Noroviruses as a Cause of Diarrhea in Immunocompromised Pediatric Hematopoietic Stem Cell and Solid Organ Transplant Recipients.

Case reports describe significant norovirus gastroenteritis morbidity in immunocompromised patients. We evaluated norovirus pathogenesis in prospectively enrolled solid organ (SOT) and hematopoietic stem cell transplant (HSCT) patients with diarrhea who presented to Texas Children's Hospital and submitted stool for enteric testing. Noroviruses were detected by real-time reverse transcription polymerase chain reaction. Clinical outcomes of norovirus diarrhea and non-norovirus diarrhea patients, matched by transplanted organ type, were compared. Norovirus infection was identified in 25 (22%) of 116 patients, more frequently than other enteropathogens. Fifty percent of norovirus patients experienced diarrhea lasting ≥14 days, with median duration of 12.5 days (range 1-324 days); 29% developed diarrhea recurrence. Fifty-five percent of norovirus patients were hospitalized for diarrhea, with 27% requiring intensive care unit (ICU) admission. One HSCT recipient developed pneumatosis intestinalis. Three HSCT patients expired ≤6 months of norovirus diarrhea onset. Compared to non-norovirus diarrhea patients, norovirus patients experienced significantly more frequent ICU admission (27% vs. 0%, p = 0.02), greater serum creatinine rise (median 0.3 vs. 0.2 mg/dL, p = 0.01), and more weight loss (median 1.6 vs. 0.6 kg, p < 0.01). Noroviruses are an important cause of diarrhea in pediatric transplant patients and are associated with significant clinical complications.

Characterization of norovirus-associated traveler's diarrhea.

BACKGROUND: Traveler's diarrhea is the most common medical complaint of international visitors to developing regions. Previous findings suggested that noroviruses (NoVs) are an underappreciated cause of traveler's diarrhea. METHODS. In the present study, we sought to define the presence of NoVs in 320 acute diarrheic stool samples collected from 299 US students who traveled to Guadalajara, Cuernavaca, or Puerto Vallarta, Mexico, during the period from 2007 through 2008. Conventional and quantitative real-time polymerase chain reaction assays were used to detect and determine NoV loads in stool samples. NoV strains were characterized by purification of viral RNA followed by sequencing of the viral capsid protein 1 gene. Sequences were compared using multiple sequence alignment, and phylogenetic trees were generated to evaluate the evolutionary relatedness of the viral strains associated with cases of traveler's diarrhea. RESULTS: NoV RNA was detected in 30 (9.4%) of 320 samples. Twelve strains belonged to genogroup I, and 18 strains belonged to genogroup II. NoV prevalence was higher in the winter season than in the summer season (23% vs 7%, respectively; P = .001). The cDNA viral loads of genogroup I viruses were found to be 500-fold higher than those of genogroup II strains. Phylogenetic analysis revealed a diverse population of NoV strains over different locations and years. CONCLUSIONS: NoV strains are important causes of traveler's diarrhea in Mexico, especially during the wintertime, and US students in Mexico may represent a suitable group for future NoV vaccine efficacy trials.

Effect of recent seasonal influenza vaccination on serum antibody responses to candidate pandemic influenza A/H5N1 vaccines: A meta-analysis.

Recent studies have suggested that among those receiving seasonal influenza vaccine (SIV), reduced immunogenicity is observed in recently vaccinated (RV; within the past season or 2) persons when compared with those not recently vaccinated (NRV). We performed a meta-analysis to assess the effect of recent immunization with SIV on serum H5 hemagglutination inhibition (HAI) antibody responses after influenza A/H5N1 vaccination using data from a series of randomized controlled trials. The primary outcome was seroconversion measured by HAI assays following receipt of 2 doses of H5N1 vaccine. The geometric mean titer (GMT) of serum HAI antibody after vaccination was the secondary outcome. Analyses were performed using propensity score (PS) matching. The PS for each individual in the meta-analysis cohort was calculated using logistic regression and covariates included age, gender, race, antigen dose, adjuvant, statin use and vaccine manufacturer. 2015 subjects enrolled in 7 clinical trials were eligible for inclusion in the meta-analysis cohort; among these, 915 (45%) were RV. 901 RV subjects were matched (1:1) with replacement to a subject who was NRV. Subjects who received SIV within the previous season were significantly less likely to seroconvert following H5N1 vaccination (adjusted odds ratio 0.76; 95%CI 0.60-0.96; p = 0.024), and the GMT was 18% higher among NRV subjects (GM ratio of HAI antibody 1.18; 95%CI 1.04-1.33; p = 0.008). Further work is needed to better define the effects of, and mechanisms contributing to, reduced immune responses to H5N1 vaccine among RV subjects.

