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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.  Factors Associated with Ceftriaxone Nonsusceptibility of Streptococcus pneumoniae: Analysis of South African National Surveillance Data, 2003 to 2010 
It is important to monitor β-lactam antimicrobial nonsusceptibility trends for Streptococcus pneumoniae to inform empirical treatment guidelines. In this study, we describe penicillin and ceftriaxone susceptibility trends using national laboratory-based pneumococcal surveillance data from 2003 to 2010. A sentinel enhanced-site patient subset (2009 to 2010) contributed to the risk factor and mortality analyses. We included 9,218 invasive pneumococcal disease (IPD) cases for trend analyses and 2,854 IPD cases for risk factor and mortality analyses. Overall, we detected no significant changes in penicillin (patients <5 years of age, P = 0.50; patients ≥5 years of age, P = 0.05) or ceftriaxone nonsusceptibility rates (patients <5 years of age, P = 0.21; patients ≥5 years of age, P = 0.60). Factors associated with ceftriaxone nonsusceptibility on multivariate analysis were an age of <5 years (<1 year of age: adjusted odds ratio [aOR], 2.87; 95% confidence interval [CI], 1.70 to 4.86; 1 to 4 years of age: aOR, 2.58; 95% CI, 1.53 to 4.35, versus 25 to 44 years of age), province (Gauteng [aOR, 2.46; 95% CI, 1.26 to 4.84], and Northern Cape [aOR, 4.52; 95% CI, 1.95 to 10.52] versus KwaZulu-Natal), β-lactam use within 24 h preceding admission (aOR, 2.52; 95% CI, 1.41 to 4.53), and 13-valent vaccine serotypes (aOR, 51.64; 95% CI, 7.18 to 371.71). Among patients ≥5 years of age with meningitis who were treated according to current guidelines, HIV-infected patients (aOR, 2.94; 95% CI, 1.32 to 6.54) and patients infected with ceftriaxone-nonsusceptible isolates (aOR, 3.17; 95% CI, 1.27 to 7.89) had increased mortality rates. Among children <5 years of age with meningitis, mortality was increased in HIV-infected patients (aOR, 3.04; 95% CI, 1.40 to 6.56) but not in those with ceftriaxone-nonsusceptible isolates. Penicillin and ceftriaxone nonsusceptibility remained stable over the study period. Ceftriaxone nonsusceptibility was associated with increased mortality among patients ≥5 years of age with meningitis. The introduction of a pneumococcal conjugate vaccine may reduce ceftriaxone-nonsusceptible meningitis.
doi:10.1128/AAC.02580-13
PMCID: PMC4068496  PMID: 24687499
3.  Population Snapshot of Streptococcus pneumoniae Causing Invasive Disease in South Africa Prior to Introduction of Pneumococcal Conjugate Vaccines 
PLoS ONE  2014;9(9):e107666.
We determined the sequence types of isolates that caused invasive pneumococcal disease (IPD) prior to routine use of pneumococcal conjugate vaccines (PCV) in South Africa. PCV-13 serotypes and 6C isolates collected in 2007 (1 461/2 437, 60%) from patients of all ages as part of on-going, national, laboratory-based surveillance for IPD, were selected for genetic characterization. In addition, all 134 non-PCV isolates from children <2 years were selected for characterization. Sequence type diversity by serotype and age category (children <5 years vs. individuals ≥5 years) was assessed for PCV serotypes using Simpson’s index of diversity. Similar genotypes circulated among isolates from children and adults and the majority of serotypes were heterogeneous. While globally disseminated clones were common among some serotypes (e.g., serotype 1 [clonal complex (CC) 217, 98% of all serotype 1] and 14 [CC230, 43%)]), some were represented mainly by clonal complexes rarely reported elsewhere (e.g., serotype 3 [CC458, 60%] and 19A [CC2062, 83%]). In children <2 years, serotype 15B and 8 were the most common serotypes among non-PCV isolates (16% [22/134] and 15% [20/134] isolates, respectively). Sequence type 7052 and 53 were most common among serotypes 15B and 8 isolates and accounted for 58% (7/12) and 64% (9/14) of the isolates, respectively. Serotype 19F, 14, 19A and 15B had the highest proportions of penicillin non-susceptible isolates. Genotypes rarely reported in other parts of the world but common among some of our serotypes highlight the importance of our data as these genotypes may emerge post PCV introduction.
doi:10.1371/journal.pone.0107666
PMCID: PMC4169438  PMID: 25233455
4.  Defining the Estimated Core Genome of Bacterial Populations Using a Bayesian Decision Model 
PLoS Computational Biology  2014;10(8):e1003788.
