Escherichia coli ST131 clones harbouring AggR and AAF/V fimbriae causing bacteremia in Mozambican children: Emergence of new variant of fimH27 subclone

Multidrug-resistant Escherichia coli ST131 fimH30 responsible for extra-intestinal pathogenic (ExPEC) infections is globally distributed. However, the occurrence of a subclone fimH27 of ST131 harboring both ExPEC and enteroaggregative E. coli (EAEC) related genes and belonging to commonly reported O25:H4 and other serotypes causing bacteremia in African children remain unknown. We characterized 325 E. coli isolates causing bacteremia in Mozambican children between 2001 and 2014 by conventional multiplex polymerase chain reaction and whole genome sequencing. Incidence rate of EAEC bacteremia was calculated among cases from the demographic surveillance study area. Approximately 17.5% (57/325) of isolates were EAEC, yielding an incidence rate of 45.3 episodes/105 children-years-at-risk among infants; and 44 of isolates were sequenced. 72.7% (32/44) of sequenced strains contained simultaneously genes associated with ExPEC (iutA, fyuA and traT); 88.6% (39/44) harbored the aggregative adherence fimbriae type V variant (AAF/V). Sequence type ST-131 accounted for 84.1% (37/44), predominantly belonging to serotype O25:H4 (59% of the 37); 95.6% (35/44) harbored fimH27. Approximately 15% (6/41) of the children died, and five of the six yielded ST131 strains (83.3%) mostly (60%; 3/5) due to serotypes other than O25:H4. We report the emergence of a new subclone of ST-131 E. coli strains belonging to O25:H4 and other serotypes harboring both ExPEC and EAEC virulence genes, including agg5A, associated with poor outcome in bacteremic Mozambican children, suggesting the need for prompt recognition for appropriate management.

Nasopharyngeal carriage of Streptococcus pneumoniae among HIV-infected and -uninfected children <5 years of age before introduction of pneumococcal conjugate vaccine in Mozambique

Nasopharyngeal carriage is a precursor for pneumococcal disease and can be useful for evaluating pneumococcal conjugate vaccine (PCV) impact. We studied pre-PCV pneumococcal carriage among HIV-infected and -uninfected children in Mozambique. Between October 2012 and March 2013, we enrolled HIV-infected children age <5 years presenting for routine care at seven HIV clinics in 3 sites, including Maputo (urban-south), Nampula (urban-north), and Manhiça (rural-south). We also enrolled a random sample of HIV-uninfected children <5 years old from a demographic surveillance site in Manhiça. A single nasopharyngeal swab was obtained and cultured following enrichment in Todd Hewitt broth with yeast extract and rabbit serum. Pneumococcal isolates were serotyped by Quellung reaction and multiplex polymerase chain reaction. Factors associated with pneumococcal carriage were examined using logistic regression. Overall pneumococcal carriage prevalence was 80.5% (585/727), with similar prevalences among HIV-infected (81.5%, 339/416) and HIV-uninfected (79.1%, 246/311) children, and across age strata. Among HIV-infected, after adjusting for recent antibiotic use and hospitalization, there was no significant association between study site and colonization: Maputo (74.8%, 92/123), Nampula (83.7%, 82/98), Manhiça (84.6%, 165/195). Among HIV-uninfected, report of having been born to an HIV-infected mother was not associated with colonization. Among 601 pneumococcal isolates from 585 children, serotypes 19F (13.5%), 23F (13.1%), 6A (9.2%), 6B (6.2%) and 19A (5.2%) were most common. The proportion of serotypes included in the 10- and 13-valent vaccines was 44.9% and 61.7%, respectively, with no significant differences by HIV status or age group. Overall 36.9% (n = 268) of children were colonized with a PCV10 serotype and 49.7% (n = 361) with a PCV13 serotype. Pneumococcal carriage was common, with little variation by geographic region, age, or HIV status. PCV10 was introduced in April 2013; ongoing carriage studies will examine the benefits of PCV10 among HIV-infected and-uninfected children.

