Effect of a serogroup A meningococcal conjugate vaccine (PsA-TT) on serogroup A meningococcal meningitis and carriage in Chad: a community study [corrected].

BACKGROUND: A serogroup A meningococcal polysaccharide-tetanus toxoid conjugate vaccine (PsA-TT, MenAfriVac) was licensed in India in 2009, and pre-qualified by WHO in 2010, on the basis of its safety and immunogenicity. This vaccine is now being deployed across the African meningitis belt. We studied the effect of PsA-TT on meningococcal meningitis and carriage in Chad during a serogroup A meningococcal meningitis epidemic. METHODS: We obtained data for the incidence of meningitis before and after vaccination from national records between January, 2009, and June, 2012. In 2012, surveillance was enhanced in regions where vaccination with PsA-TT had been undertaken in 2011, and in one district where a reactive vaccination campaign in response to an outbreak of meningitis was undertaken. Meningococcal carriage was studied in an age-stratified sample of residents aged 1-29 years of a rural area roughly 13-15 and 2-4 months before and 4-6 months after vaccination. Meningococci obtained from cerebrospinal fluid or oropharyngeal swabs were characterised by conventional microbiological and molecular methods. FINDINGS: Roughly 1·8 million individuals aged 1-29 years received one dose of PsA-TT during a vaccination campaign in three regions of Chad in and around the capital N'Djamena during 10 days in December, 2011. The incidence of meningitis during the 2012 meningitis season in these three regions was 2·48 per 100,000 (57 cases in the 2·3 million population), whereas in regions without mass vaccination, incidence was 43·8 per 100,000 (3809 cases per 8·7 million population), a 94% difference in crude incidence (p<0·0001), and an incidence rate ratio of 0·096 (95% CI 0·046-0·198). Despite enhanced surveillance, no case of serogroup A meningococcal meningitis was reported in the three vaccinated regions. 32 serogroup A carriers were identified in 4278 age-stratified individuals (0·75%) living in a rural area near the capital 2-4 months before vaccination, whereas only one serogroup A meningococcus was isolated in 5001 people living in the same community 4-6 months after vaccination (adjusted odds ratio 0·019, 95% CI 0·002-0·138; p<0·0001). INTERPRETATION: PSA-TT was highly effective at prevention of serogroup A invasive meningococcal disease and carriage in Chad. How long this protection will persist needs to be established. FUNDING: The Bill & Melinda Gates Foundation, the Wellcome Trust, and Médecins Sans Frontères.

Isolation of maternal mononuclear cells from placentas for use in in vitro functional assays.

Interest in immunoregulatory mechanisms within uteroplacental tissues, particularly in malarial infection during pregnancy, prompted us to develop a technique to extract maternal mononuclear cells from human term placentas. This method is described. The phenotypes of isolated cells were characterised for expression of CD45, CD3, CD4, CD8, CD14, CD15, CD68, CD22, CAM 5.2 and class II MHC antigens and compared with those in situ in frozen sections of the same placentas. Isolated mononuclear cell preparations were examined for contamination by fetal trophoblasts. Fetal leukocyte contamination appeared unlikely since histological sections of placental tissue, after the extraction of maternal leukocytes, showed intact chorionic villi with no disruption of fetal stem vessels. This technique produces preparations of maternal placental mononuclear cells which are representative of cells in situ, show minimal fetal cell contamination and are suitable for functional studies.

A polymerase chain reaction for the diagnosis of Haemophilus influenzae type b disease in children and its evaluation during a vaccine trial.

BACKGROUND: Determination of the etiology of pneumonia in young children is difficult because blood culture, the usual method of diagnosis, is positive in only a small proportion of cases. For this reason vaccine trials that include bacterial pneumonia as an endpoint must be large. OBJECTIVES: To determine whether a diagnostic test based on a polymerase chain reaction could be used as an alternative to conventional blood culture for diagnosis of invasive Haemophilus influenzae type b (Hib) infections in young children investigated during the course of a large vaccine trial. METHODS: DNA was extracted from blood culture supernatants and probed for the presence of Hib DNA with a PCR assay with primers derived from the cap gene locus of Hib. Results of the PCR assay were compared with those obtained by conventional culture techniques. RESULTS: Blood cultures were obtained from 1544 children with suspected pneumonia, meningitis or septicemia and from 31 healthy control children who were contacts of cases. Blood culture supernatants were tested for Hib DNA in the PCR test. The sensitivity and specificity of a positive PCR test in blood culture supernatant as against culture of Hib from any normally sterile site were 100 and 99%, respectively. Eleven children had positive Hib PCR tests on blood culture supernatants but were negative by culture. In one of these cases Hib was isolated from a lung aspirate and in two other patients H. influenzae strains other than Hib were obtained from the cerebrospinal fluid. Eight of these 11 children were in the control group. When the results of the PCR assay were used to determine vaccine efficacy, a value of 86% was obtained compared with a figure of 95% obtained when conventional culture techniques were used. CONCLUSIONS: An Hib PCR assay on blood culture supernatants proved to be sensitive and specific for the diagnosis of Hib disease in children. The distribution of PCR-positive, culture-negative cases between Hib-vaccinated and control groups paralleled that of culture-positive cases, suggesting that most of these children had been infected with Hib. A trial of a highly efficacious vaccine provides a novel way for evaluating new diagnostic tests for which there is no standard diagnostic test of 100% reliability.

