Publications by authors named "Romina Camilli"

26 Publications

  • Page 1 of 1

Global Landscape Review of Serotype-Specific Invasive Pneumococcal Disease Surveillance among Countries Using PCV10/13: The Pneumococcal Serotype Replacement and Distribution Estimation (PSERENADE) Project.

Microorganisms 2021 Apr 2;9(4). Epub 2021 Apr 2.

National Public Health Organisation, 15123 Athens, Greece.

Serotype-specific surveillance for invasive pneumococcal disease (IPD) is essential for assessing the impact of 10- and 13-valent pneumococcal conjugate vaccines (PCV10/13). The Pneumococcal Serotype Replacement and Distribution Estimation (PSERENADE) project aimed to evaluate the global evidence to estimate the impact of PCV10/13 by age, product, schedule, and syndrome. Here we systematically characterize and summarize the global landscape of routine serotype-specific IPD surveillance in PCV10/13-using countries and describe the subset that are included in PSERENADE. Of 138 countries using PCV10/13 as of 2018, we identified 109 with IPD surveillance systems, 76 of which met PSERENADE data collection eligibility criteria. PSERENADE received data from most (n = 63, 82.9%), yielding 240,639 post-PCV10/13 introduction IPD cases. Pediatric and adult surveillance was represented from all geographic regions but was limited from lower income and high-burden countries. In PSERENADE, 18 sites evaluated PCV10, 42 PCV13, and 17 both; 17 sites used a 3 + 0 schedule, 38 used 2 + 1, 13 used 3 + 1, and 9 used mixed schedules. With such a sizeable and generally representative dataset, PSERENADE will be able to conduct robust analyses to estimate PCV impact and inform policy at national and global levels regarding adult immunization, schedule, and product choice, including for higher valency PCVs on the horizon.
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http://dx.doi.org/10.3390/microorganisms9040742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066045PMC
April 2021

Serotype Distribution of Remaining Pneumococcal Meningitis in the Mature PCV10/13 Period: Findings from the PSERENADE Project.

Microorganisms 2021 Apr 1;9(4). Epub 2021 Apr 1.

Department of Clinical Microbiology, Landspitali-The National University Hospital, Hringbraut, 101 Reykjavik, Iceland.

Pneumococcal conjugate vaccine (PCV) introduction has reduced pneumococcal meningitis incidence. The Pneumococcal Serotype Replacement and Distribution Estimation (PSERENADE) project described the serotype distribution of remaining pneumococcal meningitis in countries using PCV10/13 for least 5-7 years with primary series uptake above 70%. The distribution was estimated using a multinomial Dirichlet regression model, stratified by PCV product and age. In PCV10-using sites ( = 8; cases = 1141), PCV10 types caused 5% of cases <5 years of age and 15% among ≥5 years; the top serotypes were 19A, 6C, and 3, together causing 42% of cases <5 years and 37% ≥5 years. In PCV13-using sites ( = 32; cases = 4503), PCV13 types caused 14% in <5 and 26% in ≥5 years; 4% and 13%, respectively, were serotype 3. Among the top serotypes are five (15BC, 8, 12F, 10A, and 22F) included in higher-valency PCVs under evaluation. Other top serotypes (24F, 23B, and 23A) are not in any known investigational product. In countries with mature vaccination programs, the proportion of pneumococcal meningitis caused by vaccine-in-use serotypes is lower (≤26% across all ages) than pre-PCV (≥70% in children). Higher-valency PCVs under evaluation target over half of remaining pneumococcal meningitis cases, but questions remain regarding generalizability to the African meningitis belt where additional data are needed.
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http://dx.doi.org/10.3390/microorganisms9040738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066874PMC
April 2021

Changes in Invasive Pneumococcal Disease Caused by Serotype 1 Following Introduction of PCV10 and PCV13: Findings from the PSERENADE Project.

Microorganisms 2021 03 27;9(4). Epub 2021 Mar 27.

National Centre for Immunisation Research and Surveillance and Discipline of Child and Adolescent Health, Children's Hospital Westmead Clinical School, Faculty of Medicine and Health, University of Sydney, Westmead, NSW 2145, Australia.

serotype 1 (ST1) was an important cause of invasive pneumococcal disease (IPD) globally before the introduction of pneumococcal conjugate vaccines (PCVs) containing ST1 antigen. The Pneumococcal Serotype Replacement and Distribution Estimation (PSERENADE) project gathered ST1 IPD surveillance data from sites globally and aimed to estimate PCV10/13 impact on ST1 IPD incidence. We estimated ST1 IPD incidence rate ratios (IRRs) comparing the pre-PCV10/13 period to each post-PCV10/13 year by site using a Bayesian multi-level, mixed-effects Poisson regression and all-site IRRs using a linear mixed-effects regression (N = 45 sites). Following PCV10/13 introduction, the incidence rate (IR) of ST1 IPD declined among all ages. After six years of PCV10/13 use, the all-site IRR was 0.05 (95% credibility interval 0.04-0.06) for all ages, 0.05 (0.04-0.05) for <5 years of age, 0.08 (0.06-0.09) for 5-17 years, 0.06 (0.05-0.08) for 18-49 years, 0.06 (0.05-0.07) for 50-64 years, and 0.05 (0.04-0.06) for ≥65 years. PCV10/13 use in infant immunization programs was followed by a 95% reduction in ST1 IPD in all ages after approximately 6 years. Limited data availability from the highest ST1 disease burden countries using a 3+0 schedule constrains generalizability and data from these settings are needed.
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http://dx.doi.org/10.3390/microorganisms9040696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066231PMC
March 2021

The changing epidemiology of carbapenemase-producing Klebsiella pneumoniae in Italy: toward polyclonal evolution with emergence of high-risk lineages.

