Laboratory-based diagnosis of pneumococcal pneumonia: state of the art and unmet needs.

In view of the increasing use of pneumococcal vaccines, especially in the developing world, there is a need for appropriate diagnostics to understand the aetiology of pneumonia, to define the burden of pneumococcal disease, and to monitor vaccine efficacy and effectiveness. This article summarizes a meeting on the diagnosis, detection and serotyping of pneumococcal disease organized by PATH and Fondation Mérieux (18-20 October 2009, Fondation Mérieux Conference Centre, Les Pensières, France). Workers and experts met to discuss the gaps in the microbiology-based diagnosis of Streptococcus pneumoniae disease, with special emphasis on pneumonia. The meeting was designed to evaluate the state of the art of pneumococcal diagnostics and serotyping methodologies, identify research and development needs, and propose new guidelines to public health authorities to support the introduction of vaccines. Regarding detection, the main recommendations were to encourage chest X-rays and antigen detection in urine. Large-scale studies are needed to evaluate the diagnostic utility of test algorithms that associate chest X-rays, antigen detection in urine, S. pneumoniae quantitative PCR in nasopharyngeal aspirates and sputum, and C-reactive protein or procalcitonin measurement in blood. Efforts should be focused on proteomics to identify pneumococcus-specific antigens in urine or host markers in blood expressed during pneumonia. It was recommended to develop S. pneumoniae typing capacities, to understand the epidemiology of pneumococcal disease, and to evaluate vaccine effectiveness. Simple and effective approaches are encouraged, and new technologies based on beads, microarrays or deep sequencing should be developed to determine, in a single test capsular serotype, resistance profile and genotype.

Influenza immunization in pregnancy.

Among healthy persons, two groups are notable for increased risk of serious illness and hospitalization with influenza infection: healthy women in pregnancy and their healthy infants (aged 0 to 6 months). Inactivated influenza vaccine has been used in pregnant women since the 1960s in both the United States and Canada; however, currently, only 15% of pregnant women receive the vaccine. A randomized, controlled trial has shown influenza immunization of pregnant women reduced influenza-like illness by more than 30% in both the mothers and the infants and reduced laboratory-proven influenza infections in 0- to 6-month-old infants by 63%. Physicians caring for pregnant women should be aware of the risks of influenza and of the availability of an effective and cost-saving intervention.

Incidence of Haemophilus influenzae type b meningitis in India.

BACKGROUND & OBJECTIVE: Vaccine policy depends on locally relevant disease burden estimates. The incidence of Haemophilus influenzae type b (Hib) disease is not well characterized in the South Asian region, home to 30 per cent of the world's children. There are limited data from prospective population incidence studies of Hib in Asia, and no data available from India. We therefore carried out this study to assess the burden of Hib meningitis in India. METHODS: A prospective surveillance study was carried out during 1997 and 1999 in hospitals for cases of Hib meningitis from 5 administrative areas of an Indian district (Vellore, Tamil Nadu) with 56,153 children under 5 yr of age, over a 24 month period RESULTS: Ninety seven cases of possible meningitis (> 10 WBC/microl in CSF) were reported, an annual incidence of 86 per 100,000 (95%CI 69 to 109) in 0-4 yr old children, and 357 per 100,000 in 0-11 month infants. Eighteen had proven bacterial meningitis, an annual incidence of 15.9 per 100,000. Eight CSF had Hib by culture or antigen testing, an annual incidence of 7.1 per 100,000 (95%CI 3.1 to 14.0) in children 0-59 months. In infants 0-11 months of age, the incidence of Hib meningitis was 32 per 100,000 (95%CI 16 to 67) and in the 0-23 month group it was 19 (95%CI 8 to 37). INTERPRETATION & CONCLUSION: Our data are the first minimal estimate of the incidence of Hib meningitis for Indian children. The observed incidence data are similar to European reports before Hib vaccine use, suggest substantial disease before 24 months of age, and provide data useful for policy regarding Hib immunization.

