Campaigns with oral polio vaccine may lower mortality and create unexpected results.

Three studies from Guinea-Bissau found conflicting effects of OPV-at-birth (OPV0) on child survival. One study from 2004 suggested excess male mortality among children receiving OPV0 compared with children receiving NoOPV0 during a period of shortage of OPV. However, two subsequent studies showed beneficial effects of OPV0. In 2004, two national OPV-campaigns had been conducted in Guinea-Bissau. In a reanalysis of the 2004-study, in a survival analysis the age-adjusted mortality rate of study participants was 67% (95% CI=42-81%) lower after the OPV-campaigns than before the campaigns. In the OPV0 group only 22% (655/3031 person-years (pyrs)) of follow-up time was "after" the OPV-campaigns whereas 55% (473/859 pyrs) of the time in the NoOPV0 group was post-campaign (p<0.0001, Chi2). Censoring for OPV-campaigns in the original study removed excess male mortality and made the three studies more homogeneous. Overall, there is now considerable evidence that OPV, like other live vaccines, has important beneficial non-specific effects.

Cancer survival in the Gambia, 1993-1997.

The national cancer registry of the Gambia was established in 1986 as part of the Gambia Hepatitis Intervention Study in collaboration with IARC, France; Medical Research Council (MRC) Laboratories of the UK; and the Government of the Gambia at MRC, Banjul. Registration of incident cancer cases is done by active and passive methods. For this study, the registry contributed data on survival for six cancer sites or types registered during 1993-1997. Follow-up has been carried out predominantly by active methods with median follow-up ranging between 1-6 months. The proportion of histologically verified diagnosis for various cancers ranged between 1-45%, and 54-82% of total registered cases were included for survival analysis. Complete follow-up at five years from the incidence date ranged between 81-98% for different cancers. The 5-year age-standardized relative survival for selected cancers were cervix (23%), non-Hodgkin lymphoma (22%), breast (10%), stomach (4%) and liver (3%). The 5-year relative survival by age group showed fluctuations with no definite pattern or trend emerging, and with no survivors in many age intervals.

Acquisition of antibody isotypes against Plasmodium falciparum blood stage antigens in a birth cohort.

Information on the period during which infants lose their maternally derived antibodies to malaria and begin to acquire naturally their own immune responses against parasite antigens is crucial for understanding when malaria vaccines may be best administered. This study investigated the rates of decline and acquisition of serum antibody isotypes IgG1, IgG2, IgG3, IgG4, IgM and IgA to Plasmodium falciparum antigens apical membrane antigen (AMA1), merozoite surface proteins (MSP1-19, MSP2 and MSP3) in a birth cohort of 53 children living in an urban area in the Gambia, followed over the first 3 years of life (sampled at birth, 4, 9, 18 and 36 months). Antigen-specific maternally transferred antibody isotypes of all IgG subclasses were detected at birth and were almost totally depleted by 4 months of age. Acquisition of specific antibody isotypes to the antigens began with IgM, followed by IgG1 and IgA. Against the MSP2 antigen, IgG1 but not IgG3 responses were observed in the children, in contrast with the maternally derived antibodies to this antigen that were mostly IgG3. This confirms that IgG subclass responses to MSP2 are strongly dependent on age or previous malaria experience, polarized towards IgG1 early in life and to IgG3 in older exposed individuals.

Hepatitis B viral load and risk for liver cirrhosis and hepatocellular carcinoma in The Gambia, West Africa.

The main objectives of this study were to define the occurrence and levels of hepatitis B virus (HBV) DNA in asymptomatic HBV carriers, cirrhosis patients and hepatocellular carcinoma (HCC) cases from The Gambia, and to evaluate the risk for cirrhosis or HCC associated with HBV viremia. We used sensitive real-time quantitative PCR assays to measure HBV DNA in samples from a case-control study consisting of 60 asymptomatic HBV carriers, 53 cirrhotic patients and 129 HCC cases. Logistic regression was used to estimate the risks of cirrhosis and HCC associated with HBV-DNA levels and HBV e antigenemia (HBeAg) detection (a surrogate marker for viral replication). Detectable HBV viremia and HBeAg positivity were both significantly associated with cirrhosis (increasing risk by fourfold and 11-fold respectively) and with HCC (increasing risk by sixfold and threefold respectively). HBV-DNA levels were significantly higher in both HCC cases and cirrhotic patients compared to asymptomatic carriers (P < 0.01 for both). High-level HBV DNA (>10,000 copies/mL) was strongly associated with both HCC and cirrhosis (17- and 39-fold increased risk). Lower level HBV viremia (200-10,000 copies/mL) conferred a significant risk of HCC, although the association with cirrhosis was not significant. In conclusion, we find that high HBV-DNA levels are strongly associated with the serious sequelae of HBV infection, independent of HBeAg status. While risk for cirrhosis and for HCC notably increases at HBV-DNA levels >or=10,000 copies/mL, low-level viremia was also associated with significant risk for HCC.

