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1.  Effectiveness of 7-Valent Pneumococcal Conjugate Vaccine Against Invasive Pneumococcal Disease in HIV-Infected and -Uninfected Children in South Africa: A Matched Case-Control Study 
Cohen, Cheryl | von Mollendorf, Claire | de Gouveia, Linda | Naidoo, Nireshni | Meiring, Susan | Quan, Vanessa | Nokeri, Vusi | Fortuin-de Smit, Melony | Malope-Kgokong, Babatyi | Moore, David | Reubenson, Gary | Moshe, Mamokgethi | Madhi, Shabir A. | Eley, Brian | Hallbauer, Ute | Kularatne, Ranmini | Conklin, Laura | O'Brien, Katherine L. | Zell, Elizabeth R. | Klugman, Keith | Whitney, Cynthia G. | von Gottberg, Anne | Moore, David | Verwey, Charl | Varughese, Sheeba | Archary, Moherndran | Naby, Fathima | Dawood, Khathija | Naidoo, Ramola | Elliott, Gene | Hallbauer, Ute | Eley, Brian | Nuttall, James | Cooke, Louise | Finlayson, Heather | Rabie, Helena | Whitelaw, Andrew | Perez, Dania | Jooste, Pieter | Naidoo, Dhamiran | Kularatne, Ranmini | Reubenson, Gary | Cohen, Cheryl | de Gouveia, Linda | du Plessis, Mignon | Govender, Nevashan | Meiring, Susan | Quan, Vanessa | von Mollendorf, Claire | Fortuin-de Smidt, Melony | Naidoo, Nireshni | Malope-Kgokong, Babatyi | Nokeri, Vusi | Ncha, Relebohile | Lindani, Sonwabo | von Gottberg, Anne | Spies, Barry | Sono, Lino | Maredi, Phasweni | Hamese, Ken | Moshe, Mamokgethi | Nchabeleng, Maphosane | Ngcobo, Ntombenhle | van den Heever, Johann | Madhi, Shabir | Conklin, Laura | Verani, Jennifer | Whitney, Cynthia | Zell, Elizabeth | Loo, Jennifer | Nelson, George | Klugman, Keith | O'Brien, Katherine
A 2 + 1 seven-valent pneumococcal conjugate vaccine schedule is effective against vaccine-serotype invasive pneumococcal disease (IPD) in HIV-uninfected children and HIV-exposed but -uninfected children and against all-serotype multidrug-resistant IPD in HIV-uninfected children.
Background. South Africa introduced 7-valent pneumococcal conjugate vaccine (PCV7) in April 2009 using a 2 + 1 schedule (6 and 14 weeks and 9 months). We estimated the effectiveness of ≥2 PCV7 doses against invasive pneumococcal disease (IPD) in human immunodeficiency virus (HIV)–infected and -uninfected children.
Methods. IPD (pneumococcus identified from a normally sterile site) cases were identified through national laboratory-based surveillance. Specimens were serotyped by Quellung or polymerase chain reaction. Four controls, matched for age, HIV status, and hospital were sought for each case. Using conditional logistic regression, we calculated vaccine effectiveness (VE) as 1 minus the adjusted odds ratio for vaccination.
Results. From March 2010 through November 2012, we enrolled 187 HIV-uninfected (48 [26%] vaccine serotype) and 109 HIV-infected (43 [39%] vaccine serotype) cases and 752 HIV-uninfected and 347 HIV-infected controls aged ≥16 weeks. Effectiveness of ≥2 PCV7 doses against vaccine-serotype IPD was 74% (95% confidence interval [CI], 25%–91%) among HIV-uninfected and −12% (95% CI, −449% to 77%) among HIV-infected children. Effectiveness of ≥3 doses against vaccine-serotype IPD was 90% (95% CI, 14%–99%) among HIV-uninfected and 57% (95% CI, −371% to 96%) among HIV-infected children. Among HIV-exposed but -uninfected children, effectiveness of ≥2 doses was 92% (95% CI, 47%–99%) against vaccine-serotype IPD. Effectiveness of ≥2 doses against all-serotype multidrug-resistant IPD was 96% (95% CI, 62%–100%) among HIV-uninfected children.
Conclusions. A 2 + 1 PCV7 schedule was effective in preventing vaccine-serotype IPD in HIV-uninfected and HIV-exposed, uninfected children. This finding supports the World Health Organization recommendation for this schedule as an alternative to a 3-dose primary series among HIV-uninfected individuals.
doi:10.1093/cid/ciu431
PMCID: PMC4144265  PMID: 24917657
children; HIV; pneumococcus; pneumococcal conjugate vaccine; South Africa
2.  Pneumococcal Serotypes and Serogroups Causing Invasive Disease in Pakistan, 2005–2013 
PLoS ONE  2014;9(6):e98796.