Climatic, temporal, and geographic characteristics of respiratory syncytial virus disease in a tropical island population.

Respiratory syncytial virus (RSV) is an important cause of morbidity in children worldwide, although data from equatorial regions are limited. We analysed climatic, spatial, and temporal data for children presenting to hospitals in Lombok island, Indonesia with clinical pneumonia. During the study period, 2878 children presented and 741 RSV cases were identified. In multivariate analysis with an 8-day lag, occurrence of rain was associated with 64% higher incidence of RSV disease [incidence rate ratio (IRR) 1.64, 95% confidence interval (CI) 1.13-2.38]. A 1% rise in mean relative humidity and 1 degree C increase in mean air temperature was associated with a 6% (IRR 1.06, 95% CI 1.03-1.10) and 44% (IRR 1.44, 95% CI 1.24-1.66) increase in RSV cases, respectively. Four statistically significant local clusters of RSV pneumonia were identified within the annual island-wide epidemics. This study demonstrates statistical association of monsoon-associated weather in equatorial Indonesia with RSV. Moreover, within the island-wide epidemics, localized RSV outbreaks suggest local factors influence RSV disease.

Animal models for protein pneumococcal vaccine evaluation: a summary.

This paper summarizes the discussion and viewpoints from a recent meeting regarding the use of animal models for pneumococcal protein vaccines. A wide spectrum of workers in this field met to discuss the animal species to be used in models, the approach of passive and active protection, the characteristics of challenge pneumococcal strains, inducing disease syndromes by a variety of routes of challenge, the determination of specific endpoints to assess effectiveness, the correlates of protection and other details of experimental design and analysis. Vaccine regulatory aspects were discussed. The document concludes with a series of questions that remain to be investigated.

Evaluation of the WHO clinical decision rule for streptococcal pharyngitis.

AIMS: To prospectively assess the WHO clinical decision rule (CDR) for group A beta haemolytic streptococcal (GABHS) pharyngitis in three countries. METHODS: A prospective, observational cohort study in urban outpatient clinics in Rio de Janeiro, Cairo, and Zagreb. There were 2225 children aged 2-12 years with cough, rhinorrhoea, red or sore throat; 1810 of these with sore throat were included in the analysis. RESULTS: The proportion of children presenting with sore throat and found to have GABHS pharyngitis ranged from 24.6% (Brazil) to 42.0% (Croatia). WHO CDR sensitivity was low for all sites in both age groups. In children age 5 or older, sensitivity ranged from 3.8% in Egypt to 10.8% in Brazil. In children under 5, sensitivity was low (0.0-4.6%) Specificity was high in both age groups in all countries (93.8-97.4%). CONCLUSIONS: In these populations, the current WHO CDR has high specificity, but low sensitivity; it did not detect up to 96.0% of children who have laboratory confirmed GABHS pharyngitis. A CDR with higher sensitivity should be developed for use in regions where rheumatic fever and rheumatic heart disease are still major health problems.

Antipneumolysin antibody titers in HIV-seropositive injection drug users before and after pneumococcal bacteremia.

Lower baseline antipneumolysin antibody (alpha-PLY) levels have been found in populations with a higher incidence of pneumococcal infections. To determine whether predisease alpha-PLY titer is associated with invasive pneumococcal disease in HIV-seropositive injection drug users (IDU), we utilized a prospective cohort of IDU in Baltimore to compare alpha-PLY titers before bacteremia in 28 HIV- seropositive IDU cases with alpha-PLY titers in 56 matched (CD4 and seroconversion date) HIV-seropositive IDU control subjects and 28 matched (calendar time) HIV-seronegative IDU control subjects remaining free of pneumococcal disease. We also compared the postinfection fold-rise of alpha-PLY titers in cases relative to the change in alpha-PLY titers in control subjects during the same interval; alpha-PLY titers were measured using quantitative ELISA, and functional activity was assessed using antihemolysin assays. Predisease alpha-PLY titer did not differ between cases (66 units) and HIV-seropositive control subjects (70 units, p = 0.56) or HIV-seronegative control subjects (80 units, p = 0.10). There was a significant difference in fold-rise of alpha-PLY titers postdisease between cases (1.18) and HIV-seronegative control subjects (0.76), p = 0.03. Baseline alpha-PLY titers do not differ significantly between HIV-seropositive IDU who develop pneumococcal bacteremia from HIV-seropositive and HIV-seronegative IDU control subjects remaining free of severe pneumococcal disease.