Burden of invasive pneumococcal disease among children in rural Mozambique: 2001-2012

: Invasive pneumococcal disease (IPD) is a major cause of illness and death among children worldwide. 10-valent pneumococcal conjugate vaccine (PCV10) was introduced as part of the Mozambican routine immunization program in April 2013. We characterized the IPD burden in a rural area of Mozambique before PCV introduction and estimated the potential impact of this intervention. Methods: We conducted population-based surveillance for IPD, defined as S. pneumoniae isolated from blood or cerebrospinal fluid, among children <5 years old admitted to Manhiça District Hospital, a referral hospital in a rural area with high prevalence of human immunodiciency virus infection. S. pneumoniae was identified using standard microbiologic methods and serotyped using sequential multiplex PCR or Quellung. IPD incidence was calculated among cases from a defined catchment area. Results: From January 2001 through December 2012, we isolated 768 cases of IPD, 498 (65%) of which were bacteraemic pneumonia episodes. A total of 391 (51%) were from the catchment area, yielding IPD incidence rates of 479, 390 and 107 episodes per 100,000 children-years at risk among children <12, 12-23 and 24-<60 months old, respectively. The overall IPD incidence fluctuated and showed a downward trend over time. In these same age groups, in-hospital death occurred in 48 (17%), 26 (12%), and 21 (13%) of all IPD cases, respectively. Overall 90% (543/603) of IPD isolates were available for serotyping; of those, 65% were covered by PCV10 and 83% by PCV13. Among 77 hospital deaths associated with serotyped IPD, 49% and 69% were caused by isolates included in the PCV10 and PCV13, respectively. Conclusions: We describe very high rates of IPD among children in rural Mozambique that were declining before PCV introduction. Children <1 year old have the greatest incidence and case fatality; although the rates remain high among older groups as well. Most IPD episodes and many deaths among children <5 years old will likely be prevented through PCV10 introduction in Mozambique.

Infant mortality and morbidity associated with preterm and small-for-gestational-age births in southern Mozambique: A retrospective cohort study

Background: Preterm and small for gestational age (SGA) births have been associated with adverse outcomes during the first stages of life. We evaluated the morbidity and mortality associated with preterm and SGA births during the first year of life in a rural area of Southern Mozambique. Methods: This is a retrospective cohort study using previously collected data from children born at the Manhiça District Hospital in two different periods (2003-2005 and 2010-2012). Newborns were classified as being preterm and/or SGA or as babies not fulfilling any of the previous conditions (term non-SGA). All children were followed up for a year for morbidity and mortality outcomes. Results: A total of 5574 live babies were included in the analysis. The prevalence of preterm delivery was 6.2% (345/5574); the prevalence of SGA was 14.0% (776/5542) and 2.2% (114/5542) of the children presented both conditions. During the neonatal period, preterm delivery and SGA were associated with 13 (HR: 13.0, 95% CI 4.0-42.2) and 5 times (HR: 4.5, 95% CI: 1.6-12.6) higher mortality compared to term non-SGA babies. Risk of hospitalization was only increased when both conditions were present (IRR: 3.5, 95%CI: 1.5-8.1). Mortality is also increased during the entire first year, although at a lower rate. Conclusions: Neonatal and infant mortality rates are remarkably high among preterm and SGA babies in southern Mozambique. These increased rates are concentrated within the neonatal period. Prompt identification of these conditions is needed to implement interventions aimed at increasing survival of these high-risk newborns.

Changing Trends in P. falciparum Burden, Immunity, and Disease in Pregnancy

Background: Prevention of reinfection and resurgence is an integral component of the goal to eradicate malaria. However, the adverse effects of malaria resurgences are not known. Methods: We assessed the prevalence of Plasmodium falciparum infection among 1819 Mozambican women who delivered infants between 2003 and 2012. We used microscopic and histologic examination and a quantitative polymerase-chain-reaction (qPCR) assay, as well as flow-cytometric analysis of IgG antibody responses against two parasite lines. Results: Positive qPCR tests for P. falciparum decreased from 33% in 2003 to 2% in 2010 and increased to 6% in 2012, with antimalarial IgG antibody responses mirroring these trends. Parasite densities in peripheral blood on qPCR assay were higher in 2010-2012 (geometric mean [±SD], 409±1569 genomes per microliter) than in 2003-2005 (44±169 genomes per microliter, P=0.02), as were parasite densities in placental blood on histologic assessment (50±39% of infected erythrocytes vs. 4±6%, P<0.001). The malaria-associated reduction in maternal hemoglobin levels was larger in 2010-2012 (10.1±1.8 g per deciliter in infected women vs. 10.9±1.7 g per deciliter in uninfected women; mean difference, -0.82 g per deciliter; 95% confidence interval [CI], -1.39 to -0.25) than in 2003-2005 (10.5±1.1 g per deciliter vs. 10.6±1.5 g per deciliter; difference, -0.12 g per deciliter; 95% CI, -0.67 to 0.43), as was the reduction in birth weight (2863±440 g in women with past or chronic infections vs. 3070±482 g in uninfected women in 2010-2012; mean difference, -164.5 g; 95% CI, -289.7 to -39.4; and 2994±487 g vs. 3117±455 g in 2003-2005; difference, -44.8 g; 95% CI, -139.1 to 49.5). Conclusions: Antimalarial antibodies were reduced and the adverse consequences of P. falciparum infections were increased in pregnant women after 5 years of a decline in the prevalence of malaria. (Funded by Malaria Eradication Scientific Alliance and others).