J Antimicrob Chemother 2021 Jan;76(2):355-361

Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy.

Background: Previous studies showed that the epidemic of carbapenem-resistant Klebsiella pneumoniae (CR-KP) observed in Italy since 2010 was sustained mostly by strains of clonal group (CG) 258 producing KPC-type carbapenemases. In the framework of the National Antibiotic-Resistance Surveillance (AR-ISS), a countrywide survey was conducted in 2016 to explore the evolution of the phenotypic and genotypic characteristics of CR-KP isolates.

Methods: From March to July 2016, hospital laboratories participating in AR-ISS were requested to provide consecutive, non-duplicated CR-KP (meropenem and/or imipenem MIC >1 mg/L) from invasive infections. Antibiotic susceptibility was determined according to EUCAST recommendations. A WGS approach was adopted to characterize the isolates by investigating phylogeny, resistome and virulome.

Results: Twenty-four laboratories provided 157 CR-KP isolates, of which 156 were confirmed as K. pneumoniae sensu stricto by WGS and found to carry at least one carbapenemase-encoding gene, corresponding in most cases (96.1%) to blaKPC. MLST- and SNP-based phylogeny revealed that 87.8% of the isolates clustered in four major lineages: CG258 (47.4%), with ST512 as the most common clone, CG307 (19.9%), ST101 (15.4%) and ST395 (5.1%). A close association was identified between lineages and antibiotic resistance phenotypes and genotypes, virulence traits and capsular types. Colistin resistance, mainly associated with mgrB mutations, was common in all major lineages except ST395.

Conclusions: This WGS-based survey showed that, although CG258 remained the most common CR-KP lineage in Italy, a polyclonal population has emerged with the spread of the new high-risk lineages CG307, ST101 and ST395, while KPC remained the most common carbapenemase.
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http://dx.doi.org/10.1093/jac/dkaa431DOI Listing
January 2021

Differential Responses of Human Dendritic Cells to Live or Inactivated : Impact on Cytokine Production and T Helper Expansion.

Front Immunol 2019 12;10:2622. Epub 2019 Nov 12.

Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy.

Understanding ()-host immune system interaction is crucial to meet the tremendous medical need associated with this life-threatening bacterial infection. Given the crucial role of dendritic cells (DC) in dictating immune responses upon microbial challenge, we investigated how the bacterial viability and the conservation of structural integrity influence the response of human DC to . To this end, human primary DC were stimulated with the methicillin-resistant USA300 live strain, USA300 inactivated by heat (HI), ultraviolet irradiation (UVI), or paraformaldehyde treatment (PFAI) and subsequently analyzed for cell phenotype and immune-modulatory properties. Although no differences in terms of DC viability and maturation were observed when DC were stimulated with live or inactivated bacteria, the production of IL-12, IL-23, and other cytokines differed significantly. The Th1 and Th17 expansion was also more pronounced in response to live vs. inactivated . Interestingly, cytokine production in DC treated with live and inactivated USA300 required phagocytosis, whereas blocking endosomal Toll-like receptor signaling mainly reduced the cytokine release by live and HI USA300. A further analysis of IFN-β signaling revealed the induction of a cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING)-independent and IRF3-dependent signaling pathway(s) in UVI-stimulated DC. This study underscores the capacity of human DC to discriminate between live and inactivated and, further, indicates that DC may represent a valuable experimental setting to test different inactivation methods with regard to the retention of immunoregulatory properties. These and further insights may be useful for the development of novel therapeutic and prophylactic anti- vaccine strategies.
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http://dx.doi.org/10.3389/fimmu.2019.02622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861420PMC
November 2020

Pneumococcal carriage among adults aged 50 years and older with co-morbidities attending medical practices in Rome, Italy.

Vaccine 2019 08 5;37(35):5096-5103. Epub 2019 Jul 5.

Department of Infectious Diseases, Istituto Superiore di Sanità, Rome. Electronic address:

Background: Data on Streptococcus pneumoniae carriage in adults with co-morbidities are limited. In this study we estimated the pneumococcal carriage among adults with co-morbidities and evaluated socio-demographic and clinical risk factors. The potential coverage of the current pneumococcal vaccines recommended for adults (PCV13 and PPV23) was also investigated.

Methods: A cross-sectional study on S. pneumoniae carriage among unvaccinated adults ≥50 years with co-morbidities, presenting with or without acute respiratory symptoms at general practitioners in Rome, Italy, between October 2015 and July 2016 was conducted. Pneumococcal carriage was investigated by both cultural and molecular methods. Socio-demographic variables and co-morbidities were evaluated by logistic models as possible risk factors for pneumococcal carriage.

Results: Out of 248 patients (median age: 73 yrs; IQR: 65-79), 12 (4.8%) and 83 (33.5%) individuals were found colonized using cultural or molecular methods, respectively. Potential risk factors for pneumococcal colonization as ascertained by molecular methods were: low level of education (adjusted OR = 3.71, 95% CI: 1.62-9.40), winter months (December-March vs other months, adjusted OR = 2.56, 95% CI: 1.29-5.14), and presence of chronic lung diseases (adjusted OR = 2.18, 95% CI: 1.15-4.16). The combination of serotype-specific multiplex RT-PCR and conventional PCR allowed to identify 22 serotypes/group of serotypes, of which the most common were: 24F/24A/24B, 12F/12A/12B/44/46, 6A/6B, 14, 15B/15C, and 22F/22A. Prevalence of pneumococcal carriage due to PCV13 serotypes and non-PCV13 serotypes was 23.6% and 67.3%, respectively. Prevalence of colonization due to PPV23 serotypes was estimated to be 54.6%.

