Real-time polymerase chain reaction on filter paper spotted samples: a gateway to molecular diagnosis of invasive bacterial diseases for rural areas in low-income countries.

BACKGROUND: Bacterial culture is the gold standard for the diagnosis of invasive bacterial diseases (IBDs) but molecular methods are more specific and sensitive. Fresh liquid samples (FLSs) show patent limitations for shipping and storage. We aimed to evaluate the sensitivity and specificity of real-time polymerase chain reaction (PCR) performed on dried sample spots (DSSs) obtained from different biological fluids compared with real-time PCR or culture performed on FLSs. METHODS: FLSs positive for Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Escherichia coli, Streptococcus pyogenes, Staphylococcus aureus, Bordetella pertussis and/or Pseudomonas aeruginosa were spotted on filter paper. Real-time PCR was performed on both FLSs and DSSs and results were compared. The stability of the DSS results over time was evaluated. RESULTS: Real-time PCR performed on 114 DSSs showed a specificity of 99.1% and a sensitivity of 91.2% for IBD diagnosis. A positive correlation was found between FLS cycle threshold (Ct) and DSS Ct (r=0.84; r2=0.71) with the Pearson statistical test and Bland-Altman analysis showing that 95% of the specimens were within agreeable limits. Although we observed a trend towards signal reduction over time in the DSSs, there was no statistical evidence of an increase in Ct values. Real-time PCR on DSSs was 2.2 times more sensitive than culture. CONCLUSIONS: Real-time PCR applied to DSSs may be a useful approach in different situations, such as IBD diagnosis, both for rural areas of low-income countries and family practitioners in various settings.

TREC and KREC in very preterm infants: reference values and effects of maternal and neonatal factors.

OBJECTIVE: T-cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) assays have been used for severe combined immunodeficiencies newborn screening (NBS). We assessed TREC and KREC NBS values in preterm infants and investigated if perinatal characteristics affect their values. METHODS: We performed a retrospective study collecting data from TREC and KREC NBS database and from mothers' and infants' medical charts. RESULTS: TREC and KREC values were lower in preterm infants born at 23-31 or 32-36 weeks of gestation than in term infants. Gestational age <28 weeks of gestation, leukopenia, and hypertensive disorders of pregnancy lowered TREC. Hypertensive disorders of pregnancy lowered KREC and intrapartum fever >38 °C increased it. Low TREC and KREC values were not associated to the risk of developing early-onset sepsis and late-onset sepsis. CONCLUSION: TREC and KREC levels are lower in preterm than term infants, but this did not increase the risk of neonatal sepsis.

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

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 < 0.0001. No increase in non-PCV13 serotypes was recorded. S. pneumoniae remained the most frequent pathogen identified in the post-PCV13 era in unvaccinated children with an unchanged serotype distribution: respectively 26/66 (39.4%), 25/66 (37.9%), 5/66 (7.6%), and 4/66 (6.1%) for 1, 3, 7F and 19A. On the other hand 7F and 19A disappeared in vaccinated children and serotype 1 and 3 decreased by 91.8% and 31.5%, respectively. Realtime PCR was significantly more sensitive than culture both in pleural fluid (79.7% vs 12.5%) and in blood (17.8% vs 7.4%). 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.

Culture and Real-time Polymerase Chain reaction sensitivity in the diagnosis of invasive meningococcal disease: Does culture miss less severe cases?

