BackgroundPneumococcal 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 FindingsSensitivity 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 pneumococcal serotypes in nasopharyngeal swabs was found more frequently by Realtime PCR (19/30; 63.3%) than by Multiplex-sequential PCR (3/19; 15.8%; p = 0.003;95%CL 1.87–39.97).Conclusions/SignificanceIn conclusion, both MS-PCR and Realtime PCR can be used for pneumococcal serotyping of most serotypes/serogroups directly on clinical samples from culture-negative patients but Realtime-PCR appears more sensitive.

RT-PCR allows diagnosis and serotyping of pneumococcal bacteremic community-acquired pneumonia in children and is an important tool for evaluating serotype distribution in culture-negative samples.

Neisseria meningitidis group B (MenB) is a leading cause of meningitis and sepsis. A new vaccine has been recently licensed. The aim of the present study was to evaluate the epidemiology of MenB disease in pediatric age and define the optimal age for vaccination. All patients aged 0-18 years admitted with a diagnosis of meningitis or sepsis to the 83 participating Italian pediatric hospitals were included in the study. Blood and/or cerebrospinal fluid (CSF) samples were tested by Realtime-PCR and/or culture. One hundred and thirty-six cases (mean age 5.0 years, median 2.7) of MenB disease were found. Among these, 96/136 (70.6%) were between 0 and 5 years, 61/136 (44.9%) were between 0 and 2 years. Among the latter, 39/61 (63.9%) occurred during the first year of life with highest incidence between 4 and 8 months. A case-fatality rate of 13.2% was found, with 27.8% cases below 12 months. Sepsis lethality was 24.4%. RT-PCR was significantly more sensitive than culture: 82 patients were tested at the same time by both methods, either in blood or in CSF; MenB was found by RT-PCR in blood or CSF in 81/82 cases (98.8%), culture identified 27/82 (32.9%) infections (Cohen's Kappa 0.3; McNemar's: p<10⁻⁵). The study shows that the highest incidence of disease occurs in the first year of age, with a peak between 4 and 8 months of life; 30% of deaths occur before 12 months. The results suggest that the greatest prevention could be obtained starting MenB vaccination in the first months of life; a catch-up strategy up to the fifth year of life could be considered. Our results also confirm that Realtime PCR is significantly more sensitive than culture. In those countries where only isolate positive infections are counted as cases, the incidence of MenB infection results highly underestimated.

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