Conclusions: A high prevalence of S. pneumoniae carriage was observed among adults with co-morbidities, especially among individuals affected by chronic lung diseases. These results support vaccine strategies based on the sequential administration of PCV13 and PPV23 to control potentially invasive pneumococcal strains in adults, especially in subjects with co-morbidities.
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http://dx.doi.org/10.1016/j.vaccine.2019.06.052DOI Listing
August 2019

Risk factors for Haemophilus influenzae and pneumococcal respiratory tract colonization in CVID.

J Allergy Clin Immunol 2018 12 28;142(6):1999-2002.e3. Epub 2018 Aug 28.

Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2018.08.014DOI Listing
December 2018

Esx Factors Control Human Dendritic Cell Functions Conditioning Th1/Th17 Response.

Front Cell Infect Microbiol 2017 21;7:330. Epub 2017 Jul 21.

Department of Infectious Diseases, Istituto Superiore di SanitàRome, Italy.

The opportunistic pathogen () is a major cause of nosocomial- and community-acquired infections. In addition, many antibiotic-resistant strains are emerging worldwide, thus, there is an urgent unmet need to pinpoint novel therapeutic and prophylactic strategies. In the present study, we characterized the impact of infection with the pandemic methicillin-resistant USA300 strain on human primary dendritic cells (DC), key initiators and regulators of immune responses. In particular, among staphylococcal virulence factors, the function of EsxA and EsxB, two small acidic dimeric proteins secreted by the type VII-like secretion system Ess (ESAT-6-like secretion system), was investigated in human DC setting. A comparative analysis of bacterial entry, replication rate as well as DC maturation, apoptosis, signaling pathway activation and cytokine production was performed by using wild type (wt) USA300 and three isogenic mutants carrying the deletion of (Δ), (Δ), or both genes (Δ). The mutant lacking only the EsxA protein (Δ stimulated a stronger pro-apoptotic phenotype in infected DC as compared to wt USA300, Δ, and Δ strains. When the mutant carrying the deletion (Δ) was analyzed, a higher production of both regulatory and pro-inflammatory mediators was found in the infected DC with respect to those challenged with the wt counterpart and the other mutants. In accordance with these data, supernatant derived from Δ-infected DC promoted a stronger release of both IFN-γ and IL-17 from CD4 T cells as compared with those conditioned with supernatants derived from wild type USA300-, Δ-, and Δ-infected cultures. Although, the interaction of with human DC is not yet fully understood, our data suggest that both cytokine production and apoptotic process are modulated by Esx factors, thus indicating a possible role of these proteins in the modulation of DC-mediated immunity to .
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http://dx.doi.org/10.3389/fcimb.2017.00330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519619PMC
April 2018

Impact of pneumococcal conjugate vaccine (PCV7 and PCV13) on pneumococcal invasive diseases in Italian children and insight into evolution of pneumococcal population structure.

Vaccine 2017 08 14;35(35 Pt B):4587-4593. Epub 2017 Jul 14.

Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy.

Background: The use of PCV7 for children immunization was gradually implemented in the Italian regions starting from 2006 and was replaced by PCV13 in 2010-2011. In this study we aimed to assess the PCV impact on invasive pneumococcal diseases (IPD) incidence, serotype distribution and antibiotic resistance in Italian children under 5years old.

Methods: All IPD cases in children from 5 Italian regions (Emilia-Romagna, Lombardia, A. P. Bolzano, A. P. Trento, and Piemonte) reported through the nationwide surveillance system during 2008-2014 were included in this study. Pneumococcal isolates were subjected to serotyping, antibiotic susceptibility testing, and clonal analysis according to standard methods.

Results: During the study period overall IPD incidence decreased from 7.8 cases/100,000 inhabitants in 2008 to 3.0 cases/100,000 in 2014 (61% decrease, P<0.001). In particular, from 2008 to 2014, PCV7-type IPD decreased from 2.92 to 0.13 cases/100,000 inhabitants (95% decrease, P<0.001) while PCV13-non-PCV7 type IPD decreased from 3.2 to 0.89 cases/100,000 inhabitants (72% decrease, P=0.008). Conversely, non-vaccine serotype (NVS) IPD increased overtime, becoming more common than PCV13 serotype IPD in 2013-2014. Emergent NVS 24F and 12F were the most prevalent in 2014. Antibiotic resistance testing revealed an overall increasing trend in penicillin resistance, from 14% in 2008 to 23% in 2014. Erythromycin resistance showed a downward trend, from 38% in 2008 to 27% in 2014. While in 2008 PCV13 serotypes were the major responsible for antibiotic resistance, during the following years antimicrobial resistance due to NVS increased, mainly as a result of expansion of pre-existing clones.

Conclusions: Both PCVs led to a substantial decrease in vaccine-related IPD incidence in the children population. However NVS-related IPD increased, becoming the most prevalent in the last two-years period. Continuous surveillance is an essential tool to monitor evolution of pneumococcal population causing IPD in children.
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http://dx.doi.org/10.1016/j.vaccine.2017.07.010DOI Listing
August 2017

ICESpy009, a Conjugative Genetic Element Carrying mef(E) in Streptococcus pyogenes.

Antimicrob Agents Chemother 2016 07 20;60(7):3906-12. Epub 2016 Jun 20.

Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

Efflux-mediated macrolide resistance due to mef(E) and mel, carried by the mega element, is common in Streptococcus pneumoniae, for which it was originally characterized, but it is rare in Streptococcus pyogenes In S. pyogenes, mega was previously found to be enclosed in Tn2009, a composite genetic element of the Tn916 family containing tet(M) and conferring erythromycin and tetracycline resistance. In this study, S. pyogenes isolates containing mef(E), apparently not associated with other resistance determinants, were examined to characterize the genetic context of mega. By whole-genome sequencing of one isolate, MB56Spyo009, we identified a novel composite integrative and conjugative element (ICE) carrying mega, designated ICESpy009, belonging to the ICESa2603 family. ICESpy009 was 55 kb long, contained 61 putative open reading frames (ORFs), and was found to be integrated into hylA, a novel integration site for the ICESa2603 family. The modular organization of the ICE was similar to that of members of the ICESa2603 family carried by different streptococcal species. In addition, a novel cluster of accessory resistance genes was found inside a region that encloses mega. PCR mapping targeting ICESpy009 revealed the presence of a similar ICE in five other isolates under study. While in three isolates the integration site was the same as that of ICESpy009, in two isolates the ICE was integrated into rplL, the typical integration site of the ICESa2603 family. ICESpy009 was able to transfer macrolide resistance by conjugation to both S. pyogenes and S. pneumoniae, showing the first evidence of the transferability of mega from S. pyogenes.
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http://dx.doi.org/10.1128/AAC.03082-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914681PMC
July 2016

Invasive pneumococcal disease in children and adults in seven Italian regions after the introduction of the conjugate vaccine, 2008-2014.

Epidemiol Prev 2015 Jul-Aug;39(4 Suppl 1):134-8

Dip. Malattie infettive, parassitarie e immunomediate, Istituto superiore di sanità, Roma, Italy.

Objective: To describe the trend of invasive pneumococcal disease in the years 2008-2014; to verify the impact of the conjugate vaccine and monitor the occurrence of serotype replacement.

Design: Prospective observational study based on data from the national surveillance for invasive bacterial diseases coordinated by the Istituto superiore di sanità.

Setting And Participants: Seven Italian regions (A.P. Bolzano, A.P. Trento, Emilia-Romagna, Friuli-Venezia Giulia, Lombardia, Piemonte, Veneto), accounting for 43% of the national population.

Main Outcome Measures: Number of cases and incidence of invasive pneumococcal diseases: global, stratified by age groups and by serotypes included or not in the PCV13.

Results: In 2008-2014, in the 0-4 age group IPD incidence for all serotypes decreased from 7.1 to 2.9/100,000; incidence for vaccine serotypes (VT) decreased from 5.5 to 1.1/100,000, while incidence for non-vaccine serotypes (NVT) increased from 1.6 to 2.0/100,000 (2.5 in 2013). In the >64 age group, IPD incidence increased from 5.3 to 7.5/100,000; VT incidence decreased from 3.9 to 3.2 (4.9 in 2010 and 4.3 in 2013), whereas NVT incidence increased from 1.4 to 4.4/100,000.

Conclusion: Use of the conjugate vaccine has reduced the number of cases of IPD by VT in children; the increase in IPD by NVT, above all in older age groups, suggests a serotype replacement.
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May 2017

Carriage of Haemophilus influenzae is associated with pneumococcal vaccination in Italian children.

Vaccine 2015 Aug 16;33(36):4559-64. Epub 2015 Jul 16.

Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy. Electronic address:

Background: The pneumococcal population changes observed after the implementation of children immunization with pneumococcal conjugative vaccines (PCV) might have affected the composition of the microbial flora inhabiting the same ecological niche of Streptococcus pneumoniae. The aim of this study was to investigate the effect of PCV immunization, (PCV7 or PCV13), on S. pneumoniae and Haemophilus influenzae colonization in young children in Italy.

Methods: Nasopharyngeal swabs were obtained from 301 children under 6 years of age (vaccinated or unvaccinated with PCV) during the period January-April 2012. Presence of S. pneumoniae and H. influenzae was investigated using conventional cultural methods. S. pneumoniae isolates were serotyped by the Quellung reaction; capsular type of H. influenzae isolates was determined by PCR. The pattern of associations between the two species and potential risk factors were investigated by a Structural Equation Modelling (SEM) analysis.

Results: The prevalence of carriage was 31.56% and 43.18% for S. pneumoniae and H. influenzae, respectively. The majority of S. pneumoniae isolates belonged to non vaccine serotypes (non PCV13-types 81.1%) while H. influenzae isolates were all non-typeable. SEM analysis revealed a synergistic association between S. pneumoniae and H. influenzae colonization (rho: 0.27; 95%CI: 0.09-0.46; p=0.004). In addition, children vaccinated with PCV, either with PCV7 (coef 0.43; 95%CI: 0.07-0.79; p=0.021) or with PCV13 (coef: 0.45; 95%CI: 0.08-0.82; p=0.018), were more likely to be colonized by H. influenzae.

Conclusions: Pneumococcal vaccination increased H. influenzae nasopharyngeal carriage in children. This result highlights that an indirect effect of PCV vaccination can be perturbation of the nasopharyngeal flora. In the era of higher-valent pneumococcal vaccines, surveillance of carriage is crucial to monitor alterations in the bacterial ecosystem, thus preventing possible clinical problems.
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http://dx.doi.org/10.1016/j.vaccine.2015.07.009DOI Listing
August 2015

Characterization of Streptococcus pneumoniae clones from paediatric patients with cystic fibrosis.

J Med Microbiol 2014 Dec 9;63(Pt 12):1704-1715. Epub 2014 Oct 9.

Integrated Research Centre (CIR), University Campus Biomedico, Via Alvaro del Portillo 200, 00128 Rome, Italy.