A phase 1 study of the safety, reactogenicity, and immunogenicity of a Schistosoma mansoni vaccine with or without glucopyranosyl lipid A aqueous formulation (GLA-AF) in healthy adults from a non-endemic area.

BACKGROUND: Schistosomiasis caused by Schistosoma mansoni (Sm) is a chronic, debilitating and potentially deadly neglected tropical disease. The licensure of a vaccine to prevent schistosomiasis would represent a major breakthrough in public health. METHODS: The safety and immunogenicity of a candidate Sm vaccine were assessed in this phase I, double-blind, dose-escalation trial. Seventy-two healthy Sm-naïve 18-50 year olds were randomized to receive 3 doses ∼ 8 weeks apart of saline placebo, or 10 µg, 30 µg, or 100 µg of recombinant Sm-Tetraspanin-2 vaccine formulated on aluminum hydroxide adjuvant (Sm-TSP-2/Al) with or without 5 µg of glucopyranosyl lipid A aqueous formulation (GLA-AF). Clinical and serologic responses were assessed for 1 year after dose 3. RESULTS: Vaccines were safe and well-tolerated. The most common reactions were injection site tenderness and pain, and headache and fatigue. Tenderness and pain were more frequent in groups receiving vaccine with GLA-AF than placebo (p = 0.0036 and p = 0.0014, respectively). Injection site reactions among those given Sm-TSP-2/Al with GLA-AF lasted 1.22 and 1.33 days longer than those receiving Sm-TSP-2/Al without GLA-AF or placebo (p < 0.001 for both). Dose- and adjuvant-related increases in serum IgG against Sm-TSP-2 were observed. Peak IgG levels occurred 14 days after dose 3. Seroresponse frequencies were low among recipients of Sm-TSP-2/Al without GLA-AF, but higher among subjects receiving 30 µg or 100 µg of Sm-TSP-2/Al with GLA-AF. More seroresponses were observed among those given 30 µg or 100 µg of Sm-TSP-2/Al with GLA-AF compared to placebo (p = 0.023 and p < 0.001, respectively). Seroresponse frequencies were 0%, 30%, 50%, and 89%, respectively, among those given placebo, or 10 µg, 30 µg or 100 µg of Sm-TSP-2/Al with GLA-AF, suggesting a dose-response relationship for Sm-TSP-2/Al with GLA-AF (p = 0.0001). CONCLUSIONS: Sm-TSP-2/Al with or without GLA-AF was safe and well tolerated in a Sm-naïve population. A vaccine like the one under development may represent our best hope to eliminating this neglected tropical disease.

Real-time RT-PCR for norovirus screening in shellfish.

Real-time RT-PCR, combining amplification and detection of virus-specific amplicons, is a promising tool for norovirus detection in environmental or food samples such as shellfish. We developed a real-time RT-PCR assay based on one-step detection using single primer sets and probes for norovirus genogroups I and II. Seventy and seven RT-PCR units of genogroup I and II reference norovirus strains, respectively, were detected in artificially contaminated oysters. Validation of the new method on 150 archived naturally contaminated shellfish confirmed the utility of the genogroup II primer set to detect a large range of different strains circulating in France since 1995, but genogroup I strains were detected infrequently.

Respiratory tract viral infections in inner-city asthmatic adults.

