Culture of all sputum samples irrespective of quality adds value to the diagnosis of pneumococcal community-acquired pneumonia in the elderly.

Culture of expectorated sputum in the microbiological diagnosis of community-acquired pneumonia (CAP) is considered valid only if high-quality (HQ) samples are obtained, but evidence regarding pneumococcal etiology specifically is lacking. We studied 323 radiologically confirmed CAP cases in patients aged ≥ 65 years. Sputum samples were assessed for quality microscopically and cultured. Two quality criteria sets were applied to delineate HQ from low-quality (LQ) sputa: leukocytes/epithelial cells ratio > 5 and ≤ 2.5 epithelial cells/400× magnification field (HQ1), or leukocytes/epithelial cells ratio > 1 (HQ2). A sputum sample was obtained and the quality assessed in 224 cases; 47% were HQ1 and 76% HQ2. Encapsulated pneumococci (EPnc) were cultured in 25 (24%), 14 (12%), 35 (21%), and 4 (7%) of the HQ1-, LQ1-, HQ2-, and LQ2-samples, respectively. If another pneumococcal test (blood culture, urine antigen, or ≥ twofold increase in CbpA or PsaA antibodies) was positive, EPnc were cultured at similar proportions in HQ1- and LQ1-sputa; if the other test was negative, EPnc were cultured less often in LQ1- than HQ1-sputa. EPnc were found less often in LQ2- than in HQ2-sputa. Our results suggest similar specificity in LQ- and HQ-sputum cultures. All sputum samples add value to the pneumococcal CAP-diagnosis in the elderly.

Testing Pneumonia Vaccines in the Elderly: Determining a Case Definition for Pneumococcal Pneumonia in the Absence of a Gold Standard.

Clinical assessments of vaccines to prevent pneumococcal community-acquired pneumonia (CAP) require sensitive and specific case definitions, but there is no gold standard diagnostic test. To develop a new case definition suitable for vaccine efficacy studies, we applied latent class analysis (LCA) to the results from 7 diagnostic tests for pneumococcal etiology on clinical specimens from 323 elderly persons with radiologically confirmed pneumonia enrolled in the Finnish Community-Acquired Pneumonia Epidemiology study during 2005-2007. Compared with the conventional use of LCA, which is mainly to determine sensitivities and specificities of different tests, we instead used LCA as an appropriate instrument to predict the probability of pneumococcal etiology for each CAP case based on individual test profiles, and we used the predictions to minimize the sample size that would be needed for a vaccine efficacy trial. When compared with the conventional laboratory criteria of encapsulated pneumococci in culture, in blood culture or high-quality sputum culture, or urine antigen positivity, our optimized case definition for pneumococcal CAP resulted in a trial sample size that was almost 20,000 subjects smaller. We believe that the novel application of LCA detailed here to determine a case definition for pneumococcal CAP could also be similarly applied to other diseases without a gold standard.

lytA Quantitative PCR on Sputum and Nasopharyngeal Swab Samples for Detection of Pneumococcal Pneumonia among the Elderly.

Real-time quantitative PCR (qPCR) assay of sputum or nasopharyngeal specimens has shown promising results in the detection of pneumococcal community-acquired pneumonia (PncCAP). We applied qPCR for the autolysin gene (lytA) and compared sputum and nasopharyngeal swab (NPS) pneumococcal loads in elderly patients with community-acquired pneumonia (CAP), and specifically in patients with PncCAP, to those in patient groups with other respiratory diseases. We studied patients aged ≥65 years with radiologically confirmed CAP, clinical CAP not retrospectively radiologically confirmed, other acute respiratory infections, or stable chronic lung disease. Pneumococcal etiology of CAP was ascertained by using a combination of multiple diagnostic methods. We analyzed sputum and NPS specimens by lytA qPCR with 104 pneumococcal genome equivalents (GE)/ml as a cutoff for positivity. Among PncCAP patients, lytA qPCR detected pneumococci in 94% of the sputum samples and in large quantities (mean, 6.82 ± 1.02 log10 GE/ml) but less frequently in NPS (44%) and in smaller quantities (5.55 ± 0.92 log10 GE/ml). In all other patient groups, ≤10% of the sputum samples and <5% of the NPS samples were lytA qPCR positive; but when they were positive, the sputum pneumococcal loads were similar to those in the PncCAP patients, suggesting a pneumococcal etiology in these patients. This was supported by other pneumococcal assay results. Overall, sputum lytA qPCR positivity was more common in PncCAP patients than in the other patient groups, but the quantitative results were mainly similar. NPS lytA qPCR was less sensitive than sputum lytA qPCR in detecting PncCAP.