The bacterial core genome is of intense interest and the volume of whole genome sequence data in the public domain available to investigate it has increased dramatically. The aim of our study was to develop a model to estimate the bacterial core genome from next-generation whole genome sequencing data and use this model to identify novel genes associated with important biological functions. Five bacterial datasets were analysed, comprising 2096 genomes in total. We developed a Bayesian decision model to estimate the number of core genes, calculated pairwise evolutionary distances (p-distances) based on nucleotide sequence diversity, and plotted the median p-distance for each core gene relative to its genome location. We designed visually-informative genome diagrams to depict areas of interest in genomes. Case studies demonstrated how the model could identify areas for further study, e.g. 25% of the core genes with higher sequence diversity in the Campylobacter jejuni and Neisseria meningitidis genomes encoded hypothetical proteins. The core gene with the highest p-distance value in C. jejuni was annotated in the reference genome as a putative hydrolase, but further work revealed that it shared sequence homology with beta-lactamase/metallo-beta-lactamases (enzymes that provide resistance to a range of broad-spectrum antibiotics) and thioredoxin reductase genes (which reduce oxidative stress and are essential for DNA replication) in other C. jejuni genomes. Our Bayesian model of estimating the core genome is principled, easy to use and can be applied to large genome datasets. This study also highlighted the lack of knowledge currently available for many core genes in bacterial genomes of significant global public health importance.
Author Summary
Whole genome sequencing has revolutionised the study of pathogenic microorganisms. It has also become so affordable that hundreds of samples can reasonably be sequenced in an individual project, creating a wealth of data. Estimating the bacterial core genome – traditionally defined as those genes present in all genomes – is an important initial step in population genomics analyses. We developed a simple statistical model to estimate the number of core genes in a bacterial genome dataset, calculated pairwise evolutionary distances (p-distances) based on differences among nucleotide sequences, and plotted the median p-distance for each core gene relative to its genome location. Low p-distance values indicate highly-conserved genes; high values suggest genes under selection and/or undergoing recombination. The genome diagrams depict areas of interest in genomes that can be explored in further detail. Using our method, we analysed five bacterial species comprising a total of 2096 genomes. This revealed new information related to antibiotic resistance and virulence for two bacterial species and demonstrated that the function of many core genes in bacteria is still unknown. Our model provides a highly-accessible, publicly-available tool to use on the vast quantities of genome sequence data now available.
doi:10.1371/journal.pcbi.1003788
PMCID: PMC4140633  PMID: 25144616
5.  Epidemiology of Influenza Virus Types and Subtypes in South Africa, 2009–20121 
Emerging Infectious Diseases  2014;20(7):1149-1156.
Patient age and co-infections, but not disease severity, were associated with virus type and subtype.
To determine clinical and epidemiologic differences between influenza caused by different virus types and subtypes, we identified patients and tested specimens. Patients were children and adults hospitalized with confirmed influenza and severe acute respiratory illness (SARI) identified through active, prospective, hospital-based surveillance from 2009–2012 in South Africa. Respiratory specimens were tested, typed, and subtyped for influenza virus by PCR. Of 16,005 SARI patients tested, 1,239 (8%) were positive for influenza virus. Patient age and co-infections varied according to virus type and subtype, but disease severity did not. Case-patients with influenza B were more likely than patients with influenza A to be HIV infected. A higher proportion of case-patients infected during the first wave of the 2009 influenza pandemic were 5–24 years of age (19%) than were patients infected during the second wave (9%). Although clinical differences exist, treatment recommendations do not differ according to subtype; prevention through vaccination is recommended.
doi:10.3201/eid2007.131869
PMCID: PMC4073865  PMID: 24960314
influenza; influenza A; influenza B; H3N2; H1N1; types; subtypes; pneumonia; South Africa; viruses
6.  Temporal Association in Hospitalizations for Tuberculosis, Invasive Pneumococcal Disease and Influenza Virus Illness in South African Children 
PLoS ONE  2014;9(3):e91464.
Introduction
The seasonal variability in hospitalization for tuberculosis may in part relate to super-imposed bacterial or predisposing respiratory viral infections. We aimed to study the temporal association between hospitalization for culture-confirmed pulmonary tuberculosis (PTB), invasive pneumococcal disease (IPD) and influenza virus epidemics in South African children.
Methods
We undertook a retrospective analysis which examined seasonal trends, from 2005 to 2008, for hospitalization for culture-confirmed PTB and IPD among children in relation to the influenza epidemics in Soweto, South Africa. Original time-series of the influenza virus epidemics and hospitalization rates for PTB and IPD were decomposed into three components: a trend cycle component, a seasonal component and an irregular component using the X-11 seasonal adjustment method. To compare the seasonality amongst the three series, the trend and irregular components were removed and only seasonal components examined.
Results
Across the study period, the influenza virus epidemics peaked during May to July (winter) months, which was closely followed by an increase in the incidence of hospitalization for IPD (August to October) and PTB (August to November).
Discussion
Within- and between-year temporal changes associated with childhood TB hospitalization may in part be driven by factors which influence temporal changes in pneumococcal disease, including potential variability in the severity of influenza virus epidemics in temperate climates. The dynamics of the interplay between the host and these infectious agents appears to be complex and multifactorial.
doi:10.1371/journal.pone.0091464
PMCID: PMC3950213  PMID: 24618667
7.  Pneumococcal Capsular Switching: A Historical Perspective 
The Journal of Infectious Diseases  2012;207(3):439-449.
Background. Changes in serotype prevalence among pneumococcal populations result from both serotype replacement and serotype (capsular) switching. Temporal changes in serotype distributions are well documented, but the contribution of capsular switching to such changes is unknown. Furthermore, it is unclear to what extent vaccine-induced selective pressures drive capsular switching.