BACKGROUND: Respiratory tract viral infections (RTVIs) have been identified frequently in association with asthma exacerbations in children, but few studies have shown similar rates of viral infections in adults with asthma. Further studies using newer diagnostic techniques to evaluate the frequency of RTVIs in adults with acute exacerbations of asthma need to be performed. METHODS: Twenty-nine asthmatic adults were recruited from the pulmonary clinic of an urban county hospital and were followed up in a longitudinal cohort study for signs and symptoms of asthma and RTVI. One hundred twenty-two asthmatic adults presenting to the emergency department (ED) of the same hospital with acute symptoms of asthma underwent evaluation for RTVI in a cross-sectional prevalence study. In both studies, respiratory secretions and paired serum samples were collected from subjects with acute wheezing episodes and evaluated using virus culture, serologic testing, and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In the longitudinal cohort study, 138 respiratory illnesses, of which 87 were asthma exacerbations, were evaluated; 41% of all illnesses and 44% of asthma exacerbations were associated with an RTVI. In the ED study, 148 asthma exacerbations were evaluated; 55% were associated with an RTVI. An RTVI was identified in 21 (50%) of 42 of the subjects hospitalized in the ED study. Picornaviruses (rhinoviruses), coronaviruses, and influenza viruses were the most commonly identified causes of RTVI. Forty-six (60%) of the 77 picornavirus infections and 22 (71%) of the 31 coronavirus infections were identified only using RT-PCR. CONCLUSIONS: Asthmatic exacerbations in adults are frequently associated with an RTVI. Identification of such infections often requires newer diagnostic methods, such as virus-specific RT-PCR. The high frequency of RTVIs identified in association with asthmatic exacerbations in adults from the inner city suggests that strategies for the prevention of RTVI should be targeted toward this population.

B-cell activation and differentiation by HIV-1 antigens among volunteers vaccinated with VaxSyn HIV-1.

The generation of memory B cells in response to vaccination with a baculovirus-derived recombinant gp160 candidate AIDS vaccine, VaxSyn HIV-1, was investigated in 12 healthy human volunteers who were immunized with VaxSyn HIV-1, hepatitis B vaccine, or alum adjuvant alone on days 1, 28, 180, and 540. Peripheral blood mononuclear cells were collected pre- and post-immunization and cultured unstimulated or with pokeweed mitogen (PWM), VaxSyn HIV-1 (rgp160), or HIV-1 lysate (iHIV-1) for 7 days before polyclonal and HIV-1-specific IgG production in culture supernatants (SNs) were measured. No differences were seen in the spontaneous or PWM-induced IgG production in SN from vaccinees and controls. Only vaccinee SN contained higher-than-normal levels of polyclonal IgG after stimulation with either rgp160 or iHIV-1, especially after the second and third booster immunizations on days 180 and 540, respectively. There were also contemporaneous increases in HIV-1-specific antibody in SN of all vaccinees, albeit at different time points throughout the study. We conclude that VaxSyn HIV-1 induces antigen-specific B-cell responses with the generation of memory B cells in vivo that can be reactivated in vitro to deliver an anamnestic response.

Cytokines and impaired CD8+ CTL activity among elderly persons and the enhancing effect of IL-12.

We have previously demonstrated that about 70% of elderly persons exhibit deficient cytotoxic T lymphocyte (CD8+ CTL) responses against influenza viruses when compared to young persons. Since IFN-gamma, a Th1 cytokine and IL-4, a Th2 cytokine, stimulate and inhibit CD8+ CTL responses respectively, their role(s) in the age-related CTL deficiency was investigated. Lymphocytes from young adults (34 +/- 5 years old) and elderly subjects (71 +/- 1 years old) were stimulated in vitro with influenza A/H3N2, A/H1N1 or influenza B virus for 6-7 days. The CD8+ CTL activity against virus-infected autologous target cells was significantly lower among the elderly than the young subjects (P < 0.01). Following stimulation with influenza virus, IL-4 production in both age groups was similar on day 3 but significantly higher among elderly persons on day 6 (P < 0.05). In contrast, T cells from the elderly produced significantly lower IFN-gamma than did those from young persons on both days (P < 0.05). Treatment of T cells from young and elderly adults with recombinant human IL-12, a pivotal cytokine that stimulates Th1 cytokines, resulted in enhancement of CD8+ CTL activity and IFN-gamma production in a dose dependent manner (P < 0.01). IL-12-dependent enhancement of CTL activity was not always abrogated by anti-IFN-gamma antibody treatment. These results suggest that deficient influenza virus-specific CTL activity among the elderly is attributable to a Th1 to Th2 cytokine production switch. Immunotherapy with IL-12 could represent a useful approach to correct the CD8+ CTL deficiency and cytokine imbalance among elderly humans.

Ribavirin aerosol treatment of bronchiolitis associated with respiratory syncytial virus infection in infants.

