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

Antibiotic resistance and molecular characterization of Shigella isolates recovered from children less than 5 years in Manhiça, Southern Mozambique

The objective of this study was to assess antibiotic resistance and the molecular epidemiology of shigella isolates from a case-control study of diarrhoea, conducted from 2007 to 2012 in children aged less than 5 years in Manhiça district, southern Mozambique. All isolates were tested for antimicrobial susceptibility using the disc diffusion method. Polymerase chain reaction was used to detect different molecular mechanisms of antibiotic resistance. Serotyping was performed using specific antisera. The clonal relationship of Shigella flexneri and Shigella sonnei was assessed by pulsed-field gel electrophoresis (PFGE). Of the 67 shigella isolates analysed, 59 were diarrhoeal cases and eight were controls. S. flexneri (70.1%; 47/67) was the most common species, followed by S. sonnei (23.9%; 16/67). The most prevalent S. flexneri serotypes were 2a (38.3%; 18/47), 6 (19.2%; 9/47) and 1b (14.9%; 7/47). High rates of antimicrobial resistance were observed for trimethoprim-sulfametoxazole (92.5%; 62/67), tetracycline (68.7%; 46/67), chloramphenicol (53.7%; 36/67) and ampicillin (50.7%; 34/67). Multi-drug resistance (MDR) was present in 55.2% (37/67) of the isolates and was associated with a case fatality rate of 8.1% (3/37). PFGE revealed 22 clones (16 S. flexneri and 6 S. sonnei), among which P1 (31.9%; 15/47), P9 (17%; 8/47) and P2 (10.6%; 5/47) were the most prevalent clones of S. flexneri. In conclusion, S. flexneri was the most prevalent species, with MDR isolates mainly belonging to three specific clones (P1, P9 and P2). The case fatality rate observed among MDR isolates is a matter of concern, indicating the need for appropriate treatment

Epidemiology and molecular characterization of multidrug-resistant Escherichia coli isolates harboring blaCTX-M group 1 extended-spectrum β-lactamases causing bacteremia and urinary tract infection in Manhiça, Mozambique

Background: The emergence and spread of extended-spectrum β-lactamases (ESBLs), especially CTX-M, is an important public health problem with serious implications for low-income countries where second-line treatment is often unavailable. Knowledge of the local prevalence of ESBL is critical to define appropriate empirical therapeutic strategies for multidrug-resistant (MDR) organisms. This study aimed to assess and characterize the presence of ESBL and especially CTX-M-producing Escherichia coli MDR isolates from patients with urinary tract infections (UTIs) and bacteremia in a rural hospital in Mozambique. Materials and methods: One hundred and fifty-one E. coli isolates from bacteremia and UTI in children were screened for CTX-M, TEM, SHV and OXA β-lactamases by polymerase chain reaction and sequencing. Isolates carrying CTX-M group 1 β-lactamases were further studied. The resistance to other antibiotic families was determined by phenotypic and genotypic methods, the location of the blaCTX-M gene and the epidemiology of the isolates were studied, and extensive plasmid characterization was performed. Results: Approximately 11% (17/151) of E. coli isolates causing bacteremia and UTI were ESBL producers. CTX-M-15 was the most frequently detected ESBL, accounting for 75% of the total isolates characterized. The blaCTX-M gene is located in different plasmids belonging to different incompatibility groups and can be found in non-epidemiologically related isolates, indicating the high capacity of this resistance determinant to spread widely. Conclusion: Our data suggest the presence of a co-selection of third-generation cephalosporin-resistant determinants in the study area despite limited access to these antibiotics. This highlights the importance of continuous surveillance of antimicrobial resistance of both genetic elements of resistance and resistant isolates in order to monitor the emergence and trends of ESBL-producing isolates to promote adequate therapeutic strategies for the management of MDR bacterial infections

Risk factors for death among children 0–59 months of age with moderate-to-severe diarrhea in Manhiça district, southern Mozambique

Background: Despite major improvements in child survival rates, the number of deaths due to diarrhea remains unacceptably high. We aimed to describe diarrhea-associated mortality and evaluate risk factors for death among Mozambican children with moderate-to-severe diarrhea (MSD). Methods: Between December 2007 and November 2012, children under-five with MSD were enrolled in Manhiça district, as part of the Global Enteric Multicenter study (GEMS). Clinical, epidemiological, and socio-demographic characteristics were collected. Anthropometric measurements were performed and stool samples collected upon recruitment. A follow-up visit ~ 60 days post-enrolment was conducted and verbal autopsies performed in all death cases. Results: Of the 916 MSD-cases analyzed; 90% (821/916) completed 60 days follow-up and 69 patients died. The case fatality rate at follow-up was 8% (69/821), and the mortality rate 10.2 (95%CI: 7.75–13.59) deaths per 1000 persons-week at risk. Nearly half of the deaths 48% (33/69) among study participants clustered within 2 weeks of the onset of diarrhea. Typical enteropathogenic Escherichia coli (typical EPEC) and Cryptosporidium were the two pathogens associated to an increased risk of death in the univariate analysis with (HR = 4.16, p = 0.0461) and (H = 2.84, p = 0.0001) respectively. Conversely, Rotavirus infection was associated to a decreased risk of death (HR = 0.52, p = 0.0198). According to the multivariate analysis, risk factors for death included co-morbidities such as malnutrition (HR = 4.13, p < 0.0001), pneumonia/lower respiratory infection (HR = 3.51, p < 0.0001) or invasive bacterial disease (IBD) (HR = 6. 80, p = 0.0009), presenting on arrival with lethargy or overt unconsciousness (HR = 1.73, p = 0.0302) or wrinkled skin (HR = 1.71, p = 0.0393), and cryptosporidium infection (HR = 2.14, p = 0.0038). When restricting the analysis to those with available HIV results (n = 191, 22% of the total study sample), HIV was shown to be a significant risk factor for death (HR = 5.05, p = 0.0009). Verbal autopsies were conducted in 100% of study deaths, and highlighted diarrhea as the main underlying cause of death 39%, (27/69); followed by HIV/AIDS related deaths 29.0% (20/69) and sepsis 11.6% (8/69).Conclusion: Preventive strategies targeting Cryptosporidium, malnutrition and early identification and treatment of associated co-morbidities could contribute to the prevention of the majority of diarrhea associated deaths in Mozambican children