BACKGROUND: Invasive meningococcal disease (IMD) is a highly lethal disease. Diagnosis is commonly performed by culture or Realtime-PCR (qPCR). AIMS: Our aim was to evaluate, retrospectively, whether culture positivity correlates with higher bacterial load and fatal outcome. Our secondary aim was to compare culture and qPCR sensitivity. METHODS: The National Register for Molecular Surveillance was used as data source. Cycle threshold (CT), known to be inversely correlated with bacterial load, was used to compare bacterial load in different samples. RESULTS: Three-hundred-thirteen patients were found positive for Neisseria meningitidis by qPCR, or culture, or both; 41 died (case fatality rate 13.1%); 128/143 (89.5%) blood samples and 138/144 (95.8%) CSF were positive by qPCR, 37/143 (25.9%) blood samples and 45/144 (31.2%) CSF were also positive in culture. qPCR was 3.5 times (blood) or 3.1 times (CSF) more sensitive than culture in achieving a laboratory diagnosis of IMD (OR 24.4; 95% CI 12.2-49.8; p < .10-4; Cohen's κ 0.08 for blood and OR 49.0; 95% CI 19.1-133.4; p<10-4; Cohen's κ 0.02; for CSF). Positivity of culture did not correlate with higher bacterial loads in blood (mean CT 27.7±5.71, and CT 28.1±6.03, p = 0.739 respectively in culture positive or negative samples) or in CSF (mean CT 23.1±4.9 and 24.7±5.4 respectively in positive or negative CSF samples, p = 0.11).CT values in blood from patients who died were significantly lower than in patients who survived (respectively mean 18.0, range 14-23 and mean 29.6, range 16-39; p<10-17). No deaths occurred in patients with CT in blood over 23. Positive blood cultures were found in 10/25 (40%) patients who died and in 32/163 (19.6%) patients who survived, p = 0.036, OR 2.73; 95% CL 1.025-7.215), however 60% of deaths would have remained undiagnosed with the use of culture only. CONCLUSIONS: In conclusion our study demonstrated that qPCR is significantly (at least 3 times) more sensitive than culture in the laboratory confirmation of IMD. The study also demonstrated that culture negativity is not associated with lower bacterial loads and with less severe cases. On the other side, in patients with sepsis, qPCR can predict fatal outcome since higher bacterial load, evaluated by qPCR, appears strictly associated with most severe cases and fatal outcome. The study also showed that molecular techniques such as qPCR can provide a valuable addition to the proportion of diagnosed and serotyped cases of IMD.

Factors That Negatively Affect the Prognosis of Pediatric Community-Acquired Pneumonia in District Hospital in Tanzania.

Community-acquired pneumonia (CAP) is still the most important cause of death in countries with scarce resources. All children (33 months ± 35 DS) discharged from the Pediatric Unit of Itigi Hospital, Tanzania, with a diagnosis of CAP from August 2014 to April 2015 were enrolled. Clinical data were gathered. Dried blood spot (DBS) samples for quantitative real-time polymerase chain reaction (PCR) for bacterial detection were collected in all 100 children included. Twenty-four percent of patients were identified with severe CAP and 11% died. Surprisingly, 54% of patients were admitted with a wrong diagnosis, which increased complications, the need for antibiotics and chest X-rays, and the length of hospitalization. Comorbidity, found in 32% of children, significantly increased severity, complications, deaths, need for chest X-rays, and oxygen therapy. Malnourished children (29%) required more antibiotics. Microbiologically, Streptococcus pneumonia (S. p.), Haemophilus influenza type b (Hib) and Staphylococcus aureus (S. a.) were the bacteria more frequently isolated. Seventy-five percent of patients had mono-infection. Etiology was not correlated with severity, complications, deaths, oxygen demand, or duration of hospitalization. Our study highlights that difficult diagnoses and comorbidities negatively affect clinical evolution. S. p. and Hib still play a large role; thus, implementation of current vaccine strategies is needed. DBS is a simple and efficient diagnostic method for bacterial identification in countries with scarce resources.

PCV13 serotype decrease in Italian adolescents and adults in the post-PCV13 era: Herd protection from children or secular trend?