While pneumococcal conjugate vaccines have been implemented in most countries worldwide, use in Asia has lagged in part because of a lack of data on the amount of disease that is vaccine preventable in the region. We describe pneumococcal serotypes elicited from 111 episodes of invasive pneumococcal disease (IPD) from 2005 to 2013 among children and adults in Pakistan. Seventy-three percent (n = 81) of 111 IPD episodes were cases of meningitis (n = 76 in children 0–15 years and n = 5 among adults). Serotypes were determined by target amplification of DNA extracted from pneumococcal isolates (n = 52) or CSF specimens (n = 59). Serogroup 18 was the most common serogroup causing meningitis in children <5 years, accounting for 21% of cases (n = 13). The 10-valent pneumococcal conjugate vaccine (PCV 10) or PCV10- related serotypes were found in 61% (n = 47) of childhood (age 0–15 years) meningitis episodes. PCV-13 increased this coverage to 63% (one additional serotype 19A; n = 48). Our data indicate that use of PCVs would prevent a large proportion of serious pneumococcal disease.
doi:10.1371/journal.pone.0098796
PMCID: PMC4043782  PMID: 24892937
3.  The Pneumococcus 
Emerging Infectious Diseases  2005;11(2):356-357.
doi:10.3201/eid1102.041010
PMCID: PMC3320451
Streptococcus pneumoniae; pneumococcus; book review
5.  Serotype-Specific Changes in Invasive Pneumococcal Disease after Pneumococcal Conjugate Vaccine Introduction: A Pooled Analysis of Multiple Surveillance Sites 
PLoS Medicine  2013;10(9):e1001517.
In a pooled analysis of data collected from invasive pneumococcal disease surveillance databases, Daniel Feikin and colleagues examine serotype replacement after the introduction of 7-valent pneumococcal conjugate vaccine (PCV7) into national immunization programs.
Please see later in the article for the Editors' Summary
Background
Vaccine-serotype (VT) invasive pneumococcal disease (IPD) rates declined substantially following introduction of 7-valent pneumococcal conjugate vaccine (PCV7) into national immunization programs. Increases in non-vaccine-serotype (NVT) IPD rates occurred in some sites, presumably representing serotype replacement. We used a standardized approach to describe serotype-specific IPD changes among multiple sites after PCV7 introduction.
Methods and Findings
Of 32 IPD surveillance datasets received, we identified 21 eligible databases with rate data ≥2 years before and ≥1 year after PCV7 introduction. Expected annual rates of IPD absent PCV7 introduction were estimated by extrapolation using either Poisson regression modeling of pre-PCV7 rates or averaging pre-PCV7 rates. To estimate whether changes in rates had occurred following PCV7 introduction, we calculated site specific rate ratios by dividing observed by expected IPD rates for each post-PCV7 year. We calculated summary rate ratios (RRs) using random effects meta-analysis. For children <5 years old, overall IPD decreased by year 1 post-PCV7 (RR 0·55, 95% CI 0·46–0·65) and remained relatively stable through year 7 (RR 0·49, 95% CI 0·35–0·68). Point estimates for VT IPD decreased annually through year 7 (RR 0·03, 95% CI 0·01–0·10), while NVT IPD increased (year 7 RR 2·81, 95% CI 2·12–3·71). Among adults, decreases in overall IPD also occurred but were smaller and more variable by site than among children. At year 7 after introduction, significant reductions were observed (18–49 year-olds [RR 0·52, 95% CI 0·29–0·91], 50–64 year-olds [RR 0·84, 95% CI 0·77–0·93], and ≥65 year-olds [RR 0·74, 95% CI 0·58–0·95]).
Conclusions
Consistent and significant decreases in both overall and VT IPD in children occurred quickly and were sustained for 7 years after PCV7 introduction, supporting use of PCVs. Increases in NVT IPD occurred in most sites, with variable magnitude. These findings may not represent the experience in low-income countries or the effects after introduction of higher valency PCVs. High-quality, population-based surveillance of serotype-specific IPD rates is needed to monitor vaccine impact as more countries, including low-income countries, introduce PCVs and as higher valency PCVs are used.
Please see later in the article for the Editors' Summary
Editors’ Summary
Background
Pneumococcal disease–a major cause of illness and death in children and adults worldwide–is caused by Streptococcus pneumoniae, a bacterium that often colonizes the nose and throat harmlessly. Unfortunately, S. pneumoniae occasionally spreads into the lungs, bloodstream, or covering of the brain, where it causes pneumonia, septicemia, and meningitis, respectively. These invasive pneumococcal diseases (IPDs) can usually be successfully treated with antibiotics but can be fatal. Consequently, it is better to avoid infection through vaccination. Vaccination primes the immune system to recognize and attack disease-causing organisms (pathogens) rapidly and effectively by exposing it to weakened or dead pathogens or to pathogen molecules that it recognizes as foreign (antigens). Because there are more than 90 S. pneumoniae variants or “serotypes,” each characterized by a different antigenic polysaccharide (complex sugar) coat, vaccines that protect against S. pneumoniae have to include multiple serotypes. Thus, the pneumococcal conjugate vaccine PCV7, which was introduced into the US infant immunization regimen in 2000, contains polysaccharides from the seven S. pneumoniae serotypes mainly responsible for IPD in the US at that time.
Why Was This Study Done?
