Paulina Hawkins

Inflammatory Genital Infections Mitigate a Severe Genetic Bottleneck in Heterosexual Transmission of Subtype A and C HIV-1

The HIV-1 epidemic in sub-Saharan Africa is driven largely by heterosexual transmission of non-subtype B viruses, of which subtypes C and A are predominant. Previous studies of subtype B and subtype C transmission pairs have suggested that a single variant from the chronically infected partner can establish infection in their newly infected partner. However, in subtype A infected individuals from a sex worker cohort and subtype B individuals from STD clinics, infection was frequently established by multiple variants. This study examined over 1750 single-genome amplified viral sequences derived from epidemiologically linked subtype C and subtype A transmission pairs very early after infection. In 90% (18/20) of the pairs, HIV-1 infection is initiated by a single viral variant that is derived from the quasispecies of the transmitting partner. In addition, the virus initiating infection in individuals who were infected by someone other than their spouse was characterized to determine if genital infections mitigated the severe genetic bottleneck observed in a majority of epidemiologically linked heterosexual HIV-1 transmission events. In nearly 50% (3/7) of individuals infected by someone other than their spouse, multiple genetic variants from a single individual established infection. A statistically significant association was observed between infection by multiple genetic variants and an inflammatory genital infection in the newly infected individual. Thus, in the vast majority of HIV-1 transmission events in cohabiting heterosexual couples, a single genetic variant establishes infection. Nevertheless, this severe genetic bottleneck can be mitigated by the presence of inflammatory genital infections in the at risk partner, suggesting that this restriction on genetic diversity is imposed in large part by the mucosal barrier.

Serotypes and Genotypes of Invasive Streptococcus pneumoniae Before and After PCV10 Implementation in Southern Brazil

To reduce the burden of pneumococcal diseases, different formulations of pneumococcal conjugate vaccines (PCV) have been introduced in many countries. In Brazil, PCV10 has been available since 2010. We aimed to analyze the serotype and genetic composition of invasive pneumococci from Brazil in pre- and post- vaccination periods (2007–2012). Antibiotic susceptibility was determined and genotypes of macrolide and fluoroquinolone resistance were characterized. The genotypes of isolates of the most frequent serotypes were determined by multilocus sequence typing. The study included 325 isolates, which were primarily recovered from blood. The most common serotypes recovered were 14, 3, 4, 23F, 7F, 9V, 12F, 20, 19F, 8, 19A, and 5. Thirty-eight pneumococci (11.7%) were from children ≤5 years old. Considering the overall population, PCV10 and PCV13 serotype coverage was 50.1% and 64.9%, respectively. During the pre-vaccine period, isolates with serotypes belonging to the PVC10 represented 51.5% (100/194), whereas in the post vaccine they represented 48.0% (63/131). PCV13 serotypes represented 67.5% (131/194) and 59.2% (77/131) of total for pre- and post-vaccination periods, respectively. Seventy different sequence types [STs] were found, accounting for 9 clonal complexes [CCs] and 45 singletons. Eight STs (156, 180, 218, 8889, 53, 191, 770, and 4967) represented the majority (51.5%) of isolates. Fifty STs were associated with the pre-vaccination period (27 exclusive) and 43 (20 exclusive) with the post-vaccination period; 23 STs were identified in both periods. Some serotypes were particularly clonal (7F, 8, 12F, 20). Non-susceptibility to penicillin was associated with serotype 19A, CC320. Erythromycin resistance was heterogeneous when considering serotype and ST. A single serotype 23F (ST4967) isolate was resistant to levofloxacin. Continued surveillance is required to determine vaccine impact and to monitor changes in pneumococcal population biology post-PCV10 introduction in Brazil.

Timing and source of subtype-C HIV-1 superinfection in the newly infected partner of Zambian couples with disparate viruses