Comparison of different culture media and storage temperatures for the long-term preservation of Streptococcus pneumoniae in the tropics.

OBJECTIVE: The preservation of Streptococcus pneumoniae by standard freezing methods for subsequent tests--such as serotyping and antibiotic susceptibility--is not possible or is difficult in many developing countries because of the high cost of equipment, inadequate equipment maintenance, and irregular power supply. We evaluated alternative low-cost methods, by comparing different culture media and storage temperatures. METHODS: Clinical isolates of five capsular types (1, 5, 7, 19, and 23) of S. pneumoniae were preserved in rabbit blood, sheep blood, skimmed milk, or glycerol-chocolate broth, and stored at -20 degrees C or -70 degrees C. The cultures were also preserved by lyophilization or sand desiccation, followed by storage at room temperature and 4 degrees C. The viability of the preserved cultures was determined by making serial colony counts on day 0 and after 1 week, 4 weeks, 4 months and 16 months. The viability of cultures preserved by sand desiccation and storage at 4 degrees C was also determined every 6 months for up to 68 months. FINDINGS: Irrespective of the media used, cultures maintained at -20 degrees C became nonviable by the fourth month, while those maintained at -70 degrees C were still viable at 16 months. Cultures preserved by lyophilization or sand desiccation lost their viability by the fourth month when maintained at local room temperature (30-42 degrees C), but remained viable when stored at 4 degrees C for up to 68 months. CONCLUSIONS: Our results confirm that freezing at -70 degrees C, or lyophilization and storage at 4 degrees C are the ideal methods for the preservation of S. pneumoniae. In laboratories where lyophilization is not feasible, sand desiccation and storage at 4 degrees C offers an alternative low-cost method for the long-term preservation of S. pneumoniae.

A polymerase chain reaction-enzyme immunoassay for diagnosis of pneumococcal meningitis in children and adults.

BACKGROUND AND OBJECTIVES: meningitis due to Streptococcus pneumoniae is common among children and adults. In this study a polymerase chain reaction (PCR) for diagnosis of pneumococcal meningitis was evaluated prospectively. METHODS: a total of 61 cerebrospinal fluid specimens were included prospectively using defined inclusion and exclusion criteria. These samples were studied by PCR-EIA and results compared with conventional microbiological procedures and antigen detection techniques. Primers were used against the conserved region of the pneumococcal autolysin gene and the amplified product was labelled using the digoxigenin-labelled dUTP. The product was detected by an enzyme immuno assay (EIA) after hybridization with a biotin labelled probe. RESULTS: a total of 15 specimens were positive for S.pneumoniae by one or more methods used. Culture for S.pneumoniae was positive in 13 specimens, PCR-EIA was positive in 11 of these specimens with an additional pickup of 2 specimens and latex agglutination (LA) positive only in one. INTERPRETATION AND CONCLUSIONS: sensitivity and specificity of 84.6 and 95.8 per cent respectively were observed with PCR-EIA. It seems to be a good tool for the diagnosis of pneumococcal meningitis especially in cases of partially treated pyogenic meningitis.

Extensive swelling after booster doses of acellular pertussis-tetanus-diphtheria vaccines.