Haemophilus influenzae type b disease among children in rural Mozambique: impact of vaccine introduction.

OBJECTIVE: Haemophilus influenzae type b (Hib) conjugate vaccine has dramatically reduced invasive Hib disease worldwide. Yet, data on protection against pneumonia and among children with HIV are limited. We evaluated the impact of Hib conjugate vaccine introduction in 2009 in a rural, high-HIV prevalence area in Mozambique. STUDY DESIGN: From 2006-2011, we conducted hospital-based surveillance for invasive Hib disease and clinical pneumonia (classified as severe and very severe) among children <5 years of age. Incidences calculated using population denominators were compared between baseline (2006-2008) and post-Hib conjugate vaccine (2010-2011) periods. Surveillance data for radiologically-confirmed pneumonia among children <2 years of age in 2011 were compared with baseline data from 2004-2006. RESULTS: Among 50 cases of invasive Hib disease, 5 occurred after Hib conjugate vaccine introduction; 1 case-patient was age-eligible for Hib conjugate vaccine (and had received 3 doses). Four post-Hib conjugate vaccine case-patients (including Hib conjugate vaccine failure) had HIV. Among children <1 and <5 years of age, significant reductions occurred in rates of invasive Hib disease (91% and 85%, respectively) and very severe pneumonia (29% and 34%, respectively). Radiologically-confirmed pneumonia incidence fell significantly (33%) in children <2 years of age. Severe pneumonia incidence did not decline. CONCLUSIONS: We demonstrate important reductions in invasive disease and pneumonia following Hib conjugate vaccine introduction in a high-HIV area. Continued surveillance is needed to monitor long-term Hib conjugate vaccine effects, particularly among children with HIV.

Safety of the RTS, S/AS02D candidate malaria vaccine in infants living in a highly endemic area of Mozambique: a double blind randomised controlled phase I/IIb trial

Background: Malaria remains a leading global health problem that requires the improved use of existing interventions and the accelerated development of new control methods. We aimed to assess the safety, immunogenicity, and initial efficacy of the malaria vaccine RTS,S/AS02D in infants in Africa. Methods: We did a phase I/IIb double-blind randomised trial of 214 infants in Mozambique. Infants were randomly assigned to receive three doses either of RTS,S/AS02D or the hepatitis B vaccine Engerix-B at ages 10 weeks, 14 weeks, and 18 weeks of age, as well as routine immunisation vaccines given at 8, 12, and 16 weeks of age. The primary endpoint was safety of the RTS,S/AS02D during the first 6 months of the study, and analysis was by intention to treat. Secondary endpoints included immunogenicity and analysis of new Plasmodium falciparum infections during a 3-month follow up after the third dose. Time to new infections in the per-protocol cohort were compared between groups using Cox regression models. This study is registered with ClinicalTrials.gov, number NCT00197028. Findings: There were 17 children (15.9%; 95% CI 9.5-24.2) with serious adverse events in each group. In the follow-up which ended on March 6, 2007, there were 31 serious adverse events in the RTS,S/AS02D group and 30 serious adverse events in the Engerix-B group, none of which were reported as related to vaccination. There were four deaths during this same follow-up period; all of them after the active detection of infection period had finished at study month 6 (two in RTSS/AS02D group and two in the Engerix-B group). RTS,S/AS02D induced high titres of anti-circumsporozoite antibodies. 68 first or only P falciparum infections were documented: 22 in the RTS,S/AS02D group and 46 in the control group. The adjusted vaccine efficacy was 65.9% (95% CI 42.6-79.8%, p<0.0001). Interpretation: The RTS,S/AS02D malaria vaccine was safe, well tolerated, and immunogenic in young infants. These findings set the stage for expanded phase III efficacy studies to confirm vaccine efficacy against clinical malaria disease