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ABSTRACTDuring the 20thcentury, the discovery of modern vaccines and ensuing mass vaccination dramatically decreased the incidence of many infectious diseases and in some cases eliminated them. Despite this, we are now witnessing a decrease in vaccine confidence that threatens to reverse the progress made. Considering the different extents of low vaccine confidence in different countries of the world, both developed and developing, we aim to contribute to the discussion of the reasons for this, and to propose some viable scientific solutions to build or help restore vaccine confidence worldwide.

Diarrhoeal diseases cause illness and death among children younger than 5 years in low-income countries. We designed the Global Enteric Multicenter Study (GEMS) to identify the aetiology and population-based burden of paediatric diarrhoeal disease in sub-Saharan Africa and south Asia.

ABSTRACTHelicobacter pyloriis a globally important and genetically diverse gastric pathogen that infects most people in developing countries. Eradication efforts are complicated by antibiotic resistance, which varies in frequency geographically. There are very few data on resistance in African strains. Sixty-four Gambian H. pyloristrains were tested for antibiotic susceptibility. The role of rdxAin metronidazole (Mtz) susceptibility was tested by DNA transformation and sequencing; RdxA protein variants were interpreted in terms of RdxA structure. Forty-four strains (69%) were resistant to at least 8 μg of Mtz/ml. All six strains from infants, but only 24% of strains from adults, were sensitive (P= 0.0031). Representative Mtz-resistant (Mtzr) strains were rendered Mtz susceptible (Mtzs) by transformation with a functional rdxAgene; conversely, Mtzsstrains were rendered Mtzrby rdxAinactivation. Many mutations were found by Gambian H. pylorirdxAsequencing; mutations that probably inactivated rdxAin Mtzrstrains were identified and explained using RdxA protein's structure. All of the strains were sensitive to clarithromycin and erythromycin. Amoxicillin and tetracycline resistance was rare. Sequence analysis indicated that most tetracycline resistance, when found, was not due to 16S rRNA gene mutations. These data suggest caution in the use of Mtz-based therapies in The Gambia. The increasing use of macrolides against respiratory infections in The Gambia calls for continued antibiotic susceptibility monitoring. The rich variety of rdxAmutations that we found will be useful in further structure-function studies of RdxA, the enzyme responsible for Mtz susceptibility in this important pathogen.

ABSTRACTA phase II clinical study was conducted in African toddlers (aged 12 to 23 months), with subjects receiving either investigational meningococcal group A conjugate (PsA-TT), meningococcal ACWY polysaccharide (PsACWY), or Haemophilus influenzaetype b (Hib-TT) vaccine. Ten months following vaccination, the 3 study groups were further randomized to receive a dose of PsA-TT, a 1/5 dose of PsACWY, or a dose of Hib-TT vaccine. Group A serum bactericidal antibody (SBA) results have been reported previously, with PsA-TT demonstrating superior immunogenicity versus PsACWY vaccine. Immunogenicity for serogroups W135 and C was assessed by SBA assay to investigate the impact of multiple doses in this age group. Blood samples were taken prior to vaccination, 28 days and 40 weeks post-primary vaccination, and 7 and 28 days post-booster vaccination with a 1/5 dose of PsACWY. Subjects who had previously received a full dose of PsACWY had W135 SBA geometric mean titers (GMTs) of 26.1 and 4.4 at 7 and 28 days post-booster vaccination with a 1/5 PsACWY dose, respectively, whereas the W135 SBA GMTs of naïve subjects at these time points following vaccination with a 1/5 dose of PsACWY were 861.1 and 14.6, respectively. Similar differences were observed for serogroup C, with SBA GMTs of 99 and 5.9 at 7 and 28 days post-booster vaccination with a 1/5 dose of PsACWY, respectively, for naïve subjects, compared to 4.1 and 3.2 for previously vaccinated subjects. Immunologic hyporesponsiveness for groups C and W135 was observed following a full dose of PsACWY vaccine at 12 to 23 months of age and a 1/5 dose of PsACWY 10 months later compared to the case for PsACWY-naïve subjects receiving a 1/5 dose of PsACWY vaccine.

