Hepatocellular transplantation in acute hepatic failure and targeting genetic markers to hepatic cells.

Orthotopic liver transplantation (OLT) represents the only therapeutic option for many patients with end-stage liver disease as well as many inborn genetic errors of hepatic metabolism. Despite dramatic progress in methods for OLT, the utilization of this procedure is limited by its considerable morbidity and mortality, by a chronic shortage of organs for transplant, and by difficulty arranging funding for many patients. Many children with fulminant hepatic failure do not receive OLT because this technology is unavailable or unaffordable. Hepatocellular transplantation (HCT), in which isolated, heterologous hepatocytes from a donor liver would be infused into the diseased organ in order to provide essential hepatic functions, could provide a much needed therapeutic alternative to OLT in the treatment of some causes of hepatic insufficiency. Experiments in animals have demonstrated that several genetic deficiencies of hepatic metabolism as well as experimental induced hepatic failure in animals can be reversed by HCT. Despite this experience, HCT has never been attempted in human subjects. This protocol represents the first proposed clinical trial of HCT. We are proposing a clinical trial in which HCT would be attempted as a therapeutic intervention in children with acute hepatic failure who have no other medical or surgical options. This proposal is intended to establish surgical methods for HCT and to evaluate the feasibility of this procedure for treating hepatic disease in humans. It is our expectation that HCT may provide short-term support for patients awaiting organ availability, a "bridge to recovery" allowing patients with fulminant hepatic failure to recover, or a long-term repopulation of the patient's liver with healthy donor cells. One of the major limitations of many animal studies in HCT is that, since the donor hepatocytes are often indistinguishable from those of the host, it has often been difficult to demonstrate a clear correlation between engraftment and the therapeutic effect. In order to verify engraftment independent of the therapeutic response, we propose to "mark" the donor hepatocytes by transducing these cells with a recombinant retroviral vector (LNL6) carrying a marker gene (NEO-R, neomycin phosphoribosyl transferase). The presence of this marker will enhance the ability to identify transplanted cells in the host using assays for the NEO-R gene or transcribed NEO-R mRNA. The LNL6 vector has been approved for human use and has been used as a marker gene for transplanted cells in human subjects without any reported adverse effects. We would like to emphasize that this is a proposal with therapeutic intent.(ABSTRACT TRUNCATED AT 400 WORDS)

The diagnosis of CMV pneumonitis in lung and heart/lung transplant patients by PCR compared with traditional laboratory criteria.

Polymerase chain reaction (PCR) amplification of CMV DNA recovered from bronchial alveolar lavage (BAL) and peripheral blood samples was compared with tissue culture, cytology, and/or histology for the earlier detection of CMV pneumonitis in 12 recipients of single-lung or heart/lung transplants. In patients with confirmed CMV pneumonitis, cytological evidence of CMV disease in BAL samples was detected 38 +/- 14 days posttransplantation, while tissue culture and PCR-positive results were noted as early as 30 +/- 4.0 days and 18 +/- 4.6 days, respectively. While PCR was positive earlier than culture in a number of cases, culture-positive results were subsequently obtained in each case, consistent with earlier detection of viral replication by PCR as opposed to detection of latent virus. CMV was detected by PCR in 6 of 24 blood samples from patients with confirmed or suspected CMV pneumonitis, while results of all 24 blood samples were negative when assayed by tissue culture. PCR-based testing was more sensitive than traditional tests, allowing detection of viral replication earlier than tissue culture in the posttransplant period. PCR could provide a powerful means of monitoring the immunocompromised patients in whom preemptive therapeutic intervention for CMV disease is desirable.

Progressive hearing loss in infants with asymptomatic congenital cytomegalovirus infection.

Congenital cytomegalovirus (CMV) infection is a major public health problem because 30,000 to 40,000 neonates with the infection are born each year in the United States. Although 90% of the congenitally infected infants are asymptomatic at birth, evidence is accumulating that these infants are at risk for audiologic, neurologic, and developmental sequelae. The current study describes the audiologic outcome of 59 infants with asymptomatic congenital CMV infection compared with 26 control infants. Eight of 59 infected infants had congenital sensorineural hearing loss (SNHL) but none of the control subjects did. Longitudinal audiologic assessments revealed that 5 of the 8 infants had further deterioration of their SNHL; a ninth infant with initially normal hearing experienced a unilateral SNHL during the first year of life, with further deterioration subsequently. The frequency of SNHL was similar for infected infants born to mothers with recurrent CMV infections during pregnancy (2 of 9) and for those born to mothers who experienced primary CMV infections (5 of 26). There was a significant difference between the occurrence of hearing loss in infected infants with normal computed tomographic scans (2 of 40) compared with those with either periventricular radiolucencies (4 of 13) or calcifications (1 of 3). Children with SNHL often have no identified cause of the loss; thus, it is likely that many of these children had asymptomatic congenital CMV infection. Given the progressive nature of SNHL associated with asymptomatic congenital CMV infection, longitudinal audiologic assessments are mandatory.

