Transthoracic lung aspiration for the aetiological diagnosis of pneumonia: 25 years of experience from The Gambia.

Pneumonia remains the leading cause of death in young children worldwide. Global pneumonia control depends on a good understanding of the aetiology of pneumonia. Percutaneous transthoracic aspiration culture is much more sensitive than blood culture in identifying the aetiological agents of pneumonia. However, the procedure is not widely practised because of lack of familiarity with it and concerns about potential adverse events. We review the diagnostic usefulness and safety of this procedure over 25 years of its use in research and routine practice at the UK Medical Research Council (MRC), The Gambia, and give a detailed description of the procedure itself. Published materials were identified from the MRC's publication database and systematic searches using the PubMed/Medline and Google search engines. Data from a current pneumonia aetiology study in the unit are included together with clinical experience of staff practising at the unit over the period covered in this review. A minimum of 500 lung aspirates were performed over the period of review. Lung aspiration produces a greater yield of diagnostic bacterial isolates than blood culture. It is especially valuable clinically when pathogens not covered by standard empirical antibiotic treatment, such as Mycobacterium tuberculosis and Staphylococcus aureus, are identified. There have been no deaths following the procedure in our setting and a low rate of other complications, all transient. Lung aspiration is currently the most sensitive method for diagnosing pneumonia in children. With appropriate training and precautions it can be safely used for routine diagnosis in suitable referral hospitals.

Antibody and T-cell responses during acute and convalescent stages of invasive pneumococcal disease.

OBJECTIVE: To understand the pattern of immune responses to pneumococcal proteins during invasive disease as a guide to their development as vaccine candidates. METHODS: The antibody concentration and avidity, as well as frequency of interferon-gamma (IFN-γ)-, interleukin-10 (IL-10)-, and tumor necrosis factor-alpha (TNF-α)-containing CD4+ T-lymphocytes in response to pneumolysin, pneumococcal surface protein A (PspA), and choline-binding protein A (CbpA), during and after invasive pneumococcal disease (IPD) in 20 children were compared to those of 20 healthy matched controls. RESULTS: During the acute phase of IPD, the concentrations of antibodies against these three pneumococcal proteins were lower, whereas the frequencies of IL-10- and TNF-α-producing CD4+ T-cells were higher, compared to values obtained during convalescence and in healthy controls (p < 0.01). In addition, the concentrations of antibodies against the capsular polysaccharides for the serotypes isolated from these patients, were all below the detection level of the assay during both the acute and convalescent phases of IPD. CONCLUSION: These data indicate that the recognition of these antigens by the immune system occurs in variable proportions according to the stage of infection, implying the important role of these in the pathogenesis of IPD, and support their usefulness in vaccine development.

Epidemiological studies of large resistance plasmids in Haemophilus.

The distribution of large conjugative Haemophilus influenzae plasmids in the nasopharyngeal haemophili of a group of people and in a large collection of 541 H. influenzae type b (Hib) isolates was studied. A newly developed PCR-based assay was used to detect the plasmids. The target sequences were chosen from sequence analysis of part of p1056, a large multiresistance plasmid isolated from a clinical Hib isolate, 1056. Fifty-nine per cent of people were found to carry beta-lactamase-positive (beta-lac(+)), ampicillin-resistant (ampR) haemophili with detectable plasmid sequences. Of these, 83% were in Haemophilus parainfluenzae and 17% were in H. influenzae. In the collection of 541 Hib, antibiotic resistance [beta-lac(+)ampR, beta-lac(+)ampR plus tetracycline resistance (tetR) or tetR] was highly correlated with large plasmids. It was found that 2.3% of the isolates contained large cryptic plasmids (i.e. these isolates were susceptible to antibiotics). The distribution of plasmids between invasive and carried Hib did not differ significantly (25 of 245 and 23 of 276, respectively). Isolates with large plasmids occur at high frequency in the nasopharynx of the normal human population and consist of two populations in Hib, one associated with specific antibiotic resistance traits and the other cryptic. These plasmids do not appear to influence the invasiveness of Hib.