Host genetic contribution to the cellular immune response to Chlamydia trachomatis: Heritability estimate from a Gambian twin study.

If the cellular immune response to Chlamydia trachomatis is subject to genetic influences, the degree and mechanisms of such genetic control may have important implications for vaccine development. We estimated the relative contribution of host genetics to the total variation in lymphoproliferative responses to C. trachomatis antigen by analyzing these responses in 64 Gambian twin pairs from trachoma endemic areas. Zygosity was determined by restriction fragment length polymorphism analysis of minisatellite probes and microsatellite typing. Proliferative responses to serovar A elementary body antigen were estimated in monozygotic (MZ) and dizygotic (DZ) twin pairs. We found a stronger correlation and lower within-pair variability in these responses in MZ than in DZ twin pairs. The heritability estimate was 0.39 (P = 0.07) suggesting that host genetic factors contributed 39% of the variation. A better understanding of these genetic influences will contribute to the elucidation of preventive therapies for ocular C. trachomatis infection and may identify important mechanisms in protection for rational vaccine construction.

Neurological morbidity among human T-lymphotropic-virus-type-1-infected individuals in a rural West African population.

BACKGROUND: Community-based neurological data about human T lymphotropic virus type 1 (HTLV-1) morbidity in sub-Saharan Africa are scarce. OBJECTIVES: To ascertain the prevalence of neurological morbidity, in particular tropical spastic paraparesis (TSP), among HTLV-1-infected subjects and to compare TSP prevalence in HTLV-1-infected with that in non-infected subjects in a rural West African population. METHODS: A cross-sectional study of HTLV-1-infected cases and controls (ratio 4:1) from a rural community (population approximately 10 000, HTLV-1 prevalence 7.7%). One neurologist masked to HTLV-1 serological status assessed all subjects. Clinical criteria were employed to diagnose TSP. RESULTS: From 205 eligible cases and controls, 139 were recruited with a mean age of 56 years, and 113 (81%) were HTLV-1-infected. 108/139 (78%) were female, and 8/113 HTLV-1 infected cases (7.1%) had a definite or probable TSP (all females; mean age 67 years) compared with 0/26 controls. Two with TSP were co-infected with HIV-2. Complaints of back pain and leg weakness were more common in HTLV-1-infected individuals (p = 0.03, p = 0.02), but no single symptom distinguished between subjects with and without TSP. CONCLUSION: We report a prevalence of TSP among HTLV-1-infected persons in this rural West African setting of 7.1%. There are difficulties excluding other potential aetiologies here.

Application of a novel, rapid, and sensitive oligonucleotide ligation assay for detection of cancer-predicting mutations in the precore and basal core promoter of hepatitis B virus.

Hepatocellular carcinoma (HCC) and cirrhosis are important causes of mortality worldwide. Persistent hepatitis B virus (HBV) infection is a major cause of these diseases. Double mutations in the basal core promoter (BCP) (A1762T and G1764A) and precore (pre-C) (G1896A) regions of the virus are associated with progression to HCC. The current study is aimed at developing a simple method for screening and detecting BCP and pre-C mutations in HBV carriers. We have developed and validated an oligonucleotide ligation assay (OLA) to detect point mutations in the HBV core gene. We have applied OLA methods to samples from HBV-infected carriers recruited from the Gambia Liver Cancer Study (GLCS) comprising asymptomatic HBsAg carriers, patients with cirrhosis, and patients with HCC. We observed an 89.3% and 95.8% concordance between the OLA and DNA sequencing for BCP and pre-C mutations, respectively. OLA detected the mutations in single-strain infections and in infections with mixtures of wild-type and mutant viruses under conditions where sequencing detected only the single dominant strains. BCP mutations were detected in 75.7% of patients with advanced liver disease (cirrhosis/HCC) compared to 47.6% of asymptomatic carriers, while pre-C mutations were detected in 34.5% of advanced liver disease patients and in 47.6% of asymptomatic HBsAg carriers. There was a significant association between the presence of BCP mutations and advanced liver disease. In conclusion, OLA is a simple, economical, and reliable assay for detection of pre-C and BCP mutations. Its application can lead to improvement in diagnosis and clinical care in regions where HBV is endemic.

