High throughput quantitative colorimetric microneutralization assay for the confirmation and differentiation of West Nile Virus and St. Louis encephalitis virus.

An automated colorimetric micro-neutralization assay (CmNt) was developed for confirmation and differentiation of West Nile Virus (WNV)-positive human sera as a higher throughput alternative to the standard six-well plaque-reduction neutralization test (PRNT). CmNt was performed in high-capacity 96-well micro-titer plates and required 4-6 days to complete. Inhibition of infection was determined by reduced neutral red-dye retention and conveniently recorded by a colorimetric plate reader. Human sera previously confirmed by PRNT as either negative (N = 52), WNV positive (N = 81), or St. Louis encephalitis virus positive (N = 12) were tested by CmNt; interpreted results were virtually identical to PRNT with a reduced turnaround time and higher throughput. Additionally, a handful of dengue virus positive and negative specimens (four each) were tested by CmNt; interpreted results were identical to PRNT.

Persistence of rubella antibodies after 2 doses of measles-mumps-rubella vaccine.

BACKGROUND: Since 1990, most schoolchildren in the United States have received a second dose of measles-mumps-rubella vaccine (MMR2) at kindergarten entry. Elimination of endemic rubella virus circulation in the United States was declared in 2004. The objective of the current study was to evaluate the short- and long-term rubella immunogenicity of MMR2. METHODS: At enrollment in 1994-1995, children (n = 307) in a rural Wisconsin health maintenance organization received MMR2 at age 4-6 years. A comparison group of older children (n = 306) was vaccinated at age 9-11 years. Serum specimens were collected during a 12-year period. Rubella antibody levels were evaluated by plaque-reduction neutralization (lowest detectable titer, 1:10). RESULTS: Before administration of MMR2 in the kindergarten group, 9% of subjects were seronegative, 60% had the lowest detectable titer, and the geometric mean titer (GMT) was 1:13. One month after administration of MMR2, 1% were seronegative, 6% had the lowest detectable titer, and the GMT was 1:42. Four-fold boosts occurred in 62% of subjects, but only 0.3% were immunoglobulin M positive. Twelve years after MMR2 administration, 10% were seronegative, 43% had the lowest detectable titer, and the GMT was 1:17. The middle-school group showed similar patterns. CONCLUSIONS: Rubella antibody response to MMR2 was vigorous, but titers decreased to pre-MMR2 levels after 12 years. Because rubella is a highly epidemic disease, vigilance will be required to assure continued elimination.

Persistence of mumps antibodies after 2 doses of measles-mumps-rubella vaccine.

BACKGROUND: Since 1990, most US schoolchildren have received a second dose of measles-mumps-rubella vaccine (MMR2) at kindergarten entry. The objective of the present study was to evaluate the short- and long-term mumps immunogenicity of MMR2. METHODS: At enrollment in 1994-1995, children (n=308) in a rural Wisconsin health maintenance organization received MMR2 at age 4-6 years. A comparison group of older children (n=308) was vaccinated at age 9-11 years. Serum samples were collected over 12 years. Mumps antibody levels were evaluated by plaque-reduction neutralization (lowest detectable titer, 10). RESULTS: Before MMR2, the geometric mean titer (GMT) for the younger group was 33; no subject was seronegative, but 16% had the lowest detectable titer. In response to MMR2, the GMT tripled to 97, and the proportion with low titers diminished to 3%. Four-fold boosts occurred among 54%, but only 3% were positive for immunoglobulin M. Twelve years after MMR2, the GMT declined to 46, the proportion with titers

Seroprevalence of measles antibody in the US population, 1999-2004.