Influenza C virus infection in military recruits--symptoms and clinical manifestation.

Due to the lack of rapid diagnostic tests, clinical features of Influenza C virus infections are poorly characterized. Respiratory infections in military recruits in eastern Finland were monitored between July 2004 and December 2005 in order to study the epidemiology and clinical picture of infections caused by this virus. Blood samples were obtained at entry and at the end of the military service, and during each episode of respiratory infection to measure antibody responses against 10 viral and 2 bacterial pathogens. If possible, sputum samples were collected during the acute phase of respiratory infection episodes. Symptoms of the episodes were recorded for comparison of the clinical picture caused by various infectious agents. Infection with influenza C virus was detected in 38 of 892 young men during their service. The virus usually caused a mild upper respiratory tract infection. Most typical clinical features of influenza C virus infection were cough, rhinitis, and hoarseness. A striking difference to infections caused by influenza A virus was the lack of fever. Influenza C virus is an important cause of a respiratory tract infection in army conscripts. Infections with this virus are usually mild but can be complicated in some cases.

Incidence and etiology of community-acquired pneumonia in the elderly in a prospective population-based study.

BACKGROUND: We conducted a prospective population-based epidemiological study to prepare a setting for documentation of the efficacy of novel vaccines against pneumococcal (Pnc) community-acquired pneumonia (CAP) in the elderly. Specific objectives were to demonstrate setting feasibility, to construct a case definition for Pnc CAP, and to estimate its incidence. METHODS: We prospectively enrolled patients with clinical and radiological findings compatible with CAP at municipal on-call clinics serving an elderly population (age ≥ 65 y) of approximately 29,500. Sputum, urine, nasopharyngeal swab (NPS), and blood samples were analyzed using diverse methods for the identification of Pnc (culture, PCR, antigen tests, serology) and of other pathogens. The following case definition for Pnc CAP was derived: encapsulated Pnc in blood culture or in high-quality sputum culture or at least 2 of the following: positive urine Pnc antigen; ≥ 2-fold increase in serum anti-PsaA or anti-CbpA antibodies; encapsulated Pnc culture or LytA PCR in either sputum or NPS. RESULTS: We enrolled 490 clinical CAP patients during the 2-y follow-up, 53% of all clinical CAP patients in the source population; 323 were radiologically confirmed. The incidence of radiologically confirmed CAP was 5.5/1000 person-y (95% confidence interval (CI) 4.9-6.1) and 10.5/1000 person-y when adjusted for non-captured patients. The proportion of radiologically confirmed CAP caused by Pnc was estimated at 17%; i.e. 0.95/1000 person-y (95% CI 0.7-1.2) and 1.8 when adjusted for non-captured patients. CONCLUSIONS: We developed and documented a feasible methodology for capturing endpoints in a vaccine trial for the prevention of pneumonia. CAP incidence in the elderly population remains considerable and Streptococcus pneumoniae was one of the most commonly detected causative agents.

Decline in temperature and humidity increases the occurrence of influenza in cold climate.

BACKGROUND: Both temperature and humidity may independently or jointly contribute to the risk of influenza infections. We examined the relations between the level and decrease of temperature, humidity and the risk of influenza A and B virus infections in a subarctic climate. METHODS: We conducted a case-crossover study among military conscripts (n = 892) seeking medical attention due to respiratory symptoms during their military training period and identified 66 influenza A and B cases by PCR or serology. Meteorological data such as measures of average and decline in ambient temperature and absolute humidity (AH) during the three preceding days of the onset (hazard period) and two reference periods, prior and after the onset were obtained. RESULTS: The average temperature preceding the influenza onset was -6.8 ± 5.6°C and AH 3.1 ± 1.3 g/m3. A decrease in both temperature and AH during the hazard period increased the occurrence of influenza so that a 1°C decrease in temperature and 0.5 g decrease per m3 in AH increased the estimated risk by 11% [OR 1.11 (1.03 to 1.20)] and 58% [OR 1.58 (1.28 to 1.96)], respectively. The occurrence of influenza infections was positively associated with both the average temperature [OR 1.10 per 1°C (95% confidence interval 1.02 to 1.19)] and AH [OR 1.25 per g/m3 (1.05 to 1.49)] during the hazard period prior to onset. CONCLUSION: Our results demonstrate that a decrease rather than low temperature and humidity per se during the preceding three days increase the risk of influenza episodes in a cold climate.