Vaccination with PCV7 was subsequently introduced in several other high- and middle-income countries, and IPD caused by the serotypes included in the vaccine declined substantially in children and in adults (because of reduced bacterial transmission and herd protection) in the US and virtually all these countries. However, increases in IPD caused by non-vaccine serotypes occurred in some settings, presumably because of “serotype replacement.” PCV7 prevents both IPD caused by the serotypes it contains and carriage of these serotypes. Consequently, after vaccination, previously less common, non-vaccine serotypes can colonize the nose and throat, some of which can cause IPD. In July 2010, a World Health Organization expert consultation on serotype replacement called for a comprehensive analysis of the magnitude and variability of pneumococcal serotype replacement following PCV7 use to help guide the introduction of PCVs in low-income countries, where most pneumococcal deaths occur. In this pooled analysis of data from multiple surveillance sites, the researchers investigate serotype-specific changes in IPD after PCV7 introduction using a standardized approach.
What Did the Researchers Do and Find?
The researchers identified 21 databases that had data about the rate of IPD for at least 2 years before and 1 year after PCV7 introduction. They estimated whether changes in IPD rates had occurred after PCV7 introduction by calculating site-specific rate ratios–the observed IPD rate for each post-PCV7 year divided by the expected IPD rate in the absence of PCV7 extrapolated from the pre-PCV7 rate. Finally, they used a statistical approach (random effects meta-analysis) to estimate summary (pooled) rate ratios. For children under 5 years old, the overall number of observed cases of IPD in the first year after the introduction of PCV7 was about half the expected number; this reduction in IPD continued through year 7 after PCV7 introduction. Notably, the rate of IPD caused by the S. pneumonia serotypes in PCV7 decreased every year, but the rate of IPD caused by non-vaccine serotypes increased annually. By year 7, the number of cases of IPD caused by non-vaccine serotypes was 3-fold higher than expected, but was still smaller than the decrease in vaccine serotypes, thereby leading to the decrease in overall IPD. Finally, smaller decreases in overall IPD also occurred among adults but occurred later than in children 2 years or more after PCV7 introduction.
What Do These Findings Mean?
These findings show that consistent, rapid, and sustained decreases in overall IPD and in IPD caused by serotypes included in PCV7 occurred in children and thus support the use of PCVs. The small increases in IPD caused by non-vaccine serotypes that these findings reveal are likely to be the result of serotype replacement, but changes in antibiotic use and other factors may also be involved. These findings have several important limitations, however. For example, PCV7 is no longer made and extrapolation of these results to newer PCV10 and PCV13 formulations should be done cautiously. On the other hand, many of the serotypes causing serotype replacement after PCV7 are included in these higher valency vaccines. Moreover, because the data analyzed in this study mainly came from high-income countries, these findings may not be generalizable to low-income countries. Nevertheless, based on their analysis, the researchers make recommendations for the collection and analysis of IPD surveillance data that should allow valid interpretations of the effect of PCVs on IPD to be made, an important requisite for making sound policy decisions about vaccination against pneumococcal disease.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001517.
The US Centers for Disease Control and Prevention provides information for patients and health professionals on all aspects of pneumococcal disease and pneumococcal vaccination, including personal stories
Public Health England provides information on pneumococcal disease and on pneumococcal vaccines
The World Health Organization also provides information on pneumococcal vaccines
The not-for-profit Immunization Action Coalition has information on pneumococcal disease, including personal stories
MedlinePlus has links to further information about pneumococcal infections (in English and Spanish)
The International Vaccine Access Center at Johns Hopkins Bloomberg School of Public Health has more information on introduction of pneumococcal conjugate vaccines in low-income countries
doi:10.1371/journal.pmed.1001517
PMCID: PMC3782411  PMID: 24086113
6.  Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract 
PeerJ  2013;1:e97.