ABSTRACTThe polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae; it affects complement resistance and shields the bacterium from phagocytes. Certain capsular serotypes appear to be better able to cause invasive disease than others. Serotypes 1 and 5 are common causes of invasive disease but are rarely isolated from healthy carriers, whereas serotypes 6B and 23F are more frequently isolated from carriage than invasive disease. We have recently shown that serotypes 6B and 19F differ in resistance to complement C3 deposition and opsonophagocytic killing. In this study we assessed the complement resistance and susceptibility to opsonophagocytosis of several other serotypes targeted by the pneumococcal conjugate vaccines. Clinical isolates of serotypes 1, 4, 5, 14, 18C, and 23F were tested along reference strains of corresponding capsular types. The concentration of anticapsular antibodies required for opsonophagocytic killing correlated inversely with C3 deposition on the serotype. Serotype 1 was the most resistant of the clinical isolates to C3 deposition and, along with serotypes 5 and 19F, required the highest concentration of capsule antibodies for opsonophagocytic killing, whereas serotype 23F was the most sensitive to opsonophagocytosis. Sensitivity to C3 deposition and opsonophagocytosis was associated with serotype-specific mortality of invasive pneumococcal disease, suggesting that the primary pathogens, such as serotypes 1 and 5, are more resistant to complement and require a higher concentration of capsule antibodies for opsonophagocytic killing than the opportunistic serotypes such as 6B and 23F, which are associated with a more severe disease outcome.

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae; it affects complement resistance and shields the bacterium from phagocytes. Certain capsular serotypes appear to be better able to cause invasive disease than others. Serotypes 1 and 5 are common causes of invasive disease but are rarely isolated from healthy carriers, whereas serotypes 6B and 23F are more frequently isolated from carriage than invasive disease. We have recently shown that serotypes 6B and 19F differ in resistance to complement C3 deposition and opsonophagocytic killing. In this study we assessed the complement resistance and susceptibility to opsonophagocytosis of several other serotypes targeted by the pneumococcal conjugate vaccines. Clinical isolates of serotypes 1, 4, 5, 14, 18C, and 23F were tested along reference strains of corresponding capsular types. The concentration of anticapsular antibodies required for opsonophagocytic killing correlated inversely with C3 deposition on the serotype. Serotype 1 was the most resistant of the clinical isolates to C3 deposition and, along with serotypes 5 and 19F, required the highest concentration of capsule antibodies for opsonophagocytic killing, whereas serotype 23F was the most sensitive to opsonophagocytosis. Sensitivity to C3 deposition and opsonophagocytosis was associated with serotype-specific mortality of invasive pneumococcal disease, suggesting that the primary pathogens, such as serotypes 1 and 5, are more resistant to complement and require a higher concentration of capsule antibodies for opsonophagocytic killing than the opportunistic serotypes such as 6B and 23F, which are associated with a more severe disease outcome.

We compared the performance of tuberculin skin test (TST), Quantiferon-TB Gold in-tube (QFT-GIT), and T-SPOT.TBin diagnosing latent tuberculosis (LTBI) among childhood TB contacts in a TB endemic setting with high BCG coverage. We evaluated the performance of interferon gamma release assays (IGRAs) and TST when combined in an algorithm.

Nasopharyngeal carriage of Streptococcus pneumoniaeis extremely prevalent in The Gambia. We studied the effects of vaccination with pneumococcal conjugate vaccines on the carriage of individual serotypes and on antimicrobial resistance in vaccinated children and their younger siblings.

ABSTRACTIncreasing knowledge about DosR regulon-encoded proteins has led us to produce novel Mycobacterium tuberculosisantigens for immunogenicity testing in human populations in three countries in Africa to which tuberculosis (TB) is endemic. A total of 131 tuberculin skin test-positive and/or ESAT-6/CFP10-positive, human immunodeficiency virus-negative adult household contacts of active pulmonary TB cases from South Africa (n= 56), The Gambia (n= 26), and Uganda (n= 49) were tested for gamma interferon responses to 7 classical and 51 DosR regulon-encoded M. tuberculosisrecombinant protein antigens. ESAT-6/CFP10 fusion protein evoked responses in >75% of study participants in all three countries. Of the DosR regulon-encoded antigens tested, Rv1733c was the most commonly recognized by participants from both South Africa and Uganda and the third most commonly recognized antigen in The Gambia. The four most frequently recognized DosR regulon-encoded antigens in Uganda (Rv1733c, Rv0081, Rv1735c, and Rv1737c) included the three most immunogenic antigens in South Africa. In contrast, Rv3131 induced the highest percentage of responders in Gambian contacts (38%), compared to only 3.4% of Ugandan contacts and no South African contacts. Appreciable percentages of TB contacts with a high likelihood of latent M. tuberculosisinfection responded to several novel DosR regulon-encoded M. tuberculosisproteins. In addition to significant similarities in antigen recognition profiles between the three African population groups, there were also disparities, which may stem from genetic differences between both pathogen and host populations. Our findings have implications for the selection of potential TB vaccine candidates and for determining biosignatures of latent M. tuberculosisinfection, active TB disease, and protective immunity.