Etiology of serious infections in young Gambian infants.

BACKGROUND: 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. METHODS: At a first level health facility 497 infants with symptoms that could indicate serious infection were enrolled, of whom 239 with 1 or more signs of serious infection and 55 with no signs were investigated, yielding 17 cases with positive bacterial cultures of blood and/or cerebrospinal fluid. At a nearby pediatric referral hospital 198 infants were seen and 182 were investigated, yielding 35 positive bacterial cultures. RESULTS: There were 15 culture positive cases of meningitis caused by Streptococcus pneumoniae (7), Streptococcus pyogenes (2), Enterobacter cloacae (2), Escherichia coli (1), Haemophilus influenzae type b (1), Streptococcus agalactiae (1) and Salmonella spp. (1). Six of these children died. Thirty-three infants without meningitis had positive blood cultures for Staphylococcus aureus (17), S. pneumoniae (3), Salmonella spp. (5), E. coli (3), other enterobacteria (4) and S. agalactiae (1), of whom 14 died. Nasopharyngeal aspirates from 438 children were investigated for common respiratory viruses. Respiratory syncytial virus was found in 51, influenza A in 46, influenza B in 22, parainfluenza in 26 and adenovirus in 16. Respiratory syncytial virus and influenza A isolates were found most frequently toward the end of the wet season. Nasopharyngeal carriage of S. pneumoniae and H. influenzae was studied in 320 infants recruited during the first year. Of these 184 (58%) were positive for S. pneumoniae and 141 (44%) were positive for H. influenzae, 18 of which were type b. Infants with a bacterial isolate from blood or cerebrospinal fluid were more likely than the rest to die, whereas those with a viral isolate were less likely to die. CONCLUSIONS: The most important causes of serious infections in young Gambian infants are Staphylococcus aureus, S. pneumoniae and Salmonella spp.

Vaccination with a Haemophilus influenzae type b conjugate vaccine reduces oropharyngeal carriage of H. influenzae type b among Gambian children.

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.

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.

Randomised trial of Haemophilus influenzae type-b tetanus protein conjugate vaccine [corrected] for prevention of pneumonia and meningitis in Gambian infants.

BACKGROUND: In developing countries, pneumonia and meningitis due to Haemophilus influenzae type b (Hib) are common in children under age 12 months and the mortality from meningitis is high. Protein-polysaccharide conjugate vaccines have brought Hib disease under control in industrialised countries. We did a double-blind randomised trial in The Gambia to assess the efficacy of a Hib conjugate vaccine for the prevention of meningitis, pneumonia, and other invasive diseases due to Hib. METHODS: Between March, 1993, and October, 1995, 42,848 infants were randomly allocated the conjugate vaccine Hib polysaccharide tetanus protein (PRP-T) mixed with diphtheria-tetanus-pertussis vaccine (DTP), or DTP alone at age 2 months, 3 months, and 4 months. Children who presented with signs of invasive Hib were investigated by blood culture and, where appropriate, by lumbar puncture, chest radiograph, or percutaneous lung aspirate. Children were followed up for between 5 and 36 months. FINDINGS: The median ages at which children received the study vaccine were 11 weeks, 18 weeks, and 24 weeks. 83% of children enrolled received all three doses of vaccine. 17 cases of culture-positive Hib pneumonia, 28 of Hib meningitis, and five of other forms of invasive Hib disease were detected amongst the study children. The efficacy of the vaccine for the prevention of all invasive disease after three doses was 95% (PRP-T vaccinees 1, controls 19 [95% CI 67-100]), for the prevention of Hib pneumonia after two or three doses, 100% (vaccinees 0, controls 10 [55-100]), and for the prevention of radiologically defined pneumonia at any time after enrollment, 21.1% (PRP-T vaccinees 198, controls 251 [4.6-34.9]). INTERPRETATION: PRP-T conjugate Hib vaccine prevented most cases of meningitis and pneumonia due to Hib in Gambian infants. The reduction in the overall incidence of radiologically defined pneumonia in PRP-T vaccinees suggests that about 20% of episodes of pneumonia in young Gambian children are due to Hib. The introduction of Hib vaccines into developing countries should substantially reduce childhood mortality due to pneumonia and meningitis.