We performed culture-based and PCR-based tests for pneumococcal identification and serotyping from carriage specimens collected in rural and urban Kenya. Nasopharyngeal specimens from 237 healthy children <5 years old (C-NPs) and combined nasopharyngeal/oropharyngeal specimens from 158 adults (A-NP/OPs, 118 HIV-positive) were assessed using pneumococcal isolation (following broth culture enrichment) with Quellung-based serotyping, real-time lytA-PCR, and conventional multiplexed PCR-serotyping (cmPCR). Culture-based testing from C-NPs, HIV-positive A-NP/OPs, and HIV-negative A-NP/OPs revealed 85.2%, 40.7%, and 12.5% pneumococcal carriage, respectively. In contrast, cmPCR serotypes were found in 93.2%, 98.3%, and 95.0% of these sets, respectively. Two of 16 lytA-negative C-NPs and 26 of 28 lytA-negative A-NP/OPs were cmPCR-positive for 1–10 serotypes (sts) or serogroups (sgs). A-NP/OPs averaged 5.5 cmPCR serotypes/serogroups (5.2 in HIV-positive, 7.1 in HIV-negative) and C-NPs averaged 1.5 cmPCR serotypes/serogroups. cmPCR serotypes/serogroups from lytA-negative A-NP/OPs included st2, st4, sg7F/7A, sg9N/9L, st10A, sg10F/10C/33C, st13, st17F, sg18C/18A/18B/18F, sg22F/22A, and st39. Nine strains of three non-pneumococcal species (S. oralis, S. mitis, and S. parasanguinis) (7 from A-OP, 1 from both A-NP and A-OP, and 1 from C-NP) were each cmPCR-positive for one of 7 serotypes/serogroups (st5, st13, sg15A/15F, sg10F/10C/33C, sg33F/33A/37, sg18C/18A/18B/18F, sg12F/12A/12B/ 44/46) with amplicons revealing 83.6–99.7% sequence identity to pneumococcal references. In total, 150 cmPCR amplicons from carriage specimens were sequenced, including 25 from lytA-negative specimens. Amplicon sequences derived from specimens yielding a pneumococcal isolate with the corresponding serotype were identical or highly conserved (>98.7%) with the reference cmPCR amplicon for the st, while cmPCR amplicons from lytA-negative specimens were generally more divergent. Separate testing of 56 A-OPs and 56 A-NPs revealed that ∼94% of the positive cmPCR results from A-NP/OPs were from OP microbiota. In contrast, A-NPs yielded >2-fold more pneumococcal isolates than A-OPs. Verified and suspected non-pneumococcal cmPCR serotypes/serogroups appeared to be relatively rare in C-NPs and A-NPs compared to A-OPs. Our findings indicate that non-pneumococcal species can confound serotype-specific PCR and other sequence-based assays due to evolutionarily conserved genes most likely involved in biosynthesis of surface polysaccharide structures.
doi:10.7717/peerj.97
PMCID: PMC3698467  PMID: 23825797
Pneumococcal serotype-specific loci; Mitis group streptococci; Oropharyngeal and nasopharyngeal flora; PCR for serotype deduction
7.  Optimization of Multiple Pathogen Detection Using the TaqMan Array Card: Application for a Population-Based Study of Neonatal Infection 
PLoS ONE  2013;8(6):e66183.
Identification of etiology remains a significant challenge in the diagnosis of infectious diseases, particularly in resource-poor settings. Viral, bacterial, and fungal pathogens, as well as parasites, play a role for many syndromes, and optimizing a single diagnostic system to detect a range of pathogens is challenging. The TaqMan Array Card (TAC) is a multiple-pathogen detection method that has previously been identified as a valuable technique for determining etiology of infections and holds promise for expanded use in clinical microbiology laboratories and surveillance studies. We selected TAC for use in the Aetiology of Neonatal Infection in South Asia (ANISA) study for identifying etiologies of severe disease in neonates in Bangladesh, India, and Pakistan. Here we report optimization of TAC to improve pathogen detection and overcome technical challenges associated with use of this technology in a large-scale surveillance study. Specifically, we increased the number of assay replicates, implemented a more robust RT-qPCR enzyme formulation, and adopted a more efficient method for extraction of total nucleic acid from blood specimens. We also report the development and analytical validation of ten new assays for use in the ANISA study. Based on these data, we revised the study-specific TACs for detection of 22 pathogens in NP/OP swabs and 12 pathogens in blood specimens as well as two control reactions (internal positive control and human nucleic acid control) for each specimen type. The cumulative improvements realized through these optimization studies will benefit ANISA and perhaps other studies utilizing multiple-pathogen detection approaches. These lessons may also contribute to the expansion of TAC technology to the clinical setting.
doi:10.1371/journal.pone.0066183
PMCID: PMC3689704  PMID: 23805203
9.  Call to Action on World Pneumonia Day 
Emerging Infectious Diseases  2012;18(11):1889-93.
doi:10.3201/eid1811.121217
PMCID: PMC3559175  PMID: 23092708
pneumonia; health care; World Health Organization; WHO; World Pneumonia Day; bacteria; Global Action Plan for Prevention and Control of Pneumonia; Global Alliance for Vaccines and Immunizations
10.  Shifting Genetic Structure of Invasive Serotype 19A Pneumococci in the United States 
The Journal of Infectious Diseases  2011;203(10):1360-1368.
(See the editorial commentary by Tyrrell, on pages 1345-7.)
Background. Following 7-valent conjugate vaccine introduction in the United States in 2000, invasive serotype (sero19A) pneumococcal disease (IPD) emerged rapidly. Sero19A IPD incidence increased slightly during 2005–2008 (from 2.3 cases to 2.5 cases per 100,000 population), whereas sero19A penicillin resistance (defined as a minimum inhibitor concentration [MIC] ≥2 μg/mL) increased significantly (from 28.7% to 43.7%). To better understand changes, we characterized sero19A isolates recovered during 2004–2008.
Methods. We performed antimicrobial susceptibility testing on all 2767 sero19A IPD isolates identified through the Centers for Disease Control Active Bacterial Core surveillance during 2004–2008. We genotyped 1804 (96.3%) of 1874 sero19A isolates recovered during 2005–2007 and all 148 year 2008 sero19A isolates from children <5 years of age.
