Classification of measles breakthrough cases in an elimination setting using a comprehensive algorithm of laboratory results: why sensitive and specific IgM assays are important.

OBJECTIVE: In 2006, a measles outbreak occurred in Catalonia (Spain), six years after endemic measles was declared eliminated. This study aimed to classify 19 confirmed measles breakthrough cases (BC) using a high-performance avidity assay developed in 2010. METHODS: Serum specimens were tested by indirect IgG, indirect IgM, capture IgM enzyme immunoassay, an endpoint-titer IgG avidity assay, and a plaque reduction neutralization assay. Serology and RNA detection results were combined in an algorithm for measles confirmation and classification of breakthrough cases and analyzed with clinical and epidemiological data. RESULTS: Of 19 samples, thirteen (68%) were conclusive with the classification of BCs, and six (32%) had false-positive IgM results on an indirect-format assay; they were classified as rash and fever illness of undetermined etiology. BCs were primary vaccine failures (seven or 54%), secondary vaccine failures (four or 31%), and two (15%) could not be classified. CONCLUSIONS: In measles elimination settings, high-performing assays and a comprehensive algorithm of laboratory results (IgG, IgM, and RNA detection), including IgG avidity and PRN results when necessary, can assist in accurate laboratory confirmation and classification of suspected measles cases for surveillance. Highly specific IgM assays are required to minimize the number of false-positive results.

Vaccine-associated varicella and rubella infections in severe combined immunodeficiency with isolated CD4 lymphocytopenia and mutations in IL7R detected by tandem whole exome sequencing and chromosomal microarray.

In areas without newborn screening for severe combined immunodeficiency (SCID), disease-defining infections may lead to diagnosis, and in some cases, may not be identified prior to the first year of life. We describe a female infant who presented with disseminated vaccine-acquired varicella (VZV) and vaccine-acquired rubella infections at 13 months of age. Immunological evaluations demonstrated neutropenia, isolated CD4 lymphocytopenia, the presence of CD8(+) T cells, poor lymphocyte proliferation, hypergammaglobulinaemia and poor specific antibody production to VZV infection and routine immunizations. A combination of whole exome sequencing and custom-designed chromosomal microarray with exon coverage of primary immunodeficiency genes detected compound heterozygous mutations (one single nucleotide variant and one intragenic copy number variant involving one exon) within the IL7R gene. Mosaicism for wild-type allele (20-30%) was detected in pretransplant blood and buccal DNA and maternal engraftment (5-10%) demonstrated in pretransplant blood DNA. This may be responsible for the patient's unusual immunological phenotype compared to classical interleukin (IL)-7Rα deficiency. Disseminated VZV was controlled with anti-viral and immune-based therapy, and umbilical cord blood stem cell transplantation was successful. Retrospectively performed T cell receptor excision circle (TREC) analyses completed on neonatal Guthrie cards identified absent TREC. This case emphasizes the danger of live viral vaccination in severe combined immunodeficiency (SCID) patients and the importance of newborn screening to identify patients prior to high-risk exposures. It also illustrates the value of aggressive pathogen identification and treatment, the influence newborn screening can have on morbidity and mortality and the significant impact of newer genomic diagnostic tools in identifying the underlying genetic aetiology for SCID patients.

Monitoring progress toward measles elimination by genetic diversity analysis of measles viruses in China 2009-2010.

With the achievement of high coverage for routine immunization and supplementary immunization activities (SIAs), measles incidence in mainland China reached its lowest level in 2010. The proportion of measles cases in the vaccination-targeted population decreased during 2007-2010 after the SIAs. More than 60% of measles cases were in adults or infants, especially in the coastal and eastern provinces during 2009 and 2010. A total 567 isolates of measles virus were obtained from clinical specimens from 27 of 31 provinces in mainland China during 2009 and 2010. Except for two vaccine-associated cases, one genotype D4 strain, two genotype D9 strains, and four genotype D11 strains, the other 558 strains were genotype H1 cluster H1a. Genotype H1 has been the only endemic genotype detected in China since surveillance began in 1993. Only genotype H1 was found in mainland China during 1993-2008, except for one detection of genotype H2. More recently, multiple genotypes of imported measles were detected even with the background of endemic genetotype H1 viruses. Analysis of the 450-nucleotide sequencing window of the measles virus N gene showed that the overall genetic diversity of the recent geneotype H1 strains decreased between 2008 and 2010. The lower genetic diversity of H1 strains suggested that enhanced vaccination may have reduced the co-circulating lineages of endemic genotype H1 strains in mainland China.

