Richard L. Hodinka

Post-transplant lymphoproliferative disease in children

Abstract: Epstein–Barr virus (EBV)‐driven post‐transplant lymphoproliferative disease (PTLD) is an important cause of morbidity and mortality following transplantation, and it occurs more frequently in children than in adults. Of 22 (5%) children at our institution who developed tissue‐proven PTLD 1–60u2003months (mean 16.5u2003months) following organ transplant, 11 died: nine of these 22 patients developed PTLD between 1989 and 1993, and seven (78%) died; the remaining 13 developed PTLD between 1994 and 1998, and four (31%) died (p =u200a0.08). All nine patients who developed PTLD <u20036u2003months after transplant died, but 11 of 13 patients who manifested disease ≥u200a6u2003months after transplant survived (p =u200a0.0002). Ten of 11 (91%) survivors, but only two of eight (25%) children who died, had serologic evidence of EBV infection at the time of PTLD diagnosis (pu2003=u20030.04). EBV seroconversion identified patients at risk for developing PTLD, but also characterized patients with sufficient immune function to survive EBV‐related lymphoid proliferation. In situ hybridization for EBER1 mRNA was diagnostically helpful because it detected EBV in tissue sections of all 20 patients with B‐cell PTLD, including those with negative serology.

The clinical utility of viral quantitation using molecular methods

BACKGROUNDnThe quantitation of viral nucleic acids in biological fluids has become increasingly desirable over the past several years. To this end, a number of quantitative molecular procedures have been developed.nnnOBJECTIVESnThe objective was to review the current literature on the molecular techniques used in the quantitation of viral nucleic acids and to assess the appropriateness of these methods for clinical use.nnnRESULTSnAssays involving both target and signal amplification are now available for the accurate and precise quantitation of viral burden in infected patients. These methods include quantitative polymerase chain reaction (PCR), branched chain signal amplification (bDNA), nucleic acid sequence-based amplification (NASBA) and the SHARP signal and hybrid capture systems. Our understanding of the natural history and pathogenesis of viruses such as the human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) may be greatly facilitated by accurate determinations of viral and infected cell burden. Quantitation of viral load in infected individuals may also be useful to assess disease progression, monitor the efficacy of therapy and to predict treatment failure and the emergence of drug-resistant viruses.nnnCONCLUSIONnPrecise, accurate and reproducible quantitation of viral load is now feasible. Molecular assays for viral quantitation should have a considerable impact on medical research and clinical care.

Challenges and Opportunities in Developing Respiratory Syncytial Virus Therapeutics

Two meetings, one sponsored by the Wellcome Trust in 2012 and the other by the Global Virology Foundation in 2013, assembled academic, public health and pharmaceutical industry experts to assess the challenges and opportunities for developing antivirals for the treatment of respiratory syncytial virus (RSV) infections. The practicalities of clinical trials and establishing reliable outcome measures in different target groups were discussed in the context of the regulatory pathways that could accelerate the translation of promising compounds into licensed agents. RSV drug development is hampered by the perceptions of a relatively small and fragmented market that may discourage major pharmaceutical company investment. Conversely, the public health need is far too large for RSV to be designated an orphan or neglected disease. Recent advances in understanding RSV epidemiology, improved point-of-care diagnostics, and identification of candidate antiviral drugs argue that the major obstacles to drug development can and will be overcome. Further progress will depend on studies of disease pathogenesis and knowledge provided from controlled clinical trials of these new therapeutic agents. The use of combinations of inhibitors that have different mechanisms of action may be necessary to increase antiviral potency and reduce the risk of resistance emergence.