Results. Resistant clonal complex (CC) 320/27119A increased from 20.9% (115 of 550) to 32.9% (208 of 633; P < .001) of IPD isolates during 2005–2007, which paralleled increased sero19A penicillin resistance (from 28.7% [163 of 567 isolates] to 39.5% [261 of 661 isolates]; P < .001). Total IPD due to 320/27119A increased during 2005–2007 and increased from 2.1 to 3.6 cases per 100,000 population during 2005–2008 in children <5 years of age. The penicillin-susceptible/intermediate, putative vaccine-escape CC69519A increased from 7.5% (41 of 550) to 13.6% (85 of 633) of sero19A isolates during 2005–2007 (P = .002).
Conclusions. Sero19A rates may have plateaued; however, clonal shifts are increasing resistance. Increased IPD caused by CC320/27119A and CC69519A could reflect additional selective advantages in addition to resistance.
doi:10.1093/infdis/jir052
PMCID: PMC3080895  PMID: 21398395
11.  Application of TaqMan Low-Density Arrays for Simultaneous Detection of Multiple Respiratory Pathogens▿ 
Journal of Clinical Microbiology  2011;49(6):2175-2182.
The large and growing number of viral and bacterial pathogens responsible for respiratory infections poses a challenge for laboratories seeking to provide rapid and comprehensive pathogen identification. We evaluated a novel application of the TaqMan low-density array (TLDA) cards for real-time PCR detection of 21 respiratory-pathogen targets. The performance of the TLDA was compared to that of individual real-time PCR (IRTP) assays with the same primers and probes using (i) nucleic acids extracted from the 21 pathogen strains and 66 closely related viruses and bacteria and (ii) 292 clinical respiratory specimens. With spiked samples, TLDA cards were about 10-fold less sensitive than IRTP assays. By using 292 clinical specimens to generate 2,238 paired individual assays, the TLDA card exhibited 89% sensitivity (95% confidence interval [CI], 86 to 92%; range per target, 47 to 100%) and 98% specificity (95% CI, 97 to 99%; range per target, 85 to 100%) overall compared to IRTP assays as the gold standard with a threshold cycle (CT) cutoff of 43. The TLDA card approach offers promise for rapid and simultaneous identification of multiple respiratory pathogens for outbreak investigations and disease surveillance.
doi:10.1128/JCM.02270-10
PMCID: PMC3122721  PMID: 21471348
12.  Setting Research Priorities to Reduce Global Mortality from Childhood Pneumonia by 2015 
PLoS Medicine  2011;8(9):e1001099.
Igor Rudan and colleagues report the results of their consensus building exercise that identified health research priorities to help reduce child mortality from pneumonia.
doi:10.1371/journal.pmed.1001099
PMCID: PMC3181228  PMID: 21980266
13.  Identifying Host Genetic Risk Factors in the Context of Public Health Surveillance for Invasive Pneumococcal Disease 
PLoS ONE  2011;6(8):e23413.
Host genetic factors that modify risk of pneumococcal disease may help target future public health interventions to individuals at highest risk of disease. We linked data from population-based surveillance for invasive pneumococcal disease (IPD) with state-based newborn dried bloodspot repositories to identify biological samples from individuals who developed invasive pneumococcal disease. Genomic DNA was extracted from 366 case and 732 anonymous control samples. TagSNPs were selected in 34 candidate genes thought to be associated with host response to invasive pneumococcal disease, and a total of 326 variants were successfully genotyped. Among 543 European Americans (EA) (182 cases and 361 controls), and 166 African Americans (AA) (53 cases and 113 controls), common variants in surfactant protein D (SFTPD) are consistently underrepresented in IPD. SFTPD variants with the strongest association for IPD are intronic rs17886286 (allelic OR 0.45, 95% confidence interval (CI) [0.25, 0.82], with p = 0.007) in EA and 5′ flanking rs12219080 (allelic OR 0.32, 95%CI [0.13, 0.78], with p = 0.009) in AA. Variants in CD46 and IL1R1 are also associated with IPD in both EA and AA, but with effects in different directions; FAS, IL1B, IL4, IL10, IL12B, SFTPA1, SFTPB, and PTAFR variants are associated (p≤0.05) with IPD in EA or AA. We conclude that variants in SFTPD may protect against IPD in EA and AA and genetic variation in other host response pathways may also contribute to risk of IPD. While our associations are not corrected for multiple comparisons and therefore must be replicated in additional cohorts, this pilot study underscores the feasibility of integrating public health surveillance with existing, prospectively collected, newborn dried blood spot repositories to identify host genetic factors associated with infectious diseases.
doi:10.1371/journal.pone.0023413
PMCID: PMC3156135  PMID: 21858107
15.  Revisiting Pneumococcal Carriage by Use of Broth Enrichment and PCR Techniques for Enhanced Detection of Carriage and Serotypes▿  
Journal of Clinical Microbiology  2010;48(5):1611-1618.
