Low frequency of enterohemorrhagic, enteroinvasive and diffusely adherent Escherichia coli in children under 5 years in rural Mozambique: a case-control study

BACKGROUND NlmCategory: BACKGROUND content: Diarrheagenic Escherichia coli (DEC) are among the leading pathogens associated with endemic diarrhea in low income countries. Yet, few epidemiological studies have focused the contribution of enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC) and diffusely adherent E. coli (DAEC). - Label: METHODS NlmCategory: METHODS content: "We assessed the contribution of EHEC, EIEC and DAEC isolated from stool samples from a case-control study conducted in children aged <\xE2\x80\x895\xE2\x80\x89years in Southern Mozambique between December 2007 and November 2012. The isolates were screened by conventional PCR targeting stx1 and stx2 (EHEC), ial and ipaH (EIEC), and daaE (DAEC) genes." - Label: RESULTS NlmCategory: RESULTS content: "We analyzed 297 samples from cases with less-severe diarrhea (LSD) matched to 297 controls, and 89 samples from cases with moderate-to-severe diarrhea (MSD) matched to 222 controls, collected between November 3, 2011 and November 2, 2012. DEC were more common among LSD cases (2.7%, [8/297] of cases vs. 1.3% [4/297] of controls; p\xC2\xA0=\xE2\x80\x890.243]) than in MSD cases (0%, [0/89] of cases vs. 0.4%, [1/222]\xC2\xA0of controls; p\xC2\xA0=\xE2\x80\x891.000). Detailed analysis revealed low frequency of EHEC, DAEC or EIEC and no association with diarrhea in all age strata. Although the low frequency, EIEC was predominant in LSD cases aged 24-59\xE2\x80\x89months (4.1% for cases vs. 0% for controls), followed by DAEC in similar frequency for cases and controls in infants (1.9%) and lastly EHEC from one control. Analysis of a subset of samples from previous period (December 10, 2007 and October 31, 2011) showed high frequency of DEC in controls compared to MSD cases (16.2%, [25/154] vs. 11.9%, [14/118], p\xC2\xA0=\xE2\x80\x890.383, respectively). Among these, DAEC predominated, being detected in 7.7% of cases vs. 17.6% of controls aged 24-59\xE2\x80\x89months, followed by EIEC in 7.7% of cases vs. 5.9% of controls for the same age category, although no association was observed. EHEC was detected in one sample from cases and two from controls." - Label: CONCLUSIONS NlmCategory: CONCLUSIONS content: Our data suggests that although EHEC, DAEC and EIEC are less frequent in endemic diarrhea in rural Mozambique, attention should be given to their transmission dynamics (e.g. the role on sporadic or epidemic diarrhea) considering that the role of asymptomatic individuals as source of dissemination remains unknown

Invasive bacterial disease trends and characterization of group B streptococcal isolates among young infants in southern Mozambique, 2001-2015

BACKGROUND: Maternal group B streptococcal (GBS) vaccines under development hold promise to prevent GBS disease in young infants. Sub-Saharan Africa has the highest estimated disease burden, although data on incidence and circulating strains are limited. We described invasive bacterial disease (IBD) trends among infants <90 days in rural Mozambique during 2001-2015, with a focus on GBS epidemiology and strain characteristics. METHODS: Community-level birth and mortality data were obtained from Manhica's demographic surveillance system. IBD cases were captured through ongoing surveillance at Manhica district hospital. Stored GBS isolates from cases underwent serotyping by multiplex PCR, antimicrobial susceptibility testing, and whole genome sequencing. RESULTS: There were 437 IBD cases, including 57 GBS cases. Significant declines in overall IBD, neonatal mortality, and stillbirth rates were observed (P<0.0001), but not for GBS (P = 0.17). In 2015, GBS was the leading cause of young infant IBD (2.7 per 1,000 live births). Among 35 GBS isolates available for testing, 31 (88.6%) were highly related serotype III isolates within multilocus sequence types (STs) 17 (68.6%) or 109 (20.0%). All seven ST109 isolates (21.9%) had elevated minimum inhibitory concentration (MIC) to penicillin (>/=0.12 mug/mL) associated with penicillin-binding protein (PBP) 2x substitution G398A. Epidemiologic and molecular data suggest this is a well-established clone. CONCLUSION: A notable young infant GBS disease burden persisted despite improvements in overall maternal and neonatal health. We report an established strain with pbp2x point mutation, a first-step mutation associated with reduced penicillin susceptibility within a well-known virulent lineage in rural Mozambique. Our findings further underscores the need for non-antibiotic GBS prevention strategies