BackgroundHIV-1 superinfection occurs at varying frequencies in different at risk populations. Though seroincidence is decreased, in the negative partner of HIV-discordant couples after joint testing and counseling in the Zambia Emory HIV Research Project (ZEHRP) cohort, the annual infection rate remains relatively high at 7-8%. Based on sequencing within the gp41 region of each partners virus, 24% of new infections between 2004 and 2008 were the result of transmission from a non-spousal partner. Since these seroconvertors and their spouses have disparate epidemiologically-unlinked viruses, there is a risk of superinfection within the marriage. We have, therefore, investigated the incidence and viral origin of superinfection in these couples.ResultsSuperinfection was detected by heteroduplex mobility assay (HMA), degenerate base counting of the gp41 sequence, or by phylogenetic analysis of the longitudinal sequences. It was confirmed by full-length env single genome amplification and phylogenetic analysis. In 22 couples (44 individuals), followed for up to five years, three of the newly infected (initially HIV uninfected) partners became superinfected. In each case superinfection occurred during the first 12 months following initial infection of the negative partner, and in each case the superinfecting virus was derived from a non-spousal partner. In addition, one probable case of intra-couple HIV-1 superinfection was observed in a chronically infected partner at the time of his seroconverting spouses initial viremia. Extensive recombination within the env gene was observed following superinfection.ConclusionsIn this subtype-C discordant couple cohort, superinfection, during the first year after HIV-1 infection of the previously negative partner, occurred at a rate similar to primary infection (13.6% [95% CI 5.2-34.8] vs 7.8% [7.1-8.6]). While limited intra-couple superinfection may in part reflect continued condom usage within couples, this and our lack of detecting newly superinfected individuals after one year of primary infection raise the possibility that immunological resistance to intra-subtype superinfection may develop over time in subtype C infected individuals.

First report of Streptococcus pneumoniae serotype 6D in South America

Streptococcus pneumoniae includes the two serotypes 6A and 6B as well as two recently discovered serotypes, 6C and 6D, in which the wciNα gene is replaced by wciNβ within the cps locus. Serotype 6D occurrence in Asia (2, 4), the Fiji islands (5) and Europe (7, 9) has recently been reported. To our knowledge, we describe here the first description of serotype 6D isolates identified within the Americas. Using the CDC serogroup 6 serotyping scheme (8), we identified 155 serogroup 6 isolates within a larger collection (n = 693) taken from nasopharyngeal carriage in children <2 years old in Peru (2007 to 2009; 541 strains) (unpublished data) and invasive pneumococcal disease (IPD) surveillance in children in Lima hospitals (2006 to 2009; 152 strains) (reference 10 and unpublished data). Among the 155 serogroup 6 isolates, 26, 105, and 22 isolates were identified as serotypes, 6A, 6B, and 6C, respectively (Table 1). We tentatively identified two 6D isolates on the basis of positive reactions with factor sera for serotype 6B (factor 6c) and serotype 6C (factor 6d) and a negative reaction with factor serum for serotype 6A (factor 6b). We verified the serologic results for all 155 serogroup 6 isolates using a recently developed PCR scheme that efficiently resolves all four serogroup 6 serotypes (6). This scheme discriminates between serotypes 6A/6C and 6B/6D using wciP allele-specific reactions. The presence or absence of wciNβ determined by a second reaction subsequently allowed resolution into all four serotypes (Table 1). Table 1. Quellung and PCR results for 155 serogroup 6 isolates The two 6D isolates, both from carriage, shared the new multilocus sequence typing (MLST) profile ST6148 (ST6148, 8-8-263-12-6-104-14) and were both fully susceptible to a variety of antimicrobial agents by broth microdilution. ST6148 is unrelated to previously described 6D genotypes on the MLST website (http://spneumoniae.mlst.net/, last accessed on 5 January 2011). It is, however, highly related to serotype 6A and 6B isolates both from carriage and invasive disease that were characterized from this same study, differing only at the recP allele. These data, together with data published elsewhere, show identical or related multilocus sequence types shared between serotype 6C or 6D strains containing wciNβ and 6A or 6B strains that contain wciNα (3, 4, 7). This suggests that the two different wciN genes are frequently horizontally transferred between different serogroup 6 strains in nature to effect intraserogroup 6 capsular switch events. On the basis of these genotype observations it also circumstantially appears likely that in some cases wciP is cotransferred with wciN, which could effect, for example, a serotype 6A to serotype 6D capsular switch. In summary, we believe that this is the first reported detection of serotype 6D among pneumococcal isolates recovered in South America or elsewhere in the western hemisphere, despite the fact that our U.S.-based surveillance has been vigilant for its appearance using serologic and PCR-based approaches for 6D detection (3, 8). The work presented here validates serotype 6D as the 92nd serotype identifiable by using CDC pneumococcal typing sera (Table 1). Serotype 6C was not effectively targeted by the pneumococcal 7-valent conjugate vaccine according to recent IPD surveillance data (3); however, there are no existing comparable data for serotype 6D. It is hoped that the recently implemented 13-valent conjugate vaccine (1) will effectively target all serogroup 6 pneumococcal diseases.