Inhibitory effect of cerebrospinal fluid on the growth of meningococci and pneumococci.

Initial cerebrospinal fluid (CSF) samples obtained from patients with pneumococcal meningitis usually contain far greater numbers of bacteria than initial CSF samples obtained from patients with meningococcal meningitis. Normal CSF was found to have an inhibitory effect on the growth of group A meningococci but not on type 1 pneumococci. The inhibitory effect of normal CSF was abolished by dialysis, indicating that the inhibitory factor has a low molecular weight. Heating normal CSF to 62.5 degrees C for 30 min resulted in a considerable reduction in the inhibitory effect, indicating that the inhibitory factor is heat labile.

SDS-PAGE analysis of membrane proteins of group A Neisseria meningitidis isolated before and during an epidemic of group A meningococcal disease in northern Nigeria.

Meningococcal membrane protein patterns were studied by SDS-PAGE analysis of group A meningococci isolated before and during a large epidemic of group A meningococcal disease in northern Nigeria. No difference was found in the membrane protein patterns of strains isolated before or during the 3 years of the epidemic. Isolates obtained from cases and carriers had similar membrane protein patterns.

An epidemic of meningococcal infection at Zaria, Northern Nigeria. 3. Meningococcal carriage.

Meningococcal carriage was studied in household contacts of patients with group A meningococcal disease and in controls. The carriage rate of group A meningococci among 1,098 household contacts was low (3.8%) and only slightly higher than the carriage rate found among 416 controls (2.6%). However, higher carrier rates were found among those in close contact with a patient. Carriage was found most frequently among children and young adults and was commoner in adult females than in adult males. Sulphonamides had no effect on carriage and all 60 strains tested in vitro were resistant to sodium sulphadiazine at a concentration of 10 micrograms/ml.

Single injection treatment of meningococcal meningitis. 2. Long-acting chloramphenicol.

A single injection of a long-acting oily preparation of chloramphenicol (Tifomycine) was compared with a five-day course of crystalline and procaine penicillin in the treatment of 131 adult patients with meningococcal meningitis. The clinical response to treatment was similar in the two groups of patients. Serial lumbar punctures showed a parallel fall in CSF cell count, protein and lactate and all posttreatment cultures were sterile. Single injection chloramphenicol treatment was cheaper and much easier to administer than penicillin. Long-acting chloramphenicol is thus an effective form of treatment for meningococcal meningitis and is likely to prove of particular value in the management of epidemics in areas with limited medical resources.

Single injection treatment of meningococcal meningitis. 1. Long-acting penicillin.

A single injection of a long-acting preparation of penicillin (Triplopen) was compared with a five-day course of crystalline and procaine penicillin in the treatment of meningococcal meningitis. The clinical response of patients treated with Triplopen was very similar to that of patients treated with crystalline penicillin and much more convenient to administer. However, four patients treated with Triplopen had a positive CSF culture 48 or 72 hours after their injection. One injection of Triplopen cannot, therefore, be recommended as an entirely safe form of treatment for meningococcal meningitis unless patients can be carefully followed.

An epidemic of meningococcal infection at Zaria, Northern Nigeria. 1. General epidemiological features.

In 1977 Zaria, in Northern Nigeria, was affected by a severe epidemic of group A meningococcal infection, 1,257 patients being admitted to hospital with the disease during a three-month period. The epidemic started towards the end of the dry season when it was hot, dry and dusty and finished shortly after the onset of the rains. The over-all attack rate was 3.6 per 1,000 but this varied considerably from area to area within the town. Few cases occurred amongst those belonging to the upper social classes. The disease was seen most frequently amongst those from five to 14 years old and there was a strong male preponderance. The over-all mortality was 8.3% but mortality was much higher (40.6%) amongst 67 patients with acute meningococcaemia.