Behaviour change and competitive exclusion can explain the diverging HIV-1 and HIV-2 prevalence trends in Guinea-Bissau.

The aim of this study was to determine whether a temporary rise in sexual risk behaviour during war in Guinea-Bissau could explain the observed trends in HIV-1 and HIV-2 prevalence, and to explore the possible contribution of competitive elimination of HIV-2 by HIV-1. A simulation model of the heterosexual transmission of sexually transmitted infections was parameterized using demographic, behavioural and epidemiological data from rural Guinea-Bissau, and fitted to the observed HIV-1 and HIV-2 trends with and without a historic rise in risk behaviour. The observed trends could only be simulated by assuming a temporary rise in risk behaviour. Around 30% of the projected decline in HIV-2 prevalence from a peak of 8.7% to 4.3% in 2010 was due to competitive elimination by HIV-1. Importantly for public health, HIV-1 prevalence was predicted to continue increasing and to become the dominant HIV type by 2010. Data collection is required to validate this prediction.

A comparison of ex vivo cytokine production in venous and capillary blood.

We performed a randomized study of the immunological effects of an early measles vaccine given at 4.5 months of age and aimed to obtain venous samples from the infants at baseline and 6 weeks later. If this was not feasible, a capillary sample was obtained. We analysed baseline samples from the first 50 children enrolled in the study to investigate the potential differences in ex vivo cytokine production between venous blood and capillary blood. We also obtained paired venous and capillary blood samples from 11 adult volunteers. Whole blood was stimulated with lipopolysaccharide (LPS) [a Toll-like receptor (TLR)-4 ligand], (S)-(2, 3-bis (palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH, trihydrochloride (PAM3Cys) (a TLR-2 ligand), phytohaemagglutinin (PHA) or purified protein derivative (PPD). Cytokine concentrations in the supernatants were assessed by a multiplexed assay and were compared between venous and capillary samples in both infants and adults. The production of both the pro- and the anti-inflammatory cytokines, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10, was higher in cultures of capillary blood compared with venous blood. This was found in non-stimulated control samples as well as in blood stimulated with PAM3Cys and PPD. Adults produced more IL-5 in venous blood than in capillary blood upon PHA stimulation. We found no other difference in the levels of IL-5 or IFN-gamma between venous and capillary blood. In capillary blood we found sex differences in response to PHA but this was not the case in venous blood. We found significant differences in the production of cytokines between venous and capillary blood. Such differences should be taken into account when setting up immuno-epidemiological studies.

Similar long-term vaccine efficacy of two versus three doses of HBV vaccine in early life.

WHO currently recommends three vaccinations against hepatitis B to provide optimal protection against infection and carriage. However, immunological theory and mathematical modelling suggest that similar protection could be induced with two doses, and trials among adolescents and adults have shown comparable rates for both primary seroprotection and geometric mean titres following vaccination. We determined vaccine efficacy among 60 children who only received two doses of hepatitis B vaccine as infants and among 463 children who had received three doses after 4-7 years of follow-up. Vaccine efficacy among the two-dose group was 86.3% against anti-HBc positivity (infection) and 92.3% against HBsAg positivity (carriage), which was similar to the vaccine efficacy found among the participants who had received three doses. To confirm this comparable vaccine efficacy a randomised controlled non-inferiority trial with long-term follow-up is needed.

An adaptation of recombinant vaccinia-based ELISPOT and intracellular cytokine staining for a comparative measurement of cellular immune responses in HIV-1 and HIV-2 infections in West Africa.