BACKGROUND: Endemic measles transmission was declared eliminated in the United States in 2000. To ensure that elimination can be maintained, high population immunity must be sustained and monitored. Testing for measles antibody was included in the National Health and Nutrition Examination Survey (NHANES), a nationally representative survey, conducted during 1999-2004. METHODS: A measles-specific immunoassay was used to measure the seroprevalence of measles antibody in NHANES participants 6-49 years of age. For analysis, participants were grouped by birth cohort. RESULTS: During 1999-2004, the rate of measles seropositivity in the population overall was 95.9% (95% confidence interval [CI], 95.1%-96.5%). The highest seroprevalence of measles antibody was in non-Hispanic blacks (98.6% [95% CI, 98.0%-99.1%]). Those born during 1967-1976 had significantly lower levels of measles antibody (92.4% [95% CI, 90.8%-93.9%]) than did the other birth cohorts. Independent predictors of measles seropositivity in the 1967-1976 birth cohort were non-Hispanic/black race/ethnicity, more than a high school education, having health insurance, and birth outside the United States. CONCLUSIONS: Measles seropositivity was uniformly high in the US population during 1999-2004. Nearly all population subgroups had evidence of measles seropositivity levels greater than the estimated threshold necessary to sustain measles elimination. Non-Hispanic whites and Mexican Americans born during 1967-1976 had the lowest measles seropositivity levels and represent populations that might be at increased risk for measles disease if the virus were reintroduced into the United States.

Persistence of measles antibodies after 2 doses of measles vaccine in a postelimination environment.

OBJECTIVE: To evaluate the persistence of measles antibodies after 2 doses of measles vaccine in a setting where exposure to wild-type measles was unlikely. Measles was declared eliminated from the United States in 2000, an achievement attributed to effective implementation of a routine 2-dose vaccination policy. Some have questioned whether measles transmission could resume if immunity wanes in the absence of boosting from wild-type measles. DESIGN: Prospective, observational, volunteer cohort study. SETTING: Rural Wisconsin health maintenance organization. PARTICIPANTS: Children who received the second measles vaccine dose at kindergarten (aged 4-6 years) or middle school (aged 10-12 years) in 1994 or 1995. Serum samples were collected periodically during a 10-year period for the kindergarten group and a 5-year period for the middle school group. INTERVENTION: Second dose of measles vaccine. MAIN OUTCOME MEASURE: Measles antibody levels were assessed by plaque-reduction neutralization: titers less than 8 mIU/mL were considered seronegative and suggestive of susceptibility to measles, and titers of 120 mIU/mL or less were considered low and suggestive of potential susceptibility. RESULTS: During the study period, no measles was reported in the study area. Voluntary attrition reduced the study population from 621 at enrollment to 364 (58.6%) by study end. Before the second dose, 3.1% (19/621) had low titers, of whom 74% (14/19) were antibody-negative, with geometric mean titers being significantly higher in kindergarteners (1559 mIU/mL) than in middle schoolers (757 mIU/mL) and rates of negativity significantly lower (1.0% [3/312] vs 3.6% [11/309]). One month after the second dose, 0.2% (1/612) had low titers and none was seronegative, with geometric mean titers being significantly higher in kindergarteners (2814 mIU/mL) than in middle schoolers (1672 mIU/mL). By study end, 4.9% (18/364) had low titers and none was seronegative, with no significant difference in geometric mean titers between kindergarteners (641 mIU/mL) and middle schoolers (737 mIU/mL) when both groups were aged 15 years. Projections suggest that the proportion of persons with low antibody levels may increase over time. CONCLUSIONS: Measles antibody persisted in all vaccinees available for follow-up 10 years after a second dose of vaccine, with no seronegative results detected. Declining titers suggest the need for vigilance in ensuring disease protection for the vaccinated population.

Rubella immunity levels in the United States population: has the threshold of viral elimination been reached?

After the 1989-1991 rubella resurgence, rubella vaccination efforts targeted children and women of childbearing age. Utilizing National Health and Nutrition Examination Survey data collected during 1988-1994 and 1999-2004, we assessed whether US levels of rubella seropositivity are consistent with rubella elimination and whether changes are consistent with immunization efforts. Serum samples with rubella antibody levels > or =10 IU tested by rubella immunoglobulin G enzyme immunoassay were considered to be positive. In 1999-2004, the overall age-adjusted rubella seropositivity level was 91.3% (95% confidence interval [CI], 90.5%-92.1%), a significant increase from 88.1% (95% CI, 86.9%-89.1%) in 1988-1994 (P<.001). Among children, seropositivity was highest in children 6-11 years of age (96.2%), followed by adolescents 12-19 years of age (93.7%). Both groups showed significant increases in immunity levels, in comparison with those in 1988-1994 (P<.001). Among adults, seropositivity among women increased (from 88.9% to 91.5%; P=.015), and there was no change among men (from 87.8% to 88.0%; P=.84). In 1999-2004, population rubella immunity levels were at or above the modeled threshold for elimination of rubella virus transmission. Increases in immunity levels are consistent with vaccination efforts.