Investigation of a mumps outbreak among university students with two measles-mumps-rubella (MMR) vaccinations, Virginia, September-December 2006.

Following the clinical diagnosis of the first case of mumps on September 22, 2006 at the University of Virginia (UVA), 52 suspected cases were identified through active surveillance for mumps by the end of December 2006. Samples were collected from 47 students who presented with parotitis despite a documented history of two doses of measles, mumps, and rubella (MMR) vaccine. Six of 47 serum samples (13%) were positive for mumps IgM, and 46/47 specimens were positive for mumps IgG. Endpoint titration of acute phase serum samples from laboratory-confirmed cases did not provide evidence that elevated serum IgG is a consistent marker for infection among cases due to secondary vaccine failure. Buccal swab samples from 39 of the 47 students were tested by real-time reverse transcription-polymerase chain reaction (RT-PCR) and/or viral culture. Mumps virus or mumps RNA was detected in 12 of 39 buccal samples (31%). Genetic analysis of the virus from the outbreak at UVA indicated that the outbreak was not linked to the large mumps outbreak in the Midwestern US that occurred earlier in 2006. Our findings support the use of viral detection to improve laboratory diagnosis of mumps among persons who have received two doses of MMR.

Molecular epidemiology of measles virus.

Genetic characterization of wild-type measles viruses provides a means to study the transmission pathways of the virus and is an essential component of laboratory-based surveillance. Laboratory-based surveillance for measles and rubella, including genetic characterization of wild-type viruses, is performed throughout the world by the WHO Measles and Rubella Laboratory Network, which serves 166 countries in all WHO regions. In particular, the genetic data can help confirm the sources of virus or suggest a source for unknown-source cases as well as to establish links, or lack thereof, between various cases and outbreaks. Virologic surveillance has helped to document the interruption of transmission of endemic measles in some regions. Thus, molecular characterization of measles viruses has provided a valuable tool for measuring the effectiveness of measles control programs, and virologic surveillance needs to be expanded in all areas of the world and conducted during all phases of measles control.

Genetic variability and mRNA editing frequencies of the phosphoprotein genes of wild-type measles viruses.

The sequences of the nucleoprotein (N) and hemagglutinin (H) genes are routinely used for molecular epidemiologic studies of measles virus (MV). However, the amount of genetic diversity contained in other genes of MV has not been thoroughly evaluated. In this report, the nucleotide sequences of the phosphoprotein (P) genes from 34 wild-type strains representing 15 genotypes of MV were analyzed and found to be almost as variable as the H genes but less variable than the N genes. Deduced amino acid sequences of the three proteins encoded by the P gene, P, V and C, demonstrated considerably higher variability than the H proteins. Phylogenetic analysis showed the same tree topography for the P gene sequences as previously seen for the N and H genes. RNA editing of P gene transcripts affects the relative ratios of P and V proteins, which may have consequences for pathogenicity. Wild-type isolates produced more transcripts with more than one G insertion; however, there was no significant difference in the use of P and V open reading frames, suggesting that the relative amounts of P and V proteins in infected cells would be similar for both vaccine and wild-type strains.

Rubella and measles seroprevalence among women of childbearing age, Argentina, 2002.

To assess rubella and measles susceptibility among women of childbearing age we conducted a cross-sectional seroprevalence study in four cities and one rural area in Argentina. A convenience sample of women aged 15-49 years seeking care in public health-care institutions was selected (n=2804). Serum specimens were tested for rubella and measles IgG antibody titres. The overall susceptibility to rubella and measles was 8.8 and 12.5% respectively. Seroprevalence differences were found for both rubella (P<0.001) and measles (P=0.002) across sites. Rubella seroprevalence was higher in women aged >or=40 years than in younger women (P=0.04). Measles seroprevalence tended to increase with age (P<0.001). Approximately 15% of women aged 15-29 years were not immune to measles. No risk factors were associated with rubella seronegativity; however, age (P<0.001) and having less than four pregnancies (P<0.001) were factors associated with measles seronegativity. Our findings support the introduction of supplemental immunization activities targeting adolescents and young adults to prevent congenital rubella syndrome and measles outbreaks over time.