An efficient and quantitative tool for rapid assessment of human immunodeficiency virus (HIV)-induced cellular immune responses is important for resource-limited settings, such as in sub-Saharan Africa. Modifications are required to previously reported methods for evaluating ex-vivo antigen-specific cellular responses based on direct recombinant vaccinia virus (rVV) stimulation of peripheral blood mononuclear cells (PBMCs) by enzyme linked immunosorbent assay (ELISPOT) and by flow cytometry intracellular cytokine assay (ICA). We made such modifications in order to detect specific responses and compared quantitative cellular immune responses in HIV-1 and HIV-2 infected Gambians. The sensitivity of the rVV-based ELISPOT assay was on average 1.25 interferon (IFN)-gamma spot forming cells (SFC) per 50 000 PBMCs specific for either infection, and 5 IFN-gamma-secreting CD8+ T cells/50 000 in the ICA. The level of IFN-gamma SFC detected by ELISPOT and by ICA were correlated (P < 0.02). ICA detected pol-specific responses in 88% and 67% of HIV-1 and HIV-2 subjects, respectively, and gag-specific responses in more than 80% of both infections. Lower proportions of responders were obtained with ELISPOT, for which pol responses were present in 60% of HIV-1 and 46% of HIV-2 infected patients, and gag responses in 55% and 69%, respectively. The assays did not show any significant difference in cellular immune responses between HIV-1 and HIV-2 infected subjects with CD4% >or= 20%. These outcomes are comparable with results obtained using standard techniques and thus this method is a suitable, rapid and less expensive assessment of cellular immunity.

Long-term protection against carriage of hepatitis B virus after infant vaccination.

BACKGROUND: Carriage of hepatitis B virus (HBV) is a major risk factor for liver cirrhosis and hepatocellular carcinoma. Infant vaccination has been effective in preventing horizontal transmission during early childhood. It is unknown whether protection is maintained into early adulthood. METHODS: In 1984, early childhood vaccination was introduced in 2 rural Gambian villages. In 2003, serological assessment of 81.5% of 1,350 eligible participants 1-24 years old was done, to determine vaccine efficacy against infection and carriage. RESULTS: Overall vaccine efficacy against infection and carriage was 83.4% (95% confidence interval [CI], 79.8%-86.6%) and 96.5% (85% CI, 93.9%-98.9%), respectively. Vaccine efficacy against infection was similar when restricted to primary responders (85.3%), but a significant effect of peak antibody concentration was found. Both vaccine efficacy and levels of hepatitis B surface antibody (anti-HBs) decreased with age, resulting in a vaccine efficacy against infection and carriage among 20-24-year-old participants of 70.9% (95% CI, 60.4%-80.5%) and 91.1% (95% CI, 75.8%-100%), respectively. Fifteen years after vaccination, fewer than half of the vaccinees had detectable anti-HBs. The prevalence of carriage in the unvaccinated population was similar to the prevalence 20 years earlier. CONCLUSIONS: HBV vaccination early during life can provide long-lasting protection against carriage, despite decreasing antibody levels. The role played by subclinical boosting and the necessity of a booster need to be evaluated.

Hepatitis B surface antigenaemia and alpha-foetoprotein detection from dried blood spots: applications to field-based studies and to clinical care in hepatitis B virus endemic areas.

In many resource-limited regions with endemic hepatitis B virus (HBV), there is limited infrastructure to collect, process, transport, and store blood samples for identification of persons with chronic HBV infection or with hepatocellular carcinoma (HCC). We describe the application of a simple technique using commercially available kits for detection of HBV surface antigen (HBsAg) and alpha-foetoprotein (AFP) in dried blood spots (DBS) collected on filter paper. Study participants included subjects with and without chronic HBV infection and subjects with HCC or cirrhosis. Three to five blood drops were dried on filter paper. Dried blood (equivalent to 20 muL) was eluted and tested for HBsAg by Determine(TM) HBsAg and for AFP by counter-current immuno-electrophoresis and radio-immunoassay (RIA). The primary analysis focused on comparison of DBS results to serum testing results as the gold standard. The sensitivity of DBS for detecting chronic HBV infection was 96% (98-98) with specificity of 100% (CI 99-100). Sensitivity of DBS in detecting AFP compared with serum RIA was 73% (60-86) with specificity of 90% (81-98). Both HBsAg and AFP recovery were unaffected when DBS were left at room temperature (30-33 degrees C) and under humid conditions for up to 28 days prior to elution. We conclude that DBS can be reliably used as an economical and logical alternative for detection of HBsAg in chronically infected patients and for AFP-based diagnosis of HCC in clinical situations which preclude adequate collection and processing of blood samples. Both research-oriented field studies and routine clinical care may benefit from application of these techniques in resource-limited settings.