Carriage prevalence of Salmonella enterica serotype Typhi in gallbladders of adult autopsy cases from Mozambique

INTRODUCTION: Typhoid fever is an important public health problem in many low-income countries where asymptomatic carriers play an important role in its dissemination. The bacterium causing typhoid fever can live in the gallstones of asymptomatic persons after the infection. These carriers are reservoirs of S. Typhi, are highly contagious, and spread the disease through the secretion of bacteria in feces and urine. The aim of this study was to determine the carrier rate in an area of Mozambique. METHODOLOGY: The presence of S. Typhi was analyzed in gallbladder samples obtained from 99 adult corpses (in-hospital deaths) from Mozambique by gold-standard culture and polymerase chain reaction (PCR). RESULTS: Only one sample was positive with the culture. However, nine additional samples were positive by PCR and confirmed by DNA sequencing. Thus, the prevalence of S. Typhi was 10.1% (10/99). CONCLUSIONS: We report a high prevalence of S. Typhi in gallbladders among adult autopsy cases from Mozambique

Characterization of Vaginal Escherichia coli Isolated from Pregnant Women in Two Different African Sites

The relevance of vaginal colonization of pregnant women by Escherichia coli is poorly understood, despite these strains sharing a similar virulence profile with other extraintestinal pathogenic E. coli producing severe obstetric and neonatal infections. We characterized the epidemiology, antimicrobial susceptibility and virulence profiles of 84 vaginal E. coli isolates from pregnant women from Rabat (Morocco) and Manhica (Mozambique), two very distinct epidemiological settings. Low levels of antimicrobial resistance were observed to all drugs tested, except for trimethoprim-sulfamethoxazole in Manhica, where this drug is extensively used as prophylaxis for opportunistic HIV infections. The most prevalent virulence factors were related to iron acquisition systems. Phylogroup A was the most common in Rabat, while phylogroups E and non-typeable were the most frequent in Manhica. Regardless of the apparently "low virulence" of these isolates, the frequency of infections is higher and the outcomes more devastating in constrained-resources conditions, especially among pregnant women and newborns

Impact of rotavirus vaccination on diarrheal hospitalizations in children younger than 5 years of age in a rural southern Mozambique