The role of Streptococcus pneumoniae in cystic fibrosis (CF) is poorly understood. The pneumococcal population has changed over time after the introduction of the heptavalent conjugate vaccine (PCV7) and, more recently, the 13-valent conjugate vaccine (PCV13). Although serotypes and clones causing invasive pneumococcal disease or colonizing healthy children have been extensively analysed, little is known so far on the serotypes and clones of pneumococci in CF patients. The aim of this work was to investigate serotypes, antibiotic susceptibilities, genotypes and biofilm production of CF pneumococcal isolates. Overall, 44 S. pneumoniae strains collected from 32 paediatric CF patients from January 2010 to May 2012 in a large Italian CF Centre were tested for antimicrobial susceptibility testing by Etest, serotyped by the Quellung reaction and genotyped by a combination of different molecular typing methods, including pbp gene restriction profiling, pspA restriction profiling and sequencing, PFGE and multilocus sequence typing. Biofilm production by pneumococcal strains was also assessed. Penicillin non-susceptibility was 16 %. High resistance rates (>56 %) were observed for erythromycin, clindamycin and tetracycline. The most frequent serotype recovered was serotype 3 (31.8 %). The coverage of PCV7 and PCV13 was 6.8 and 47.7 %, respectively. More than 80 % of CF strains belonged to Pneumococcal Molecular Epidemiology Network (PMEN) reference clones, the most common being Netherlands(3)-ST180 (28.2 %), and Greece(21)-30/ST193 (15.4 %). All strains produced biofilm in vitro, although with large variability in biofilm formation efficiency. No correlation was found between biofilm levels and serotype, clone or antibiotic resistance. The high isolation rate of antibiotic-resistant serotype 3 pneumococci from CF patients suggests that PCV13 could increase protection from pneumococcal colonization and infection.
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http://dx.doi.org/10.1099/jmm.0.072199-0DOI Listing
December 2014

Identification of Streptococcus pneumoniae serotype 11E, serovariant 11Av and mixed populations by high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy and flow cytometric serotyping assay (FCSA).

PLoS One 2014 26;9(6):e100722. Epub 2014 Jun 26.

Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

Background: Recent studies have identified Streptococcus pneumoniae serotype 11E and serovariant 11Av among isolates previously typed as 11A by classical serotyping methods. Serotype 11E and serovariant 11Av differ from serotype 11A by having totally or partially inactive wcjE, a gene in cps locus coding for an O-acetyl transferase. Serotype 11E is rare among carriage isolates but common among invasive isolates suggesting that it survives better during invasion. Aim of this work was to investigate the epidemiology of serotype 11A in a pneumococcal collection using a new serotyping approach based on High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) spectroscopy to distinguish serotypes 11A and 11E.

Methods: A collection of 48 (34 invasive and 14 carriage) S. pneumoniae isolates from Italy, previously identified as serotype 11A by the Quellung reaction, were investigated by wcjE sequencing, HR-MAS NMR spectroscopy and the reference flow cytometric serotyping assay (FCSA) based on monoclonal antibodies.

Results: HR-MAS NMR spectra from serotypes 11A and 11E showed different NMR peaks indicating that HR-MAS NMR could be used to distinguish these serotypes, although HR-MAS NMR could not distinguish serotype 11Av from serotype 11E unambiguously. Thirty-eight isolates were confirmed to be serotype 11A, 8 isolates with a mutated wcjE were serotype 11E, 1 isolate belonged to serovariant 11Av, and 1 isolate was a mixed population 11A/11Av. All 11E isolates were identified among invasive isolates.

Conclusions: We proved that HR-MAS NMR can be of potential use for pneumococcal serotyping. The detection of serotype 11E among invasive isolates in our collection, supports previous epidemiological studies suggesting that mutations in wcjE can represent a mechanism promoting pneumococcal survival during invasion. The discovery of a spectrum of immunochemical diversity within established serotypes should stimulate efforts to develop new serotyping approaches.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0100722PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072641PMC
February 2015

Pneumococcal carriage in young children one year after introduction of the 13-valent conjugate vaccine in Italy.

PLoS One 2013 4;8(10):e76309. Epub 2013 Oct 4.

Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

Background: In mid 2010, the 7-valent pneumococcal conjugate vaccine (PCV7) was replaced by the 13-valent conjugate vaccine (PCV13) for childhood immunization in Italy. Our objective in this study was to obtain a snapshot of pneumococcal carriage frequency, colonizing serotypes, and antibiotic resistance in healthy children in two Italian cities one year after PCV13 was introduced.

Methods: Nasopharyngeal swabs were obtained from 571 children aged 0-5 years from November 2011-April 2012. Pneumococcal isolates were serotyped and tested for antimicrobial susceptibility. Penicillin and/or erythromycin non-susceptible isolates were analyzed by Multi Locus Sequence Typing (MLST).

Results: Among the children examined, 81.2% had received at least one dose of PCV7 or PCV13 and 74.9% had completed the recommended vaccination schedule for their age. Among the latter, 57.3% of children had received PCV7, 27.1% PCV13, and 15.6% a combination of the two vaccines. The overall carriage rate was 32.9%, with children aged 6-35 months the most prone to pneumococcal colonization (6-23 months OR: 3.75; 95% CI: 2.19-6.43 and 24-35 months OR: 3.15, 95%CI: 2.36-4.22). A total of 184 pneumococcal isolates were serotyped and divided into PCV7 (5.4%), PCV13 (18.0%), and non-PCV13 (82.0%) serotypes. Serotypes 6C, 24F, and 19A were the most prevalent (10.3%, 8.6%, and 8.1%, respectively). The proportion of penicillin non-susceptible (MIC >0.6 mg/L) isolates was 30.9%, while 42.3% were erythromycin resistant. Non-PCV13 serotypes accounted for 75.4% and 70.8% of the penicillin and erythromycin non-susceptible isolates, respectively.

Conclusions: Our results revealed low rates of PCV7 and PCV13 serotypes in Italian children, potentially due to the effects of vaccination. As the use of PCV13 continues, its potential impact on vaccine serotypes such as 19A and cross-reactive serotypes such as 6C will be assessed, with this study providing a baseline for further analysis of surveillance isolates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076309PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790677PMC
July 2014

Increase of pneumococcal serotype 19A in Italy is due to expansion of the piliated clone ST416/CC199.