Serious respiratory illness associated with rhinovirus infection in a pediatric population

BACKGROUNDnRhinoviruses have long been associated with mild upper respiratory illness in both adults and children. However, the role of rhinoviruses as lower respiratory tract pathogens has not been fully characterized. Previous data suggests that rhinoviruses may cause severe lower respiratory illness in young children or infants.nnnOBJECTIVESnThe present study describes the clinical presentations, severity of illness and outcomes for a large cohort of pediatric patients with documented rhinovirus infections.nnnSUBJECTS AND METHODSnA retrospective chart review was done on 93 pediatric patients from whom 101 nasopharyngeal or endotracheal specimens were positive by viral culture for a rhinovirus. All patients were hospitalized or seen in the pediatric emergency department at The Childrens Hospital of Philadelphia between 1 January, 1990 and 31 May, 1996.nnnRESULTSnOf the 93 patients, 52 were male and 41 female. The age range was 0 days to 18 years with 25 (27%) less than 3 months, 42 (45%) between 3 and 12 months and 26 (28%) over the age of 12 months. Clinical presentations on evaluation in the emergency department or admission included 78 (84%) patients with acute respiratory illness, 13 (17%) with fever and suspected sepsis and 11 (12%) with other complaints. Reported physical findings on examination included one or more lower respiratory symptoms or signs of acute distress and fever greater than or equal to 38.1 degrees C. A total of 64 (69%) children were noted to have significant past medical histories, including 28 (44%) with prematurity or complicated neonatal courses, 11 (17%) with prior reactive airways, 8 (12%) with congenital cardiac disease and 7 (11%) with neurologic disorders. Of the patients, 29 (31%) were considered to be otherwise healthy children with no underlying dysfunctions. The mean duration of hospitalization for 69 patients admitted with respiratory illness who did not develop subsequent unrelated complications was 3.7 days. No significant bacterial or fungal pathogens were identified in 91% of the cases.nnnCONCLUSIONSnThis study shows that rhinoviruses were associated with severe lower respiratory illness and hospitalization in a large pediatric population and that rhinovirus infection was a complicating factor in those patients with underlying or predisposing conditions.

Detection of hepatitis C virus antibodies and specific hepatitis C virus ribonucleic acid sequences in cord bloods from a heterogeneous prenatal population

OBJECTIVEnOur aim was to quantify the prevalence of at-risk pregnancies for maternal-fetal hepatitis C virus transmission in a heterogeneous prenatal population by detection of both hepatitis C virus-specific antibody and hepatitis C virus ribonucleic acid sequences in cord bloods from their deliveries.nnnSTUDY DESIGNnAn anonymous serosurvey of 1432 consecutive umbilical cord blood samples were analyzed for hepatitis C virus antibodies with a second-generation enzyme immunoassay with all hepatitis C virus antibody-positive samples batched and analyzed for both human immunodeficiency virus antibodies and hepatitis C virus ribonucleic acid sequences by polymerase chain reaction.nnnRESULTSnForty-seven of the samples (3.2%) were positive for hepatitis C virus antibodies; seropositivity rates differed significantly by socioeconomic status but not by race. Significantly more of the antibody-positive women underwent cesarean section for delivery (31.9% vs 21.9%, p = 0.03). Three (6.4%) hepatitis C virus antibody-positive samples were also human immunodeficiency virus-antibody positive, whereas nine (19.1%) were hepatitis C virus ribonucleic acid positive.nnnCONCLUSIONnAs many as 19% of hepatitis C virus antibody-positive women in this study also had hepatitis C virus ribonucleic acid isolated from their delivery cord blood samples, which may indicate an increased risk of vertical hepatitis C virus transmission in those pregnancies. Hepatitis C virus-specific antibody and ribonucleic acid detection may also be markers for other pregnancy complications that result in higher rates of cesarean section for these women.

Recent H3N2 Influenza Virus Clinical Isolates Rapidly Acquire Hemagglutinin or Neuraminidase Mutations When Propagated for Antigenic Analyses

ABSTRACT Prior to serological testing, influenza viruses are typically propagated in eggs or cell culture. Recent human H3N2 strains bind to cells with low avidity. Here, we isolated nine primary H3N2 viral isolates from respiratory secretions of children. Upon propagation in vitro, five of these isolates acquired hemagglutinin or neuraminidase mutations that increased virus binding to cell surfaces. These mutations can potentially confound serological assays commonly used to identify antigenically novel influenza viruses.