Association between anti-capsular IgG levels at birth and risk of invasive group B streptococcus disease in Finnish newborns: a retrospective case-control study.

BACKGROUND: Group B streptococcus is a major cause of neonatal disease. Natural history studies have linked maternally transferred anti-group B streptococcus capsular polysaccharide antibodies with protection against infant group B streptococcus disease. Previous studies of capsular polysaccharide antibody concentration in European populations have used maternal (not infant) sera and a non-standardised assay. This study aimed to evaluate anti-capsular polysaccharide IgG concentrations associated with protection against invasive group B streptococcus disease in Finnish infants. METHODS: In this retrospective case-control study, we used cord sera from the Finnish DIPP study repository, which was obtained between Jan 1, 1995, and Dec 31, 2017. We included infants aged 6 months or younger with group B streptococcus infection (cases) and healthy infants (controls). We enrolled infants with invasive neonatal group B streptococcus (55 cases) and matched controls (229 controls) aged 6 months or younger after identification from Finnish health registers. We measured anti-capsular polysaccharide IgG (serotypes Ia-V) concentration using a standardised immunoassay and we estimated its relationship to disease risk using a Bayesian model. We used the derived risk-concentration curve to predict potential efficacy of six-valent group B streptococcus capsular polysaccharide vaccine (GBS6) based on previously reported immunogenicity data. FINDINGS: Most (32 [58%] of 55 cases) group B streptococcus cases were due to serotype III and anti-serotype III streptococcus capsular IgG concentrations were higher in serotype III-matched controls than in cases (p<0·001). 0·120-0·266 µg/mL serotype III-specific IgG was estimated to confer 75-90% risk reduction against serotype III disease. A universal risk-concentration curve, aggregating results across all six serotypes, yielded similar results. Application of this curve to GBS6 immunogenicity data predicted maternal immunisation to be more than 80% efficacious for prevention of infant group B streptococcus disease. INTERPRETATION: Higher neonatal anti-capsular polysaccharide serum IgG concentration at birth correlated with reduced risk of infant group B streptococcus disease in Finland. Based on these results, a maternal group B streptococcus capsular conjugate vaccine currently in development is predicted to be efficacious. FUNDING: Pfizer.

Respiratory viral infections among children with community-acquired pneumonia and pleural effusion.

Pleural effusion (PE), a complication of community-acquired pneumonia (CAP), is usually attributed to a bacterial infection. Nonetheless, viral infections have not been investigated routinely. We searched for bacterial and viral infections among 277 children hospitalized with CAP. Among these children 206 (74%) had radiographic confirmation, of whom 25 (12%) had PE. The aetiology was established in 18 (72%) PE cases: bacterial (n = 5; 28%), viral (n = 9; 50%), and viral-bacterial (n = 4; 22%) infections were found. Infection by rhinovirus (n = 3), enterovirus, Streptococcus pneumoniae (n = 2 each), Haemophilus influenzae, Moraxella catarrhalis, Mycoplasma pneumoniae, influenza A virus, and respiratory syncytial virus (RSV) (n = 1 each) were detected as probable sole infections. Parainfluenza virus 1/3 + influenza A virus and RSV + influenza A virus (n = 1 each) were identified as mixed viral-viral infections. Probable viral non-bacterial infection was identified in a third of the cases with CAP and PE. It is advisable to investigate viral as well as bacterial infections among children with CAP and PE.

Genotypic and phenotypic characterization of carriage and invasive disease isolates of Neisseria meningitidis in Finland.