In a double-blind study, bronchiolitis associated with respiratory syncytial virus infection in 12 randomly selected patients treated with ribavirin aerosol improved more rapidly than in 14 control patients given saline aerosol (P = .044, Wilcoxon rank sum test, two-tailed). An estimated 10 mg of ribavirin per kilogram of body weight was administered in daily 12-hour treatments over a five-day period. Respiratory syncytial virus disappeared from secretions at about the same rate in treated and control patients. There was no local or systemic intolerance, and there was no evidence of hematologic or other organ toxicity in the ribavirin-treated patients.

Influenza A virus outbreak in a neonatal intensive care unit.

BACKGROUND: Nosocomial infections with influenza virus are rarely recognized in neonatal intensive care units (NICU). An outbreak of influenza A virus infection in the NICU of an urban county hospital during the 1997 to 1998 influenza season is reported. METHODS: Clinical and virologic data were recorded in all symptomatic NICU patients after influenza A infection was diagnosed in one infant in October, 1997. RESULTS: Influenza A/H3N2 was isolated from two of four symptomatic infants. The application of rapid diagnostic techniques for the characterization of influenza virus infection allowed the timely institution of basic infection control measures, limiting this outbreak. Resistance to amantadine was documented for the first time in this patient population by reverse transcription-PCR within 48 h of treatment in one case. CONCLUSIONS: Prevention by immunization is a priority in those caring for high risk NICU patients.

Development of an immunomagnetic capture reverse transcription-PCR assay for the detection of Norwalk virus.

Norwalk virus (NV) is the prototype human virus of the family Caliciviridae. A rapid immunomagnetic capture/reverse transcription-(IMC/RT-)PCR assay was developed for the detection of NV. Immunomagnetic capture (IMC) utilizes paramagnetic beads coupled to a virus-specific antibody and allows separation of virus from contaminating materials and virus concentration in a single step. The detection limit of the developed assay was approximately 250-750 genomic equivalents/ml of 10% stool suspension. The detection limit of the assay was not altered by the presence of excess hepatitis A virus (HAV), although non-specific binding of HAV to the paramagnetic beads was observed. A panel of 100 stools from experimental human infections was screened for NV using a previously described heat release method, an antigen ELISA, or IMC/RT-PCR. NV was detected in 65/100 of these samples by IMC/RT-PCR compared to only 38/99 by heat release and 31/95 by antigen detection ELISA. All samples that were negative by IMC were also negative by both heat release and antigen ELISA. The number of samples in which RT-PCR was inhibited was greatly reduced by the use of IMC/RT-PCR compared to the heat release method (1/100 and 16/95 samples inhibited, respectively). The ability of IMC to concentrate virus (> or =2000-fold greater than heat release) and effectively remove inhibitory substances gives this assay distinct advantages over both the heat release and antigen ELISAs.

Unanticipated diagnoses found at autopsy in an urban public teaching hospital.

The authors set out to evaluate the use of the autopsy in an urban public teaching hospital setting during the AIDS era. Demographic and length of hospital stay data were obtained from weekly mortality review reports on all patients dying on the medicine service between 1/1/92 and 12/31/93. Clinical and autopsy diagnoses were compared for those patients who had autopsies. The autopsy rate was 16% (152/974). Significant, unsuspected diagnoses were found in 35% (53/152) of the cases, with infections, pulmonary emboli, and myocardial infarctions being most common. Human immunodeficiency virus-infected patients had a greater percentage of unsuspected findings (55%, 23/42), and many of these also were from an infectious etiology. The authors conclude that valuable, unsuspected information frequently can be obtained from autopsies in this clinical setting.

Distribution of Norwalk virus within shellfish following bioaccumulation and subsequent depuration by detection using RT-PCR.