Funding Information: We thank the participants in this study and their parents for allowing the collection of samples and data. The authors would also like to thank all Centro de Investigação em Saúde de Manhiça (CISM) staff particularly those supporting Diarrheal Disease Research Area and Manhiça District Hospital. Core funding for CISM is provided by the Spanish Agency for International Cooperation and Development (AECID). ISGlobal receives support from the Spanish Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. The GEMS study was supported by the Bill & Melinda Gates Foundation (Project OPP 38874). The impact of rotavirus study was supported by GAVI funds through Centers for Disease Control and Prevention Foundation (CDCF), Atlanta & World Health Organization, Regional Offices for Africa (WHO AFRO). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention and World Health Organization. The authors declare no conflict of interest. Funding Information: We thank the participants in this study and their parents for allowing the collection of samples and data. The authors would also like to thank all Centro de Investigação em Saúde de Manhiça (CISM) staff particularly those supporting Diarrheal Disease Research Area and Manhiça District Hospital. Core funding for CISM is provided by the Spanish Agency for International Cooperation and Development (AECID). ISGlobal receives support from the Spanish Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. The GEMS study was supported by the Bill & Melinda Gates Foundation (Project OPP 38874). The impact of rotavirus study was supported by GAVI funds through Centers for Disease Control and Prevention Foundation (CDCF), Atlanta & World Health Organization, Regional Offices for Africa (WHO AFRO). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Publisher Copyright: © 2022Background: Rotavirus vaccine (Rotarix®) was introduced in Mozambique through its Expanded Program of Immunization in September 2015. We assessed the impact of rotavirus vaccination on childhood gastroenteritis-associated hospitalizations post-vaccine introduction in a high HIV prevalence rural setting of southern Mozambique. Methods: We reviewed and compared the trend of hospitalizations (prevalence) and incidence rates of acute gastroenteritis (AGE), and rotavirus associated-diarrhea (laboratory confirmed rotavirus) in pre- (January 2008–August 2015) and post-rotavirus vaccine introduction periods (September 2015–December 2020), among children <5 years of age admitted to Manhiça District Hospital. Results: From January 2008 to December 2020, rotavirus vaccination was found to contribute to the decline of the prevalence of AGE from 19% (95% CI: 18.14–20.44) prior to the vaccine introduction to 10% (95% CI: 8.89–11.48) in the post-introduction period, preventing 40% (95 % IE: 38–42) and 84% (95 % IE: 80–87) of the expected AGE and laboratory confirmed rotavirus cases, respectively, among infants. Similarly, the overall incidence of rotavirus was 11.8-fold lower in the post-vaccine introduction period (0.4/1000 child-years-at-risk [CYAR]; 95% CI: 0.3–0.6) compared with the pre-vaccination period (4.7/1000 CYAR; 95% CI: 4.2–5.1) with the highest reduction being observed among infants (16.8-fold lower from the 15.1/1000 CYAR in the pre-vaccine to 0.9/1000 CYAR in the post-vaccine eras). Conclusions: We documented a significant reduction in all-cause diarrhea hospitalizations and rotavirus positivity after vaccine introduction demonstrating the beneficial impact of rotavirus vaccination in a highly vulnerable population.publishersversionpublishe

Escherichia coli ST131 clones harbouring AggR and AAF/V fimbriae causing bacteremia in Mozambican children: Emergence of new variant of fimH27subclone

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

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

BACKGROUND: 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 Manhica 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

Molecular Characterisation of Cryptosporidium spp. in Mozambican Children Younger than 5 Years Enrolled in a Matched Case-Control Study on the Aetiology of Diarrhoeal Disease.

Cryptosporidium is a leading cause of childhood diarrhoea and associated physical and cognitive impairment in low-resource settings. Cryptosporidium-positive faecal samples (n = 190) from children aged ≤ 5 years enrolled in the Global Enteric Multicenter Study (GEMS) in Mozambique detected by ELISA (11.5%, 430/3754) were successfully PCR-amplified and sequenced at the gp60 or ssu rRNA loci for species determination and genotyping. Three Cryptosporidium species including C. hominis (72.6%, 138/190), C. parvum (22.6%, 43/190), and C. meleagridis (4.2%, 8/190) were detected. Children ≤ 23 months were more exposed to Cryptosporidium spp. infections than older children. Both C. hominis and C. parvum were more prevalent among children with diarrhoeal disease compared to those children without it (47.6% vs. 33.3%, p = 0.007 and 23.7% vs. 11.8%, p = 0.014, respectively). A high intra-species genetic variability was observed within C. hominis (subtype families Ia, Ib, Id, Ie, and If) and C. parvum (subtype families IIb, IIc, IIe, and IIi) but not within C. meleagridis (subtype family IIIb). No association between Cryptosporidium species/genotypes and child's age was demonstrated. The predominance of C. hominis and C. parvum IIc suggests that most of the Cryptosporidium infections were anthroponotically transmitted, although zoonotic transmission events also occurred at an unknown rate. The role of livestock, poultry, and other domestic animal species as sources of environmental contamination and human cryptosporidiosis should be investigated in further molecular epidemiological studies in Mozambique.This research was funded by the Bill and Melinda Gates Foundation through the Center for Vaccine Development at the University of Maryland, School of Medicine who coordinated GEMS, grant number 38874 (GEMS) and OPP1033572 (GEMS1A). Additional funding was obtained from the Health Institute Carlos III (ISCIII), Ministry of Economy and Competitiveness (Spain), grant number PI16CIII/00024, from the Fundo Nacional de Investigacão, Ministry of Science and Technology (Mozambique), grant number 245-INV, and from the USAID Country Office of Mozambique, grant number AID-656-F-16-00002.N