BACKGROUND: Diphtheria and tetanus toxoid combined with acellular pertussis (DTaP) vaccines are less reactogenic than diphtheria and tetanus toxoid combined with whole cell pertussis (DTwP) vaccines. However, local reactions increase in rate and severity with each successive DTaP dose, and swelling of the entire injected limb has been reported after booster doses. METHODS: We reviewed reports of swelling of the entire thigh or upper arm after the fourth and fifth dose, respectively, of DTaP vaccines administered in the National Institutes of Health multicenter comparative DTaP studies. Relationships were explored among reports of severe swelling, rates of other reactions, quantity of vaccine contents, and prevaccination and postvaccination antibody levels to pertussis toxin, tetanus toxin, and diphtheria toxin. RESULTS: Entire thigh swelling was an unsolicited reaction reported in 20 (2%) of the 1015 children who received 4 consecutive doses of the same DTaP vaccine. The reaction was associated with 9 of the 12 DTaP vaccines evaluated. Although there were no reports of swelling of the entire upper arm in 121 children given a fifth dose of the same DTaP, 4 (2.7%) of 146 recipients of 5 doses of a mixed schedule of DTaP vaccines experienced such swelling. Rates of other reactions were higher in children with entire thigh swelling than in those without. Of the children with entire thigh swelling, 60% had local pain, and 60% had erythema. All swelling subsided spontaneously without sequelae. There was a significant linear association between the rates of entire thigh swelling after dose 4 and diphtheria toxoid content in the DTaP products. Lesser degrees of swelling (>50 mm but less than entire limb) correlated with pertussis toxoid content after dose 4 and aluminum content after dose 5. No relationship was established between levels of serum antibody to diphtheria, tetanus, or pertussis toxin and rates of swelling of the whole thigh. CONCLUSIONS: Booster doses of DTaP vaccines can cause entire limb swelling, which is usually associated with redness and pain. Our data suggest that this extensive swelling reaction may be more common with vaccines containing high diphtheria toxoid content.

Persistent rhinorrhoea in rural Indian children: prevalence and consequences.

To determine whether persistent rhinorrhoea constitutes a significant problem requiring intervention, 17 rural day care centres (Balwadis) in Tamilnadu, India, were visited. Among 414 children in the Balwadis 92 (22 per cent) children with persistent rhinorrhoea (15 days duration or longer) were identified. Demographic and clinical data and nasopharyngeal swabs for bacterial culture were obtained from 56 such children and 91 age-matched controls from the same Balwadi. Type of housing or nutritional status did not appear to be significant risk factors. There was a significantly higher number of children aged 5-15 years in the household of cases as compared to controls (1.23 +/- 1.08 vs. 0.83 +/- 0.95, p = 0.02). Other illnesses were noted in 25 (44.6 per cent) cases and seven (7.7 per cent) controls (OR 11.5; CI, 4.13-33.4; p < 0.00001). Notably, chronic ear discharge was noted in 6 (11.7 per cent) cases but in none of the controls (p = 0.007). Streptococcus pneumoniae was isolated from nasopharyngeal swabs in 42/49 (85.7 per cent) cases and 44/80 (55 per cent) controls (p < 0.001) and H. influenzae from seven cases and five controls; S. pneumoniae was isolated in all children with chronic ear discharge and H. influenzae from one child. Serotypes of pneumococci commonly associated with otitis media, i.e., types 6, 14, 19, and 23 were isolated from 25 (51 per cent) cases and 16 (20 per cent) controls (OR 4.17; 95% CI, 1.78-9.85; p < 0.001). Persistent rhinorrhoea, presumably due to pneumococcus, is a common condition among rural Indian children and appears to be associated with chronic otitis media.

Age- and cause-specific childhood mortality in Lombok, Indonesia, as a factor for determining the appropriateness of introducing Haemophilus influenzae type b and pneumococcal vaccines.

