Realtime PCR Is More Sensitive than Multiplex PCR for Diagnosis and Serotyping in Children with Culture Negative Pneumococcal Invasive Disease

Background: Pneumococcal serotyping is usually performed by Quellung reaction, considered the gold standard test. However the method cannot be used on culture-negative samples. Molecular methods can be a useful alternative. The aim of the study was to evaluate the use of Multiplex-sequential-PCR (MS-PCR) or Realtime-PCR on blood samples for diagnosis and serotyping of invasive pneumococcal disease (IPD) in a pediatric clinical setting. Methodology/Principal Findings: Sensitivity and specificity of MS-PCR and Realtime-PCR have been evaluated both on 46 well characterized pneumococcal isolates and on 67 clinical samples from children with culture-negative IPD. No difference in sensitivity and specificity between MS-PCR and Realtime PCR was found when the methods were used on isolates: both methods could type 100 % isolates and the results were always consistent with culture-based methods. On the contrary, when used on clinical samples 43/67 (64.2%) were typeable by MS-PCR and 61/67 (91.0%) by Realtime-PCR (p = 0.0004,K Cohen 0.3, McNemar’s p,0.001). Non-typeability by MS-PCR was associated in 18/20 cases (90.0%) with low bacterial load. The difference between the two methods was present both when they were used on normally sterile fluids (respectively 31/ 33 (93.9%) typeable samples for Realtime-PCR and 24/33 (72.7%) for MS-PCR, p = 0.047, 95%CL 0.03–0.98; K Cohen 0.3; McNemar’s p = 0.0016) and when they were used on nasopharyngeal swabs (respectively 30/34 (88.2%) typeable samples for Realtime-PCR and 19/34 (55.9%) for MS-PCR, p = 0.007, 95%CL 0.04–0.66); the presence of multiple pneumococca

Pneumococcal DNA is not detectable in the blood of healthy carrier children by real-time PCR targeting the lytA gene

The diagnosis of invasive pneumococcal disease (IPD) is currently based on culture methods, which lack sensitivity, especially after antibiotic therapy. Molecular methods have improved sensitivity and do not require viable bacteria; however, their use is complicated by reports of low specificity with some assays. The present study investigated the specificity of a real-time PCR targeting lytA for the detection of IPD. A group of 147 healthy children, aged 6 months to 16 years (mean 6.4 years, median 4.9 years, interquartile range 6.4 years), who were in hospital for routine examinations, were tested for pneumococcal carrier status and for the presence of detectable pneumococcal DNA in their blood by real-time PCR targeting the pneumococcal lytA gene. In addition, 35 culture-positive biological samples were analysed. Urine was examined for the presence of pneumococcal DNA and C-polysaccharide antigen. Carriage was detected in 77 of the 147 subjects (52.4 %); however, regardless of carrier status, none of the subjects had a positive result from blood. Analysis of the culture-positive biological samples yielded positive results in 100 % (15/15) of cerebrospinal fluid samples and 95 % (19/20) of blood samples. All urine samples from healthy carriers were negative for DNA, whilst antigenuria was detected in 44/77 carriers (57.1 %). In conclusion, real-time PCR is both sensitive and specific and can be a useful tool in the routine diagnosis of IPD. Its sensitivity, which surpasses that of other methods for this purpose, does not come at the cost of reduced specificity

Significant impact of pneumococcal conjugate vaccination on pediatric parapneumonic effusion: Italy 2006-2018

Abstract Etiology and serotyping of parapneumonic effusion (PPE) and the impact of vaccination was evaluated over a 12-year period, before and after the PCV13 introduction (2011) for Italian children From 0 to 16 years of age. Five hundred and two children were evaluated; 226 blood and 356 pleural fluid samples were obtained and tested using Realtime-PCR and culture. In the pre-PCV13 era S. pneumoniae was the most frequent pathogen identified (64/90; 71.1%) with a large predominance of serotypes 1 (42.4%), 3 (23.7%), 7F (5.1%) and 19A (11.9%). The impact of vaccination, calculated on children 0–8 years of age, demonstrated a significant reduction of PPE: with an incidence rate of 2.82 (95%CL 2.32–3.41) in the pre-PCV13 era and an age-standardized rate (ASR) of 0.66 (95% CL 0.37–1.99) in the post-PCV13 era, p  In conclusion, our findings indicate that routine immunization with PCV13 has significantly reduced the burden of childhood PPE in vaccinated children, without increasing PPE due to other bacteria and without serotype shift. Moreover, the impact of PCV13 may be underestimated due to the increase in pneumococcal surveillance in Italy. Data has also shown that Real-time PCR is an essential tool to better define the etiology of PPE and to monitor vaccination plans. Longer studies will be necessary to evaluate the role of herd protection in PPE prevention

Primer and probe sets for pneumococcal serotyping by Realtime PCR.

<p>(Primers included in the MS-PCR analysis were the following: 1, 3, 4, 5, 6A/B, 7F/A, 7C/B, 8, 9V/A, 10A, 11A/D/F, 12A/B/F, 14, 15A, 15B/C, 16F, 17F, 18A/B/C/F, 19A, 19F, 20, 22A/F, 23F, 31, 33A/F, 34, 35B, 35F, 38F <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009282#pone.0009282-Azzari2" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009282#pone.0009282-Pai1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009282#pone.0009282-Pai2" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009282#pone.0009282-Pimenta1" target="_blank">[38]</a>.</p

Number of typeable or non-typeable samples obtained from patients with culture-negative invasive pneumococcal infections as evidenced by Multiplex Sequential PCR or Realtime-PCR (sensitivity of Realtime vs Multiplex Sequential PCR p = 0.0004; 95%CL 1.98–17.05).

<p>Number of typeable or non-typeable samples obtained from patients with culture-negative invasive pneumococcal infections as evidenced by Multiplex Sequential PCR or Realtime-PCR (sensitivity of Realtime vs Multiplex Sequential PCR p = 0.0004; 95%CL 1.98–17.05).</p

Presence of single or multiple pneumococcal serogroups/serotypes in nasopharyngeal swabs as evidenced by Multiplex Sequential PCR or Realtime-PCR (frequency of multiple serotypes/serogroup evaluated with Realtime PCR vs Multiple Sequential PCR p = 0.003; 95%CL = 1.87–39.97).

<p>Presence of single or multiple pneumococcal serogroups/serotypes in nasopharyngeal swabs as evidenced by Multiplex Sequential PCR or Realtime-PCR (frequency of multiple serotypes/serogroup evaluated with Realtime PCR vs Multiple Sequential PCR p = 0.003; 95%CL = 1.87–39.97).</p

Potential serotype coverage of three pneumococcal conjugate vaccines against invasive pneumococcal infection in Italian children

Since the introduction of the 7-valent vaccine, invasive pneumococcal disease have greatly decreased; however, changes in the distribution of pneumococcal serotypes have recently highlighted the need for vaccines with wider coverage. The aim of the work was to assess the potential serotype coverage of three pneumococcal conjugate vaccines (7-, 10- and 13-valent) against bacteremic pneumococcal pneumonia and meningitis/sepsis in Italian children