Pneumococcal serotypes in children with acute and chronic inflammatory diseases of the lungs and in healthy children

The serotyping of 350 Streptococcus pneumoniae strains isolated from patients with acute and chronic diseases of the respiratory organs and from healthy children was carried out with the use of the agglutination test. 145 pleural fluid samples were tested for the presence of the pneumococcal antigen and its K-type. The prevailing pneumococcal serotypes were 6, 1, 19, 12 and 9 in acute pneumonia and 6, 19, 42, 3 and 12 in chronic pneumonia, including, respectively, 64.8% and 62% of all typed strains. Cases of pneumonia with complications in the form of pleuritis were mostly induced by serotypes 3, 1, 12 and 14, while in the destructive forms of pneumonia K-types 3, 14, 9 and 12 were isolated. Examinations of healthy pre-school children have shown 16.1% of them to be transitory pneumococcus carriers. Multiple biological examinations carried out during 1.5 years revealed that in this period 42.4% of the children were pneumococcus carriers.     

The Relevance of a Novel Quantitative Assay to Detect up to 40 Major Streptococcus pneumoniae Serotypes Directly in Clinical Nasopharyngeal and Blood Specimens

For epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664) from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, quantitative assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution.     

Accelerated laboratory diagnosis of bacterial pneumonia

The effectiveness of countercurrent immunoelectrophoresis (CIE) used for the accelerated differential diagnosis of pneumococcal, staphylococcal, mycoplasmal and Legionella infections in cases of pneumonia has been shown. The presence of correlation between the results obtained in the bacterial study of sputa and bronchial washings and in CIE has been revealed, which gives grounds for recommending CIE for the accelerated diagnosis of pneumococcal pneumonia on the basis of the analysis of sputa, bronchial washings and blood sera obtained from patients. In Legionella infection the passive hemagglutination test with antigenic diagnostica has proved to be more effective for accelerated diagnosis than CIE.     

Diagnostic value of different laboratory methods in the diagnosis of pneumonia caused by Haemophilus influenzae type B

Comparative analysis of the diagnostic value of different laboratory methods in the diagnosis of H. influenzae b (Hib) pneumonia in children (bacteriological method, latex agglutination, counter immunoelectrophoresis, the passive hemagglutination test and the enzyme immunoassay (EIA) was carried out. EIA proved to be the most informative method for the diagnosing Hib pneumonia. EIA makes it possible to detect specific Hib antigens in different clinical materials in 48.8% of cases, as well as high titers of antibodies to mis infective agent in 61.7% of cases. The authors propose the unified criteria of the laboratory diagnosis of Hib infection in children.     

Pneumococcus serotypes in acute and chronic inflammatory lung disease (1978-1980)

The comparison of the antigenic structure of the polysaccharide capsule in 342 pneumococcal strains isolated in Leningrad from the bronchial contents of patients with acute and chronic inflammatory pulmonary diseases indicated that during 1978-1980 a change in the composition of the prevailing groups of serotypes occurred every year. The comparison of the groups of prevailing pneumococcal serotypes isolated from children and adults has revealed no statistically significant differences in the specific prominence of different types, with the exception of serotype 15. Serotypes 6, 7 and 2 have been found to occur most frequently in acute pneumonia, and serotypes 23, 3, 9, 6, 15, 29 and 34 in chronic inflammatory pulmonary diseases. The preliminary data on the differences in the composition of the prevailing pneumococcal serotypes causing acute pneumonia in Leningrad and Kaunas have been obtained.     

An easy method for detection of nasopharyngeal carriage of multiple Streptococcus pneumoniae serotypes

In this paper, a simplified method for detection of pneumococcal carriage and for revealing the presence of several serotypes in a nasopharyngeal sample is evaluated. Enrichment broth was used for transportation and for the initial culturing of samples. All specimens were examined directly by the capsular reaction test for the presence of any of the 91 known pneumococcal serotypes. Sub-culturing on blood agar was used for isolation of the pneumococcal strains detected in the primary broth culture. A total of 693 nasopharyngeal swabs were obtained among children, their parents and employees in day care centres. Pneumococci were observed in 363 samples and 36 of these (9.9%) contained more than one serotype (multiple carriages). Two persons carried 3 different serotypes simultaneously. A significant increase in the positive sampling rate (5.8%) was achieved by using the simplified method compared to conventional streaking of the swabs directly on blood agar (p<0.0001).     