Genetic regulation of immune responses to vaccines in early life.

Infant immunization is the most cost-effective strategy to prevent infectious diseases in childhood, but is limited by immaturity of the immune system. To define strategies to improve vaccine immunogenicity in early life, the role of genetic and environmental factors in the control of vaccine responses in infant twins was studied. Immune responses to BCG, polio, hepatitis B, diphtheria, pertussis and tetanus vaccines were measured at 5 months of age in 207 Gambian twin pairs recruited at birth. Intrapair correlations for monozygous and dizygous pairs were compared to estimate the environmental and genetic components of variation in responses. High heritability was observed for antibody (Ab) responses to hepatitis B (77%), oral polio (60%), tetanus (44%) and diphtheria (49%) vaccines. Significant heritability was also observed for interferon-gamma and interleukin-13 responses to tetanus, pertussis and some BCG vaccine antigens (39-65%). Non-HLA genes played a dominant role in responses to Ab-inducing vaccines, whereas responses to BCG were predominantly controlled by genes within the HLA class II locus. Genetic factors, particularly non-HLA genes, significantly modulate immune responses to infant vaccination. The identification of the specific genes involved will provide new targets for the development of vaccines and adjuvants for young infants that work independently of HLA.

Mobility and the spread of human immunodeficiency virus into rural areas of West Africa.

BACKGROUND: In eastern and southern Africa, the human immunodeficiency virus (HIV) epidemic appeared first in urban centres and then spread to rural areas. Its overall prevalence is lower in West Africa, with the highest levels still found in cities. Rural areas are also threatened, however, because of the population's high mobility. We conducted a study in three different communities with contrasting infection levels to understand the epidemiology of HIV infection in rural West Africa. METHOD: A comparative cross-sectional study using a standardized questionnaire and biological tests was conducted among samples in two rural communities of Senegal (Niakhar and Bandafassi, 866 and 952 adults, respectively) and a rural community of Guinea-Bissau (Caio, 1416 adults). We compared the distribution of population characteristics and analysed risk factors for HIV infection in Caio at the individual level. RESULTS: The level of HIV infection was very low in Niakhar (0.3%) and Bandafassi (0.0%), but 10.5% of the adults in Caio were infected, mostly with HIV type 2 (HIV-2). Mobility was very prevalent in all sites. Short-term mobility was found to be a risk factor for HIV infection among men in Caio (adjusted odds ratio (aOR) = 2.06; 95% CI: 1.06-3.99). Women from Caio who reported casual sex in a city during the past 12 months were much more likely to be infected with HIV (aOR = 5.61 95% CI: 1.56-20.15). Short-term mobility was associated with risk behaviours at all sites. CONCLUSIONS: Mobility appears to be a key factor for HIV spread in rural areas of West Africa, because population movement enables the virus to disseminate and also because of the particularly risky behaviours of those who are mobile. More prevention efforts should be directed at migrants from rural areas who travel to cities with substantial levels of HIV infection.

A Gambian TNF haplotype matches the European HLA-A1,B8,DR3 and Chinese HLA-A33,B58,DR3 haplotypes.

Caucasians carry TNFA-308*2 in the 8.1 ancestral haplotype (AH) (HLA-A1,B8,DR3). In Gambians, TNFA-308*2 occurs without HLA-B8 or -DR3, suggesting an independent effect of TNFA-308 on disease. Hence we sought a segment of the 8.1 AH in Gambians. BAT1 (intron 10)*2 was selected as a specific marker of the haplotype and was found with TNFA-308*2 in Gambians. Samples homozygous at TNFA-308 and BAT1 (intron 10) demonstrated identity between the African TNFA-308*2 haplotype, the 8.1AH and the Asian diabetogenic 58.1AH (HLA-A33,B58,DR3) across a region spanning BAT1, ATP6G, IKBL, LTA, TNFA, LTB, LST-1 and AIF-1. Conservation of this block in geographically distinct populations suggests a common evolutionary origin and challenges current views of the role of TNFA-308*2 in disease.