ABSTRACTMycobacterium africanum, first described in Senegal in 1968, causes up to half of the smear-positive pulmonary tuberculosis cases in West Africa, but it has not been found in other geographical areas except among recent West African migrants. The reasons for the geographic restriction of M. africanumare unknown. We used molecular tools to determine the population structure of the Mycobacterium tuberculosiscomplex in a cohort study of consecutive smear-positive tuberculosis cases in The Gambia. We collected and genotyped 386 clinical isolates using spoligotype analysis and PCRs for large sequence polymorphisms (LSPs) and compared the genotype patterns to the patterns in an international database. The results of spoligotyping and LSP analysis for the study population were also compared to determine the correlation between them. The main lineages within the Mycobacterium tuberculosiscomplex identified in The Gambia included M. africanumtype I (38.4%), characterized by an LSP in region of difference 702 (RD702; West African type 2). Among the M. tuberculosissensu stricto isolates, lineages characterized by RD182 and by RD174 were the most common. We also detected a gradient in the prevalence of M. africanumthat extended from neighboring Guinea-Bissau. The genotypic diversity of the spoligotype patterns was greater among the isolates of M. africanumthan among the isolates of M. tuberculosis. We postulate that M. africanumbecame endemic in West Africa first, before the introduction of different lineages within M. tuberculosissensu stricto.

The incidence of community-acquired bacteremia (CAB) in Africa is several-fold higher than in industrialized countries. We report here the incidence of invasive bacterial infections in rural Gambia and compare the clinical characteristics of children with pneumococcal infection with those of children with extraintestinal nontyphoidal salmonella infection (NTS) or other bacterial infections.

ABSTRACTOverlapping peptides of Mycobacterium tuberculosisantigens ESAT-6 and CFP-10 offer increased specificity over the purified protein derivative skin test when they were used in an ex vivo enzyme-linked immunospot (ELISPOT) assay for gamma interferon detection for the diagnosis of M. tuberculosisinfection from recent exposure. We assessed whether equivalent results could be obtained for a fusion protein of the two antigens and whether a combined readout would offer increased sensitivity in The Gambia. We studied the ELISPOT assay results for 488 household contacts of 88 sputum smear-positive tuberculosis (TB) cases. The proportions of subjects positive by each test and by the tests combined were assessed across an exposure gradient, defined according to sleeping proximity to a TB case. Eighty-eight (18%) subjects were positive for CFP-10 peptides, 148 (30%) were positive for ESAT-6 peptides, 161 (33%) were positive for both peptides, and 168 (34%) were positive for the fusion protein; 188 (39%) subjects had either a positive result for a peptide or a positive result for the fusion protein. There was reasonable agreement between the peptide and the protein results (kappa statistic = 0.78) and no significant discordance (P= 0.38). There was a strong correlation between the fusion protein and combined peptide spot counts (r= 0.9), and responses to the peptide and the proteins all increased significantly according to M. tuberculosisexposure. The proportion of subjects positive for either the pool of peptides or the fusion protein offered maximum sensitivity, being significantly higher than the proportion of subjects positive for ESAT-6 peptides alone (P= 0.007). A fusion protein of ESAT-6 and CFP-10 is equivalent to overlapping peptides for the diagnosis of latent M. tuberculosisinfection. Use of a combination of peptides and fusion protein offers improved sensitivity.