Subacute sclerosing panencephalitis in the differential diagnosis of encephalitis.

The authors describe five cases of subacute sclerosing panencephalitis (SSPE) identified through the California Encephalitis Project that emphasize the importance of considering SSPE in the differential diagnosis of encephalitis, particularly among pediatric patients. SSPE was not suspected in the differential diagnosis of three of the cases until results of measles testing were known. The diagnosis of SSPE is often not considered by clinicians because of its rarity in the United States and the nonspecific clinical manifestations at onset.

Negative impact of clinical misdiagnosis of measles on health workers' confidence in measles vaccine.

We conducted a survey to determine the accuracy of the clinical diagnosis of measles in Zimbabwe. Between December 1996 and February 1997, we collected blood samples and clinical and demographic information from a sample of 105 children with a clinical diagnosis of measles. A clinical case of measles was defined as a person with a history of fever, rash for three or more days, and either cough, coryza, or conjunctivitis. A laboratory-confirmed case of measles or rubella had IgM antibodies against measles virus or rubella virus respectively. A total of 91% of children met the clinical case definition. Among those who met the clinical case definition for measles, 72% were IgM-positive for measles virus only, 23% were IgM-positive for rubella virus only, 3% were IgM-positive for both measles and rubella viruses, and 2% were IgM-negative for both viruses. This study demonstrates the importance of considering selective laboratory confirmation of measles in periods of high disease incidence when the effectiveness of the vaccine is questioned.

Comparative detection of measles and rubella IgM and IgG derived from filter paper blood and serum samples.

We compared the use of serum and filter paper blood spots as specimen sources for the detection of measles- and rubella-specific IgM and IgG. We collected capillary blood into microtainer tubes and onto filter paper spots from 60 children and 60 healthy adults. The blood was collected from 12-15-month-old children approximately 3 weeks after primary vaccination with measles, mumps, rubella vaccine, and the sample-pairs were tested for measles-specific IgM and IgG antibodies by using a capture antibody EIA and an indirect EIA, respectively. We tested sample-pairs from a subset of participants for rubella- specific IgM and IgG antibodies by using commercially available capture IgM (Captia) and indirect IgG (Wampole) assays. The concordance of results from serum and filter paper blood spots was high for all assays: 98% for measles IgM, 93% for measles IgG, 94% for rubella IgM, and 93% for rubella IgG, and increased to between 96-100% for all four assays when indeterminate samples were excluded. The correlation coefficients for EIA signals were 0.99 and 0.77 for measles IgM and IgG, respectively, and 0.92 and 0.94 for rubella IgM and IgG, respectively. The cut-off values used for filter paper samples were the same as those used for serum samples for all tests except for the rubella IgM assay. The use of filter paper blood spots is a promising future option for the detection of measles- and rubella-specific antibodies.

Evaluation of an early two-dose measles vaccination schedule.

Vaccination at 6 months of age followed by routine revaccination is recommended when exposure of infants to measles is likely. Dade County, Florida, began this early two-dose schedule during a large epidemic in 1986-1987 (i.e., 22% of cases occurred in infants aged 6-11 months). This schedule was continued routinely in high-risk areas. The effect of an early two-dose schedule on measles prevention in the county was examined by comparing measles vaccination coverage and epidemiology before (1985-1987) and after (1988-1996) the schedule became routine. To assess serologic response, seroprevalence of measles antibody among children aged 4-6 years in 1995 was examined. To evaluate vaccine effectiveness, a case-control study was conducted among preschool-aged children. Among those aged 2 years, vaccination coverage with > or =1 dose increased from 75% to 94% in 1996. The number of annual cases declined, and endemic measles transmission reportedly ended after 1993. Seroprevalence of plaque reduction neutralization antibody (titer > 1:120) among those receiving vaccination according to an early two-dose schedule and a single dose at age > or =12 months was 94% (95% confidence interval: 89, 98) and 98% (95% confidence interval: 95, 100). In these groups, vaccine effectiveness was comparably high. Early two-dose measles vaccination is associated with improved coverage and a comparably high level of humoral immunity and clinical protection as a single dose at age > or =12 months. This strategy can be useful in areas at high risk for measles among infants.

Induction of cytokine mRNA in peripheral blood mononuclear cells of infants after the first dose of measles vaccine.