The measurement of pneumococcal carriage in the nasopharyngeal reservoir is subject to potential confounders that include low-density and multiple-strain colonization. To compare different methodologies, we picked a random sampling of 100 nasopharyngeal specimens recovered from infants less than 2 years of age who were previously assessed for pneumococcal carriage and serotypes by a conventional method that used direct plating from the transport/storage medium (50 specimens were culture negative and 50 specimens were culture positive for pneumococci). We used a broth enrichment approach and a conventional PCR approach (with and without broth enrichment) to determine pneumococcal carriage and serotypes, and the results were compared to the initial conventional culture-based results. Additionally, we used a lytA-targeted real-time PCR for pneumococcal detection. Broth enrichment for both the culture-based and the PCR-based methods enhanced the isolation of pneumococci and detection of serotype diversity, with the most effective serotype deduction method being one that used broth enrichment prior to sequential multiplex PCR. Similarly, we also found that broth enrichment followed by the lytA-specific real-time PCR was the most sensitive for the detection of apparent pneumococcal carriage. The broth enrichment, conventional multiplex PCR, and real-time PCR approaches used in this study were effective in detecting pneumococcal carriage in the 50 specimens that were negative by conventional direct plating from transport medium (range of numbers of positive specimens, 8/50 to 22/50 [16 to 44%]), and the three different serotyping approaches that used broth enrichment increased the number of serotype identifications from the 100 specimens (12 to 29 additional serotype identifications to be positive). A PCR-based approach that employed a broth enrichment step appeared to best enhance the detection of mixed serotypes and low-density pneumococcal carriage.
doi:10.1128/JCM.02243-09
PMCID: PMC2863911  PMID: 20220175
16.  PCR-Based Quantitation and Clonal Diversity of the Current Prevalent Invasive Serogroup 6 Pneumococcal Serotype, 6C, in the United States in 1999 and 2006 to 2007▿  
Journal of Clinical Microbiology  2008;47(3):554-559.
Following introduction of the 7-valent pneumococcal conjugate vaccine to the United States, rates of invasive pneumococcal disease (IPD) caused by serotype 6A declined among all age groups, while rates of IPD caused by newly identified serotype 6C increased slightly among persons 5 years of age and older. Conventionally serotyped 6A isolates (CS6As) from active population-based surveillance during 1999 and 2006 to 2007 were classified as serotypes 6A and 6C by an expedient and highly accurate serotype 6C-specific PCR assay developed during this study. PCR testing of 636 year 1999, 2006, and 2007 CS6As revealed 6C proportions of 35/214 (16.4%), 141/218 (64.7%), and 141/204 (69.1%), respectively. These results agreed with those from a previously devised monoclonal antibody-based serotyping system (346 CS6As compared). Type 6C IPD incidence significantly increased during 2006 and 2007 compared to during 1999 (0.57 to 0.58 cases per 100,000 and 0.22 cases per 100,000, respectively; 164% increase from 1999 to 2007 [95% confidence interval, 87 to 270%]), while rates of IPD due to types 6A and 6B markedly decreased. In 2007, 31.2% of 6C isolates were not susceptible to penicillin. Serotype 6C is now the predominant serotype associated with serogroup 6 IPD in the United States and is often penicillin nonsusceptible. We performed multilocus sequence typing (MLST) on a limited sampling of 6C isolates with different antimicrobial susceptibility profiles. MLST of 42 6C isolates revealed 12 genotypes distributed among six distinct genetic groups. Fifteen 6C isolates shared one of four different MLST types with 6C-negative CS6As. MLST results suggest 6C strains arose from independent recombination events involving only serotype 6A and 6C parental strains.
doi:10.1128/JCM.01919-08
PMCID: PMC2650953  PMID: 19116353
17.  In Vitro Evaluation of the Antimicrobial Activity of Ceftaroline against Cephalosporin-Resistant Isolates of Streptococcus pneumoniae▿  
Increasing pneumococcal resistance to extended-spectrum cephalosporins warrants the search for novel agents with activity against such resistant strains. Ceftaroline, a parenteral cephalosporin currently in phase 3 clinical development, has demonstrated potent in vitro activity against resistant gram-positive organisms, including penicillin-resistant Streptococcus pneumoniae. In this study, the activity of ceftaroline was evaluated against highly cefotaxime-resistant isolates of pneumococci from the Active Bacterial Core surveillance program of the Centers for Disease Control and Prevention and against laboratory-derived cephalosporin-resistant mutants of S. pneumoniae. The MICs of ceftaroline and comparators were determined by broth microdilution. In total, 120 U.S. isolates of cefotaxime-resistant (MIC ≥ 4 μg/ml) S. pneumoniae were tested along with 18 laboratory-derived R6 strains with known penicillin-binding protein (PBP) mutations. Clinical isolates were characterized by multilocus sequence typing, and the DNAs of selected isolates were sequenced to identify mutations affecting pbp genes. Ceftaroline (MIC90 = 0.5 μg/ml) had greater in vitro activity than penicillin, cefotaxime, or ceftriaxone (MIC90 = 8 μg/ml for all comparators) against the set of highly cephalosporin-resistant clinical isolates of S. pneumoniae. Ceftaroline was also more active against the defined R6 PBP mutant strains, which suggests that ceftaroline can overcome common mechanisms of PBP-mediated cephalosporin resistance. These data indicate that ceftaroline has significant potency against S. pneumoniae strains resistant to existing parenteral cephalosporins and support its continued development for the treatment of infections caused by resistant S. pneumoniae strains.