Mucosal and systemic immune responses to plasmid protein pgp3 in patients with genital and ocular Chlamydia trachomatis infection.

The circulating and cervical B cell responses to Chlamydia trachomatis plasmid protein pgp3 were characterized in children and adults with ocular or genital chlamydial infection using the enzyme-linked immunospot assay (ELISPOT) and ELISA. No pgp3-specific ASCs were detected in healthy controls, but predominantly IgA ASCs were detected in UK adults with uncomplicated cervicitis or urethritis (P = 0.03, 0.019). In patients with extragenital complications or pelvic inflammatory disease a mixed response with more IgG and IgM ASCs was evident, suggesting a breach of mucosal immune compartmentalization with more extensive infection. In women with chlamydial cervicitis, ASCs secreting predominantly IgA, but also IgG, to pgp3 were present in cervix at presentation, with a frequency 30-50 times higher than blood. Cervical ASC numbers, especially IgG, fell markedly six weeks after antibiotic treatment. We detected principally IgA pgp3-specific antibody secreting cells (ASCs) in children resident in a Gambian endemic area, with a trend towards suppression of IgA responses during intense trachomatous inflammation (P = 0.06), as previously reported for other chlamydial antigens, and in keeping with the findings in genital disease. These data provide a rationale for further studies of immune responses to pgp3 in humans and animal models of chlamydia-induced disease, and its potential use in diagnostic assays and protective immunization strategies.

Severe respiratory syncytial virus infection in early life is associated with increased type 2 cytokine production in Gambian children.

BACKGROUND: Severe respiratory syncytial virus (RSV) infection in early childhood has been associated with subsequent wheezing and atopy. The aim of this study was to test if severe RSV infection in early life was associated with an increase in type 2 cytokine production and atopy in Gambian children 5 years later. METHODS: A cohort of children with severe RSV infection during the first year of life ('cases', n = 66) and without ('controls', n = 122) was followed-up at 5 years of age. Immediate hypersensitivity to common allergens, airway reactivity, serum IgE concentration and the production of IFN-gamma, IL-5 and IL-13 by lymphocytes activated in vitro with RSV F-G or control antigens was determined. RESULTS: After adjustment for confounders, cases produced significantly higher concentrations of IL-13 in response to RSV F-G and of IL-5 and IL-13 in response to tuberculin. Cases were more likely to have presented with a wheezy lower respiratory tract infection in the first 3 years of life (adjusted odds ratio = 9.9; 95% CI 1.6-61.0), but not thereafter. Cases and controls had similar skin response to allergens, airway reactivity and serum IgE concentrations. CONCLUSION: Severe RSV infection in early life is associated with a higher production of type 2 cytokines in Gambian children at 5 years of age. However this does not appear to result in increased risk of atopy or clinical allergy at that age.

Measles virus strains circulating in Central and West Africa: Geographical distribution of two B3 genotypes.

Africa remains one of the major reservoirs of measles infection. Molecular epidemiological studies have permitted different measles virus isolates to be grouped into clades and genotypes; the major group, which has been identified as indigenous to Africa, is clade B. The viruses from epidemics in the Gambia (1993) and in the Cameroon (2001) were examined. In both studies, the homogeneity of the virus isolates within the epidemic as shown by sequence analysis revealed less than 0.2% variation of nucleotides between isolates. The measles viruses isolated in 1983 in Yaoundé, Cameroon, were designated as the B1 genotype. However, in 2001 only viruses belonging to the B3 genotype were found in this city. The viruses in the Gambia (1993) were also of the B3 genotype. However, these viruses could be distinguished from each other at the antigenic level and by comparative sequence analysis. The B3 Cameroon (2001) viruses were related to the proposed B3.1 subgroup, whereas the Gambian (1993) isolates corresponded to the B3.2 subgroup. The geographical distribution for the period 1993-2001 of these two viruses shows that B3.1 is found from the Sudan to Nigeria and Ghana extending south to the Cameroon, whereas the B3.2 genotype is found in West Africa. In Nigeria and Ghana, the viruses co-circulate. The identification of these viruses will permit more meaningful epidemiological studies after the proposed increase in measles vaccination coverage.