The relationship between carriage and the development of invasive meningococcal disease is not fully understood. We investigated the changes in meningococcal carriage in 892 military recruits in Finland during a nonepidemic period (July 2004 to January 2006) and characterized all of the oropharyngeal meningococcal isolates obtained (n = 215) by using phenotypic (serogrouping and serotyping) and genotypic (porA typing and multilocus sequence typing) methods. For comparison, 84 invasive meningococcal disease strains isolated in Finland between January 2004 and February 2006 were also analyzed. The rate of meningococcal carriage was significantly higher at the end of military service than on arrival (18% versus 2.2%; P < 0.001). Seventy-four percent of serogroupable carriage isolates belonged to serogroup B, and 24% belonged to serogroup Y. Most carriage isolates belonged to the carriage-associated ST-60 clonal complex. However, 21.5% belonged to the hyperinvasive ST-41/44 clonal complex. Isolates belonging to the ST-23 clonal complex were cultured more often from oropharyngeal samples taken during the acute phase of respiratory infection than from samples taken at health examinations at the beginning and end of military service (odds ratio [OR], 6.7; 95% confidence interval [95% CI], 2.7 to 16.4). The ST-32 clonal complex was associated with meningococcal disease (OR, 17.8; 95% CI, 3.8 to 81.2), while the ST-60 clonal complex was associated with carriage (OR, 10.7; 95% CI, 3.3 to 35.2). These findings point to the importance of meningococcal vaccination for military recruits and also to the need for an efficacious vaccine against serogroup B isolates.

Nasopharyngeal carriage of Streptococcus pneumoniae and pneumococcal urine antigen test in healthy elderly subjects.

BACKGROUND: Children frequently carry Streptococcus pneumoniae (pneumococcus) in their nasopharynx, even when healthy. Lower carriage rates have been reported in adults and only sparse data are available for the elderly. We sampled healthy elderly subjects for nasopharyngeal carriage to assess the prevalence of pneumococcal carriage using various assays. METHODS: A deep nasopharyngeal swab sample was taken from 590 healthy elderly subjects aged ≥ 65 y. The samples were stored in STGG (skim milk-tryptone-glucose-glycerol) medium and cultured directly and after incubation in enrichment broth using routine identification methods. Real-time polymerase chain reaction (PCR) assays specific for pneumolysin and pneumococcal surface antigen A genes was performed on the same samples. Urine was also collected and assayed using the commercial Binax Streptococcus pneumoniae NOW urine antigen test. RESULTS: The prevalence of pneumococcal carriage in healthy elderly persons was 1.5% for encapsulated pneumococci and 5.3% for all presumptive pneumococci. The use of the enrichment broth did not increase the yield of positives. PCR assays gave higher numbers of positives, but pneumolysin PCR in particular gave probable false-positive results. Only 1 urine antigen test was positive, and this was in a person not carrying pneumococcus. CONCLUSIONS: Nasopharyngeal carriage of pneumococci in the elderly was rare. Identification of presumptive pneumococci in culture requires further confirmation, e.g. by serotyping. The urine antigen test was not affected by concurrent carriage. Low carriage prevalence suggests that encapsulated pneumococci detected in a respiratory tract sample during sickness may be the true cause of disease, since contamination from asymptomatic nasopharyngeal carriage seems unlikely.

Procalcitonin is useful in identifying bacteraemia among children with pneumonia.

Empirical antibiotic use is prescribed in managing children with pneumonia worldwide. We assessed the usefulness of procalcitonin (PCT) and interferon-alpha (IFN-alpha) in differentiating viral from bacterial pneumonia. Among 159 hospitalized children, pneumonia was diagnosed based on clinical complaints plus pulmonary infiltrate. Aetiology was investigated for 9 viruses and 4 atypical and 3 typical bacteria. PCT and IFN-alpha were measured in the serum sample collected on admission. Eight patients had bacteraemic infections, 38 had non-bacteraemic typical infections, and 19 patients had atypical bacterial infections. Viral and unknown aetiology was established in 57 (36%) and 34 (21%) cases, respectively. Three patients with bacterial infection without collected blood culture were excluded. IFN-alpha (IU/ml) was detectable in 20 (13%) cases. The difference among median PCT values of the bacteraemic (4.22; 1.56-7.56), non-bacteraemic typical bacterial (1.47; 0.24-4.07), atypical bacterial (0.18; 0.06-1.03) and only viral (0.65; 0.11-2.22) subgroups was significant (p = 0.02). PCT was > or =2 ng/ml in 52 (33%) cases. The presence of IFN-alpha was associated with PCT <2 ng/ml (90% vs. 64%, p = 0.02). The negative predictive value (95% confidence interval) of PCT > or =2 ng/ml was 95% (89-100%), 89% (78-100%), 93% (85-100%) for differentiation of bacteraemic from viral, atypical bacterial and non-bacteraemic typical bacterial infection, respectively, and 58% (49-68%) for differentiation between bacterial and viral infection. PCT may be useful in identifying bacteraemia among children hospitalized with community-acquired pneumonia. IFN-alpha was uncommonly detected.