Methods. Serotype and multilocus sequence typing data for 426 pneumococci dated from 1937 through 2007 were analyzed. Whole-genome sequence data for a subset of isolates were used to investigate capsular switching events.
Results. We identified 36 independent capsular switch events, 18 of which were explored in detail with whole-genome sequence data. Recombination fragment lengths were estimated for 11 events and ranged from approximately 19.0 kb to ≥58.2 kb. Two events took place no later than 1960, and the imported DNA included the capsular locus and the nearby penicillin-binding protein genes pbp2x and pbp1a.
Conclusions. Capsular switching has been a regular occurrence among pneumococcal populations throughout the past 7 decades. Recombination of large DNA fragments (>30 kb), sometimes including the capsular locus and penicillin-binding protein genes, predated both vaccine introduction and widespread antibiotic use. This type of recombination has likely been an intrinsic feature throughout the history of pneumococcal evolution.
doi:10.1093/infdis/jis703
PMCID: PMC3537446  PMID: 23175765
Capsule; serotype; switching; pneumococcus
8.  Severe Influenza-associated Respiratory Infection in High HIV Prevalence Setting, South Africa, 2009–2011 
Emerging Infectious Diseases  2013;19(11):1766-1774.
Data on influenza epidemiology in HIV-infected persons are limited, particularly for sub-Saharan Africa, where HIV infection is widespread. We tested respiratory and blood samples from patients with acute lower respiratory tract infections hospitalized in South Africa during 2009–2011 for viral and pneumococcal infections. Influenza was identified in 9% (1,056/11,925) of patients enrolled; among influenza case-patients, 358 (44%) of the 819 who were tested were infected with HIV. Influenza-associated acute lower respiratory tract infection incidence was 4–8 times greater for HIV-infected (186–228/100,000) than for HIV-uninfected persons (26–54/100,000). Furthermore, multivariable analysis showed HIV-infected patients were more likely to have pneumococcal co-infection; to be infected with influenza type B compared with type A; to be hospitalized for 2–7 days or >7 days; and to die from their illness. These findings indicate that HIV-infected persons are at greater risk for severe illnesses related to influenza and thus should be prioritized for influenza vaccination.
doi:10.3201/eid1911.130546
PMCID: PMC3837669  PMID: 24209781
influenza; HIV; AIDS; adults; children; pneumonia; pneumococcal; South Africa; viruses; vaccination; lower respiratory tract infection; respiratory infections; co-infection; bacteria; pneumoccocus
9.  Sequential Triplex Real-Time PCR Assay for Detecting 21 Pneumococcal Capsular Serotypes That Account for a High Global Disease Burden 
Journal of Clinical Microbiology  2013;51(2):647-652.
We developed and validated a real-time PCR assay consisting of 7 triplexed reactions to identify 11 individual serotypes plus 10 small serogroups representing the majority of disease-causing isolates of Streptococcus pneumoniae. This assay targets the 13 serotypes included within the 13-valent conjugate vaccine and 8 additional key serotypes or serogroups. Advantages over other serotyping assays are described. The assay will be expanded to 40 serotypes/serogroups. We will provide periodic updates at our protocol website.
doi:10.1128/JCM.02927-12
PMCID: PMC3553924  PMID: 23224094
10.  Rapid pneumococcal evolution in response to clinical interventions 
Science (New York, N.Y.)  2011;331(6016):430-434.
Epidemiological studies of the naturally transformable bacterial pathogen Streptococcus pneumoniae have previously been confounded by high rates of recombination. Sequencing 240 isolates of the PMEN1 (Spain23F-1) multidrug-resistant lineage enabled base substitutions to be distinguished from polymorphisms arising through horizontal sequence transfer. Over 700 recombinations were detected, with genes encoding major antigens frequently affected. Among these were ten capsule switching events, one of which accompanied a population shift as vaccine-escape serotype 19A isolates emerged in the USA following the introduction of the conjugate polysaccharide vaccine. The evolution of resistance to fluoroquinolones, rifampicin and macrolides was observed to occur on multiple occasions. This study details how genomic plasticity within lineages of recombinogenic bacteria can permit adaptation to clinical interventions over remarkably short timescales.
doi:10.1126/science.1198545
PMCID: PMC3648787  PMID: 21273480
11.  Clonal Analysis of Neisseria meningitidis Serogroup B Strains in South Africa, 2002 to 2006: Emergence of New Clone ST-4240/6688 
Journal of Clinical Microbiology  2012;50(11):3678-3686.