Consumption of raw bivalve mollusks contaminated with pathogens from human feces continues to present a human health risk. The purpose of this study was to monitor the uptake, localization, and removal of Norwalk virus (NV) in shellfish (oyster and clam) tissues by analyzing virus distribution in selected dissected tissues. Live shellfish were allowed to bioaccumulate different input titers of NV for time periods from 4 to 24 h. In some experiments, depuration by shellfish that bioaccumulated NV and Escherichia coli bacteria was allowed to proceed for 24 or 48 hours. Dissected stomach (St), digestive diverticula (DD), adductor muscle (AM), and hemolymph cells (HC) tissues were assayed for NV by the reverse transcription polymerase chain reaction (RT-PCR) method. An internal RNA standard control was added to the RT-PCR to identify the presence of inhibitors to RT-PCR. NV titers in DD tissues before and after depuration were estimated using quantitative RT-PCR end-point dilution. NV was found in the alimentary tract (DD or St) at all concentrations of input virus, but was present more frequently after exposure to higher levels of virus. NV was detected in AM and HC only following exposure to higher levels of virus. In experiments where depuration by oysters was continued for 48 h, depuration of bacteria was efficient (95% reduction of bacteria), but minimal (7%) reduction of NV titers from DD tissues was detected. These findings indicate that NV can localize both within and outside the alimentary tract of shellfish, and NV is poorly depurated using conditions favorable for E. coli depuration.

Safety and immunogenicity of a recombinant nonglycosylated erythrocyte binding antigen 175 Region II malaria vaccine in healthy adults living in an area where malaria is not endemic.

Erythrocyte binding antigen region II (EBA-175) is a conserved antigen of Plasmodium falciparum that is involved in binding of the parasite to the host's erythrocytes. We evaluated the safety and immunogenicity of a recombinant EBA-175 vaccine with aluminum phosphate adjuvant in healthy young adults living in the United States. Eighteen subjects/group received ascending doses (5, 20, 80, or 160 µg) of the vaccine at 0, 1, and 6 months; 8 subjects received placebo. Most of the injection site and systemic reactions were mild to moderate in intensity. After 2 or 3 doses of the vaccine at any concentration, antibody levels measured by enzyme-linked immunosorbent assay were significantly higher than those for the placebo group. Sera from subjects who received 3 doses of the vaccine at any concentration inhibited the growth of erythrocyte-stage P. falciparum at low levels compared to sera from placebo recipients or preimmune sera. In conclusion, the EBA-175 vaccine with adjuvant was safe and immunogenic in malaria-naïve subjects.

Safety, reactogenicity and immunogenicity of Francisella tularensis live vaccine strain in humans.

We evaluated the safety, reactogenicity and immunogenicity of escalating doses of a new Francisella tularensis Live Vaccine Strain (LVS) lot by scarification (SCAR) or subcutaneously (SQ) in humans. Subjects (N=10/group) received one dose of LVS via SCAR at 10(5),10(7) or 10(9)cfu/ml or SQ at 10(2), 10(3),10(4) or 10(5)cfu/ml; 14 subjects received placebo. All doses/routes were well tolerated. When compared to placebo, vaccination with 10(7) SCAR and 10(9) SCAR resulted in significantly higher serologic response frequencies, as measured by ELISA for IgG, IgM, IgA and microagglutination; whereas vaccination with 10(5) SCAR, 10(7) SCAR 10(9) SCAR and 10(5) SQ elicited a significantly higher interferon-gamma response frequency.

Classification of respiratory tract picornavirus isolates as enteroviruses or rhinoviruses by using reverse transcription-polymerase chain reaction.

The classification of picornaviruses isolated from respiratory secretions as human rhinoviruses (HRVs) or enteroviruses (EVs) by using reverse transcription-polymerase chain reaction was compared to that derived from acid lability testing. Of the 135 clinical isolates examined, 91 were found to be HRVs and 44 were EVs. There was 100% concordance between the two classification methods. Reverse transcription-polymerase chain reaction is an effective alternative to traditional methods for differentiating HRVs from EVs.

Pneumonia caused by Mycoplasma pneumoniae and the TWAR agent.

Mycoplasma pneumoniae and the TWAR agent account for a good proportion of pneumonias acquired in the community among older children and young adults. Recovery from these infections is common, although serious complications may occur. Each is associated with fever, nonproductive cough, and headache. The full clinical manifestations of TWAR agent infection are only now beginning to be defined. Diagnosis of M pneumoniae can be by serology and/or culture capabilities. The TWAR agent cannot be routinely isolated and serologic tests are available only in research laboratories. Response to tetracycline antibiotics has been established for each agent and hospitalization is rarely needed. Erythromycin and other macrolide antibiotics have good activity against M pneumoniae and can be employed as an alternative to tetracycline antibiotics for this pathogen.