Nasopharyngeal carriage of pneumococci in Gambian children and in their families.

BACKGROUND: Nasopharyngeal carriage of pneumococci is prevalent among children in developing countries but little is known about the relationship of nasopharyngeal carriage to invasive disease or about the way in which pneumococci spread within households. OBJECTIVES: To determine the prevalence of nasopharyngeal carriage in healthy and sick Gambian children and to investigate transmission within households. METHODS: Nasopharyngeal swabs were obtained by the per nasal route and cultured for pneumococci on selective media. Pneumococci were serotyped with the use of latex particles coated with type-specific antisera. RESULTS: Pneumococci were isolated from the nasopharynx of 73 (90.1%) of 81 children with invasive pneumococcal disease, 86 (76.1%) of 113 healthy, age-matched control children and 911 (85.1%) of 1071 sick children. Pneumococci belonging to serotypes 1, 14 and 12 were isolated significantly more frequently from cases than from matched controls. In 43 (76.8%) of 56 children with invasive disease, pneumococci isolated from the nasopharynx and from the blood or other sterile site belonged to the same serotype. Pneumococci of the same serotype as the bacterium responsible for invasive disease in a child were obtained from 72 (8.5%) of 843 family members, most frequently from young siblings of the case patients. CONCLUSION: Nasopharyngeal carriage of pneumococci is more prevalent among young Gambian children than among adults and invasive infections are probably acquired more frequently from siblings than from parents. However, further studies are needed to confirm this hypothesis with more discriminating markers than polysaccharide serotyping.

A study of risk factors for pneumococcal disease among children in a rural area of west Africa.

BACKGROUND: Pneumoccal infection is one of the leading causes of pneumonia, meningitis and septicaemia in developing countries. We have investigated possible risk factors for pneumococcal disease among children living in a rural area of The Gambia. METHODS: A prospective case-control study was conducted in which children with pneumococcal infection were identified from among children attending out-patient and under-fives clinics and matched according to age with healthy children selected randomly from the local community. A questionnaire was used to investigate possible nutritional, medical, socioeconomic and environmental risk factors for pneumococcal disease. RESULTS: An increased risk of pneumococcal disease was associated with poor weight gain, a history of serious illness in the previous 6 months, exposure to cigarette smoke or being carried on mother's back while cooking. The risk of pneumococcal disease was reduced among children whose mothers had a personal source of income. CONCLUSIONS: The incidence of pneumococcal disease could be reduced by improving nutrition and taking steps to identify and rehabilitate those children whose weight is faltering or falling. Encouraging mothers to develop greater financial independence may also be beneficial. Reduced exposure to smoke should be promoted by improving ventilation in kitchens, introducing more efficient and less polluting stoves, keeping children away from smoky environments and discouraging parental smoking.

PIP: Pneumococcal infection is a leading cause of pneumonia, meningitis, and septicemia in developing countries. The authors investigated possible risk factors for pneumococcal disease during 1989-91 among children living in the rural Upper River Division of The Gambia. A prospective case-control study approach was used in which 80 children with pneumococcal infection were matched according to age with 159 healthy children randomly selected from the local community. The subjects were of mean age 14.0-14.2 months. A questionnaire was used to identify possible nutritional, medical, socioeconomic, and environmental risk factors for pneumococcal disease. The study found an increased risk of pneumococcal disease to be associated with poor weight gain, a history of serious illness during the previous 6 months, exposure to cigarette smoke, or being carried upon a mother's back while she cooks. The risk of pneumococcal disease was reduced among children whose mothers had a personal source of income. The authors suggest reducing the incidence of pneumococcal disease by improving nutrition and growth monitoring, encouraging mothers to develop greater financial independence, and reducing children's exposure to smoke.