Using age and cause-specific childhood mortality in Lombok, Indonesia, as a factor for determining the appropriateness of introducing Haemophilus influenzae type b (Hib) and pneumococcal vaccines, the study describes a cross-sectional, hamlet-level mortality survey in 40 of 305 villages in Lombok Island, Indonesia. Causes of death were assessed with a standardized verbal-autopsy questionnaire. One thousand four hundred ninety-nine births and 141 deaths occurring among children aged less than 2 years were identified, with 43% of deaths occurring during the first 2 months of life. The infant mortality rate was 89 (95% CI: 75, 104) per 1,000 live-births. All mortality rates are reported per 1,000 live-births. To examine children whose deaths could potentially have been prevented through vaccination with Hib or pneumococcal vaccine, deaths due to acute respiratory infection (ARI) and central nervous system (CNS) infections among children, aged 2-23 months, were analyzed. ARI and CNS infections caused 58% (mortality rate: 31 per 1,000 live-births; 95% CI: 23, 41) and 17% (mortality rate: 9 per 1,000 live-births; 95% CI: 5, 16), respectively, of all deaths within this age group. Between the ages of 2 and 23 months, 5% of all babies born alive died of ARI, and another 1% died of CNS infections. Our results indicate that current efforts to reduce childhood mortality should focus on reducing ARI and meningitis. These efforts should include evaluating the impact of Hib and pneumococcal vaccines within the routine Expanded Programme on Immunization system.

Effectiveness of influenza vaccine in health care professionals: a randomized trial.

CONTEXT: Data are limited and conflicting regarding the effectiveness of influenza vaccine in health care professionals. OBJECTIVE: To determine the effectiveness of trivalent influenza vaccine in reducing infection, illness, and absence from work in young, healthy health care professionals. DESIGN: Randomized, prospective, double-blind, controlled trial over 3 consecutive years, from 1992-1993 to 1994-1995. SETTING: Two large teaching hospitals in Baltimore, Md. PARTICIPANTS: Two hundred sixty-four hospital-based health care professionals without chronic medical problems were recruited; 49 participated for 2 seasons; 24 participated for 3 seasons. The mean age was 28.4 years, 75% were resident physicians, and 57% were women. INTERVENTION: Participants were randomly assigned to receive either an influenza vaccine or a control (meningococcal vaccine, pneumococcal vaccine, or placebo). Serum samples for antibody assays were collected at the time of vaccination, 1 month after vaccination, and at the end of the influenza season. Active weekly surveillance for illness was conducted during each influenza epidemic period. MAIN OUTCOME MEASURES: Serologically defined influenza infection (4-fold increase in hemagglutination-inhibiting antibodies), days of febrile respiratory illness, and days absent from work. RESULTS: We conducted 359 person-winters of serologic surveillance (99.4% follow-up) and 4746 person-weeks of illness surveillance (100% follow-up). Twenty-four(13.4%) of 179 control subjects and 3 (1.7%) of 180 influenza vaccine recipients had serologic evidence of influenza type A or B infection during the study period. Vaccine efficacy against serologically defined infection was 88% for influenza A (95% confidence interval [CI], 47%-97%; P=.001) and 89% for influenza B (95% CI, 14%-99%; P=.03). Among influenza vaccinees, cumulative days of reported febrile respiratory illness were 28.7 per 100 subjects compared with 40.6 per 100 subjects in controls (P=.57) and days of absence were 9.9 per 100 subjects vs 21.1 per 100 subjects in controls (P=.41). CONCLUSIONS: Influenza vaccine is effective in preventing infection by influenza A and B in health care professionals and may reduce reported days of work absence and febrile respiratory illness. These data support a policy of annual influenza vaccination of health care professionals.

Molecular characterization of a globally distributed lineage of serotype 12F Streptococcus pneumoniae causing invasive disease.