ABSTRACTHelicobacter pyloriis a genetically diverse bacterial species that chronically infects human stomachs and sometimes causes severe gastroduodenal disease. Studies of polymorphic DNA sequences can suggest geographic origins of individual strains. Here, we describe a 180-bp insertion (ins180), which is just after the translation stop of a gene of unknown function, near the promoter of jhp0152-jhp0151two-component signal transduction genes in strain J99, and absent from this site in strain 26695. This ins180 insertion was found in 9 of 9 Gambian (West African), 9 of 20 (45%) South African, and 9 of 40 (23%) Spanish strains but in only 2 of 20 (10%) North American strains and none of 20 Lithuanian, 20 Indian, and 20 Japanese strains. Four South African isolates that lacked ins180 and that belonged to an unusual outlier group contained a 480-bp insertion at this site (ins480), whereas none of 181 other strains screened contained ins480. In further tests 56% (10 of 18) of strains from African Americans but only 17% (3 of 18) of strains from Caucasian Americans carried ins180 (P< 0.05). Thus, the H. pyloristrains of modern African Americans seem to retain traces of African roots, despite the multiple generations since their ancestors were taken from West Africa. Fragmentary ins180-like sequences were found at numerous sites in H. pylorigenomes, always between genes. Such sequences might affect regulation of transcription and could facilitate genome rearrangement by homologous recombination. Apparent differences between African-American and Caucasian-American H. pylorigene pools may bear on our understanding of H. pyloritransmission and disease outcome.

In breast-feeding populations, immunization during pregnancy with pneumococcal polysaccharide offers a potentially useful approach to preventing pneumococcal disease in young infants.

Unrelenting high morbidity and mortality have mandated that immunogenic vaccines be used to combat pneumococcal disease in infants.

Despite improvements in infant mortality rates in many developing countries including The Gambia, neonatal mortality remains high and many neonatal deaths are caused by infection. The study described in this paper was conducted to determine the bacterial and viral etiology of serious infections in Gambian infants younger than 91 days old.

SummaryAs part of a treatment trial of cerebral malaria, blood cultures were done in 276 Gambian children, aged between 1 and 9 years, with cerebral malaria. Fourteen (5%) of these were positive. The organisms isolated were Staphylococcus aureus(6), coliforms (4), Pseudomonas spp. (2), Salmonella spp. (1) and Streptococcus spp. (1). Thirteen of these children survived, most without appropriate antibiotic treatment. Most of the retrieved organisms were therefore suspected to be contaminants. Bacteraemia complicating cerebral malaria is not common in The Gambia, and routine antibiotic treatment of children with cerebral malaria is not warranted.

Streptococcus pneumoniaeis a major cause of morbidity and mortality in young children in the developing world. The recent development of pneumococcal polysaccharide/protein conjugate vaccines may make possible prevention of this infection. However, little is known about the epidemiology of invasive pneumococcal disease in children in the developing world.

Children in developing countries who present with malnutrition often have infections, particularly pneumonia, at the time of presentation. We evaluated the initial antibiotic management of 144 Gambian children who presented for the first time with malnutrition and who had clinical or radiologic evidence of pneumonia. They were enrolled in a double blind trial of trimethoprim-sulfamethoxazole vs.chloramphenicol. Most children in the study underwent detailed investigations of bacterial and viral etiology as part of another study. The study drug was administered for a week along with oral metronidazole, vitamins and standardized nutritional therapy. Treatment failure was defined as the need for change to parenteral antibiotics during treatment, failure to respond to a week of treatment with the study drug or relapse during the following 2 weeks. There were no differences between the treatment groups in the clinical indicators of severity, etiology or radiologic findings. Thirty-three children were excluded from the analysis because of tuberculosis, inappropriate enrollment or inadequate follow-up. Of the 111 children remaining, 32 (16 in each arm of the study) failed treatment. Clinical failure was not related…

When 117 strains of Yersiniawere grown in serial broth culture at 22 or 30 C, 44 (38%) formed a broad‐spectrum, mannose‐resistant hemagglutinin (MR/Y‐HA) associated with thick (8 nm) channelled (MR/Y) fimbriae; eight other strains (7%) formed a narrow‐spectrum, mannose‐resistant hemagglutinin (MR/K‐like HA) associated with thin (4 nm) non‐channelled (type 3‐like) fimbriae. The distribution of these two fimbrial hemagglutinins in different Yersiniaspecies is discussed and their properties are compared with those of other reported mannose‐resistant hemagglutinins. The thick fimbriae of four Yersiniaspecies were antigenically similar as judged by immunoelectronmicroscopy. Likewise, the thin fimbriae of three Yersiniaspecies were antigenically similar, though different from the thick fimbriae.