Immunization with a pneumococcal capsular polysaccharide vaccine during pregnancy.

The feasibility of preventing invasive pneumococcal infections during the first few months of life by immunization during pregnancy has been investigated. One hundred and fifty Gambian women were immunized with either a 23-valent pneumococcal polysaccharide vaccine or a meningococcal polysaccharide vaccine during the last trimester of pregnancy. Pregnant women showed a good antibody response to five of the six pneumococcal polysaccharides tested (types 1, 3, 5, 6, 14 and 19) but not to type 6 polysaccharide. Mean cord blood/maternal blood IgG antibody ratios varied from 24% (type 1) to 49% (type 3) and differed substantially between individual mother/infant pairs. Pneumococcal antibody levels were higher at birth in infants of women immunized with pneumococcal polysaccharide vaccine than in control infants. However, these antibodies disappeared rapidly during the first few months of life and it is uncertain how much clinical protection against pneumococcal infection maternal immunization would have provided.

Haemophilus influenzae type b disease in the western region of The Gambia: background surveillance for a vaccine efficacy trial.

In preparation for a field trial of an Haemophilus influenzae type b conjugate vaccine in the Western Region of The Gambia, a 3-year prospective study was undertaken to determine the incidence of Hib disease and the vaccination status of affected children. One hundred and eighty-two children with invasive Hib disease were found; 141 (77%) had meningitis, 31 (17%) pneumonia and 10 (6%) other forms of invasive disease. The estimated annual incidence rates for all invasive Hib diseases were 274 and 73 per 100,000 in children aged < 1 and < 5 years, respectively. For meningitis, the rate was 222 per 100,000 per year in children aged < 1 year. Children with meningitis were significantly younger than those with pneumonia (median age 7 months, interquartile range [IQR] 5-9, vs 12 months, IQR 6-15 (P = 0.002)) and younger than those with other forms of Hib disease. Of 142 children for whom vaccination status was known, 18 had received no DPT, 36 had received one, 40 had received two and 48 had received three doses. This study confirmed the high incidence of systemic Hib disease among Gambian children and the need to vaccinate at an early age. It provided the background epidemiological data required for the successful planning of an Hib vaccine trial which is now in progress.

Pneumococcal disease among children in a rural area of west Africa.

BACKGROUND: The pneumococcus is a frequent cause of pneumonia and other serious infections among young children in developing countries. Defining the pattern of pneumococcal infection in these countries is important so that, with the advent of pneumococcal conjugate vaccines, rational vaccination policies can be developed. METHODS: Children younger than 5 years of age who attended clinics in a rural area of The Gambia, West Africa, were screened by assistants during a 2-year period. Children with predefined features suggestive of a diagnosis of pneumonia, meningitis or septicemia were referred to the Medical Research Council Field Station at Basse for investigation. RESULTS: Of 2898 children investigated 103 cases of invasive pneumococcal disease (70 definite and 33 probable) were identified, suggesting that the incidence of this infection in the study community is at least 554/100,000/year in children younger than 1 year of age and 240/100,000/year in those younger than 5 years, rates many times higher than those found in industrialized societies. The mean age of presentation was 15 months; more boys than girls were affected. Cases of pneumonia were encountered 8 times more frequently than those of meningitis. Antibiotic resistance was rarely found and cases of pneumonia, but not meningitis, responded well to treatment. Case-fatality rates in children with pneumonia and meningitis were 1 and 55%, respectively. The most prevalent pneumococcal serotypes were types 6, 14, 19, 1 and 5. CONCLUSION: About 60% of invasive pneumococcal infection in children in this community could potentially be prevented by a nine-valent pneumococcal conjugate vaccine (types 1, 4, 5, 6B, 9, 14, 18, 19F and 23) given at the ages of 2, 3 and 4 months.

Meningococcal antibody titres in infants of women immunised with meningococcal polysaccharide vaccine during pregnancy.