Semiquantitative measurement of cytomegalovirus DNA in lung and heart-lung transplant patients by in vitro DNA amplification.

We report the cases of two lung transplant recipients (one heart-lung and one single lung) who eventually developed cytomegalovirus (CMV) pneumonitis after documentation of increasing CMV DNA titers in sequential bronchoalveolar lavage (BAL) specimens by polymerase chain reaction (PCR) amplification. To our knowledge, this is the first report that semiquantitation of PCR-amplified DNA can detect an increase in CMV DNA titer in BAL specimens prior to the onset of clinical symptoms or detection of infection by traditional techniques in lung transplant patients. The results obtained in these two cases suggest that DNA titer measurement on sequential BAL samples may differentiate latency from active viral replication and, thus, provide an opportunity for clinical intervention before the development of overt clinical symptoms.

Effect of rapid diagnosis on management of influenza A infections.

BACKGROUND: Few studies have examined the impact of rapid viral diagnostic tests on patient management. OBJECTIVE: To assess the effect of rapid diagnosis of influenza A infections on patient management. METHODS: The medical records of children with respiratory infections who were evaluated at a children's hospital between July 1, 1995, and June 30, 1997, were reviewed. Children (n = 56) evaluated in the Emergency Department (ED) who had a positive influenza A enzyme immunoassay (EIA) were compared with two control groups for the likelihood of admission, antibiotic use and duration of hospitalization and antibiotic administration. RESULTS: Patients discharged from the ED with a positive EIA test were less likely to receive antibiotics than those with a negative EIA test (20% vs. 53%; P = 0.04). Patients admitted to the hospital with a positive EIA test were as likely to receive antibiotics as those without a rapid diagnosis, but the duration of antibiotic administration was significantly shorter in the group with a positive EIA test (3.5 vs. 5.4 days; P = 0.03). Patients with a positive EIA test also were more likely to receive antiviral therapy than either control group (25% vs. 0 and 1.8%; P < 0.001). CONCLUSIONS: The detection of influenza A by EIA has a positive impact on medical management by decreasing antibiotic use in pediatric patients evaluated in an ED, by decreasing the duration of antibiotic use in hospitalized patients and by encouraging antiviral therapy.

Parainfluenza virus type 4 infections in pediatric patients.

BACKGROUND: The purpose of this study was to describe the clinical characteristics and epidemiology of parainfluenza virus type 4 infections in pediatric patients. METHODS: Hospital records of 13 patients from whom parainfluenza virus type 4 was isolated were retrospectively reviewed. RESULTS: Parainfluenza virus type 4 infection was associated with upper respiratory tract disease, severe lower respiratory tract disease requiring hospitalization in 10 of 13 patients and aseptic meningitis. Nine of the 10 hospitalized patients were < 24 months old (mean age, 8.3 months) and required hospitalization for 4 to 25 days (mean, 5.5 days). CONCLUSIONS: Serious illnesses associated with parainfluenza virus type 4 infections are more common than previously recognized.

Cytomegalovirus urinary excretion and long term outcome in children with congenital cytomegalovirus infection. Congenital CMV Longitudinal Study Group.