Occurrence of p-fimbriae in enteropathogenic E coli (EPEC) and in E. coli strains isolated from patients with urinary tract infections]

The aim of this study was to gain knowledge of prevalence of P+ clones among EPEC strains isolated from children with diarrhoea and E. coli strains isolated from urine. Three hundred eighty four E. coli strains isolated from children with diarrhoea were tested. They belonged to 11 serotypes (018, 025, 026, 044, 055, 0111, 0114, 0119, 0124, 0125, and 0128). Nine hundred thirty colonies of E. coli from Mac Conkey's agar plated quantitatively with urine samples of 178 individuals suffering from urinary tract infections were also tested. All strains were assayed by mannose-resistant active haemagglutination test (MRHA) and by slide agglutination using self prepared latex reagent for detection of P fimbriae. Out of 384 E. coli strains tested 122 (31.8%) showed presence of adhesins detected by mannose-resistant active haemagglutination test (MRHA) and in 90 (23.3%) out of all tested strains the presence of P fimbriae was found. The highest percentage of P fimbriae prevalence was found in E. coli belonging to the following serotypes: 018 (in 68.9% strains), 025 (in 29.2% strains), and 0125 (in 25.0% strains). This type of fimbriae was also detected in serotypes 026 (9.1%), 044 (8.7%), 055 (5.6%), and 0119 (in 2 strains out of 5 isolated). Out of 933 colonies of E. coli, isolated from 178 urine samples, 434 (46.5%) colonies gave positive results in MRHA test, including 133 positive in latex test for P fimbriae. These studies showed that for MRHA adhesins, including P fimbriae, a parallel examination of higher number of E. coli was necessary.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical and Microbiological Factors Associated with High Nasopharyngeal Pneumococcal Density in Patients with Pneumococcal Pneumonia

BackgroundWe aimed to study if certain clinical and/or microbiological factors are associated with a high nasopharyngeal (NP) density of Streptococcus pneumoniae in pneumococcal pneumonia. In addition, we aimed to study if a high NP pneumococcal density could be useful to detect severe pneumococcal pneumonia.MethodsAdult patients hospitalized for radiologically confirmed community-acquired pneumonia were included in a prospective study. NP aspirates were collected at admission and were subjected to quantitative PCR for pneumococcal DNA (Spn9802 DNA). Patients were considered to have pneumococcal etiology if S. pneumoniae was detected in blood culture and/or culture of respiratory secretions and/or urinary antigen test.ResultsOf 166 included patients, 68 patients had pneumococcal DNA detected in NP aspirate. Pneumococcal etiology was noted in 57 patients (84%) with positive and 8 patients (8.2%) with negative test for pneumococcal DNA (p<0.0001). The median NP pneumococcal density of DNA positive patients with pneumococcal etiology was 6.83 log₁₀ DNA copies/mL (range 1.79–9.50). In a multivariate analysis of patients with pneumococcal etiology, a high pneumococcal density was independently associated with severe pneumonia (Pneumonia Severity Index risk class IV-V), symptom duration ≥2 days prior to admission, and a medium/high serum immunoglobulin titer against the patient’s own pneumococcal serotype. NP pneumococcal density was not associated with sex, age, smoking, co-morbidity, viral co-infection, pneumococcal serotype, or bacteremia. Severe pneumococcal pneumonia was noted in 28 study patients. When we studied the performance of PCR with different DNA cut-off levels for detection of severe pneumococcal pneumonia, we found sensitivities of 54–82% and positive predictive values of 37–56%, indicating suboptimal performance.ConclusionsPneumonia severity, symptom duration ≥2 days, and a medium/high serum immunoglobulin titer against the patient’s own serotype were independently associated with a high NP pneumococcal density. NP pneumococcal density has limited value for detection of severe pneumococcal pneumonia.     