To better characterize the cytokine response to measles virus vaccine, we examined the levels of IL-2, IL-4, IL-5, IL-10, IL-12 and gamma-interferon (gamma-IFN) in measles virus-stimulated peripheral blood mononuclear cells from 18 donors before and 2 weeks after vaccination. Donors were grouped as seropositive or seronegative on the basis of measles-specific IgM antibody present at 2 weeks postvaccination. After vaccination, similar levels of upregulation of IL-2 and gamma-IFN mRNA were observed in the two groups. The majority of donors in both groups did not exhibit an increase in measles specific IL-4 or IL-10 mRNA after vaccination. IL-12 mRNA was not induced by measles virus in any of the donors. A statistically significant upregulation of IL-5 mRNA was observed among seropositive (9/13) compared with seronegative (1/5) donors after vaccination (P=0.09, one tailed Fisher's test). The observed measles specific induction of IL-5 mRNA is suggestive of a possible association between IL-5 production and an antibody response to measles virus.

Molecular characterization of the polymerase gene and genomic termini of Nipah virus.

In 1998, Nipah virus (NV) emerged in peninsular Malaysia, causing fatal encephalitis in humans and a respiratory disease in swine. NV is most closely related to Hendra virus (HV), a paramyxovirus that was identified in Australia in 1994, and it has been proposed that HV and NV represent a new genus within the family Paramyxoviridae. This report describes the analysis of the sequences of the polymerase gene (L) and genomic termini of NV as well as a comparison of the full-length, genomic sequences of HV and NV. The L gene of NV is predicted to be 2244 amino acids in size and contains the six domains found within the L proteins of all nonsegmented, negative-stranded (NNS) RNA viruses. However, the GDNQ motif found in most NNS RNA viruses was replaced by GDNE in both NV and HV. The 3' and 5' termini of the NV genome are nearly identical to the genomic termini of HV and share sequence homology with the genomic termini of other members of the subfamily Paramyxovirinae. At 18,246 nucleotides, the genome of NV is 12 nucleotides longer than the genome of HV and they have the largest genomes within the family Paramyxoviridae. The comparison of the structures of the genomes of HV and NV is now complete and this information will help to establish the taxonomic position of these novel viruses within the family Paramyxoviridae.

Genetic characterization of contemporary wild-type measles viruses from Vietnam and the People's Republic of China: identification of two genotypes within clade H.

Genetic characterization was conducted on 17 wild-type measles viruses isolated near Hanoi, Vietnam, during 1998 as well as on eight viruses isolated in the Hunan, Hainan, Shandong, and Anhui provinces of the People's Republic of China during 1995, 1998, and 1999. Previous studies had shown that, compared to wild-type measles viruses found in other parts of the world, wild-type viruses from China were genetically distinct and comprised a new clade of viruses, clade H. In this study, sequence analyses of the nucleotides coding for the COOH terminal 150 amino acids of the nucleoprotein (N) and the entire hemagglutinin (H) protein indicated that although all of the viruses from Vietnam were members of clade H, they were clearly distinct from the Chinese viruses. With the exception of MVi/Beijing.China/94/1, the Vietnamese viruses differed from all of the Chinese viruses by at least 3.5 and 2.5% at the nucleotide level for the N and H genes, respectively. These data suggest that clade H should be divided into two genotypes with the Chinese viruses placed in genotype H1 and the Vietnamese viruses in genotype H2. Sequence analysis of measles viruses imported into the United States from either China or Vietnam demonstrated that this designation of genotypes will be helpful in future measles surveillance activities.

Molecular biology of Hendra and Nipah viruses.

The structure and genetic organization of Hendra and Nipah viruses places them in the subfamily Paramyxovirinae. However, low homology with other subfamily members and several novel biological and molecular features such as genome length and F(0 )cleavage site suggest classification in a new genus within the Paramyxovirinae.

National serologic survey of measles immunity among persons 6 years of age or older, 1988-1994.