Evidence of human herpesvirus 6 infection in 4 immunocompetent patients with encephalitis.

We describe 4 patients with encephalitis due to possible reactivation of human herpesvirus 6 (HHV-6) infection who were enrolled in the California Encephalitis Project. All were immunocompetent and had HHV-6 loads determined in cerebrospinal fluid specimens. Tests for detection of HHV-6 should be considered for individuals with encephalitis.

Safety and immunogenicity of concomitant versus nonconcomitant administration of hepatitis B, tetanus-diphtheria, and measles-mumps-rubella vaccines in healthy eleven- to twelve-year-olds.

PURPOSE: To study safety and immunogenicity of concomitant administration of hepatitis B (HB), Tetanus-diphtheria (Td), and Measles-mumps-rubella (MMR) vaccines in healthy 11-12-year-olds. METHOD: One hundred ninety-seven healthy 11-12-year-olds from the general community were randomized in an unblinded study to concomitant vaccination with Recombivax HB brand of HB vaccine and Td and MMR vaccines or to HB given at a separate time from Td and MMR vaccination. Primary analyses were for induction of protective immunity and safety. Secondary endpoints were mean geometric antibody titers for HB and a boosting effect for Td and MMR. Differences in immunogenicity were analyzed using the one-sided Pearson's Chi-square test. RESULTS: Concomitant vaccination for HB, Td, and MMR had a significantly increased incidence of headache, red eyes, and rash compared with nonconcomitant vaccination. There was no significant difference in incidence of serious adverse events, rates of protective immunity, or in secondary endpoints. CONCLUSIONS: Concomitant HB, Td, and MMR vaccination in 11-12-year-olds is as safe and immunogenic as nonconcomitant vaccination.

Kaposi's sarcoma-associated herpesvirus glycoprotein K8.1 is dispensable for virus entry.

Kaposi's sarcoma-associated herpesvirus (KSHV) is considered the etiologic agent of Kaposi's sarcoma and several lymphoproliferative disorders. Recently, the KSHV genome was cloned into a bacterial artificial chromosome and used to construct a recombinant KSHV carrying a deletion of the viral interferon regulatory factor gene (F. C. Zhou, Y. J. Zhang, J. H. Deng, X. P. Wang, H. Y. Pan, E. Hettler, and S. J. Gao, J. Virol. 76:6185-6196, 2002). The K8.1 glycoprotein is a structural component of the KSHV particle and is thought to facilitate virus entry by binding to heparan sulfate moieties on cell surfaces. To further address the role of K8.1 in virus infectivity, a K8.1-null recombinant virus (BAC36DeltaK8.1) was constructed by deletion of most of the K8.1 open reading frame and insertion of a kanamycin resistance gene cassette within the K8.1 gene. Southern blotting and diagnostic PCR confirmed the presence of the engineered K8.1 gene deletion. Transfection of the wild-type genome (BAC36) and mutant genome (BAC36DeltaK8.1) DNAs into 293 cells in the presence or absence of the complementing plasmid (pCDNAK8.1A), transiently expressing the K8.1A gene, produced infectious virions in the supernatants of transfected cells. These results demonstrated that the K8.1 glycoprotein is not required for KSHV entry into 293 cells.

Acyclovir responsive brain stem disease after the Ramsay Hunt syndrome.

We report an immunocompetent patient with the Ramsay Hunt syndrome (RHS) followed days later by brainstem disease. Extensive virological studies proved that varicella zoster virus (VZV) was the causative agent. Treatment with intravenous acyclovir resulted in prompt resolution of all neurological deficits except peripheral facial palsy. This case demonstrates that after geniculate zoster, brainstem disease may develop even in an immunocompetent individual and effective antiviral therapy can be curative.