BACKGROUND: Cytomegalovirus (CMV) is the most frequent cause of congenital infection, and both symptomatic and asymptomatic infants may have long term sequelae. Children with congenital CMV infection are chronically infected and excrete CMV in the urine for prolonged periods. However, the effect of prolonged viral replication on the long term outcome of these children is unknown. OBJECTIVE: To determine whether duration of CMV excretion is associated with outcome at 6 years of life in symptomatic and asymptomatic congenitally infected children. METHODS: Longitudinal cohort study. Children congenitally infected with CMV were identified at birth and followed prospectively in a study of long term effects of congenital CMV infection. The relationship between duration of CMV urinary excretion and growth, neurodevelopment and presence and progression of sensorineural hearing loss (SNHL) at 6 years of age was determined. RESULTS: There was no significant difference in the duration of viral urinary excretion between children born with asymptomatic (median, 4.55 years) and symptomatic (median, 2.97 years) congenital CMV infection (P = 0.11). Furthermore there was no association between long term growth or cognitive outcome and duration of viral excretion. However, a significantly greater proportion of children who excreted CMV for <4 years had SNHL and progressive SNHL compared with children with CMV excretion >4 years (P = 0.019, P = 0.009, respectively). CONCLUSIONS: Children congenitally infected with CMV are chronically infected for years, but the duration of CMV urinary excretion is not associated with abnormalities of growth, or neurodevelopmental deficits. However, SNHL and progressive SNHL were associated with a shorter duration of CMV excretion.

Diagnosis, surveillance, and epidemiologic evaluation of viral infections in pediatric cardiac transplant recipients with the use of the polymerase chain reaction.

BACKGROUND: Viral infections, particularly those caused by cytomegalovirus, are a major cause of postoperative morbidity and mortality in heart transplant recipients. These infections have classically been diagnosed by history, physical examination, peripheral viral cultures, and serologic studies. These methods are often time-consuming and lack sensitivity. Positive viral cultures from the heart are rarely obtained, and viral myocarditis and acute cellular rejection are unable to be differentiated histologically. We have therefore used the polymerse chain reaction to diagnose possible viral infection in pediatric heart transplant recipients with findings consistent with acute unexplained rejection. METHODS: Polymerase chain reaction was used as an aid to diagnose cytomegalovirus infection of cardiac tissue obtained by right ventricular endomyocardial biopsy and follow its long-term course. In addition, polymerase chain reaction was used to diagnose infection of the heart by other viruses in patients with clinical and histologic evidence of rejection, especially those with unexplained late rejection or chronic rejection. Polymerase chain reaction primers were designed to amplify nucleic acid sequences from cytomegalovirus, parvovirus, adenovirus, herpes simplex virus, Epstein-Barr virus, and the RNA viruses of the Enterovirus family. RESULTS: Forty patients underwent serial right ventricular endomyocardial biopsy (129 samples) for rejection surveillance with positive results obtained in 41 samples (32%) from 21 patients. Viral genome amplified included cytomegalovirus in 16 samples, adenovirus in 14, enterovirus in 6, parvovirus in 3, and herpes simplex virus in 2. In 13 of the 21 patients positive for viral genome (62%), endomyocardial biopsy histologic scores were consistent with multifocal moderate to severe rejection (Internal Society for Heart and Lung Transplantation scores of 3A or greater). CONCLUSIONS: Polymerase chain reactions may be used as a rapid and sensitive method to evaluate postoperative viral infections in heart transplant recipients, especially in those with late-onset rejection or chronic rejection. Polymerase chain reaction may also be useful in the serial analysis of cytomegalovirus status in transplant recipients. The use of multiple viral primers improves the diagnostic evaluation of these patients and may lead to a better understanding of the epidemiologic characteristics of posttransplantation viral infections and the cause of late or chronic rejection.

Disinfection of eyelid speculums for retinopathy of prematurity examination.

OBJECTIVE: To evaluate the effectiveness of 70% isopropyl alcohol swabs in disinfecting eyelid speculums after examination for retinopathy of prematurity. METHODS: Two phases. Phase 1: 46 autoclave-sterilized eyelid speculums randomized into either a cleaned or control group following examination for retinopathy of prematurity. Speculums in the cleaned group were disinfected with a 70% isopropyl alcohol swab while control speculums were not cleaned. Bacterial and fungal cultures were then obtained. Phase 2: 20 autoclave-sterilized eyelid speculums inoculated with a clinically relevant dilution of adenovirus serotype 5 or herpes simplex virus type 2. Inoculated speculums were randomized into either a cleaned or control group. RESULTS: Phase 1: 17 (70.8%) of 24 cultures from the cleaned group yielded bacteria compared with 21 (95.5%) of 22 controls. Fungi were isolated from only 1 control and from no cleaned speculums. Phase 2: all speculums inoculated with adenovirus supported growth of the organism irrespective of cleaning with 70% isopropyl alcohol swabs. None of 5 cleaned speculums inoculated with herpes simplex virus type 2 supported viral growth, compared with 3 (60%) of 5 cultures positive for growth in the control group. CONCLUSION: Cleaning eyelid speculums with 70% isopropyl alcohol swabs provided inadequate disinfection against bacteria following examination for retinopathy of prematurity and against adenovirus in a laboratory simulation.