An easy method for detection of nasopharyngeal carriage of multiple Streptococcus pneumoniae serotypes

In this paper, a simplified method for detection of pneumococcal carriage and for revealing the presence of several serotypes in a nasopharyngeal sample is evaluated. Enrichment broth was used for transportation and for the initial culturing of samples. All specimens were examined directly by the capsular reaction test for the presence of any of the 91 known pneumococcal serotypes. Sub-culturing on blood agar was used for isolation of the pneumococcal strains detected in the primary broth culture. A total of 693 nasopharyngeal swabs were obtained among children, their parents and employees in day care centres. Pneumococci were observed in 363 samples and 36 of these (9.9%) contained more than one serotype (multiple carriages). Two persons carried 3 different serotypes simultaneously. A significant increase in the positive sampling rate (5.8%) was achieved by using the simplified method compared to conventional streaking of the swabs directly on blood agar (p < 0.0001).     

Colony blot assay: a useful method to detect multiple pneumococcal serotypes within clinical specimens

The efficacy of pneumococal conjugate vaccines in young children may be complicated by serotype replacement. We developed a colony blot assay which enables the identification of re-colonization with novel serotypes (replacement), overgrowth by minor co-colonizing serotypes or suppression of previously predominant vaccine serotype strains as a result of vaccination. This method allows the identification of multiple serotypes in a single specimen in a ratio of 1:1000. In order to demonstrate the potential of our method, we investigated the consecutive nasopharyngeal samples of 26 children who had shown a shift in pneumococcal colonization after conjugate vaccination. Mixed colonization was found once in 15 pre-vaccination samples and four times in 26 post-vaccination samples. In the remaining children 'true replacement' had presumably occurred. Hence, we conclude that the colony blot assay is an easy to apply method, which allows the identification of different pneumococcal serotypes within single clinical specimens.     

Multi-Serotype Pneumococcal Nasopharyngeal Carriage Prevalence in Vaccine Na?ve Nepalese Children,Assessed Using Molecular Serotyping

Invasive pneumococcal disease is one of the major causes of death in young children in resource poor countries. Nasopharyngeal carriage studies provide insight into the local prevalence of circulating pneumococcal serotypes. There are very few data on the concurrent carriage of multiple pneumococcal serotypes. This study aimed to identify the prevalence and serotype distribution of pneumococci carried in the nasopharynx of young healthy Nepalese children prior to the introduction of a pneumococcal conjugate vaccine using a microarray-based molecular serotyping method capable of detecting multi-serotype carriage. We conducted a cross-sectional study of healthy children aged 6 weeks to 24 months from the Kathmandu Valley, Nepal between May and October 2012. Nasopharyngeal swabs were frozen and subsequently plated on selective culture media. DNA extracts of plate sweeps of pneumococcal colonies from these cultures were analysed using a molecular serotyping microarray capable of detecting relative abundance of multiple pneumococcal serotypes. 600 children were enrolled into the study: 199 aged 6 weeks to <6 months, 202 aged 6 months to < 12 months, and 199 aged 12 month to 24 months. Typeable pneumococci were identified in 297/600 (49·5%) of samples with more than one serotype being found in 67/297 (20·2%) of these samples. The serotypes covered by the thirteen-valent pneumococcal conjugate vaccine were identified in 44·4% of samples containing typeable pneumococci. Application of a molecular serotyping approach to identification of multiple pneumococcal carriage demonstrates a substantial prevalence of co-colonisation. Continued surveillance utilising this approach following the introduction of routine use of pneumococcal conjugate vaccinates in infants will provide a more accurate understanding of vaccine efficacy against carriage and a better understanding of the dynamics of subsequent serotype and genotype replacement.     

23-valent pneumococcal vaccine failure in a patient who developed pneumonia: a case report

We report a clinical failure of a pneumococcal vaccine in a patient who developed pneumococcal pneumonia. In 2008, an 85-year-old Italian woman was admitted to the Respiratory Disease Unit of a hospital in Southern Italy. The 23-valent pneumococcal vaccine had been administered to the patient 50 days earlier. The chest x-ray disclosed a right basal bronchopneumonic focus. Streptococcus Pneumoniae serotype 19A, a strain included in the 23-valent pneumococcal vaccine, was isolated from the sputum. There is a need for more efficacious conjugated vaccines covering the majority of the pneumococcal serotypes that cause serious illness in older children and adults worldwide.     