Molecular diversity of Giardia duodenalis in children under 5 years from the Manhiça district, Southern Mozambique enrolled in a matched case-control study on the aetiology of diarrhoea.

Giardia duodenalis is an enteric parasite commonly detected in children. Exposure to this organism may lead to asymptomatic or symptomatic infection. Additionally, early-life infections by this protozoan have been associated with impaired growth and cognitive function in poor resource settings. The Global Enteric Multicenter Study (GEMS) in Mozambique demonstrated that G. duodenalis was more frequent among controls than in diarrhoeal cases (≥3 loosing stools in the previous 24 hours). However, no molecular investigation was conducted to ascertain the molecular variability of the parasite. Therefore, we describe here the frequency and genetic diversity of G. duodenalis infections in children younger than five years of age with and without diarrhoea from the Manhiça district in southern Mozambique enrolled in the context of GEMS. Genomic DNA from 757 G. duodenalis-positive stool samples by immunoassay collected between 2007-2012, were reanalysed by multiplex PCR targeting the E1-HP and C1-P21 genes for the differentiation of assemblages A and B. Overall, 47% (353) of the samples were successfully amplified in at least one locus. Assemblage B accounted for 90% (319/353) of all positives, followed by assemblage A (8%, 29/353) and mixed A+B infections (1%, 5/353). No association between the presence of a given assemblage and the occurrence of diarrhoea could be demonstrated. A total of 351 samples were further analysed by a multi-locus sequence genotyping (MLSG) approach at the glutamate dehydrogenase (gdh), ß-giardin (bg) and triose phosphate isomerase (tpi) genes. Overall, 63% (222/351) of samples were genotyped and/or sub-genotyped in at least one of the three markers. Sequence analysis revealed the presence of assemblages A (10%; 23/222) and B (90%; 199/222) with high molecular diversity at the nucleotide level within the latter; no mixed infections were identified under the MLSG scheme. Assemblage A sequences were assigned to sub-assemblages AI (0.5%, 1/222), AII (7%, 15/222) or ambiguous AII/AIII (3%, 7/222). Within assemblage B, sequences were assigned to sub-assemblages BIII (13%, 28/222), BIV (14%, 31/222) and ambiguous BIII/BIV (59%, 132/222). BIII/BIV sequences accumulated the majority of the single nucleotide polymorphisms detected, particularly in the form of double peaks at chromatogram inspection. This study demonstrated that the occurrence of gastrointestinal illness (diarrhoea) was not associated to a given genotype of G. duodenalis in Mozambican children younger than five years of age. The assemblage B of the parasite was responsible for nine out of ten infections detected in this paediatric population. The extremely high genetic diversity observed within assemblage B isolates was compatible with an hyperendemic epidemiological scenario where infections and reinfections were common. The obtained molecular data may be indicative of high coinfection rates by different G. duodenalis assemblages/sub-assemblages and/or genetic recombination events, although the exact contribution of both mechanisms to the genetic diversity of the parasite remains unknown.GEMS was funded by the Bill and Melinda Gates Foundation through the Center for Vaccine Development at the University of Maryland, School of Medicine who coordinated GEMS-1 and GEMS-1A, with grant number 38874 (MML) and OPP1033572 (MML), respectively. DC received funding from the Health Institute Carlos III, Ministry of Economy and Competitiveness (Spain), under project PI16CIII/00024.TN received funding from the Fundo Nacional de Investigacão, Ministry of Science and Technology (Mozambique) under project 245-INV. Additional funding was obtained from the USAID Country Office of Mozambique under the Fixed Amount Award Grant No. AID-656-F-16-00002 (IM). CISM is supported by the Government of Mozambique and the Spanish Agency for International Development Cooperation (AECID). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial

BACKGROUND: The efficacy and safety of the RTS,S/AS01 candidate malaria vaccine during 18 months of follow-up have been published previously. Herein, we report the final results from the same trial, including the efficacy of a booster dose. METHODS: From March 27, 2009, until Jan 31, 2011, children (age 5-17 months) and young infants (age 6-12 weeks) were enrolled at 11 centres in seven countries in sub-Saharan Africa. Participants were randomly assigned (1:1:1) at first vaccination by block randomisation with minimisation by centre to receive three doses of RTS,S/AS01 at months 0, 1, and 2 and a booster dose at month 20 (R3R group); three doses of RTS,S/AS01 and a dose of comparator vaccine at month 20 (R3C group); or a comparator vaccine at months 0, 1, 2, and 20 (C3C [control group]). Participants were followed up until Jan 31, 2014. Cases of clinical and severe malaria were captured through passive case detection. Serious adverse events (SAEs) were recorded. Analyses were by modified intention to treat and per protocol. The coprimary endpoints were the occurrence of malaria over 12 months after dose 3 in each age category. In this final analysis, we present data for the efficacy of the booster on the occurrence of malaria. Vaccine efficacy (VE) against clinical malaria was analysed by negative binomial regression and against severe malaria by relative risk reduction. This trial is registered with ClinicalTrials.gov, number NCT00866619. FINDINGS: 8922 children and 6537 young infants were included in the modified intention-to-treat analyses. Children were followed up for a median of 48 months (IQR 39-50) and young infants for 38 months (34-41) after dose 1. From month 0 until study end, compared with 9585 episodes of clinical malaria that met the primary case definition in children in the C3C group, 6616 episodes occurred in the R3R group (VE 36.3%, 95% CI 31.8-40.5) and 7396 occurred in the R3C group (28.3%, 23.3-32.9); compared with 171 children who experienced at least one episode of severe malaria in the C3C group, 116 children experienced at least one episode of severe malaria in the R3R group (32.2%, 13.7 to 46.9) and 169 in the R3C group (1.1%, -23.0 to 20.5). In young infants, compared with 6170 episodes of clinical malaria that met the primary case definition in the C3C group, 4993 episodes occurred in the R3R group (VE 25.9%, 95% CI 19.9-31.5) and 5444 occurred in the R3C group (18.3%, 11.7-24.4); and compared with 116 infants who experienced at least one episode of severe malaria in the C3C group, 96 infants experienced at least one episode of severe malaria in the R3R group (17.3%, 95% CI -9.4 to 37.5) and 104 in the R3C group (10.3%, -17.9 to 31.8). In children, 1774 cases of clinical malaria were averted per 1000 children (95% CI 1387-2186) in the R3R group and 1363 per 1000 children (995-1797) in the R3C group. The numbers of cases averted per 1000 young infants were 983 (95% CI 592-1337) in the R3R group and 558 (158-926) in the R3C group. The frequency of SAEs overall was balanced between groups. However, meningitis was reported as a SAE in 22 children: 11 in the R3R group, ten in the R3C group, and one in the C3C group. The incidence of generalised convulsive seizures within 7 days of RTS,S/AS01 booster was 2.2 per 1000 doses in young infants and 2.5 per 1000 doses in children. INTERPRETATION: RTS,S/AS01 prevented a substantial number of cases of clinical malaria over a 3-4 year period in young infants and children when administered with or without a booster dose. Efficacy was enhanced by the administration of a booster dose in both age categories. Thus, the vaccine has the potential to make a substantial contribution to malaria control when used in combination with other effective control measures, especially in areas of high transmission. FUNDING: GlaxoSmithKline Biologicals SA and the PATH Malaria Vaccine Initiative