Seasonal patterns of viral and bacterial infections among children hospitalized with community-acquired pneumonia in a tropical region.

Community-acquired pneumonia (CAP) is a common cause of morbidity among children. Evidence on seasonality, especially on the frequency of viral and bacterial causative agents is scarce; such information may be useful in an era of changing climate conditions worldwide. To analyze the frequency of distinct infections, meteorological indicators and seasons in children hospitalized for CAP in Salvador, Brazil, nasopharyngeal aspirate and blood were collected from 184 patients aged < 5 y over a 21-month period. Fourteen microbes were investigated and 144 (78%) cases had the aetiology established. Significant differences were found in air temperature between spring and summer (p = 0.02) or winter (p < 0.001), summer and fall (p = 0.007) or winter (p < 0.001), fall and winter (p = 0.002), and on precipitation between spring and fall (p = 0.01). Correlations were found between: overall viral infections and relative humidity (p = 0.006; r = 0.6) or precipitation (p = 0.03; r = 0.5), parainfluenza and precipitation (p = 0.02; r = -0.5), respiratory syncytial virus (RSV) and air temperature (p = 0.048; r = -0.4) or precipitation (p = 0.045; r = 0.4), adenovirus and precipitation (p = 0.02; r = 0.5), pneumococcus and air temperature (p = 0.04; r = -0.4), and Chlamydia trachomatis and relative humidity (p = 0.02; r = -0.5). The frequency of parainfluenza infection was highest during spring (32.1%; p = 0.005) and that of RSV infection was highest in the fall (36.4%; p < 0.001). Correlations at regular strength were found between several microbes and meteorological indicators. Parainfluenza and RSV presented marked seasonal patterns.

The role of respiratory viral infections among children hospitalized for community-acquired pneumonia in a developing country.

We report an investigation for 16 bacteria and viruses among 184 children hospitalized with pneumonia in Salvador, Brazil. Etiology was established in 144 (78%) cases. Viral, bacterial, and mixed infections were found in 110 (60%), 77 (42%), and 52 (28%) patients, respectively. Rhinovirus (21%) and Streptococcus pneumoniae (21%) were the most common pathogens. Our results demonstrate the importance of viral and pneumococcal infections among those patients.

Cerebrospinal Fluid Cathelicidin Correlates With the Bacterial Load and Outcomes in Childhood Bacterial Meningitis.

BACKGROUND: Large cerebrospinal fluid (CSF) bacterial load in bacterial meningitis (BM) relates to poor outcome. However, the antimicrobial peptide cathelicidin seems important to host defense. We studied how cathelicidin concentrations and bacterial load in CSF relate in childhood BM and to what extent they may predict the disease outcome. METHODS: The patient data originated from a large prospective clinical trial in Latin America in 1996-2003 in which the CSF samples were collected on admission (CSF1) and 12-24 hours later (CSF2). The cathelicidin concentrations were measured by enzyme-linked immunosorbent assay and the CSF bacterial load by real-time polymerase chain reaction. This analysis comprised 76 children with meningitis caused by Haemophilus influenzae type b (n = 44), Streptococcus pneumoniae (n = 28) or Neisseria meningitidis (n = 4). RESULTS: The cathelicidin concentration correlated with the bacterial genome count in both samples (CSF1: ρ = 0.531, P < 0.001; CSF2: ρ = 0.553, P < 0.001). A high CSF1 ratio of cathelicidin to the bacterial genome count was associated with fewer audiologic sequelae (odds ratio: 0.11, 95% confidence interval: 0.02-0.61, P = 0.01) and more favorable neurologic outcomes (odds ratio: 3.95, 95% confidence interval: 1.22-12.8, P = 0.02), but not with better survival. CONCLUSIONS: In conclusion, CSF cathelicidin and the bacterial load were closely related in childhood BM. A high initial cathelicidin-to-bacterial genome count ratio predicted better outcomes in survivors.