Impact of Preceding Flu-Like Illness on the Serotype Distribution of Pneumococcal Pneumonia

Background

Even though the pathogenicity and invasiveness of pneumococcus largely depend on capsular types, the impact of serotypes on post-viral pneumococcal pneumonia is unknown.

Methods and Findings

This study was performed to evaluate the impact of capsular serotypes on the development of pneumococcal pneumonia after preceding respiratory viral infections. Patients with a diagnosis of pneumococcal pneumonia were identified. Pneumonia patients were divided into two groups (post-viral pneumococcal pneumonia versus primary pneumococcal pneumonia), and then their pneumococcal serotypes were compared. Nine hundred and nineteen patients with pneumococcal pneumonia were identified during the study period, including 327 (35.6%) cases with post-viral pneumococcal pneumonia and 592 (64.4%) cases with primary pneumococcal pneumonia. Overall, serotypes 3 and 19A were the most prevalent, followed by serotypes 19F, 6A, and 11A/11E. Although relatively uncommon (33 cases, 3.6%), infrequently colonizing invasive serotypes (4, 5, 7F/7A, 8, 9V/9A, 12F, and 18C) were significantly associated with preceding respiratory viral infections (69.7%, P<0.01). Multivariate analysis revealed several statistically significant risk factors for post-viral pneumococcal pneumonia: immunodeficiency (OR 1.66; 95% CI, 1.10–2.53), chronic lung diseases (OR 1.43; 95% CI, 1.09–1.93) and ICI serotypes (OR 4.66; 95% CI, 2.07–10.47).

Conclusions

Infrequently colonizing invasive serotypes would be more likely to cause pneumococcal pneumonia after preceding respiratory viral illness, particularly in patients with immunodeficiency or chronic lung diseases.     

Screening adenoviruses in stool samples: evaluation of a genus-specific monoclonal antibody based enzyme immunoassay

To screen fecal samples for adenovirus antigens a genus-specific monoclonal antibody based enzyme immunoassay was developed. In a comparative analysis with commercial latex agglutination test, high sensitivity was demonstrated. The assay did not detect other viruses usually found in faeces suggesting its specificity. One hundred and eighty stool samples collected in Baranya County were tested and 13 (7.2%) of them showed reactivity. The application of our immunoassay combined with other, more sophisticated methods may help us to determine the serotype specificity of these adenovirus isolates and assess the importance of adenoviruses in viral gastroenteritis.     

Serotype 1 and 8 Pneumococci Evade Sensing by Inflammasomes in Human Lung Tissue

Streptococcus pneumoniae is a major cause of pneumonia, sepsis and meningitis. The pore-forming toxin pneumolysin is a key virulence factor of S. pneumoniae, which can be sensed by the NLRP3 inflammasome. Among the over 90 serotypes, serotype 1 pneumococci (particularly MLST306) have emerged across the globe as a major cause of invasive disease. The cause for its particularity is, however, incompletely understood. We therefore examined pneumococcal infection in human cells and a human lung organ culture system mimicking infection of the lower respiratory tract. We demonstrate that different pneumococcal serotypes differentially activate inflammasome-dependent IL-1β production in human lung tissue and cells. Whereas serotype 2, 3, 6B, 9N pneumococci expressing fully haemolytic pneumolysins activate NLRP3 inflammasome-dependent responses, serotype 1 and 8 strains expressing non-haemolytic toxins are poor activators of IL-1β production. Accordingly, purified haemolytic pneumolysin but not serotype 1-associated non-haemolytic toxin activates strong IL-1β production in human lungs. Our data suggest that the evasion of inflammasome-dependent innate immune responses by serotype 1 pneumococci might contribute to their ability to cause invasive diseases in humans.     