doi:10.1128/AAC.01324-08
PMCID: PMC2630653  PMID: 19015339
18.  Antibiotic Resistance Patterns in Invasive Group B Streptococcal Isolates 
Antibiotics are used for both group B streptococcal (GBS) prevention and treatment. Active population-based surveillance for invasive GBS disease was conducted in four states during 1996–2003. Of 3813 case-isolates, 91.0% (3471) were serotyped, 77.1% (2937) had susceptibility testing, and 46.6% (3471) had both. All were sensitive to penicillin, ampicillin, cefazolin, cefotaxime, and vancomycin. Clindamycin and erythromycin resistance was 12.7% and 25.6%, respectively, and associated with serotype V (P < .001). Clindamycin resistance increased from 10.5% to 15.0% (X2 for trend 12.70; P < .001); inducible clindamycin resistance was associated with the erm genotype. Erythromycin resistance increased from 15.8% to 32.8% (X2 for trend 55.46; P < .001). While GBS remains susceptible to beta-lactams, resistance to alternative agents such as erythromycin and clindamycin is an increasing concern.
doi:10.1155/2008/727505
PMCID: PMC2637368  PMID: 19223967
19.  Integrating Host Genomics with Surveillance for Invasive Bacterial Diseases  
Emerging Infectious Diseases  2008;14(7):1138-1140.
We tested the feasibility of linking Active Bacterial Core surveillance, a prospective, population-based surveillance system for invasive bacterial disease, to a newborn dried blood spot (nDBS) repository. Using nDBS specimens, we resequenced CD46, putative host gene receptor for Neisseria meningitidis, and identified variants associated with susceptibility to this disease.
doi:10.3201/eid1407.071287
PMCID: PMC2600343  PMID: 18598642
Active bacterial core surveillance; Neisseria meningitidis; blood spots; host gene; CD46; dispatch
20.  Changes in Serotypes and Antimicrobial Susceptibility of Invasive Streptococcus pneumoniae Strains in Cleveland: a Quarter Century of Experience▿  
Journal of Clinical Microbiology  2008;46(3):982-990.
The serotypes and susceptibilities to penicillin, macrolides, and clindamycin of 1,655 invasive isolates of Streptococcus pneumoniae recovered between 1979 and 2004 were determined. A precipitous decrease of 61% in the number of isolates was found following 2000, the year of 7-valent protein-conjugated pneumococcal vaccine (PCV7) introduction (139 versus 55 per 2-year period prior to versus after 2000; P < 0.001). This decrease was 84% in children <5 years old (80 versus 13 per 2-year period; P < 0.001) and 18 to 23% in other age groups (P, not significant). PCV7 serotypes decreased by 76% overall (103 versus 25 per 2-year period; P < 0.001) and by 92% in children <5 years old (65 versus 5 per 2-year period; P < 0.001), with significant decreases in six of the seven PCV serotypes. Other serotypes, except for type 19A, decreased from 32 to 22 per 2-year period, while type 19A increased from 4 to 8 per 2-year period, although none of these changes reached significance. Drug resistance emerged slowly, with the first penicillin-intermediate strain isolated in 1980 and the first macrolide/lincosamide-resistant strain isolated in 1984. The first penicillin-resistant strain was isolated in 1993. Resistance increased steadily thereafter until 2003-2004, when 51.1% of isolates were penicillin nonsusceptible and 53.3% were macrolide resistant. Clindamycin resistance remained low until 2003-2004, when 26.7% of strains were resistant; this was associated with the emergence of multidrug-resistant type 19A strains. This study documents the emergence of resistance over a quarter century among invasive pneumococci in the Cleveland area, as well as the reduction in disease caused by PCV7 serotypes following the introduction of PCV7 in 2000.
doi:10.1128/JCM.02321-07
PMCID: PMC2268364  PMID: 18234877
21.  Pre- and Postvaccination Clonal Compositions of Invasive Pneumococcal Serotypes for Isolates Collected in the United States in 1999, 2001, and 2002 
Journal of Clinical Microbiology  2006;44(3):999-1017.