Seventy five Gambian women were immunised with a single dose of a group A+group C meningococcal polysaccharide vaccine during the last trimester of pregnancy. IgG antibody titres were measured in mothers and in their infants by an enzyme-linked immunosorbent assay (ELISA). All women had a good response to vaccination and maternal antibodies were high at the time of delivery (23.2 micrograms/ml for group A antibodies and 14.3 micrograms/ml for group C antibodies). However, only a proportion of this antibody crossed the placenta; cord blood:maternal antibody ratios were 30% for group A antibody and 44% for group C antibody, respectively. Considerable variability in cord blood:maternal blood ratios was seen between individuals. This could not be related to age, parity, or ethnic group. Mean group A and group C cord blood:maternal blood ratios were lower in women with serological evidence of syphilis than in seronegative women, and diminished transfer of group A antibody was noted in women with active malarial infection of the placenta. Antibody titres declined rapidly in infants and by the age of 3-4 months these had reached control values. Maternal immunisation may give infants some protection against group A and group C meningococcal disease but only during the first few months of life.

Carriage of group B Streptococci in pregnant Gambian mothers and their infants.

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 of 2 months. Type V was the commonest serotype among GBS isolates from mothers and infants; type III strains were uncommon. The rarity of GBS disease in Gambian infants may be due to low rates of maternal carriage with the more virulent GBS serotypes.

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.

Importance of enteric bacteria as a cause of pneumonia, meningitis and septicemia among children in a rural community in The Gambia, West Africa.

Two thousand eight hundred ninety-eight children younger than 5 years old were investigated during a 2-year period in a rural area of The Gambia for possible pneumonia, meningitis or septicemia. After clinical examination and appropriate investigations, 1014 children were diagnosed as having pneumonia, 31 as having meningitis and 100 as having septicemia. Nine hundred seven children had a final diagnosis of malaria including 702 who satisfied the World Health Organization criteria for a diagnosis of pneumonia. A bacterial etiology was established in 115 (11%) patients with a final diagnosis of pneumonia, in 25 (81%) with meningitis and in 29 (29%) with suspected septicemia. Overall the pneumococcus was the leading pathogen identified among children with pneumonia and meningitis and ranked third among those with septicemia. However, during the wet season, when malaria transmission was highest, 50% of blood culture isolates obtained from children satisfying the World Health Organization criteria for a diagnosis of pneumonia were Salmonella or coliform species, and the pneumococcus and Haemophilus influenzae type b accounted for only 43% of isolates. Thus enteric bacteria may be as important as those bacteria more usually associated with respiratory disease among children presenting with a clinical picture of pneumonia during the wet season. This finding has important implications for case management and surveillance for antibiotic resistance.

The dominance of a multiresistant strain of Neisseria gonorrhoeae among prostitutes and STD patients in The Gambia.

OBJECTIVE: To study the epidemiology of antibiotic resistant strains of Neisseria gonorrhoeae from sexually transmitted disease clinics in The Gambia. MATERIALS AND METHODS: One hundred and sixty five strains of N gonorrhoeae were tested for their antibiotic susceptibility, auxotype, serotype, and plasmid content. RESULTS: Of the total population 84 (51%) were non-penicillinase producing (nonPPNG) and 81 (49%) penicillinase-producing N gonorrhoeae (PPNG). There were 16 serovars, five auxotypes and 33 auxotype/serovar (A/S) classes in the total population and the nonPPNG. Among PPNG only five serovars, two auxotypes and nine A/S classes were found. One A/S class predominated, NR/IB-7 (86 isolates), of which 66 (77%) were PPNG and the remainder were chromosomally-mediated resistant N gonorrhoeae (CMRNG). These strains also showed reduced susceptibility to ciprofloxacin, ceftriaxone and tetracycline and were evenly distributed among patient groups. CONCLUSION: We have identified a relatively homogeneous gonococcal population with a core group of isolates exhibiting high levels of antibiotic resistance.