From August 1999 through July 2002, hyperinvasive Neisseria meningitidis serogroup B (MenB) clonal complexes (CCs), namely, ST-32/ET-5 (CC32) and ST-41/44/lineage 3 (CC41/44), were predominant in the Western Cape Province of South Africa. This study analyzed MenB invasive isolates from a national laboratory-based surveillance system that were collected from January 2002 through December 2006. Isolates were characterized by pulsed-field gel electrophoresis (PFGE) (n = 302), and multilocus sequence typing (MLST) and PorA and FetA typing were performed on randomly selected isolates (34/302, 11%). In total, 2,400 cases were reported, with the highest numbers from Gauteng Province (1,307/2,400, 54%) and Western Cape Province (393/2,400, 16%); 67% (1,617/2,400) had viable isolates and 19% (307/1,617) were identified as serogroup B. MenB incidence remained stable over time (P = 0.77) (average incidence, 0.13/100,000 population [range, 0.10 to 0.16/100,000 population]). PFGE (302/307, 98%) divided isolates (206/302, 68%) into 13 clusters and 96 outliers. The largest cluster, B1, accounted for 25% of isolates (76/302) over the study period; its prevalence decreased from 43% (20/47) in 2002 to 13% (8/62) in 2006 (P < 0.001), and it was common in the Western Cape (58/76, 76%). Clusters B2 and B3 accounted for 10% (31/302) and 6% (19/302), respectively, and showed no significant change over time and were predominant in Gauteng. Randomly selected isolates from clusters B1, B2, and B3 belonged to CC32, CC41/44, and the new CC4240/6688, respectively. Overall, 15 PorA and 12 FetA types were identified. MenB isolates were mostly diverse with no single dominant clone; however, CC32 and CC41/44 accounted for 35% and the new CC4240/6688 was the third most prevalent clone.
doi:10.1128/JCM.01079-12
PMCID: PMC3486271  PMID: 22972827
12.  Risk Factors for Multidrug-Resistant Invasive Pneumococcal Disease in South Africa, a Setting with High HIV Prevalence, in the Prevaccine Era from 2003 to 2008 
Antimicrobial Agents and Chemotherapy  2012;56(10):5088-5095.
The emergence of multidrug-resistant (MDR) Streptococcus pneumoniae complicates disease management. We aimed to determine risk factors associated with MDR invasive pneumococcal disease (IPD) in South Africa and evaluate the potential for vaccination to reduce disease burden. IPD data collected by laboratory-based surveillance from 2003 through 2008 were analyzed. Multidrug resistance was defined as nonsusceptibility to any three or more different antibiotic classes. Risk factors for multidrug resistance were evaluated using multivariable logistic regression. Of 20,100 cases of IPD identified, 3,708 (18%) had MDR isolates, with the proportion increasing from 16% (461/2,891) to 20% (648/3,326) (P < 0.001) over the study period. Serotypes included in the 13-valent pneumococcal conjugate vaccine (PCV13) accounted for 94% of MDR strains. Significant risk factors for MDR IPD included PCV13 (1,486/6,407; odds ratio [OR] of 6.3; 95% confidence interval [CI] of 5.0 to 7.9) and pediatric (3,382/9,980; OR of 12.8; 95% CI of 10.6 to 15.4) serotypes, age of <5 (802/3,110; OR of 2.0; 95% CI of 1.8 to 2.3) or ≥65 (39/239; OR of 1.5; 95% CI of 1.0 to 2.2) years versus age of 15 to 64 years, HIV infection (975/4,636; OR of 1.5; 95% CI of 1.2 to 1.8), previous antibiotic use (242/803; OR of 1.7; 95% CI of 1.4 to 2.1), previous hospital admissions (579/2,450; OR of 1.2; 95% CI of 1.03 to 1.4), urban location (883/4,375; OR of 2.0; 95% CI of 1.1 to 3.5), and tuberculosis treatment (246/1,021; OR of 1.2; 95% CI of 1.03 to 1.5). MDR IPD prevalence increased over the study period. The effect of many of the MDR risk factors could be reduced by more judicious use of antibiotics. Because PCV13 serotypes account for most MDR infections, pneumococcal vaccination may reduce the prevalence of multidrug resistance.
doi:10.1128/AAC.06463-11
PMCID: PMC3457358  PMID: 22802256
13.  Population Snapshot of Invasive Serogroup B Meningococci in South Africa from 2005 to 2008 
Journal of Clinical Microbiology  2012;50(8):2577-2584.
In South Africa, serogroup B meningococcal disease is sporadic. The aim of this study was to characterize serogroup B strains causing invasive meningococcal disease (IMD) in South Africa from 2005 to 2008. Isolates, collected through a national, laboratory-based surveillance program for IMD, were characterized by multilocus sequence typing (MLST). Two thousand two hundred thirty-four cases were reported, of which 1,447 had viable isolates. Intermediate resistance to penicillin was observed in 2.8% (41/1,447) of all strains. Serogroup B was the second most common serogroup (17%, 251/1,447) and increased from 14% (58/414) in 2005 to 25% (72/290) in 2008 (P < 0.001); however, incidence remained stable during the study period (average incidence, 0.13/100,000 population) (P = 0.54). Serogroup B was predominantly characterized by three clonal complexes, namely, ST-41/44/lineage 3, ST-32/ET-5, and the new complex ST-4240/6688, which accounted for 27% (65/242), 23% (55/242), and 16% (38/242) of isolates, respectively. ST-4240/6688 was more prevalent among young children (<5 years) than other clonal complexes (27/37 [73%] versus 108/196 [55%]; P = 0.04). In the most densely populated province of South Africa, Gauteng, the prevalence of ST-32/ET-5 increased from 8% (2/24) in 2005 to 38% (9/24) in 2008 (P = 0.04). Capsular switching was observed in 8/242 (3%) strains. The newly assigned clonal complex ST-4240/6688 was more common in young children.
doi:10.1128/JCM.00401-12
PMCID: PMC3421525  PMID: 22593593
14.  The multidrug-resistant PMEN1 pneumococcus is a paradigm for genetic success 
Genome Biology  2012;13(11):R103.