Stress-induced subclinical reactivation of varicella zoster virus in astronauts.

Varicella zoster virus (VZV) becomes latent in human ganglia after primary infection. VZV reactivation occurs primarily in elderly individuals, organ transplant recipients, and patients with cancer and AIDS, correlating with a specific decline in cell-mediated immunity to the virus. VZV can also reactivate after surgical stress. The unexpected occurrence of thoracic zoster 2 days before space flight in a 47-year-old healthy astronaut from a pool of 81 physically fit astronauts prompted our search for VZV reactivation during times of stress to determine whether VZV can also reactivate after non-surgical stress. We examined total DNA extracted from 312 saliva samples of eight astronauts before, during, and after space flight for VZV DNA by polymerase chain reaction: 112 samples were obtained 234-265 days before flight, 84 samples on days 2 through 13 of space flight, and 116 samples on days 1 through 15 after flight. Before space flight, only one of the 112 saliva samples from a single astronaut was positive for VZV DNA. In contrast, during and after space flight, 61 of 200 (30%) saliva samples were positive in all eight astronauts. No VZV DNA was detected in any of 88 saliva samples from 10 healthy control subjects. These results indicate that VZV can reactivate subclinically in healthy individuals after non-surgical stress.

Multifocal varicella-zoster virus vasculopathy without rash.

A 51-year-old woman with CREST syndrome (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) developed stepwise progressive focal neurological deficits without zoster rash. Multifocal ischemic infarcts were seen on magnetic resonance imaging, and cerebral angiography revealed focal stenosis of arteries affecting the intracranial circulation. A brain biopsy was nondiagnostic. Virological etiology of the disease was verified by the detection of varicella-zoster virus antibody in cerebrospinal fluid and by reduced serum-cerebrospinal fluid varicella-zoster virus IgG ratios (compared with normally high ratios of total IgG and albumin). Treatment with intravenous acyclovir stabilized but did not significantly improve her neurological deficits.

Measles and mumps vaccination as a model to investigate the developing immune system: passive and active immunity during the first year of life.

Evaluations of neutralizing antibody responses in 6-, 9- and 12-month-old infants given measles or mumps vaccine indicated that 6-month-old infants had diminished humoral immune responses associated with passive antibody effects, but also had an intrinsic deficiency in antiviral antibody production, which was independent of passive antibody effects. In contrast, lower neutralizing antibody titers in 9-month-olds were related only to passive antibody effects. Measles and mumps-specific T-cell proliferation and interferon-gamma (IFNgamma) production were induced by vaccination at 6, 9 or 12 months, regardless of passive neutralizing antibodies or age. These observations suggest a need to refine concepts about passive antibody interference and primary vaccine failure, taking into account the sensitization of antiviral T-cells, which occurs in the presence of passive antibodies and is observed in infants who do not develop active humoral immunity. A second dose of measles vaccine given at 12-15 months enhanced antiviral T-cell responses to measles in infants who were vaccinated at 6 or 9 months, and produced higher seroconversion rates. Since T-cell immunity is elicited under the cover of passive antibodies, the youngest infants benefit from the synergistic protection mediated by maternal antibodies and their own capacity to develop sensitized antiviral T-cells, which prime for subsequent exposures to the viral antigens. Conceptually, maternal immunization approaches with vaccines that can be given to women of child-bearing age before pregnancy, or that are safe for administration during pregnancy, should enhance passive antibody protection. Rather than being detrimental to infant adaptive immune responses, maternal vaccination can be coupled effectively with vaccine regimens that elicit priming of antiviral immune responses in infants during the first year of life.

In search of encephalitis etiologies: diagnostic challenges in the California Encephalitis Project, 1998-2000.