Cytologic Manifestations of Respiratory Syncytial Virus Pneumonia in Bronchoalveolar Lavage Fluid

BACKGROUNDnRespiratory syncytial virus (RSV) pneumonia in immunocompromised patients, especially bone marrow transplant recipients, is associated with high mortality. Early diagnosis in these cases is important because antiviral therapy with ribavirin is effective in reducing mortality.nnnCASEnA 45-year-old male with multiple myeloma who underwent autologous peripheral stem cell transplantation subsequently developed bilateral pulmonary infiltrates. A bronchoalveolar lavage specimen demonstrated the cytologic changes associated with RSV pneumonia. Infection with RSV was confirmed by indirect immunofluorescence, enzyme immunoassay and, later, on histology and electron microscopy at autopsy.nnnCONCLUSIONnRecognition of the cytologic changes associated with RSV pneumonia in immunodeficient patients can be life saving since this would initiate confirmatory immunologic studies and therapy.

Detection of Human Cytomegalovirus by Polymerase Chain Reaction

Human cytomegalovirus (HCMV) can produce life-threatening disease in immunosuppressed AIDS patients and organ transplant recipients. Congenital HCMV is the leading infectious cause of birth defects, including blindness, deafness, mental retardation, and death. HCMV can be transmitted in utero, during delivery, or postnatally through physical contact, blood transfusion, and organ transplantation. There are few treatments for HCMV disease. However, ganciclovir (DHPG) has been beneficial in HCMV-infected transplant patients.

Identification of Epstein-Barr Virus Lytic and Latent RNA Transcripts in Post-transplant Lymphoproliferative Disorder

Thirty-three specimens from 25 transplant recipients with Epstein-Barr virus-associated lymphoproliferative disease were studied by in situ hybridization for 2 lytic and 4 latent Epstein-Barr virus transcripts. All specimens were found to contain at least 1 latent transcript while 28 were positive for at least 1 lytic transcript. The amount of Epstein-Barr virus infection and lytic activity varied with histopathology and number of involved sites. Patients with localized polymorphous disease contained the lowest number of infected cells with an almost equal lytic:latent ratio. Disseminated polymorphous and single and multisite monomorphous specimens showed a large latent cell population. Minimal lytic activity was seen in single site monomorphous specimens, but disseminated monomorphous specimens showed the highest levels of lytic transcripts. Most post-transplant lymphoproliferative disorder specimens demonstrate lytic Epstein-Barr virus transcripts, although the majority of cells contain latent Epstein-Barr virus. Lytic activity is highest in patients with disseminated disease. Lytic Epstein-Barr virus infection may aid in the development and maintenance of lymphoproliferative disorders in transplant recipients.

Isolation and characterization of an infectious molecular clone of the MN strain of HIV-1.

Infectious molecular clones of the human immunodeficiency virus (HIV) have been very important tools for the analysis of regulatory gene functions and the study of differential cell tropism. We have cloned and characterized a proviral sequence of HIVmn from mn strain infected H9 cells. This clone, called KP1, was found to be infectious for different cell lines and human peripheral blood lymphocytes (PBL). KP1 proviral DNA was detected in HUT-78 cells and human PBL by polymerase chain reaction (PCR) analysis after infection of these cells with cell-free supernatants from KP1 transfected human rhabdomyosarcoma (RD) cells. To the best of our knowledge, this is the first report of an infectious molecular clone of HIVmn which is a representative of one of the most prevalent strains of HIV-1 in North America and Europe. Biologically active clones of a broadly antigenic strain such as HIVmn will be extremely useful in therapeutic approaches for AIDS.