Background
Streptococcus pneumoniae, also called the pneumococcus, is a major bacterial pathogen. Since its introduction in the 1940s, penicillin has been the primary treatment for pneumococcal diseases. Penicillin resistance rapidly increased among pneumococci over the past 30 years, and one particular multidrug-resistant clone, PMEN1, became highly prevalent globally. We studied a collection of 426 pneumococci isolated between 1937 and 2007 to better understand the evolution of penicillin resistance within this species.
Results
We discovered that one of the earliest known penicillin-nonsusceptible pneumococci, recovered in 1967 from Australia, was the likely ancestor of PMEN1, since approximately 95% of coding sequences identified within its genome were highly similar to those of PMEN1. The regions of the PMEN1 genome that differed from the ancestor contained genes associated with antibiotic resistance, transmission and virulence. We also revealed that PMEN1 was uniquely promiscuous with its DNA, donating penicillin-resistance genes and sometimes many other genes associated with antibiotic resistance, virulence and cell adherence to many genotypically diverse pneumococci. In particular, we describe two strains in which up to 10% of the PMEN1 genome was acquired in multiple fragments, some as long as 32 kb, distributed around the recipient genomes. This type of directional genetic promiscuity from a single clone to numerous unrelated clones has, to our knowledge, never before been described.
Conclusions
These findings suggest that PMEN1 is a paradigm of genetic success both through its epidemiology and promiscuity. These findings also challenge the existing views about horizontal gene transfer among pneumococci.
doi:10.1186/gb-2012-13-11-r103
PMCID: PMC3580495  PMID: 23158461
15.  Inferior quantitative and qualitative immune responses to pneumococcal conjugate vaccine in infants with nasopharyngeal colonization by Streptococcus pneumoniae during the primary series of immunization 
Vaccine  2011;29(40):6994-7001.
Background
Heightened immunogenicity, measured one month after the primary series of pneumococcal conjugate vaccine (PCV), in African children was previously hypothesized to be due to increased rates of nasopharyngeal pneumococcal colonization during early infancy.
Methods
We analyzed the effect of selected vaccine-serotype (6B, 19F and 23F) nasopharyngeal colonization prior to the first PCV dose or when colonized for the first time prior to the second or third (2nd/3rd) PCV dose on serotype quantitative and qualitative antibody responses.
Results
Colonization prior to receiving the first PCV was associated with lower geometric mean antibody concentrations (GMCs) one month after the third dose of PCV and six months later to the colonizing-serotype. Colonized infants also had lower geometric mean titers (GMTs) on opsonophagocytosis activity assay (OPA) and a lower proportion had titers ≥8 against the colonizing serotypes (19F and 23F) post vaccination. Colonization occurring only prior to the 2nd/3rd PCV dose was also associated with lower GMCs and OPA GMTs to the colonizing-serotype. The effect of colonization with serotypes 19F and 23F prior to PCV vaccination had a greater effect on a lower proportion of colonized infants having OPA titers ≥8 than the effect of colonization on the lower proportion with antibody ≥0.35 μg/ml.
Conclusion
Infant nasopharyngeal colonization at any stage before completing the primary series of PCV vaccination was associated with inferior quantitative and qualitative antibody responses to the colonizing-serotype.
doi:10.1016/j.vaccine.2011.07.035
PMCID: PMC3167924  PMID: 21787822
Streptococcus pneumoniae; pneumococcal conjugate vaccine; HIV; immunogenicity; colonization; hypo-responsiveness
16.  Trimethoprim-Sulfamethoxazole Prophylaxis and Antibiotic Nonsusceptibility in Invasive Pneumococcal Disease 
Among 5,043 invasive pneumococcal disease (IPD) isolates identified through South African national surveillance from 2003 to 2007, we estimated the effect of trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis on antimicrobial resistance. Patients on TMP-SMX prophylaxis were more likely to have a pneumococcal isolate nonsusceptible to TMP-SMX, penicillin, and rifampin. TMP-SMX nonsusceptibility was associated with nonsusceptibility to penicillin, erythromycin, and rifampin and multidrug resistance. This study informs empirical treatment of suspected IPD in patients with a history of TMP-SMX use.
doi:10.1128/AAC.05813-11
PMCID: PMC3294938  PMID: 22232291
17.  Clinical Validation of Multiplex Real-Time PCR Assays for Detection of Bacterial Meningitis Pathogens 
Journal of Clinical Microbiology  2012;50(3):702-708.
Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA and serogroup-specific genes in the cap locus for N. meningitidis serogroups A, B, C, W135, X, and Y. However, the assay sensitivity for serogroups B, W135, and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays for serogroup B synD, W135 synG, and Y synF showed 100% specificity for detecting N. meningitidis species, with high sensitivity (serogroup B synD, 99% [75/76]; W135 synG, 97% [38/39]; and Y synF, 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup B synD, W135 synG, and Y synF assays, respectively, a significant improvement compared to results for the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of (i) N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA (NHS assay); (ii) N. meningitidis serogroups A, W135, and X (AWX assay); and (iii) N. meningitidis serogroups B, C, and Y (BCY assay). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for the singleplex assay. Pairwise comparison of real-time PCR between multiplex and singleplex assays showed that cycle threshold values of the multiplex assay were similar to those for the singleplex assay. There were no substantial differences in sensitivity and specificity between these multiplex and singleplex real-time PCR assays.
doi:10.1128/JCM.06087-11
PMCID: PMC3295090  PMID: 22170919
18.  Persistent High Burden of Invasive Pneumococcal Disease in South African HIV-Infected Adults in the Era of an Antiretroviral Treatment Program 
PLoS ONE  2011;6(11):e27929.
Background
Highly active antiretroviral treatment (HAART) programs have been associated with declines in the burden of invasive pneumococcal disease (IPD) in industrialized countries. The aim of this study was to evaluate trends in IPD hospitalizations in HIV-infected adults in Soweto, South Africa, associated with up-scaling of the HAART program from 2003 to 2008.
Methods
Laboratory-confirmed IPD cases were identified from 2003 through 2008 through an existing surveillance program. The period 2003-04 was designated as the early-HAART era, 2005–06 as the intermediate-HAART era and 2007–08 as the established-HAART era. The incidence of IPD was compared between the early-HAART and established-HAART eras in HIV-infected and–uninfected individuals.
Results
A total of 2,567 IPD cases among individuals older than 18 years were reported from 2003 through 2008. Overall incidence of IPD (per 100,000) did not change during the study period in HIV-infected adults (207.4 cases in the early-HAART and 214.0 cases in the established-HAART era; p = 0.55). IPD incidence, actually increased 1.16-fold (95% CI: 1.01; 1.62) in HIV-infected females between the early-and established-HAART eras (212.1 cases and 246.2 cases, respectively; p = 0.03). The incidence of IPD remained unchanged in HIV-uninfected adults across the three time periods.
Conclusion
Despite a stable prevalence of HIV and the increased roll-out of HAART for treatment of AIDS patients in our setting, the burden of IPD has not decreased among HIV-infected adults. The study indicates a need for ongoing monitoring of disease and HAART program effectiveness to reduce opportunistic infections in African adults with HIV/AIDS, as well as the need to consider alternate strategies including pneumococcal conjugate vaccine immunization for the prevention of IPD in HIV-infected adults.
doi:10.1371/journal.pone.0027929
PMCID: PMC3225377  PMID: 22140487
19.  Conserved Mutations in the Pneumococcal Bacteriocin Transporter Gene, blpA, Result in a Complex Population Consisting of Producers and Cheaters 
mBio  2011;2(5):e00179-11.
ABSTRACT
All fully sequenced strains of Streptococcus pneumoniae possess a version of the blp locus, which is responsible for bacteriocin production and immunity. Activation of the blp locus is stimulated by accumulation of the peptide pheromone, BlpC, following its secretion by the ABC transporter, BlpA. The blp locus is characterized by significant diversity in blpC type and in the region of the locus containing putative bacteriocin and immunity genes. In addition, the blpA gene can represent a single large open reading frame or be divided into several smaller fragments due to the presence of frameshift mutations. In this study, we use a collection of strains with blp-dependent inhibition and immunity to define the genetic changes that bring about phenotypic differences in bacteriocin production or immunity. We demonstrate that alterations in blpA, blpC, and bacteriocin/immunity content likely play an important role in competitive interactions between pneumococcal strains. Importantly, strains with a highly conserved frameshift mutation in blpA are unable to secrete bacteriocins or BlpC, but retain the ability to respond to exogenous peptide pheromone produced by cocolonizing strains, stimulating blp-mediated immunity. These “cheater” strains can only coexist with bacteriocin-producing strains that secrete their cognate BlpC and share the same immunity proteins. The variable outcome of these interactions helps to explain the heterogeneity of the blp pheromone, bacteriocin, and immunity protein content.
IMPORTANCE
Streptococcus pneumoniae resides in a polymicrobial environment and competes for limited resources by the elaboration of small antimicrobial peptides called bacteriocins. A conserved cluster of genes in the S. pneumoniae genome is involved in the production of bacteriocins and their associated protective immunity proteins through secretion of a signaling pheromone. In this study, we show that a significant number of strains have lost the ability to secrete bacteriocins and signaling pheromones due to a specific mutation in a dedicated transporter protein. Because the regulatory and immunity portion of the locus is retained, these “cheater” strains can survive in the face of invasion from a bacteriocin-producing strain without the cost of bacteriocin secretion. The outcome of such interactions depends on each strain’s repertoire of pheromone, immunity protein, and bacteriocin genes, such that intrastrain competition drives the diversity in bacteriocin, immunity protein, and pheromone content.
doi:10.1128/mBio.00179-11
PMCID: PMC3171984  PMID: 21896678
20.  An Unusual Pneumococcal Sequence Type Is the Predominant Cause of Serotype 3 Invasive Disease in South Africa▿  
Journal of Clinical Microbiology  2009;48(1):184-191.