J Med Microbiol 2013 Aug 30;62(Pt 8):1220-1225. Epub 2013 May 30.

Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

The emergence of Streptococcus pneumoniae serotype 19A, following use of the heptavalent pneumococcal conjugate vaccine (PCV7), has been favoured by multiple antibiotic resistance of this serotype and by other unknown factors. The aim of this study was to examine 19A isolates from invasive pneumococcal diseases (IPD) obtained before and after PCV7 implementation to ascertain which characteristics, including the presence of pili, might have favoured the emergence of this serotype in Italy. All S. pneumoniae isolates from IPD collected at the Italian National Institute of Health in the years 2001-2003 and 2006-2009 were serotyped. The 19A isolates were submitted to antimicrobial susceptibility testing by Etest and were genotyped by a combination of pulsed field gel electrophoresis (PFGE) and Multi Locus Sequence Typing (MLST). The presence of the pilus islets PI-1 and PI-2 was detected by PCR assays targeting a marker gene in each islet. The proportion of 19A isolates from IPD significantly increased from 4 % in 2001-2003 to 12 % in 2006-2009. This was largely due to the expansion of a clone characterized by sequence type (ST) 416, clonal complex (CC) 199, already present in Italy before PCV7 implementation. This clone included isolates susceptible to penicillin and containing PI-1 genes. Other CCs contributed to the emergence of serotype 19A: CC63 and CC193, already present in 2001-2003, and new-emerging CCs or clones such as CC230, CC320 and ST5204, that include drug-resistant and/or pilus-positive isolates. The expansion of serotype 19A in Italy might have been favoured not only by antibiotic resistance, but also by other bacterial factors such as the presence of pili.
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http://dx.doi.org/10.1099/jmm.0.061242-0DOI Listing
August 2013

Point mutations in wchA are responsible for the non-typability of two invasive Streptococcus pneumoniae isolates.

Microbiology (Reading) 2012 Feb 27;158(Pt 2):338-344. Epub 2011 Oct 27.

Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

Non-typable Streptococcus pneumoniae (NTPn) strains are typically isolated from nasopharyngeal carriage or from conjunctivitis. Since the isolation of NTPn from invasive disease is rare, we characterized the genetic basis of the non-typability of two isolates obtained in Italy from two cases of bacteraemic pneumonia. MLST revealed that both NTPn belonged to ST191, which, according to the MLST database, is associated with serotype 7F. Sequencing of the capsular locus (cps) confirmed the presence of a 7F cps in both strains and revealed the existence of distinct single point mutations in the wchA gene (a glycosyltransferase), both leading to the translation of proteins truncated at the C terminus. To verify that these mutations were responsible for the non-typability of the isolates, a functional 7F WchA was overexpressed in both NTPn. The two NTPn along with their WchA-overexpressing derivatives were analysed by transmission electron microscopy and by high-resolution magic angle spinning NMR spectroscopy. Both NTPn were devoid of a polysaccharide capsule, and WchA overexpression was sufficient to restore the assembly of a serotype 7F capsule on the surface of the two NTPn. In conclusion, we identified two new naturally occurring point mutations that lead to non-typability in the pneumococcus, and demonstrated that WchA is essential for the biosynthesis of the serotype 7F capsule.
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http://dx.doi.org/10.1099/mic.0.054270-0DOI Listing
February 2012

Genetic resistance elements carrying mef subclasses other than mef(A) in Streptococcus pyogenes.

Antimicrob Agents Chemother 2011 Jul 18;55(7):3226-30. Epub 2011 Apr 18.

Department of Infectious, Parasitic, and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

In Streptococcus pyogenes, efflux-mediated erythromycin resistance is associated with the mef gene, represented mostly by mef(A), although a small portion of strains carry different mef subclasses. We characterized the composite genetic elements, including mef subclasses other than mef(A), associated with other resistance genes in S. pyogenes isolates. Determination of the genetic elements was performed by PCR mapping. The strains carrying mosaic mef(A/E), in which the 5' region was identical to mef(A) and the 3' region was identical to mef(E), also carried tet(O). The two genes were found enclosed in an element similar to S. pyogenes prophage Φm46.1, designated the Φm46.1-like element. In S. pyogenes strains carrying mef(E) and tet(M), mef(E) was included in a typical mega element, and in some strains, it was physically associated with tet(M) in the composite element Tn2009. S. pyogenes strains carrying mef(I) also carried catQ; the two genes were linked in a fragment representing a portion of the 5216IQ complex of Streptococcus pneumoniae, designated the defective IQ element. In the only isolate carrying a novel mef gene, this was associated with catQ and tet(M) in a genetic element similar to the 5216IQ complex of S. pneumoniae (5216IQ-like complex), suggesting that the novel mef is in fact a variant of mef(I). This study demonstrates that the composite elements containing mef are shared between S. pyogenes and S. pneumoniae and suggests that it is important to distinguish the mef subclass on the basis of the genetic element containing it.
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http://dx.doi.org/10.1128/AAC.01713-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122394PMC
July 2011

Complete genome sequence of a serotype 11A, ST62 Streptococcus pneumoniae invasive isolate.

BMC Microbiol 2011 Feb 1;11:25. Epub 2011 Feb 1.

Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

Background: Streptococcus pneumoniae is an important human pathogen representing a major cause of morbidity and mortality worldwide. We sequenced the genome of a serotype 11A, ST62 S. pneumoniae invasive isolate (AP200), that was erythromycin-resistant due to the presence of the erm(TR) determinant, and carried out analysis of the genome organization and comparison with other pneumococcal genomes.