Evidence for Clonal Expansion After Antibiotic Selection Pressure: Pneumococcal Multilocus Sequence Types Before and After Mass Azithromycin Treatments

BACKGROUND A clinical trial of mass azithromycin distributions for trachoma created a convenient experiment to test the hypothesis that antibiotic use selects for clonal expansion of preexisting resistant bacterial strains. METHODS Twelve communities in Ethiopia received mass azithromycin distributions every 3 months for 1 year. A random sample of 10 children aged 0-9 years from each community was monitored by means of nasopharyngeal swab sampling before mass azithromycin distribution and after 4 mass treatments. Swab specimens were tested for Streptococcus pneumoniae, and isolates underwent multilocus sequence typing. RESULTS Of 82 pneumococcal isolates identified before treatment, 4 (5%) exhibited azithromycin resistance, representing 3 different sequence types (STs): 177, 6449, and 6494. The proportion of isolates that were classified as one of these 3 STs and were resistant to azithromycin increased after 4 mass azithromycin treatments (14 of 96 isolates [15%]; P = .04). Using a classification index, we found evidence for a relationship between ST and macrolide resistance after mass treatments (P < .0001). The diversity of STs-as calculated by the unbiased Simpson index-decreased significantly after mass azithromycin treatment (P = .045). CONCLUSIONS Resistant clones present before mass azithromycin treatments increased in frequency after treatment, consistent with the theory that antibiotic selection pressure results in clonal expansion of existing resistant strains.

Mobile Elements and Chromosomal Changes Associated with MLS Resistance Phenotypes of Invasive Pneumococci Recovered in the United States

Pneumococcal macrolide resistance is usually expressed as one of two phenotypes: the M phenotype conferred by the mef gene or the MLSB phenotype caused by modification of ribosomal targets, most commonly mediated by an erm methylase. Target-site modification leading to antibiotic resistance can also occur due to sequence mutations within the 23S rRNA or the L4 and L22 riboproteins. We screened 4,535 invasive isolates resistant to erythromycin and 18 invasive isolates nonsusceptible to quinupristin-dalfopristin (Q-D) to deduce the potential mechanisms involved. Of 4,535 erythromycin-resistant isolates, 66.2% were polymerase chain reaction (PCR)-positive for mef alone, 17.8% for ermB alone, and 15.1% for both mef and ermB. Thirty-seven isolates (0.9%) were PCR negative for both determinants. Of these, 3 were positive for ermA (subclass ermTR) and 25 had chromosomal mutations. No chromosomal mutations (in 23S rRNA, rplD, or rplV) nor any of the macrolides/lincosamides/streptogramin (MLS) resistance genes screened for (ermT, ermA, cfr, lsaC, and vgaA) were found in the remaining nine isolates. Of 18 Q-D nonsusceptible isolates, 14 had chromosomal mutations and one carried both mef and ermB; no chromosomal mutations or other resistance genes were found in 3 isolates. Overall, we found 28 mutations, 13 of which have not been previously described in Streptococcus pneumoniae. The role of these mutations remains to be confirmed by transformation assays.

Streptococcus pneumoniae Serotypes 9 and 14 Circulating in Brazil over a 23-Year Period Prior to Introduction of the 10-Valent Pneumococcal Conjugate Vaccine: Role of International Clones in the Evolution of Antimicrobial Resistance and Description of a Novel Genotype

ABSTRACT Antimicrobial-resistant pneumococcal strains have been detected worldwide since the 1960s. In Brazil, the first penicillin-nonsusceptible pneumococci (PNSP) were reported in the 1980s, and their emergence and dissemination have been mainly attributed to serogroup 9 and serotype 14 strains, especially those highly related to recognized international clones. In the present study, antimicrobial susceptibility testing and multilocus sequence typing were performed on 315 pneumococcal isolates belonging to serogroup 9 (n = 99) or serotype 14 (n = 216), recovered from patients or asymptomatic carriers between 1988 and 2011 in Brazil, in order to trace changes in antimicrobial resistance and genotypes prior to the full introduction of the pneumococcal conjugate vaccine in the country. Over the 23-year study period, the PNSP levels increased, and four clonal complexes (CC156, CC66, CC15, and CC5401) have played important roles in the evolution and dissemination of pneumococcal isolates belonging to serogroup 9 and serotype 14, as well as in the emergence of antimicrobial resistance, in the pre-pneumococcal-vaccination era. The earliest PNSP strains detected in this study belonged to serotype 9N/ST66 and were single locus variants of the international clone Tennessee14-18 ST67 (CC66). The first serotype 14 PNSP isolates were identified in 1990 and were related to the England14-9 ST9 (CC15) clone. Serotype 14 PNSP variants of the Spain9V-3 ST156 clone with elevated penicillin MICs and nonsusceptibility to other beta-lactams were detected in 1995 and showed an increasing trend over the years. The results also indicated that introduction of ST156 in our region was preceded by the emergence of trimethoprim-sulfamethoxazole resistance and by the dissemination of ST162. In addition to the presence of successful international clones, a novel regional serotype 14 genotype (CC5401) has emerged in 1996.