Factors associated with cytomegalovirus excretion in hospitalized children.

Hospital employees, especially pregnant women, have expressed concern over exposure to patients excreting cytomegalovirus (CMV). However, the vast majority of CMV excreters are asymptomatic and are not identified while hospitalized. As part of a 2-year, longitudinal study of nosocomial transmission of CMV in a children's hospital, the prevalence rates of CMV excretion by patients in different areas of the hospital were determined. The average prevalence rates were neonatal intensive care unit, 5.7%; intermediate care nurseries, 4%; premature nursery, 6.3%; normal newborn nursery, 3%; hematology-oncology, 3.7%; pediatric intensive care unit, 3.6%; general pediatric ward, 6%; and a chronic care unit, 16%. During this 2-year study, 315 patients admitted to the chronic care unit were investigated to determine which clinical factors might help predict children likely to excrete CMV. With the use of multiple logistic regression analysis of variables, prior excretion of CMV, multiple hospital admissions, female sex, and Hispanic ethnic background were correlated with excretion. Although not identified as significant factors, a history of premature, bronchopulmonary dysplasia and positive results of CMV serologic studies were more commonly associated with excretion. CMV excretion occurred in all areas within a children's hospital, and certain factors were associated with excretion, but it is unlikely that this information could adequately identify "safe" patients or safe areas within a hospital. This study reinforces the need to follow proper precautions with all patients.

Method of blood processing affects the prevalence of cytomegalovirus excretion in newborn nurseries.

The potential risk of exposure by hospital personnel and noninfected patients to patients excreting the cytomegalovirus (CMV) may be increased in areas of high prevalence of CMV excretion, such as the neonatal nursery. These personnel are particularly sensitized to the adverse effects of intrauterine CMV infection because they frequently care for the severely affected congenitally infected infants. Multiple transfusions with conventionally processed blood have been associated with high excretion rates in sick preterm neonates. During a 4-year period, four separate methods of blood preparation were used. Two of these methods (CMV-seronegative blood and frozen deglycerolized red blood cells) have been previously shown to be effective in reducing posttransfusion CMV infection. We were able to show that excretion rates in neonates were significantly lower when CMV-seronegative blood and frozen deglycerolized red blood cells were used than when conventionally processed blood and saline-washed red blood cells were used. In areas where posttransfusion CMV infection has been identified as a problem, the increased expense of specially processed blood can be justified, not only to reduce transfusion-associated disease but also to minimize exposure of the hospital staff and noninfected patients to patients excreting CMV.

Congenital cytomegalovirus infection.

Congenital cytomegalovirus (CMV) infection is the most common congenital infection in humans, infecting 30,000 to 40,000 newborns each year in the United States and leaving more than 9,000 of these children with permanent neurological sequelae. Recognition of the importance of this public health problem has been growing, and recent advances in surveillance, early detection, treatment and even prevention are presented in this review.

Cytomegalovirus infection in the pregnant mother, fetus, and newborn infant.

Cytomegalovirus (CMV) is a common virus that infects persons of all ages, races, and backgrounds. Originally described as a rare cause of "cytomegalic inclusion disease," CMV is now known to cause a broad spectrum of illness in the fetus and newborn, with most infections being asymptomatic at birth. This article discusses the epidemiology and diagnosis of CMV infection in pregnant women, the fetus, and the newborn, including recent advances in antenatal diagnosis. In addition, the challenges of treatment and prevention of congenital CMV are explored.

Ophthalmologic findings in children with congenital cytomegalovirus infection.