Results: The genome sequence of S. pneumoniae AP200 is 2,130,580 base pair in length. The genome carries 2216 coding sequences (CDS), 56 tRNA, and 12 rRNA genes. Of the CDSs, 72.9% have a predicted biological known function. AP200 contains the pilus islet 2 and, although its phenotype corresponds to serotype 11A, it contains an 11D capsular locus. Chromosomal rearrangements resulting from a large inversion across the replication axis, and horizontal gene transfer events were observed. The chromosomal inversion is likely implicated in the rebalance of the chromosomal architecture affected by the insertions of two large exogenous elements, the erm(TR)-carrying Tn1806 and a functional prophage designated φSpn_200. Tn1806 is 52,457 bp in size and comprises 49 ORFs. Comparative analysis of Tn1806 revealed the presence of a similar genetic element or part of it in related species such as Streptococcus pyogenes and also in the anaerobic species Finegoldia magna, Anaerococcus prevotii and Clostridium difficile. The genome of φSpn_200 is 35,989 bp in size and is organized in 47 ORFs grouped into five functional modules. Prophages similar to φSpn_200 were found in pneumococci and in other streptococcal species, showing a high degree of exchange of functional modules. φSpn_200 viral particles have morphologic characteristics typical of the Siphoviridae family and are capable of infecting a pneumococcal recipient strain.

Conclusions: The sequence of S. pneumoniae AP200 chromosome revealed a dynamic genome, characterized by chromosomal rearrangements and horizontal gene transfers. The overall diversity of AP200 is driven mainly by the presence of the exogenous elements Tn1806 and φSpn_200 that show large gene exchanges with other genetic elements of different bacterial species. These genetic elements likely provide AP200 with additional genes, such as those conferring antibiotic-resistance, promoting its adaptation to the environment.
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http://dx.doi.org/10.1186/1471-2180-11-25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055811PMC
February 2011

New genetic element carrying the erythromycin resistance determinant erm(TR) in Streptococcus pneumoniae.

Antimicrob Agents Chemother 2008 Feb 10;52(2):619-25. Epub 2007 Dec 10.

Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

erm(A) subclass erm(TR), a common macrolide resistance determinant in Streptococcus pyogenes but quite rare in Streptococcus pneumoniae, was found in a clinical S. pneumoniae isolate (AP200) from Italy. In this isolate, erm(TR) was found included in a genetic element approximately 56 kb in size that did not appear to be conjugative but could be transferred by transformation. An erm(TR)-containing DNA fragment of approximately 10 kb was sequenced and 12 open reading frames (ORFs) were identified. Upstream of erm(TR), a regulatory protein of the TetR family and the two components of an efflux pump of the ABC type were found. Downstream of erm(TR), there were ORFs homologous to a spectinomycin phosphotransferase, transposases, and a relaxase. Since the genomic sequence of S. pyogenes MGAS10750 carrying erm(TR) became available, comparison between the erm(TR)-containing genetic elements in AP200 and in MGAS10750 was performed. The region flanking erm(TR) in MGAS10750 showed identity with AP200 for 10 ORFs out of 12. PCR mapping using primers designed on the sequence of MGAS10750 confirmed that AP200 carries a genetic element similar to that of MGAS10750. In AP200 the genetic element was inserted inside an ORF homologous to spr0790 of S. pneumoniae R6, coding for a type I restriction modification system. Homologies between the insertion sites in AP200 and MGAS10750 consisted of eight conserved nucleotides, of which three were duplicated, likely representing target site duplication. The structure of the erm(TR)-carrying genetic element shows characteristics of a transposon/prophage remnant chimera. In AP200 this genetic element was designated Tn1806.
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http://dx.doi.org/10.1128/AAC.01081-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2224770PMC
February 2008

The mef(E)-carrying genetic element (mega) of Streptococcus pneumoniae: insertion sites and association with other genetic elements.

Antimicrob Agents Chemother 2006 Oct;50(10):3361-6

Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

The structure of the macrolide efflux genetic assembly (mega) element, its genomic locations, and its association with other resistance determinants and genetic elements were investigated in 16 Streptococcus pneumoniae isolates carrying mef(E), of which 1 isolate also carried tet(M) and 4 isolates also carried tet(M) and erm(B). All isolates carried a mega element of similar size and structure that included the operon mef(E)-msr(D) encoding the efflux transport system. Among tetracycline-susceptible isolates, six different integration sites were identified, five of which were recognized inside open reading frames present in the R6 genome. In the five isolates also carrying tet(M), mega was inserted in different genetic contexts. In one isolate, it was part of previously described Tn916-like element Tn2009. In another isolate, mega was inserted in a transposon similar to Tn2009 that also included an erm(B) element. This new composite transposon was designated Tn2010. Neither Tn2009 nor Tn2010 could be transferred by conjugation to pneumococcal or enterococcal recipients. In the three isolates in which mega was not physically linked with tet(M), this gene was associated with erm(B) in transposon Tn3872, a Tn916-like element. Homologies between the chromosomal insertions of these composite transposons and sequences of multidrug-resistant pneumococcal genomes in the databases indicate the presence of preferential sites for the integration of composite Tn916-like elements carrying multiple resistance determinants in S. pneumoniae.
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http://dx.doi.org/10.1128/AAC.00277-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1610078PMC
October 2006

Zinc metalloproteinase genes in clinical isolates of Streptococcus pneumoniae: association of the full array with a clonal cluster comprising serotypes 8 and 11A.

Microbiology (Reading) 2006 Feb;152(Pt 2):313-321

Dipartimento di Biologia Molecolare, Università di Siena, and UOC Batteriologia, Azienda Ospedaliera Universitaria Senese, Siena, Italy.