We reviewed pneumococcal serotype 3 cases reported from 2000 through 2005 to a laboratory-based surveillance system for invasive pneumococcal disease in South Africa. The prevalence of serotype 3 invasive isolates was compared to their prevalence in carriage isolates to determine the odds of invasiveness due to serotype 3 among South African children. Three groups of serotype 3 strains were characterized by pulsed-field gel electrophoresis (PFGE) or Box element PCR (BOX-PCR), randomly selected invasive isolates from one province, isolates from a carriage study involving children in the same province, and antimicrobial-resistant invasive isolates collected nationally. Examples of the PFGE types identified were further characterized by multilocus sequence typing. In total, 15,980 viable isolates causing invasive disease were submitted, of which 661 (4%) were serotype 3, mostly from adults (85% [489/575]). Fewer serotype 3 isolates were nonsusceptible to antimicrobial agents tested (40/661 [6%]) than non-serotype 3 isolates (8,480/15,319 [55%]) (P < 0.001). Compared to non-serotype 3 cases, there was no association with HIV coinfection (2,212/2,569 [86%] versus 72/78 [92%]; P = 0.1) or increased case fatality ratio (1,190/4,211 [28%] versus 54/154 [35%]; P = 0.7). Serotype 3 in children had a low but statistically insignificant invasive disease potential (odds ratio [OR] of 0.15; 95% confidence interval [CI] of 0.01 to 1.06). Strains were grouped into 3 PFGE clusters, with the largest, cluster A, representing 54% (84/155), including 14 isolates confirmed as sequence type 458 (ST458). It was confirmed that 3 isolates from cluster B, which represented only 12% (18/155) of the isolates, were the serotype 3 global strain, ST180. We have therefore identified ST458 as predominating in South Africa, but with an invasive potential similar to that of the predominant global clone ST180.
doi:10.1128/JCM.01011-09
PMCID: PMC2812282  PMID: 19889905
21.  Genotypic Comparison of Invasive Neisseria meningitidis Serogroup Y Isolates from the United States, South Africa, and Israel, Isolated from 1999 through 2002▿  
Journal of Clinical Microbiology  2009;47(9):2787-2793.
The proportion of meningococcal disease in the United States, South Africa, and Israel caused by Neisseria meningitidis serogroup Y (NmY) was greater than the worldwide average during the period 1999-2002. Genotypic characterization of 300 NmY isolates by multilocus sequence typing, 16S rRNA gene sequencing, and PorA variable region typing was conducted to determine the relationships of the isolates from these three countries. Seventy different genotypes were found. Two groups of ST-23 clonal complex isolates accounted for 88% of the U.S. isolates, 12% of the South African isolates, and 96% of the isolates from Israel. The single common clone (ST-23/16S-19/P1.5-2,10-1) represented 57, 5, and 35% of the NmY isolates from the United States, South Africa, and Israel. The predominant clone in South Africa (ST-175/16S-21/P1.5-1,2-2), and 11 other closely related clones made up 77% of the South African study isolates and were not found among the isolates from the United States or Israel. ST-175 was the predicted founder of the ST-175 clonal complex, and isolates of ST-175 and related sequence types have been described previously in other African countries. Continued active surveillance and genetic characterization of NmY isolates causing disease in the United States, South Africa, and Israel will provide valuable data for local and global epidemiology and allow monitoring for any expansion of existing clonal complexes and detection of the emergence of new virulent clones in the population.
doi:10.1128/JCM.00091-09
PMCID: PMC2738070  PMID: 19571028
22.  Invasive Haemophilus influenzae Disease, Europe, 1996–2006 
Emerging Infectious Diseases  2010;16(3):455-463.
Incidence and case-fatality ratios are higher for non–type b than for type b infection.
An international collaboration was established in 1996 to monitor the impact of routine Haemophilus influenzae type b (Hib) vaccination on invasive H. influenzae disease; 14 countries routinely serotype all clinical isolates. Of the 10,081 invasive H. influenzae infections reported during 1996–2006, 4,466 (44%, incidence 0.28 infections/100,000 population) were due to noncapsulated H. influenzae (ncHi); 2,836 (28%, 0.15/100,000), to Hib; and 690 (7%, 0.036/100,000), to non–b encapsulated H. influenzae. Invasive ncHi infections occurred in older persons more often than Hib (median age 58 years vs. 5 years, p<0.0001) and were associated with higher case-fatality ratios (12% vs. 4%, p<0.0001), particularly in infants (17% vs. 3%, p<0.0001). Among non-b encapsulated H. influenzae, types f (72%) and e (21%) were responsible for almost all cases; the overall case-fatality rate was 9%. Thus, the incidence of invasive non–type b H. influenzae is now higher than that of Hib and is associated with higher case fatality.
doi:10.3201/eid1603.090290
PMCID: PMC3322004  PMID: 20202421
Haemophilus influenzae; Hib; conjugate vaccines; serotype replacement; epidemiology; surveillance; outcome; bacteria; podcast; research
23.  Molecular Characterization of Emerging Non-Levofloxacin-Susceptible Pneumococci Isolated from Children in South Africa▿  
Journal of Clinical Microbiology  2009;47(5):1319-1324.