Comparison of serological assays using pneumococcal proteins or polysaccharides for detection of Streptococcus pneumoniae infection in children with community-acquired pneumonia.

The aim of this study was to compare the results of serological assays using pneumococcal proteins or polysaccharides for the detection of pneumococcal infection in childhood pneumonia. Serological assays measured IgG against eight pneumococcal proteins (Ply,CbpA,PspA1,PspA2,PcpA,PhtD,StkP-C,PcsB-N), C-polysaccharide [in the whole study population, n = 183], or 19 pneumococcal capsular polysaccharides (1,2,4,5,6B,7F,8,9 V,10A,11A,12F,14,15B,17F,18C,19F,20,23F,33F) [only in a subgroup of patients, n = 53] in paired serum samples of children aged <5 years-old hospitalized with clinical and radiological diagnosis of community-acquired pneumonia. We also performed an inhibition of binding test with the anti-capsular polysaccharide assay in order to confirm the specificity of the antibody responses detected. Invasive pneumococcal pneumonia was investigated by blood culture and PCR (ply-primer). Among 183 children, the anti-protein assay detected antibody response in 77/183(42.1%) patients and the anti-C-polysaccharide assay in 28/183(15.3%) patients. In a subgroup of 53 children, the anti-protein assay detected response in 32/53(60.4%) patients, the anti-C-polysaccharide assay in 11/53(20.8%) patients, and the anti-capsular polysaccharide in 25/53(47.2%) patients. Simultaneous antibody responses against ≥2 different capsular polysaccharides were detected in 11/53(20.8%) patients and this finding could not be explained by cross-reactivity between different serotypes. Among 13 patients with invasive pneumococcal pneumonia, the sensitivity of the anti-protein assay was 92.3%(12/13), of the anti-C-polysaccharide assay 30.8%(4/13), and of the anti-capsular polysaccharide assay 46.2%(6/13). The serological assay using pneumococcal proteins is more sensitive for the detection of pneumococcal infection in children with pneumonia than the assay using pneumococcal polysaccharides. Future studies on childhood pneumonia aetiology should consider applying serological assays using pneumococcal proteins.

Evaluation of the BinaxNOW® Streptococcus pneumoniae antigen test on fresh, frozen and concentrated urine samples in elderly patients with and without community-acquired pneumonia.

We evaluated the BinaxNOW® urine antigen test in elderly. For fresh un-concentrated urine samples, the sensitivity for pneumococcal pneumonia was 63% and specificity 97%. After freezing and concentration, the results comparable to positive control line in intensity at 60 min gave high sensitivity (81%) with no loss in specificity (96%).

A Decrease in Temperature and Humidity Precedes Human Rhinovirus Infections in a Cold Climate.

Both temperature and humidity may independently or jointly contribute to the risk of human rhinovirus (HRV) infections, either through altered survival and spread of viruses in the environment or due to changes in host susceptibility. This study examined the relationship between short-term variations in temperature and humidity and the risk of HRV infections in a subarctic climate. We conducted a case-crossover study among conscripts (n = 892) seeking medical attention due to respiratory symptoms during their military training and identified 147 HRV cases by real-time PCR. An average temperature, a decline in daily ambient temperature and absolute humidity (AH) during the three preceding days of the onset (hazard period) and two reference periods (a week prior and after the onset) were obtained. The average daily temperature preceding HRV infections was -9.9 ± 4.9 °C and the average AH was 2.2 ± 0.9 g/m³. An average (odds ratios (OR) 1.07 (95% confidence interval (CI) 1.00-1.15)) and maximal (OR 1.08 (1.01-1.17)) change in temperature increased the risk of HRV infections by 8% per 1 °C decrease. An average (OR 1.20 (CI 1.03-1.40)) and maximal decrease (OR 1.13 (CI 0.96-1.34)) in AH increased the risk of HRV infection by 13% and 20% per 0.5 g/m³ decrease. A higher average temperature during the three preceding days was positively associated with HRV infections (OR 1.07 (CI 1.00-1.15)). A decrease rather than low temperature and humidity per se during the preceding few days increases the risk of HRV infections in a cold climate. The information is applicable to populations residing in cold climates for appropriate personal protection and prevention of adverse health effects.

Real-time pneumolysin polymerase chain reaction with melting curve analysis differentiates pneumococcus from other alpha-hemolytic streptococci.