Pneumococci display large zinc metalloproteinases on the surface, including the IgA protease, which cleaves human IgA1 in the hinge region, the ZmpC proteinase, which cleaves human matrix metalloproteinase 9 (MMP-9), and two other proteinases, ZmpB and ZmpD, whose substrates have not yet been identified. Surface metalloproteinases are antigenic and have been linked to virulence. The genes encoding these proteinases reside in three distinct loci: two loci specific for zmpB and zmpC, and a third, the iga locus, containing iga and zmpD. Data obtained by this and other groups have shown that pneumococcal metalloproteinase genes are transcribed and yield mature and enzymatically active proteins. Since the presence of the four proteinase genes is variable in the pneumococcal strains whose genomes have been sequenced, the presence of these genes in a collection of 218 pneumococcal isolates, mostly from invasive disease, was investigated. The data showed that zmpB and iga were present in all the isolates examined, while zmpC and zmpD were present in a variable proportion of the isolates (in 18 and 49 %, respectively). Interestingly, isolates carrying both zmpC and zmpD were found to belong mainly to two serotypes (sts), 8 and 11A. By molecular typing, st 8 and st 11A isolates appeared to belong to the same clonal cluster. The presence of these two additional metalloproteinases could contribute to the fitness of particular pneumococcal clones.
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http://dx.doi.org/10.1099/mic.0.28417-0DOI Listing
February 2006

Pulsed field gel electrophoresis and random amplified polymorphic DNA molecular characterization of Ralstonia pickettii isolates from patients with nosocomial central venous catheter related bacteremia.

New Microbiol 2005 Apr;28(2):145-9

Clinica di Malattie Infettive, Dipartimento Medicina Sperimentale e Scienze Biochimiche, University of Perugia, Italy.

Over a period of 18 months 3 clusters of central venous catheter-related Ralstonia pickettii bacteremia occurred in 3 different units of the same hospital. In order to investigate the relatedness of the clinical isolates we studied 15 strains using pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) techniques. The combined analysis of the results obtained by these two methods led us to conclude that all the patients except one were infected by a single clone comprising two variants circulating in the units. Only one case was due to a different strain, probably originating outside the ward. PFGE and RAPD appear to be discriminatory techniques to study the clonal relationship among the isolates and can represent a good tool to perform the epidemiological investigation of an outbreak.
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April 2005

Evolution of erythromycin resistance in Streptococcus pneumoniae in Italy.

J Antimicrob Chemother 2005 Feb 13;55(2):256-9. Epub 2005 Jan 13.

Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

Objectives: To evaluate erythromycin resistance in recent invasive isolates of Streptococcus pneumoniae in Italy, to study the phenotypic and genotypic characteristics of the isolates, and to compare data with those obtained in a previous survey.

Methods: Invasive pneumococcal isolates were obtained from 56 laboratories throughout the country, in 2001-2003. Isolates were serotyped and antimicrobial susceptibilities determined by Sensititre panels and Etest. A new PCR was performed to detect erythromycin resistance genes. Typing methods for selected erythromycin-resistant isolates included PFGE and multilocus sequence typing (MLST).

Results: One hundred and fifty-five isolates out of 444 (34.9%) were resistant to erythromycin: 95 isolates (21.4%) carried erm(B), 56 (12.6%) carried mef(A) and three carried both genes. One isolate, carrying neither erm(B) nor mef(A), showed a point mutation in domain V of the 23S rRNA genes. The mef(A)-positive isolates carried subtype mef(A) (47 isolates), subtype mef(E) (nine isolates), and both subtype mef(E) and erm(B) (three isolates). All subtype mef(A) strains, except two, belonged to serotype 14, appeared to be clonally related by PFGE and related to the England14-9 clone by MLST. The two isolates belonging to other serotypes showed different genetic backgrounds.

Conclusions: Erythromycin resistance in S. pneumoniae has increased in the last few years in Italy. erm(B) is still the predominant resistance determinant; however, the increase in erythromycin resistance (34.9% versus 28.8% of the previous years) is mainly due to an increase in the proportion of isolates carrying the efflux pump mef(A), whereas the proportion of isolates carrying erm(B) has not changed.
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http://dx.doi.org/10.1093/jac/dkh551DOI Listing
February 2005

Polymerase chain reaction in the diagnosis and prognosis of Mediterranean visceral leishmaniasis in immunocompetent children.

Pediatrics 2002 Feb;109(2):E27

Istituto di Patologia Infettiva e Virologia, Ospedale dei Bambini G. di Cristina, Università di Palermo, Italy.

Objective: To assess the usefulness of a polymerase chain reaction (PCR) assay amplifying the small subunit rRNA coding region of Leishmania species performed on peripheral blood (PB) and bone marrow (BM) aspirates for the diagnosis and follow-up of visceral leishmaniasis (VL) in children living in the Mediterranean basin.

Design: A prospective study was conducted on children consecutively hospitalized over a 1-year period at our Infectious Diseases Department in Sicily (Italy) presenting with fever, hepatosplenomegaly, and/or pancytopenia and a positive Leishmania serology (> or =1:40).

Results: Among the 14 patients hospitalized with signs and symptoms suggestive of the disease and a positive serology, we identified 10 cases of Mediterranean VL. PCR performed on PB and BM aspirates was positive in all cases and concordant with microscopy and/or culture performed on BM. Leishmania DNA was cleared from PB a median of 6 days after the start of treatment; during follow-up (median: 9 months; range: 6-12 months) 1 child relapsed. In this case, BM PCR remained positive with rapid reappearance of a positive signal also in PB.

Conclusions: PB PCR allows a rapid and noninvasive parasitologic diagnosis of Mediterranean VL among immunocompetent children and is at least as sensitive as a diagnosis made on the basis of BM aspirates. The lack of disappearance from BM and the reappearance of positive PCR on PB is predictive of clinical relapse. Qualitative and semiquantitative PCR may be the standard method for monitoring response to therapy in immunocompetent children.
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http://dx.doi.org/10.1542/peds.109.2.e27DOI Listing
February 2002