Meningococcal carriage, meningococcal disease and vaccination.

Group A meningococcal carriage rates were determined 6 months before and 6 and 18 months after a mass vaccination campaign with a combined group A and group C meningococcal polysaccharide vaccine in a rural area of The Gambia. During the first pre-vaccination survey, performed during an outbreak of meningococcal disease, the carriage rate was high (16%). The carriage rate remained high during a second survey made 6 months after a vaccination campaign that covered approximately 90% of the study population. A year later very few group A meningococcal carriers were found. Membrane protein patterns of isolates obtained before and after vaccination were similar. We conclude that vaccination had little influence on the carriage rate of group A meningococci but that this was influenced by changes in herd immunity or by other unidentified factors.

Sequential bacteriological findings in the cerebrospinal fluid of Nigerian patients with pneumococcal meningitis.

Sequential bacteriological observations were made on the cerebrospinal fluid (CSF) of 28 patients with pneumococcal meningitis treated with high doses of penicillin for 2 weeks. The organism was isolated from the CSF of four patients 48 h or more after the start of treatment and from a further patient 48 h after treatment was stopped. Positive cultures were obtained in spite of the demonstration in the CSF of penicillin at a concentration well above the minimum inhibitory concentration for the organism isolated. Persistence of bacteria and their products in the CSF of patients with pneumococcal meningitis contrasts with the rapid clearance of bacteria from the CSF of patients with meningococcal meningitis and may contribute to the difference in the prognosis of these forms of meningitis.

Factors influencing susceptibility to meningococcal disease during an epidemic in The Gambia, West Africa.

A study was made of factors that influenced susceptibility to group A meningococcal disease during an epidemic that affected The Gambia, West Africa during the dry season of 1982-83. No explanations were found for the distribution of cases between villages or within affected villages. Socio-economic status, crowding, nutrition and previous exposure to meningococcal disease all appeared to be unimportant. Examination of serum samples obtained before the outbreak from a few children who subsequently became patients and from an equal number of age-matched controls from the same village showed a higher mean serum IgA value in children who became patients than in controls. There were not, however, any significant differences found in the concentrations of IgG, IgM, complement or meningococcal antibody between the two groups. Four children who developed culture-proven group A meningococcal disease had raised titres of bactericidal antibody to the epidemic strain 2-3 months before their illnesses. Our findings suggest that some important risk factors for group A meningococcal disease remain to be identified.

Genetic linkage of mild malaria to the major histocompatibility complex in Gambian children: study of affected sibling pairs.

PIP: Case-control studies have indicated that genes for the major histocompatibility complex influence the presentation and outcome of severe Plasmodium falciparum disease. To assess the role of genetic factors in mild malaria, an analysis was conducted in 217 pairs of Gambian twins (mean age, 5.3 years) concordant for this phenotype. The twins were monitored weekly during three rainy seasons (1991-93) for fever and P. falciparum infection. This surveillance produced a total of 40 pairs of twins who were concordant for clinical malaria; none had severe disease. In the 22 of these 40 families with complete information, 11 had two shared alleles (expected value, 5.5), 10 shared one allele (expected value, 11.0), and 1 shared no allele (expected value, 5.5). If a locus is genetically linked to disease, affected siblings will share a higher than expected number of alleles identical by descent at that locus. Sharing of major histocompatibility complex alleles was not increased among the 13 pairs of dizygous twins who were discordant for malaria. These findings confirm the importance of genetic factors to the risk of uncomplicated malaria.^ieng

Investigation of different group A immunoassays following one dose of meningococcal group A conjugate vaccine or A/C polysaccharide vaccine in adults.

A double-blind, randomized, controlled phase I study to assess the safety, immunogenicity, and antibody persistence of a new group A conjugate vaccine (PsA-TT) in volunteers aged 18 to 35 years was previously performed. Subjects received one dose of either the PsA-TT conjugate vaccine, meningococcal A/C polysaccharide vaccine (PsA/C), or tetanus toxoid vaccine. The conjugate vaccine was shown to be safe and immunogenic as demonstrated by a standardized group A-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and by a serum bactericidal antibody (SBA) assay using rabbit complement (rSBA). This report details further analysis of the sera using four additional immunologic assays to investigate the relationship between the different immunoassays. The immunoassays used were an SBA assay that used human complement (hSBA), a group A-specific IgG multiplexed bead assay, and two opsonophagocytic antibody (OPA) assays which used two different methodologies. For each vaccine group, geometric mean concentrations or geometric mean titers were determined for all assays before and 4, 24, and 48 weeks after vaccination. Pearson's correlation coefficients were used to assess the relationship between the six assays using data from all available visits. An excellent correlation was observed between the group A-specific IgG concentrations obtained by ELISA and those obtained by the multiplexed bead assay. hSBA and rSBA titers correlated moderately, although proportions of subjects with putatively protective titers and those demonstrating a > or = 4-fold rise were similar. The two OPA methods correlated weakly and achieved only a low correlation with the other immunoassays. The correlation between hSBA and group A-specific IgG was higher for the PsA-TT group than for the PsA/C group.