CONTEXT: Measles incidence in the United States is at a record low, and indigenous transmission has been interrupted in each year since 1996, suggesting that measles is no longer endemic. A national estimate of measles immunity and an understanding of predictors of measles susceptibility are essential for assuring sustained elimination of endemic disease. OBJECTIVE: To assess patterns of immunity and to determine predictors of susceptibility to measles. DESIGN/SETTING: Sera and data on participants from the third National Health and Nutrition Examination Survey (1988-1994) (NHANES III) were examined. NHANES III was a cross-sectional survey of a representative sample of the civilian, noninstitutionalized population of the United States. POPULATION: 20,100 persons 6 years of age or older were tested for measles-specific immunoglobulin G (IgG) antibody by an enzyme immunoassay. MAIN OUTCOME MEASURE: Participants with serum positive for measles antibody were considered protected or immune to measles disease. RESULTS: Prevalence of measles immunity was 93%. Nearly all persons (99%) born in the prevaccine era (before 1957) were immune. Immunity declined among persons born in the vaccine era (after 1956) to 81% among those born in 1967-1976, and increased again to 89% among those born in 1977-1988. Among persons born in the vaccine era, independent predictors of measles susceptibility varied by birth cohort and included birth in the United States, residence in a noncrowded household, residence in a nonmetropolitan area, and, among males, non-Hispanic white and Mexican American race/ethnicity. Among adults 17 years of age or older, additional predictors of susceptibility included living at or above the poverty line and not currently being married. CONCLUSIONS: Population immunity among persons 6 years of age or older is very high; however, as many as 15 million persons across the United States may lack humoral immunity. While it is unclear that the susceptible population can support continuous, indigenous transmission of measles, providers should follow current recommendations to evaluate the measles susceptibility of patients born in the vaccine era and vaccinate eligible patients.

Genomic characterization of wild-type measles viruses that circulated in different states in Brazil during the 1997 measles epidemic.

Despite the marked reduction in the incidence of measles in Brazil, a measles epidemic occurred in this country in 1997. The measles cases observed during this epidemic began to reappear in large numbers in São Paulo, and spread to Rio de Janeiro and other Brazilian states. In the present study molecular biology techniques were used for the detection and genomic characterization of measles viruses from clinical samples such as urine and nasopharyngeal secretions collected in the states of Rio de Janeiro, Minas Gerais and Paraná, during the 1997 epidemic. RT-PCR and nucleotide sequence analysis of part of the carboxyl-terminal region of the nucleoprotein gene of measles viruses obtained directly from clinical samples or from infected cell cultures during this epidemic classified all as wild-type of genotype D6. As the genotype D6 was identified in different Brazilian states, this study demonstrated that this genotype was circulating in Brazil during the 1997 epidemic.

Lack of evidence of endogenous avian leukosis virus and endogenous avian retrovirus transmission to measles, mumps, and rubella vaccine recipients.

The identification of endogenous avian leukosis virus (ALV) and endogenous avian retrovirus (EAV) in chick cell-derived measles and mumps vaccines in current use has raised concern about transmission of these retroviruses to vaccine recipients. We used serologic and molecular methods to analyze specimens from 206 recipients of measles, mumps, and rubella (MMR) vaccine for evidence of infection with ALV and EAV. A Western blot assay for detecting antibodies to endogenous ALV was developed and validated. All serum samples were negative for antibodies to endogenous ALV by Western blot analysis. Peripheral blood lymphocyte samples from 100 vaccinees were further tested by polymerase chain reaction for both ALV and EAV proviral sequences; all were negative. Matching serum samples were tested by reverse transcriptase polymerase chain reaction for ALV and EAV RNA, and all 100 samples were negative, providing no evidence of viremia. These findings do not indicate the presence of either ALV or EAV infection in MMR vaccine recipients and provide support for current immunization policies.

Measles eradication: is it in our future?

Measles eradication would avert the current annual 1 million deaths and save the $1.5 billion in treatment and prevention costs due to measles in perpetuity. The authors evaluate the biological feasibility of eradicating measles according to 4 criteria: (1) the role of humans in maintaining transmission, (2) the availability of accurate diagnostic tests, (3) the existence of effective vaccines, and (4) the need to demonstrate elimination of measles from a large geographic area. Recent successes in interrupting measles transmission in the United States, most other countries in the Western Hemisphere, and selected countries in other regions provide evidence for the feasibility of global eradication. Potential impediments to eradication include (1) lack of political will in some industrialized countries, (2) transmission among adults, (3) increasing urbanization and population density, (4) the HIV epidemic, (5) waning immunity and the possibility of transmission from subclinical cases, and (6) risk of unsafe injections. Despite these challenges, a compelling case can be made in favor of measles eradication, and the authors believe that it is in our future. The question is when.