During a 2-year period 159 malnourished children ages 3 months to 5 years with radiologic evidence of pneumonia were investigated to determine the cause of their pneumonia. In addition 119 malnourished children without pneumonia, 119 well-nourished children with pneumonia and 52 well-nourished children without pneumonia were studied as controls. Percutaneous lung aspiration was performed on 35 malnourished and 59 well-nourished children with pneumonia. Bacteria were isolated from the blood, lung or pleural fluid of 28 (18) malnourished children with pneumonia, 42 (35) well-nourished children with pneumonia and from the blood of 5 (4) malnourished children without pneumonia. Streptococcus pneumoniaeand Haemophilus influenzae,which were the two organisms isolated most frequently in both groups of children with pneumonia, were found in 17 (11) malnourished and 39 (33) well-nourished children with pneumonia. Mycobacterium tuberculosiswas detected in 5 malnourished children with pneumonia. A potentially pathogenic virus was identified in 35 of malnourished children with pneumonia and 40 of well-nourished children with pneumonia, and from 25 of children without pneumonia. The viruses identified most frequently were adenovirus and respiratory syncytial virus. There were 26 viral isolates from lung aspirate specimens. Respiratory syncytial virus was more frequently isolated from well-nourished children, while herpes simplex was more often found in malnourished children. Both bacterial and viral agents were identified in 25 children. In situations where measles and kwashiorkor are uncommon, the likely bacterial causes of community-acquired pneumonia in both well-nourished and malnourished children are S. pneumoniaeand H. influenzaebut, in malnourished children, particularly those with edema, the possibility of other causes, particularly M. tuberculosis,must always be kept in mind.

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.

Respiratory syncytial virus (RSV) is a well-recognized cause of lower respiratory tract infections in early childhood in industrialized countries, but less is known about RSV infection in developing countries.

Background A major part of horizontal gene transfer that contributes to the diversification and adaptation of bacteria is facilitated by genomic islands. The evolution of these islands is poorly understood. Some progress was made with the identification of a set of phylogenetically related genomic islands among the Proteobacteria, recognized from the investigation of the evolutionary origins of a Haemophilus influenzae antibiotic resistance island, namely ICEHin1056. More clarity comes from this comparative analysis of seven complete sequences of the ICEHin1056 genomic island subfamily.Results These genomic islands have core and accessory genes in approximately equal proportion, with none demonstrating recent acquisition from other islands. The number of variable sites within core genes is similar to that found in the host bacteria. Furthermore, the GC content of the core genes is similar to that of the host bacteria (38% to 40%). Most of the core gene content is formed by the syntenic type IV secretion system dependent conjugative module and replicative module. GC content and lack of variable sites indicate that the antibiotic resistance genes were acquired relatively recently. An analysis of conjugation efficiency and antibiotic susceptibility demonstrates that phenotypic expression of genomic island-borne genes differs between different hosts.Conclusion Genomic islands of the ICEHin1056 subfamily have a longstanding relationship with H. influenzae and H. parainfluenzae and are co-evolving as semi-autonomous genomes within the 'supragenomes' of their host species. They have promoted bacterial diversity and adaptation through becoming efficient vectors of antibiotic resistance by the recent acquisition of antibiotic resistance transposons.

The prevalence of group B streptococcal (GBS) colonization was studied in 136 pregnant women and their newborn infants by collecting vaginal and rectal swabs from the mothers and throat, rectal, and umbilical swabs from their infants. Maternal and infant colonization rates were 22% and 23%, respectively. One-third of infants born to colonized mothers and 15% of infants born to noncolonized mothers had GBS isolated. Of GBS-colonized infants, 50% remained colonized at the mean age of2 months. Type V was the commonest serotype among GBS isolates from mothers and infants; type III strains were uncommon. The rarity ofGBS disease in Gambian infants may be due to low rates of maternal carriage with the more virulent GBS serotypes.

The effect of a Haemophilus influenzae type b (Hib) polyribosylribitol phosphate-tetanus toxoid conjugate vaccine (Hib/PRP-T) on oropharyngeal carriage of Hib was studied during an efficacy trial in Gambian infants. Children were vaccinated with Hib/PRP-T and diphtheria-tetanus toxoids-pertussis (DTP) or DTP alone at ages 2, 3, and 4 months. Groups of 1000 children aged 1–2 years were studied each year for 4 years. Hib was detected by production of a halo on antiserum agar plates. Carriage was significantly lower among children fully vaccinated with Hib/PRP-T given with DTP (4.4%; 95% confidence interval [CI], 3.8%–5.7%) than among children fully vaccinated with DTP alone (11.0%; 95% CI, 8.9%–13.0%) (protective effect adjusted by year = 60%; 95% CI, 44%–72%; P < .001). Hib carriage varied by year among nonvaccinated children. Hib conjugate vaccines are likely to produce a herd protective effect in underdeveloped communities, as recorded in Europe and the United States.