These studies have identified a major genetic lineage of capsule serotype 12F Streptococcus pneumoniae, which has maintained two different types of the pneumococcal surface protein A (PspA) virulence factor and caused invasive disease in geographically disjoint locations. Twenty outbreak strains from a Texas jail and Maryland day care center and 16 reference strains from Texas, Maryland, Washington, Michigan, Oklahoma, Missouri, Alaska, and Australia were examined. Although the Texas and Maryland outbreak strains were indistinguishable by IS1167 and boxA genotyping procedures, all strains examined were members of a genetically similar lineage. The microevolutionary history of pspA differed from that of the overall genetic background of the strains. Taken together, these findings suggested that the Texas and Maryland outbreaks were caused by different clones of a major genetic lineage of serotype 12F pneumococci, within which at least one PspA has been acquired via localized genetic recombination.

Serotyping of Streptococcus pneumoniae by coagglutination with 12 pooled antisera.

We report on the performance of a recently introduced commercial chessboard method using 12 antisera, in comparison with that of the 55-antiserum panel used in determining the serogroups and types (SGTs) of Streptococcus pneumoniae, both of which were carried out by a coagglutination technique. Of a total of 150 strains of S. pneumoniae studied, 135 (90%) belonged to the SGTs represented in the 23-valent pneumococcal vaccine; of these, 130 (96.3%) were identified as the same SGTs by both typing methods. The remaining five strains showed cross-reactivity with more than two pools by the chessboard method, but could be assigned to a single SGT by the Quellung test. The 96.3% concordance of the chessboard method suggests it can be adopted for determination of the SGTs of S. pneumoniae in laboratories.

Nasopharyngeal colonization of infants in southern India with Streptococcus pneumoniae.

To investigate the dynamics of nasopharyngeal colonization with Streptococcus pneumoniae, and to determine the prevalent serogroups/types (SGT) and their antimicrobial susceptibility, we studied 100 infants attending our well-baby clinic. Nasopharyngeal swab specimens were obtained at 6, 10, 14, 18 and 22 weeks and at 9 and 18 months of age and submitted for culture, serotyping and antimicrobial susceptibility testing of S. pneumoniae. Colonization with pneumococcus was seen on at least one occasion in 81 infants. The median age of acquisition was 11 weeks and the median duration of carriage was 1 3 months. The common SGTs identified were 6, 19, 14 and 15. SGT 1, which was a common invasive isolate in children in our hospital during this period, was not isolated from these children. Sequential colonization by 2, 3 or 4 SGTs was observed in 18, 5 and 2 children, respectively. Resistance to penicillin, chloramphenicol, cotrimoxazole and erythromycin was observed in 0, 13 (6%) 11 (5 %) and 5 (3 %) isolates, respectively. There was a significant difference in susceptibility to cotrimoxazole between colonizing and invasive isolates (5 % vs. 40 %, P<0.0001).

Changing trend in susceptibility pattern of Streptococcus pneumoniae to penicillin in India.

Prior to 1995 all strains of Streptococcus pneumoniae isolated at a tertiary care hospital in south India were uniformly susceptible to penicillin. However, since late 1995 strains of S. pneumoniae with intermediate resistance to penicillin have been observed. Altogether there were 25 such isolates, 9 from invasive (5 from CSF as well as blood, 1 from pleural fluid and 3 from CSF alone) and 16 from noninvasive sites (6 from throat, 6 from sputum, 3 from eye and 1 from ear) respectively, thus 4.6 per cent of S. pneumoniae showed intermediate resistance of a total of 535 strains studied so far. The minimum inhibitory concentration (MIC) values of penicillin, erythromycin, chloramphenicol and cefotaxime were determined by agar dilution method and for confirmation, E test was carried out for penicillin alone. The MIC range obtained for penicillin was between 0.125-1.0 microgram/ml. Kirby-Bauer disc diffusion method was adopted for testing of erythromycin, chloramphenicol, co-trimoxazole, cefotaxime, tetracycline and vancomycin. We observed that none of the strains with intermediate resistance to penicillin were multidrug resistant. These strains belonged predominantly to serotype 14 (n = 10), 7B (n = 9), 19A (n = 3), 7F (n = 2) and 23F (n = 1). Clonality was not observed in the 5 representative strains subjected to Box A finger printing method.