BACKGROUND AND AIM OF THE WORK: In 2010 PCV13 replaced PCV7 in the pediatric vaccination schedule for Italian children. While a strong herd effect was demonstrated for PCV7, a possible herd effect due to PCV13 is still under debate. Our aim was to evaluate differences in the distribution of pneumococcal serotypes between the pre and post-PCV13 eras in unvaccinated Italian adolescents and adults with laboratory-confirmed pneumococcal infection from 3 Italian Regions with a high rate of PCV13 vaccination of children. PATIENTS AND METHODS: Adolescents and adults admitted with laboratory-confirmed pneumococcal infection in the hospitals of 3 Italian Regions (Friuli-Venezia Giulia, Emilia Romagna, and Tuscany) between April 2006 and June 2016 were included in the study. Diagnosis of pneumococcal infection and serotyping were performed with Real Time PCR directly on normally sterile fluids or on culture isolates. RESULTS: 523 patients with laboratory-confirmed pneumococcal infection were enrolled (Male/Female ratio was 300/223, 1.3; median age 67.1, IQR 53.4-74.9). None of the patients had been vaccinated with any pneumococcal vaccine; 96.4% were serotyped. Overall, the most frequent serotypes were 3 (67/504, 13.3%), 8 (43/504, 8.5%), and 19A (38/504, 7.5%). Serotype distribution differed among age classes and clinical presentations. Overall, PCV13 serotypes accounted for 47.6% of cases: 62.3% in the pre-PCV13 era and 45.0% in the post-PCV13 era; (p=0.005 OR=2.03; CL 95%: 1.2-3.3). Serotype 7F accounted for 12/77 (15.6%) of all serotypes in the pre-PCV13 period and for 12/427 (2.8%) in the post-PCV13 period and was the only serotype significantly contributing to the difference in percentage between pre and post-PCV13 eras. CONCLUSION: Our study demonstrated a difference in percentage in serotype distribution in adolescents and adults laboratory-confirmed pneumococcal infection between the pre and post-PCV13 eras. This difference is mainly due to the decrease of serotype 7F. Thus, in order to decrease disease burden, adults and in particular the elderly should be offered a specific vaccination program.

Pneumococcal serotype distribution in adults with invasive disease and in carrier children in Italy: Should we expect herd protection of adults through infants' vaccination?

The 7-valent pneumococcal conjugate vaccine (PCV7) produced a significant herd protection in unvaccinated adult population mostly because of pneumococcus carriage decrease in vaccinated children. It is not known if the 13-valent pneumococcal vaccine can give similar effect on adults. Aims of the work were to evaluate whether the 6 additional serotypes are present in nasopharynx of children and serotype distribution in invasive pneumococcal infections (IPD) in adults. Realtime-PCR was used to evaluate pneumococcal serotypes in adults with confirmed IPD and in nasopharyngeal swabs (NP) from 629 children not vaccinated or vaccinated with PCV7 and resident in the same geographical areas. Two hundred twenty-one patients (116 males, median 67.9 years) with IPD were studied (pneumonia n = 103, meningitis n = 61 sepsis n = 50, other n = 7). Two hundred twelve were serotyped. The most frequent serotypes were 3, (31/212; 14.6%), 19A, (19/212; 9.0%), 12 (17/212; 8.0%), 7F, (14/212; 6.6%). In NP of children, the frequency of those serotypes causing over 50% of IPD in adults was very low, ranging from 0.48% for serotype 7F to 7.9% for serotype 19A. On the other side serotype 5, very frequent in NP (18.7%) caused <1% IPD. In conclusion serotypes causing IPD in adults are very rarely found in children NP. We suggest that herd protection obtainable with the additional 6 serotypes included in PCV13 may be more limited than that demonstrated with PCV7 in the past. In order to reduce the burden of disease in adults, adults should be offered a specific vaccination program with highly immunogenic PCV.

Mutant cohesin drives chromosomal instability in early colorectal adenomas.

Chromosome missegregation leads to chromosomal instability (CIN), thought to play a role in cancer development. As cohesin functions in guaranteeing correct chromosome segregation, increasing data suggest its involvement in tumorigenesis. In a screen of a large series of early colorectal adenomas, a precocious step during colorectal tumorigenesis, we identified 11 mutations in SMC1A core cohesin subunit. In addition, we sequenced the SMC1A gene in colorectal carcinomas and we found only one mutation. Finally, the transfection of the SMC1A mutations identified in early adenomas and wild-type SMC1A gene silencing in normal human fibroblasts led to CIN. Our findings that SMC1A mutations decrease from early adenomas to colorectal cancers and that mutations lead to CIN suggest that mutant cohesin could play a pivotal role during colorectal cancer development.