Prevention of secondary cases of meningococcal disease in household contacts by vaccination.

Household contacts of patients with group A meningococcal infection were vaccinated with either meningococcal vaccine or tetanus toxoid. Five of the 523 subjects who received tetanus toxoid developed meningococcal meningitis and another four probably had meningococcal disease. Only one possible case of meningococcal infection occurred among 520 contacts vaccinated with meningococcal vaccine. Vaccination had no effect on nasopharyngeal carriage of meningococci. Vaccination of household contacts of patients with group A meningococcal infections is an effective way of using limited supplies of meningococcal vaccine, though its value would be limited in an epidemic. Secondary cases of meningococcal infection often occur within a few days of the index case, and, although vaccine alone seemed to provide adequate prophylaxis in these Nigerian subjects, additional chemoprophylaxis may be needed to cover this critical period.

Measurement of growth-inhibiting (bactericidal) antibodies in patients with meningococcal meningitis and in subjects immunized with meningococcal polysaccharide vaccine by a [3H]thymidine assay.

We measured bactericidal antibodies against meningococci by a method which depends on the inhibition of [3H]thymidine uptake by bacteria in the presence of antibody and complement. A significant increase in antibody activity was found in sera from patients who had recovered from group A meningococcal meningitis (mean inhibition, 38.9 +/- 4.4%) compared with antibody activity present in sera from patients in the acute phase (mean inhibition, 7.0 +/- 2.4%) (P less than 0.001). Similarly, a significant increase in inhibitory activity against group C meningococci was observed 2 weeks after immunization with group C polysaccharide. A close correlation was obtained between the results of the thymidine assay and of a standard colony-counting method and hemagglutination assay. The assay is readily applicable to testing large numbers of samples.

Detection of Streptococcus pneumoniae DNA in blood cultures by PCR.

We have developed a PCR assay, with primers derived from the autolysin (lyt) gene, for the detection of Streptococcus pneumoniae DNA in blood cultures. The predicted fragment of 247 bp was detected in all strains of pneumococci, embracing 12 different serotypes that were tested. Although DNA extracted from four viridans streptococci spp. Streptococcus oralis, Streptococcus mitis, Streptococcus sanguis, and Streptococcus parasanguis) gave amplification products, these were quite different from the predicted fragment for S. pneumoniae. Application of the assay for diagnosis of septicemia caused by S. pneumoniae showed concordance between PCR and culture results. However, on four occasions PCR was positive in supernatants from both paired culture bottles while pneumococci were cultured from only one. Performing PCR on negative cultures in controlled studies such as vaccine trials may provide a sensitive tool for increasing case detection.

DNA fingerprinting in the epidemiology of African serogroup A Neisseria meningitidis.

The restriction endonuclease (RE) technique was used to compare 172 meningococcal group A strains collected between 1969 and 1990, mainly from countries of the so-called African Meningitis Belt, the Gambia and Ethiopia. The 64 strains from various African countries (Niger, Chad, Burkina Faso, Cameroon, Morocco, Djibouti) were distributed within 3 main restriction enzyme patterns (REPs); the 77 Gambian strains fell into 5 REPs and the 24 Ethiopian strains into 2 such patterns. Several of the main REPs were formed by clusters of closely related clones. Clones, very similar to dominating REPs of the 1960s in Niger, Burkina Faso and Cameroon, were in the 1980s found to be strongly represented in the Gambia to the extreme west of the Meningitis Belt. One of the Gambian clones from 1983-86 was identical to an Indian clone recovered in New Delhi 1986-87. Another clone was detected in 1983 in the Gambia, in 1989 again in the Gambia as well as in Ethiopia, and in 1990 in Tanzania. Our results are largely in line with those of previous studies based on modern techniques of protein and isoenzyme electrophoresis. The RE method is useful mainly for the exact genotypic differentiation of closely related clones, and seems to be a valuable complement to phenotypic tools for epidemiological mapping of Group A meningococcal infection.