extended gastrin in transgenic (MTI-G-gly) mice inhibited parietal cell loss and atrophy in stomach. We have also reported that overexpression of the full length progastrin in transgenic (hGAS) mice stimulated colon carcinogenesis in azoxymethane-treated mice. In this study, we investigated the role of progastrin for Helicobacter-associated gastric carcinogenesis in mice. Methods: hGAS, INS-GAS and non-transgenic wild type (B6 wt) mice on a C57BL/6 background were infected with H. felis for 12 or 18 months (m). We also analyzed male hGAS, INS-GAS and wild type (FVB wt) mice on a FVB/N background with H. felis infection for 7 m. Infection status was assessed by realtime PCR, microscopic evaluation and serum titer of H. felis-specific IgG antibodies. Proliferating cells and parietal cells were investigated by immunohistochemistry (IHC) with Ki-67 and HK-ATP-beta antibodies, respectively. Expression of stem cell-related genes such as CD44 &amp; DCAMKL-1 was also analyzed by IHC. Results: At 12 m post infection (p.i.), INS-GAS mice had mild corpus dysplasia and B6 wt mice had severe gastritis or metaplasia, while hGAS mice had only mild to moderate gastritis, respectively. At 18 m p.i., both INS-GAS and B6 wt mice had severe atrophic gastritis and corpus dysplasia, while hGAS mice showed only moderate gastritis with mild gastric atrophy but no corpus dysplasia. In contrast, hGAS mice had moderate to severe antral or pyloric dysplasia, while INS-GAS and B6 wt mice did not. H. felis colonization remained stable over time among 3 groups of mice. The serum titer of H. felis-specific IgG antibody as well as Th1-Th2 polarization calculated by IgG1/IgG2c subclass ratio showed no significant differences among 3 groups. At 18 m p.i., the number of Ki-67(+) cells per gastric corpus gland was the highest in INS-GAS and the lowest in hGAS mice. Parietal cell loss was remarkable in INS-GAS and B6 wt, but minimal in hGAS mice. CD44(+) and DCAMKL-1(+) cell numbers were significantly increased in INS-GAS and B6 wt, while essentially unchanged in hGAS mice. On a FVB/N background, INS-GAS mice with H. felis infection for 7 m showed gastric corpus adenocarcinoma, and infected FVB wt mice showed severe gastric atrophy with intestinal metaplasia, while infected hGAS mice showed only moderate gastritis without metaplasia or dysplasia. Conclusions: These results point to a protective effect of progastrin on Helicobacter-associated gastric corpus carcinogenesis perhaps by inhibiting the loss of parietal cells. In contrast antral pathology is increased in hGAS mice. We conclude that the different forms of gastrin have contrasting effects on Helicobacter-associated gastric corpus carcinogenesis in mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4159.

BK nephropathy in pediatric hematopoietic stem cell transplant recipients

BK nephropathy is a known cause of renal insufficiency in kidney transplant recipients. Activation of the polyoma virus may also occur in the native kidneys of non-renal allograft recipients. BK nephropathy has only been reported in a few patients after HCT, most being adult patients, and the single reported pediatric case had evidence of hemorrhagic cystitis. The response to antiviral therapy also seems to differ widely. Here, we describe two cases of BK nephropathy in the native kidneys of HCT recipients exposed to high levels of immunosuppression because of GVHD. Neither of our patients had any evidence of hemorrhagic cystitis. We present definitive renal pathology and detailed chronological evidence of the rising serum creatinine with simultaneous serum and urine BK PCR titers. In one of our cases, antiviral therapy did not seem beneficial as documented by continued renal dysfunction and elevated serum/urine BK PCR titers. Based on our report, intense immunosuppression in pediatric HCT recipients seems to be involved in the activation of BK virus and BK nephropathy should be suspected even in the absence of hematuria in HCT recipients with unexplained renal dysfunction.

A two-arm prospective trial evaluating the ability of EBV PCR to diagnose and monitor posttransplant lymphoproliferative disorder

8057 Background: EBV PCR is known to be useful in the management of patients with PTLD. However, the optimal DNA target, relative importance of peripheral blood intracellular versus free plasma EBV, and baseline profile of these EBV PCR variations in a transplant population remains unclear. This study explored these issues as well as assessed the utility of a 6 assay EBV PCR panel in the management of patients with suspected PTLD. Methods: A prospective 2-arm study was performed. Arm A consisted of 31 patients who underwent lung transplantation. Arm B consisted of 35 patients evaluated for possible PTLD. All patients underwent an initial evaluation with EBV PCR and EBV VCA antibody titer. Arm B patients were evaluated for PTLD with CT scans and biopsies of lesions. All patients in Arm A and those in Arm B with documented PTLD were then followed. For the EBV PCR panel, peripheral blood was separated into plasma and whole blood cells and then DNA was purified. Real time EBV PCR was then performed on both samples using primer sets against LMP, EBER and EBNA. Results: No patients on control Arm A developed PTLD. Seventeen (49%) patients on Arm B were found to have PTLD. Thirteen of 17 (76%) had EBV(+) PTLD. Eighteen Arm B patients were given non-PTLD diagnoses. Of 62 patients evaluable for EBV antibody titers, 60 (97%) had IgG for EBV VCA. In Arm A, only 1/31 (3%) patients developed a transient positive low level plasma EBV load. Fourteen of 31 (42%) had detectable low-level intracellular EBV. In patients with EBV(+) PTLD, 11/13 had EBV in plasma and intracellular samples, 1 had intracellular EBV only and 1 patient with isolated CNS PTLD had a negative EBV panel. The EBV PCR panel had high sensitivity (92%) and specificity (67%) for diagnosing EBV(+) PTLD. Comparing the individual PCR assays, whole blood EBER had the best sensitivity (92%) and plasma EBNA &amp; EBER had the best specificity (100%). The EBV PCR panel tracked patient's clinical course well (p=0.06) as they proceeded through treatment. EBV plasma (p=&lt;0.01) and whole blood (p=0.04) copy numbers by EBNA PCR inversely correlated with EBV VCA IgG titers. Conclusion: Peripheral blood EBV PCR is useful in the diagnosis and monitoring of patients with EBV(+) PTLD. No significant financial relationships to disclose.