The California Encephalitis Project was initiated in June 1998 to identify the causes and characterize the clinical and epidemiologic features of encephalitis in California. Testing for >or=13 agents, including herpesviruses, enteroviruses, arboviruses, Bartonella species, Chlamydia species, and Mycoplasma pneumoniae, was performed at the Viral and Rickettsial Disease Laboratory (Richmond, California). Epidemiologic and clinical information collected for each case guided further testing. From June 1998 through December 2000, 334 patients who met our case definition of encephalitis were enrolled. A confirmed or probable viral agent of encephalitis was found in 31 cases (9%), a bacterial agent was found in 9 cases (3%), and a parasitic agent was found in 2 cases (1%). A possible etiology was identified in 41 cases (12%). A noninfectious etiology was identified in 32 cases (10%), and a nonencephalitis infection was identified in 11 (3%). Despite extensive testing and evaluation, the etiology of 208 cases (62%) remained unexplained.

Varicella in Americans from NHANES III: implications for control through routine immunization.

At the time of varicella vaccine introduction in the United States, an estimated 4 million episodes of varicella occurred annually. This survey of varicella seroprevalence is the first to describe immunity to a vaccine-preventable disease prior to vaccine introduction in the United States population. The objective of this analysis is to describe patterns of naturally-acquired varicella and understand characteristics associated with infection in the varicella vaccine-naive United States population. A nationally representative cross-sectional health examination survey that included venipuncture was conducted among 21,288 U.S. participants aged 6 years and older from 1988 through 1994. Serologic evidence of varicella-zoster virus infection was measured by enzyme immunoassay of varicella-zoster virus-specific IgG antibody. The seroprevalence of IgG antibody to varicella-zoster virus increased from 86.0% in children aged 6 through 11 years to 99.6% in adults aged 40 through 49 years. Immunity to varicella remained at 99% or higher in Americans aged 50 years and older. Among persons aged 6 through 19 years, non-Hispanic black children were 40% less likely to be seropositive compared with white children (odds ratio [OR], 0.6; 95% confidence interval [CI], 0.4-0.8). Among young adults aged 20 through 39 years, women with a history of live birth (OR, 4.3; 95% CI, 2.1-8.7) and married men (OR, 2.7; 95% CI, 1.2-5.7) were more likely to have naturally-acquired immunity to varicella. This study found that, prior to use of varicella vaccine in the United States, age, race, and marital characteristics were independently associated with naturally acquired varicella. Future varicella serosurveys in Americans will provide essential information to interpret the population impact of varicella vaccine.

The protean manifestations of varicella-zoster virus vasculopathy.

Varicella-zoster virus (VZV) vasculopathy in the central nervous system (CNS) affects large and small cerebral vessels. Large-vessel disease is most common in immunocompetent individuals, whereas small-vessel disease usually develops in immunocompromised patients. In some patients, both large and small vessels are involved. Neurological features are protean. Neurological disease often occurs months after zoster and sometimes without any history of zoster rash. Magnetic resonance imaging (MRI) scanning, cerebral angiography, and examination of cerebrospinal fluid (CSF) with virological analysis are needed to confirm the diagnosis. VZV vasculopathy patients do not always have VZV DNA in CSF, but diagnosis can be confirmed by finding anti-VZV antibody in CSF, along with reduced serum/CSF ratios of VZV immunoglobulin G (IgG) compared to albumin or total IgG. When VZV vasculopathy develops months after zoster, antiviral treatment is often effective.

Patients with suspected herpes simplex encephalitis: rethinking an initial negative polymerase chain reaction result.

A statewide encephalitis diagnostic project of the California State Department of Health Services found that herpes simplex virus 1 DNA may not be detectable by molecular methods early in the clinical course of herpes simplex encephalitis.

Epstein-Barr virus myeloradiculitis and encephalomyeloradiculitis.