Fluoroquinolones are not indicated for use for the treatment of pneumonia in children; however, non-levofloxacin-susceptible Streptococcus pneumoniae (NLSSP) has emerged in South Africa among children receiving treatment for multidrug-resistant tuberculosis. This study aimed to genotypically characterize NLSSP isolates. Invasive isolates were collected through active national laboratory-based surveillance for invasive pneumococcal disease (IPD) from 2000 through 2006 (n = 19,404). Carriage studies were conducted at two hospitals for patients with tuberculosis in two provinces. Phenotypic characterization was performed by determination of MICs and serotyping. Fluoroquinolone resistance mutations were identified, and clonality was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Twelve non-levofloxacin-susceptible cases of IPD were identified, and all were in children <15 years of age. Ten isolates were serotype 19F and formed two clusters according to their PFGE profiles, antibiogram types, and fluoroquinolone resistance-conferring mutations. All nine carriage isolates from children in hospital A were NLSSP, serotype 19F, were indistinguishable by PFGE, and were related to invasive isolates in cluster 2. Of 26 child carriers in hospital B, 22 (85%) were colonized with NLSSP. The isolates were indistinguishable by PFGE, although they displayed two serotypes, serotypes 19F and 23F. The isolates were related to invasive isolates in cluster 1; however, higher levofloxacin MICs and different fluoroquinolone resistance mutations were suggestive of horizontal gene transfer. A serotype 23F carriage isolate displayed increased fitness compared with the fitness of an otherwise indistinguishable serotype 19F carriage isolate. These data suggest that a low-level non-levofloxacin-susceptible strain transformed into a highly resistant strain under antibiotic pressure and underwent capsular switching in order to have increased fitness.
doi:10.1128/JCM.02280-08
PMCID: PMC2681833  PMID: 19261799
24.  Neisseria meningitidis Intermediately Resistant to Penicillin and Causing Invasive Disease in South Africa in 2001 to 2005▿  
Journal of Clinical Microbiology  2008;46(10):3208-3214.
Neisseria meningitidis strains (meningococci) with decreased susceptibility to penicillin (MICs, >0.06 μg/ml) have been reported in several parts of the world, but the prevalence of such isolates in Africa is poorly described. Data from an active national laboratory-based surveillance program from January 2001 through December 2005 were analyzed. A total of 1,897 cases of invasive meningococcal disease were reported, with an average annual incidence of 0.83/100,000 population. Of these cases, 1,381 (73%) had viable isolates available for further testing; 87 (6%) of these isolates tested intermediately resistant to penicillin (Peni). Peni meningococcal isolates were distributed throughout all provinces and age groups, and there was no association with outcome or human immunodeficiency virus infection. The prevalence of Peni was lower in serogroup A (7/295; 2%) than in serogroup B (24/314; 8%), serogroup C (9/117; 8%), serogroup Y (22/248; 9%), or serogroup W135 (25/396; 6%) (P = 0.02). Pulsed-field gel electrophoresis grouped 63/82 Peni isolates into nine clusters, mostly according to serogroup. The clustering of patterns from Peni isolates was not different from that of penicillin-susceptible isolates. Twelve sequence types were identified among 18 isolates arbitrarily selected for multilocus sequence typing. DNA sequence analysis of the penA gene identified 26 different alleles among the Peni isolates. Intermediate penicillin resistance is thus widespread among meningococcal serogroups, has been selected in a variety of lineages, and, to date, does not appear to be associated with increased mortality. This is the first report describing the prevalence and molecular epidemiology of Peni meningococcal isolates from sub-Saharan Africa.
doi:10.1128/JCM.00221-08
PMCID: PMC2566094  PMID: 18650361
25.  Virulence Characteristics of Klebsiella and Clinical Manifestations of K. pneumoniae Bloodstream Infections 
Emerging Infectious Diseases  2007;13(7):986-993.
Differences in clinical manifestations are due to virulence factors expressed by the organism.
We studied 455 consecutive episodes of Klebsiella pneumoniae bacteremia occurring in 7 countries. Community-acquired pneumonia and an invasive syndrome of liver abscess, meningitis, or endophthalmitis occurred only in Taiwan and South Africa. Infections by K1 and K2 capsular serotype, the mucoid phenotype, and aerobactin production were important determinants of virulence. The mucoid phenotype was seen in 94% of isolates in patients with community-acquired pneumonia and in 100% of isolates that caused the invasive syndrome in Taiwan and South Africa, compared with only 2% of isolates elsewhere. Mortality of mice injected with mucoid strains (69%) was strikingly higher than that occurring in mice injected with nonmucoid strains (3%, p<0.001). Differences in clinical features of bacteremic infection with K. pneumoniae are due to the virulence factors expressed by the organism.
doi:10.3201/eid1307.070187
PMCID: PMC2878244  PMID: 18214169
Klebsiella pneumoniae; gram-negative bacteremia; virulence; epidemiology; research

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