Specific latex preparations in the etiological diagnosis of suppurative bacterial meningitis

The results of the evaluation of the diagnostic latex preparations Bactigen, manufactured by Wampole Laboratories (USA) and intended for the detection of meningococcal antigens, serogropus A, B, C, Y, pneumococcal polyantigens and type b Haemophilus influenzae antigens in the spinal fluid and blood of patients with meningococcal infection and purulent bacterial meningitides, are presented. The pathological material was studied by traditional methods and by the latex agglutination (LAG) test. 522 LAG tests were made, including 414 tests for meningococcal infection, 60 tests for pneumococcal infection and 48 tests for type b H. influenzae. The results of this study revealed that the latex preparations were highly specific with respect to type b H. influenzae antigens and meningococcal antigens (false positive reactions constituted 0.96%). The simplicity of the test and the rapid techniques making it possible to obtain results within 30-40 minutes indicate good prospects of using the LAG test in laboratory practice.     

Streptococcus pneumoniae serotypes in children with acute pneumonia and pleurisy

The study of pneumococci of different serotypes, isolated from patients with acute pneumonia and pleuritis and from healthy children was carried out. Among the pneumococcal serotypes causing pneumonia and pleuritis in children serotypes 1, 6, 19, 14 and 3 were most widely spread and constituted 62.3% of all isolated pneumococci. In young children cases of acute pneumonia and pleuritis were more often induced by serotypes 6 and 14 and in older children, by serotypes 1 and 3. In patients with uncomplicated pneumonia and pleuritis differences in the detected serotypes of pneumococci were observed, and the disease course differed in severity. Serotypes 14, 3 and 3 induced destructive processes in the lungs more often than other serotypes. Monitoring of the sensitivity of pneumococci to antibiotics showed that most of the strains retained high sensitivity to penicillin and ampicillin. In most cases the detected resistant pneumococcal strains belonged to serogroup 19.     

Incidence of Hospitalized Pneumococcal Pneumonia among Adults in Guatemala, 2008-2012

Background Streptococcus pneumoniae is a leading cause of pneumonia worldwide. However, the burden of pneumococcal pneumonia among adults in low- and middle-income countries is not well described.MethodsData from 2008–2012 was analyzed from two surveillance sites in Guatemala to describe the incidence of pneumococcal pneumonia in adults. A case of hospitalized pneumococcal pneumonia was defined as a positive pneumococcal urinary antigen test or blood culture in persons aged ≥ 18 years hospitalized with an acute respiratory infection (ARI).ResultsAmong 1595 adults admitted with ARI, 1363 (82%) had either urine testing (n = 1286) or blood culture (n = 338) performed. Of these, 188 (14%) had pneumococcal pneumonia, including 173 detected by urine only, 8 by blood culture only, and 7 by both methods. Incidence rates increased with age, with the lowest rate among 18–24 year-olds (2.75/100,000) and the highest among ≥65 year-olds (31.3/100,000). The adjusted incidence of hospitalized pneumococcal pneumonia was 18.6/100,000 overall, with in-hospital mortality of 5%.ConclusionsAn important burden of hospitalized pneumococcal pneumonia in adults was described, particularly for the elderly. However, even adjusted rates likely underestimate the true burden of pneumococcal pneumonia in the community. These data provide a baseline against which to measure the indirect effects of the 2013 introduction of the pneumococcal conjugate vaccine in children in Guatemala.     

Etiology of acute and chronic bronchopulmonary diseases in children. II. A study of the specific immune response by counterimmunoelectrophoresis and the complement fixation and passive hemagglutination reactions

Specific immune response to Streptococcus pneumoniae and Haemophilus influenzae has been studied in 158 children with acute pneumonia and pleuritis and 128 children with chronic pneumonia by countercurrent immunoelectrophoresis (CIE) and in the complement fixation (CFT) and passive hemagglutination (PHA) tests. The use of CIE leads to the detection of antibodies to H. influenzae in 23.7% of children with acute pneumonia and in 46.9% of children with chronic pneumonia. In the CFT antibodies to H. influenzae are also more often detected in children with chronic pneumonia (48%) than in those with acute respiratory infections (12.2%). In the PHA test high titers of antibodies to type b H. influenzae capsular polysaccharide occur in 11.9% of children with acute pneumonia and in 8.2% of children with chronic pneumonia.