Serotypes and genotypes of Streptococcus pneumoniae isolates from Trinidad and Tobago

SUMMARY Objectives There are currently 94 known pneumococcal capsular polysaccharide serotypes and their prevalence differs by geographic region and the period studied. Streptococcus pneumoniae infections have been diagnosed clinically in Trinidad and Tobago and other Caribbean countries, however data on the serotype and sequence type distributions in this country are limited. The objective of this study was to determine serotypes and multilocus sequence types (MLSTs) of invasive and non-invasive pneumococcal isolates from Trinidad and Tobago. Methods Ninety-eight pneumococcal isolates from several regional hospitals in the country were analyzed using both standard microbiological methods and molecular analysis. These isolates included invasive (n = 83) and selected non-invasive (n = 15) strains recovered before (n = 25) and after (n = 73) the introduction of the pneumococcal conjugate vaccine. Results More than half of the isolates (54.1%) were recovered from children under 15 years of age, with the largest proportion being from children under 2 years of age (24.5%). The most prevalent serotypes were 19F (18.4%), 6B (15.3%), 23F (14.3%), 3 (11.2%), 19A (6.1%), 6A (5.1%), 14 (5.1%), and 9V (4.1%). The most common serotype/MLST combinations were 6B/ST138 (n = 10, 10.2%), 3/ST180 (n = 5, 5.1%), 23F/ST629 (n = 5, 5.1%), 19F/ST8398 (n = 4, 4.1%), and three each of 6B/ST145, 14/9V/ST156, 9V/ST162, 19A/320, and 3/ST10440. Conclusions This report provides the first glimpse of the prevailing pneumococcal sequence types in the country. Most of the isolates represented serotypes in the 10-valent (61.2% of isolates) and 13-valent (83.7%) pneumococcal conjugate vaccines. A detailed population study is warranted to fully determine the circulating pneumococcal sequence types. Furthermore, the implementation of an effective and continuous surveillance system in Trinidad and Tobago is paramount to monitor vaccine impact.

Cross-resistance to lincosamides, streptogramins A and pleuromutilins in Streptococcus agalactiae isolates from the USA

Background Streptococcus agalactiae (Group B Streptococcus, GBS) is a leading cause of meningitis, sepsis and pneumonia in neonates in the United States. GBS also causes invasive disease in older infants, pregnant women, children and young adults with underlying medical conditions, and older adults. Resistance to lincosamides in the absence of erythromycin resistance is rare in GBS, but has been previously reported in clinical isolates, both on its own or in combination with resistance to streptogramins A and pleuromutilins (L/LSA/LSAP phenotypes). Objectives To retrospectively screen the Active Bacterial Core surveillance (ABCs) GBS isolate collection for these phenotypes in order to identify the causal genetic determinants and determine whether their frequency is increasing. Methods Based on MIC data, 65 (0.31%) isolates susceptible to erythromycin (MIC ≤0.25 mg/L) and non-susceptible to clindamycin (MIC ≥0.5 mg/L) were identified among 21 186 GBS isolates. Genomic DNA was extracted and WGS was performed. The presence of 10 genes previously associated with LSA resistance was investigated by read mapping. Results Forty-nine (75%) isolates carried the lsa (C) gene and expressed the LSAP phenotype, and 12 (18%) carried both the lnu (B) and lsa (E) genes and expressed the LSAP phenotype. The four remaining isolates were negative for all determinants investigated. Conclusions While the overall observed frequency of these phenotypes among our GBS isolates was quite low (0.31%), this frequency has increased in recent years. To the best of our knowledge, this is the first time the LSAP phenotype has been reported among GBS isolates from the USA.

Nasopharyngeal Pneumococcal Serotypes Before and After Mass Azithromycin Distributions for Trachoma

Twenty-four Ethiopian communities were randomized to receive either (1) quarterly mass azithromycin distributions for trachoma for 1 year or (2) delayed treatment. Nasopharyngeal swabs collected from separate cross-sectional population-based samples of children were processed for Streptococcus pneumoniae Mass azithromycin did not significantly alter the pneumococcal serotype distribution, and hence it would not be expected to alter vaccine coverage.