BACKGROUND: Cytomegalovirus (CMV) infection is the most common congenital viral infection in the United States, affecting 0.5% to 2% of live births. Approximately 90% of infected infants are asymptomatic at birth. We undertook this study to determine the incidence and etiology of visual impairment and other ophthalmologic abnormalities in children with congenital CMV infection. METHODS: We prospectively evaluated 42 symptomatic and 83 asymptomatic children with congenital CMV infection, along with 21 control patients. One or more comprehensive ophthalmologic examinations were performed on each patient. The frequency and etiology of visual impairment and other ophthalmologic problems were tabulated for each patient. RESULTS: Nine of 42 (22%) patients in the symptomatic group had moderate to severe visual impairment in 16 eyes. Visual impairment was primarily due to optic atrophy in 6 of 16 (37%) eyes, macular scars in 2 of 16 (13%) eyes, and cortical visual impairment in 8 of 16 (50%) eyes. In comparison, none of 83 asymptomatic patients had severe visual impairment (P <.001). One asymptomatic patient had mild unilateral visual impairment caused by a macular scar. Strabismus developed in 12 of 42 (29%) symptomatic patients compared with 1 of 83 (1.2%) asymptomatic patients (P <.001). CONCLUSIONS: Visual impairment and strabismus are common in patients with symptomatic congenital CMV infection and rare in patients with asymptomatic congenital CMV infection. Visual impairment may be caused by cortical, optic nerve, and/or retinal abnormalities. Infants with symptomatic congenital CMV infection should undergo careful ophthalmologic screening and follow-up examinations.

Dental caries in HIV-infected children: a longitudinal study.

PURPOSE: The purpose of this descriptive longitudinal clinical study was to determine primary and permanent dentition caries status in HIV-infected children, and to compare caries status with the CD4 percentage (CD4%) and immune suppression category. MATERIALS AND METHODS: 73 children up to 9 years of age with vertical HIV transmission were evaluated for caries in the primary dentition at baseline and at 6 month intervals over a 30 month period; while 19 HIV-infected children between 5 and 11 years of age had their permanent dentition evaluated for caries at baseline and at 6 month intervals over a 24 month period. Caries status was also compared with CDC CD4 percentage (> 25%, 15-24%, < 15%), and CDC immune suppression categories (immune suppression: none, moderate, severe). With primary dentition caries, comparisons were made among all children (2-9 yr-olds, N = 73), < 2 yr-olds (N = 28), 2 to 4 yr-olds (N = 20), and 5 to 9 yr-olds (N = 25), and compared with NHANES III data. Caries-free status was also determined. RESULTS: During the 30-month period, there was an almost two-fold increase in primary tooth surface caries for the 2 to 9 year-olds. Caries-free status in the primary dentition declined from 60% at baseline to 37% at the 30-month period. With 5 to 11 years-olds, DMFS and DMFT remained relatively stable, while the proportion of caries-free individuals declined from 72% at baseline to 50% at 18 months. Caries in the primary dentition was increased substantially for those in the low CDC CD4 percentage categories and CDC moderate to severe immune suppression categories. CONCLUSION: Primary dentition caries status in HIV-infected children is considerably greater than that for the US pediatric population, and increases with decreasing CD4 percentage and moderate to severe immune suppression. HIV-infected children with caries-free primary dentitions are less frequent than in the US pediatric population, and caries-free status decreases with age, lower CD4 percentage and moderate to severe immune suppression.

Detection of fungal organisms in saliva from HIV-infected children: a preliminary cytologic analysis.

PURPOSE: Fungal infections in HIV-infected individuals are associated with advancement of disease. In pediatric HIV infection, symptomatic children have a significantly higher incidence of clinical candidiasis and persistent drug-resistant candidiasis than do asymptomatic HIV-infected children. The purpose of this preliminary cytologic study was to determine the prevalence of fungal organisms in whole unstimulated saliva from children with vertically acquired HIV infection. METHODS: The subjects included 27 HIV-infected and 11 HIV-exposed, but uninfected, children. Whole unstimulated saliva was obtained for cytologic evaluation (hematoxylin and eosin, silver stains) with selected samples evaluated by electron microscopy. RESULTS: Yeast and hyphae were identified cytologically in 19% of HIV-infected (22% symptomatic HIV-infected, 11% asymptomatic HIV-infected) and 9% of HIV-exposed, but uninfected, children. Fungal organisms were found more frequently in HIV-infected with moderate (18%) and severe (27%) suppression. Fungi were more frequent with antiretroviral therapy (22%) vs no antiretroviral therapy (0%) and no antifungal therapy (20%) vs. antifungal therapy (7%). Yeast and hyphal fungal forms are more prevalent in symptomatic HIV-infection with moderate and severe suppression, and those receiving antiretroviral agents, but no antifungal medications. CONCLUSION: Fungal organisms in the saliva may reflect oral carriage or mucosal colonization, which may influence the development of clinically significant candidiasis in these immunocompromised children.