Monitoring of serotypes and their clonal associations is critical as pneumococci adapt to the selective pressures exerted by the pneumococcal seven-valent conjugate vaccine (PCV7). We genotyped 1,476 invasive isolates from the Active Bacterial Core surveillance (705 [89.8%] of the isolates were obtained from children <5 years of age, and 771 [18.4%] of the isolates were obtained from individuals >5 years of age) in 2001 and 2002 (after the introduction of PCV7). The data were compared to the results for 1,168 invasive isolates (855 [83.9%] of the isolates were from children <5 years of age) collected in 1999. Among children <5 years of age, the incidence of invasive disease due to non-PCV7 serogroups together with serogroup 19A increased (P < 0.001). Eighty-three clonal sets, representing 177 multilocus sequence types (STs), were compiled from the 3-year isolate set. Among the non-PCV7 serogroups, newly emerging clones were uncommon; and a significant expansion of already established clones occurred for serotypes 3 (ST180), 7F (ST191), 15BCF (ST199), 19A (ST199), 22F (ST433), 33F (ST662), and 38 (ST393). However, additional minor clonal types within serotypes 1, 6A, 6B, 7C, 9N, 10A, 12F, 14, 15B/C, 17F, 19A, 19F, 20, 22F, and 33F that were absent in 1999 were found during 2001 and 2002. Although 23 clonal sets exhibited multiple serotypes, for most serotypes there were either no changes or modest changes in clonal compositions since the introduction of PCV7. The only example of an identical ST shared between non-PCV7 and PCV7 or PCV7-related serotypes was ST199; however, ST199 was prevalent within serotypes 15B/C and 19A before and after PCV7 introduction. Continued genotypic surveillance is warranted, since certain clones not targeted by PCV7 are expanding, and their emergence as significant pathogens could occur with maintained vaccine pressure.
doi:10.1128/JCM.44.3.999-1017.2006
PMCID: PMC1393141  PMID: 16517889
22.  Prevalence of First-Step Mutants among Levofloxacin-Susceptible Invasive Isolates of Streptococcus pneumoniae in the United States 
By use of a PCR-restriction fragment length polymorphism assay, we screened 496 levofloxacin-susceptible invasive pneumococcal strains (MIC ≤ 2 mg/liter) for quinolone resistance-determining region mutations known to confer fluoroquinolone resistance. Among those with a levofloxacin MIC of 2 mg/liter, 16.2% of isolates recovered from nursing home residents and 6.4% from non-nursing home residents had first-step mutations.
doi:10.1128/AAC.50.4.1561-1563.2006
PMCID: PMC1426940  PMID: 16569885
23.  Clonal Association between Streptococcus pneumoniae Serotype 23A, Circulating within the United States, and an Internationally Dispersed Clone of Serotype 23F 
Journal of Clinical Microbiology  2005;43(11):5440-5444.
Streptococcus pneumoniae is an important pathogen in the United States and is associated with significant morbidity and mortality. Since the introduction of the seven-valent conjugate vaccine, a significant decline in pneumococcal disease has been reported. However, surveillance for pneumococcal disease remains essential, as the extent of cross protection against vaccine-related serotypes is still unclear. Further, any increase in non-vaccine-related serotypes also needs monitoring. We report on a new clonal association between a vaccine-related serotype, serotype 23A, obtained as part of the Active Bacterial Core surveillance, with an established internationally dispersed Pneumococcal Molecular Epidemiology Network (PMEN) clone, clone Colombia23F-26. Sixty-two isolates of serotype 23A collected from sterile sites during a 2-year period (2002 and 2003) were characterized. Twenty-one (34%) isolates were penicillin nonsusceptible, although none were fully resistant. Pulsed-field gel electrophoresis and multilocus sequence typing analysis showed that 24 (39%) of the serotype 23A isolates shared either genetic identity or high genetic relatedness with PMEN clone Colombia23F-26. Extensive variability was noted within the sequenced region of pbp2b in two penicillin-nonsusceptible isolates as well as in PMEN clone Colombia23F-26, suggesting that these isolates probably acquired penicillin resistance independently. The emergence of such new serotype and genotype associations highlights the dynamic nature of the pneumococcal population, necessitating continuous monitoring in the post-vaccine era.
doi:10.1128/JCM.43.11.5440-5444.2005
PMCID: PMC1287803  PMID: 16272467
24.  Novel Mechanism of Resistance to Oxazolidinones, Macrolides, and Chloramphenicol in Ribosomal Protein L4 of the Pneumococcus 
Two clinical Streptococcus pneumoniae isolates, identified as resistant to macrolides and chloramphenicol and nonsusceptible to linezolid, were found to contain 6-bp deletions in the gene encoding riboprotein L4. The gene transformed susceptible strain R6 so that it exhibited such resistance, with the transformants also showing a fitness cost. We demonstrate a novel bacterial mechanism of resistance to chloramphenicol and nonsusceptibility to linezolid.
doi:10.1128/AAC.49.8.3554-3557.2005
PMCID: PMC1196237  PMID: 16048983
25.  Erythromycin-nonsusceptible Streptococcus pneumoniae in Children, 1999–2001 
Emerging Infectious Diseases  2005;11(6):969-972.
After increasing from 1995 to 1999, invasive erythromycin-nonsusceptible Streptococcus pneumoniae rates per 100,000 decreased 53.6% in children from Baltimore, Maryland (US), from 1999 to 2001, which was partially attributed to strains related to the mefE-carrying England14-9 clone. The decline in infection rates was likely due to the pneumococcal 7-valent conjugate vaccine.
doi:10.3201/eid1106.050119
PMCID: PMC3367581  PMID: 15963300
Keywords: molecular epidemiology; bacteremia; Antibiotic resistance; streptococcus pneumoniae; pneumococcal; vaccine; children

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