We provide a comprehensive clinical, radiological and virological analysis of four patients with Epstein-Barr virus (EBV) infection of the nervous system. One patient developed acute myeloradiculitis, one had acute encephalomyeloradiculitis, one had acute meningoencephalomyeloradiculitis and one had a subacute meningomyeloradiculitis. The ability of EBV to affect multiple parts of the entire neuraxis from meninges and brain to the spinal cord and peripheral nerves was evidenced by combinations of stiff neck and mental status changes, as well as patterns of weakness and sensory loss due to transverse myelitis or peripheral nerve disease. The CSF of all four patients contained a pleocytosis, predominantly mononuclear with elevated levels of protein, but a normal glucose level. In the two patients with acute myeloradiculitis and subacute meningomyeloradiculitis, the MRI revealed an increased signal in the spinal cord and lumbosacral roots, but in the two patients with acute encephalomyeloradiculitis and acute meningoencephalomyeloradiculitis, the brain and spinal cord MRIs were normal. In all four patients, EBV DNA, but not cytomegalovirus (CMV), herpes simplex virus (HSV) or varicella-zoster virus (VZV) DNA, was found in the CSF. The antibody pattern in serum was consistent with recent infection, and both EBV immunoglobulin (Ig) M and IgG antibodies, but not antibodies to HSV, VZV or CMV, were found in the CSF. Finally, there were reduced serum/CSF ratios of antibody to EBV, but not to total IgG or albumin, consistent with intrathecal antibody synthesis. None of the four patients died and none had brain swelling or focal changes according to brain MRI. Residual neurological deficits were evident. The two patients with acute myeloradiculitis and acute meningomyeloradiculitis had residual lower extremity weakness, and one of these patients later developed optic neuritis. The patient with acute encephalomyeloradiculitis had a moderate flaccid paraparesis, and the patient with subacute meningomyeloradiculitis was left with sensory loss in the feet. Compared with neurological disease caused by other herpes viruses, the clinical features of acute EBV myeloradiculitis, encephalomyeloradiculitis, encephalomyeloradiculitis and subacute meningomyeloradiculitis are distinctive. Of the eight human herpesviruses, EBV and VZV produce the most protean neurological syndromes. The mechanism by which EBV produces neurological disease is unknown. More correlative pathological, virological and immunological studies are needed in EBV-associated neurological disease.

Prevalence of Kaposi sarcoma-associated herpesvirus infection in homosexual men at beginning of and during the HIV epidemic.

CONTEXT: Some studies have inferred that an epidemic of Kaposi sarcoma-associated herpesvirus (KSHV) infection in homosexual men in the United States occurred concurrently with that of human immunodeficiency virus (HIV), but there have been no direct measurements of KSHV prevalence at the beginning of the HIV epidemic. OBJECTIVES: To determine the prevalence of KSHV infection in homosexual men in San Francisco, Calif, at the beginning of the HIV epidemic in 1978 and 1979 and to examine changes in prevalence of KSHV at time points from 1978 through 1996 in light of changes in sexual behavior. DESIGN, SETTING, AND PARTICIPANTS: Analysis of a clinic-based sample (n = 398) derived from the San Francisco City Clinic Cohort (ages 18-66 years) (n = 2666 for analyses herein) and from population-based samples from the San Francisco Men's Health Study (MHS) (ages 25-54 years) (n = 825 and 252) and the San Francisco Young Men's Health Study (YMHS) (ages 18-29 years) (n = 428-976, and 557); behavioral studies were longitudinal and KSHV prevalence studies were cross-sectional. MAIN OUTCOME MEASURES: Antibodies against KSHV and HIV; sexual behaviors. RESULTS: The prevalence of KSHV infection in 1978 and 1979 was 26.5% of 235 (a random sample) overall (weighted for HIV infection) vs 6.9% (128/1842) for HIV in the San Francisco City Clinic Cohort sample. The prevalence of KSHV infection remained essentially unchanged between an MHS sample of 252 in 1984 and 1985 (29.6%) and a YMHS sample of 557 in 1995 and 1996 (26.4%), while HIV prevalence dropped from 49.5% of 825 in 1984 and 1985 (MHS) to 17.6% of 428 in 1992 and 1993 (YMHS). The proportion of men practicing unprotected receptive anal intercourse with 1 or more partners declined from 54% to 11% during the 1984 through 1993 period (MHS) with similar though slightly higher values in the YMHS in 1992 and 1993; whereas for unprotected oral intercourse it ranged between 60% and 90% in the 1984 through 1996 period (MHS and YMHS). CONCLUSIONS: Infection with KSHV was already highly prevalent in homosexual men when the HIV epidemic began in San Francisco, and its prevalence has been maintained at a nearly constant level. Any declines in the incidence of Kaposi sarcoma do not appear to be caused by a decline in KSHV transmission.