The majority of pneumococcal isolates can be identified by the conventional methods based on phenotypic characteristics. Occasionally, however, the differentiation of alpha-hemolytic streptococci from pneumococci, especially those isolated from nasopharynx, is problematic due to the discrepant results obtained by the conventional identification methods. Several gene technological methods based on the amplification of genes encoding pneumococcal virulence factors, such as pneumolysin (ply) and autolysin, have been used as additional identification methods. Recent studies have shown that especially the ply gene is frequently also present in nonpneumococcal alpha-hemolytic streptococci. In this study, we compared the commonly used phenotypic identification methods with nucleic acid-based methods, commercial AccuProbetrade mark, conventional pneumolysin polymerase chain reaction (Ply-PCR), and real-time Ply-PCR in the identification of alpha-hemolytic streptococcal strains isolated from 100 consecutive nasopharyngeal specimens. We also studied if melting curve analysis and sequencing of the amplification products of a ply gene fragment could be helpful in the identification. Our results suggest that the ply gene present in alpha-hemolytic streptococci differs from that present in pneumococcus, and that melting curve analysis would prove useful in the differentiation of these bacteria.

Isolation of pneumococcal DNA from nasopharyngeal samples for real-time, quantitative PCR: comparison of three methods.

BACKGROUND: Real-time PCR is a useful method for detecting and quantifying bacterial DNA in clinical samples. DNA extraction is a crucial step when performing quantitative PCR. METHODS: We compared three methods, QIAamp. The use of tradenames is for product identification purposes only and does not imply endorsement. DNA Mini kit, MagNAPure trade mark LC DNA Isolation Kit II together with PickPen trade mark magnetic particle transfer device, and KingFisher genomic DNA purification Kit with KingFisher mL instrument, for purification of Streptococcus pneumoniae DNA from 50 nasopharyngeal swab samples, collected into skim milk-tryptone-glucose-glycerin medium. Pneumococcal DNA was detected and quantified by real-time PCR and results were compared to culture findings. RESULTS: The 22 (44%) pneumococcal culture-positive specimens were all positive by PCR regardless of DNA extraction method used, except that one KingFisher-extracted sample was positive only when repeatedly tested. Additionally, 71%, 57%, and 82% of the culture-negative samples were positive by real-time PCR when DNA was extracted by QIAamp, MagNAPure-PickPen, and KingFisher methods, respectively. The number of genome equivalents detected by real-time PCR varied, but was mainly low in culture-negative samples. The sensitivities of culture and real-time PCR were hence compared by analyzing different dilutions of a pneumococcal suspension. Real-time PCR detected significantly higher numbers of genome equivalents than the numbers of bacteria detected by culture. CONCLUSIONS: The results indicate that the DNA extraction method used for quantitative PCR should be evaluated and that real-time PCR is more sensitive than bacterial culture for detecting pneumococcus in nasopharyngeal swab samples.

Effect of xylitol and other carbon sources on Streptococcus pneumoniae biofilm formation and gene expression in vitro.

Xylitol inhibits the growth of Streptococcus pneumoniae. In clinical trials, xylitol decreased the occurrence of acute otitis media in day-care children, but did not decrease nasopharyngeal carriage of pneumococci. We hypothesized that xylitol inhibits biofilm formation of pneumococci, and measured biofilm formation and gene expression levels of the capsule gene cpsB and two other genes: autolysin encoding gene lytA and competence gene comA in different growth media in vitro. Twenty pneumococcal isolates were grown on polystyrene plates for 18 h in test media containing 0.5% xylitol, 0.5% glucose, 0.5% xylitol and 0.5% glucose, 0.5% fructose, 0.5% xylitol and 0.5% fructose or brain heart infusion (BHI) medium supplemented with 10% horse serum. Gene expression levels were measured after 5 h of growth using a relative quantification method with calibrator normalization. Exposure to xylitol lowered OD values, which were used as an indication of biofilm, compared with BHI medium, but when the medium was supplemented with glucose or fructose, biofilm formation was enhanced and the inhibitory effect of xylitol on biofilm formation was not observed. Xylitol also lowered lytA expression levels. Changes in biofilm formation in response to different sugar compounds may partly explain the efficacy of xylitol to prevent acute otitis media in previous clinical trials.