Clinical features, risk factors, and impact of antibiotic treatment of diarrhea caused by Shigella in children less than 5 years in Manhiça District, rural Mozambique

Objectives: During the period from December 2007 to November 2012, the epidemiology of diarrhea caused by Shigella was studied among children <5 years of age residing in Manhiça District, Southern Mozambique. Materials and methods: Children from 0 to 5 years with moderate-to-severe diarrhea (MSD) and less severe diarrhea (LSD) were enrolled along with matched controls (by age, gender, and neighborhood). Age-stratified logistic regression analyses were conducted to identify clinical features and risk factors associated with Shigella positivity in cases of diarrhea. The impact of antibiotic treatment was assessed for patients with known outcome. Results: A total of 916 cases of MSD and 1979 matched controls, and 431 cases of LSD with equal number of controls were enrolled. Shigella was identified as significant pathogen in both cases of MSD and LSD compared to their respective controls. Shigella was detected in 3.9% (17/431) of LSD compared to 0.5% (2/431) in controls (P=0.001) and in 6.1% (56/916) of MSD cases compared to 0.2% (4/1979) in controls (P<0.0001), with an attributable fraction of 8.55% (95% CI: 7.86–9.24) among children aged 12–23 months. Clinical symptoms associated to Shigella among MSD cases included dysentery, fever, and rectal prolapse. Water availability, giving stored water to child, washing hands before preparing baby’s food, and mother as caretaker were the protective factors against acquiring diarrhea caused by Shigella. Antibiotic treatment on admission was associated with a positive children outcome. Conclusion: Shigella remains a common pathogen associated with childhood diarrhea in Mozambique, with dysentery being a significant clinical feature of shigellosis. Adherence to the basic hygiene rules and the use of antibiotic treatment could contribute to the prevention of most of diarrhea due to Shigella

Characterisation of extended-spectrum b-lactamases among Klebsiella pneumoniae isolates causing bacteraemia and urinary tract infection in Mozambique

The aim of this study was to determine the prevalence of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolated from urinary tract and bloodstream infections in a rural hospital in Manhiça, Mozambique. ESBLs were investigated among ceftriaxone-non-susceptible K. pneumoniae clinical isolates recovered between 2004 and 2009. Characterisation of blaCTX-M, blaSHV, blaOXA and blaTEM genes was performed by PCR and sequencing. Epidemiological relationships were established by phylogenetic analysis, repetitive extragenic palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), whilst plasmid transferability was evaluated by conjugation. In addition, the presence of class 1 and 2 integrons was studied. A total of 19 K. pneumoniae were analysed. The blaCTX-M-15 gene was found in all strains. Other ESBL genes were found concomitantly, including blaSHV-5, blaSHV-2, blaSHV-2A, blaSHV-12 and blaSHV-38. In addition, other β-lactamases such as blaTEM-1 and blaOXA-30 were also detected. REP-PCR identified 15 different epidemiological profiles. MLST analysis also showed great variability of sequence types. The blaCTX-M-15 gene showed a high transfer capacity. The presence of class 1 integrons was high. High levels of multidrug resistance were also found. In conclusion, these data show the dominance of the CTX-M-type ESBL, particularly CTX-M-15, supporting its worldwide dissemination, including in areas with limited access to third-generation cephalosporins. This finding is a matter of concern for clinical management as third-generation cephalosporins are an alternative for treating severe cases of multidrug-resistant infections in this community

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

Molecular Epidemiology of Rotavirus Strains in Symptomatic and Asymptomatic Children in Manhiça District, Southern Mozambique 2008–2019

..870-15 SC; the United States Agency for International Development (USAID), grant number AID-656-F-16-00002 and Fundo Nacional de Investiga??o (FNI), Mo?ambique, grant number 245-INV, within the context of diarrhoeal disease surveillance platform implementation. F.M PhD is supported by Calouste Gulbenkian Foundation, grant number 234066. The authors convey many thanks to all the caregivers who consented to their children?s participation in both studies (GEMS and the diarrhoeal disease platform). They would also like to thank all the professionals in the hospitals and those on field recruitment for their full dedication and effort in children enrolment and collection of data and samples whenever possible. Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Group A rotaviruses remain the leading cause of diarrhoea in children aged <5 years. Mozambique introduced rotavirus vaccine (Rotarix® ) in September 2015. We report rotavirus geno-types circulating among symptomatic and asymptomatic children in Manhiça District, Mozambique, pre-and post-vaccine introduction. Stool was collected from enrolled children and screened for ro-tavirus by enzyme-immuno-sorbent assay. Positive specimens were genotyped for VP7 (G genotypes) and VP4 (P genotypes) by the conventional reverse transcriptase polymerase chain reaction. The combination G12P[8] was more frequently observed in pre-vaccine than in post-vaccine introduction, in moderate to severe diarrhoea (34%, 61/177 vs. 0, p < 0.0001) and controls (23%, 26/113 vs. 0, p = 0.0013) and mixed genotypes (36%, 24/67 vs. 7% 4/58, p = 0.0003) in less severe diarrhoea. We observed changes in post-vaccine compared to pre-vaccine introduction, where G3P[4] and G3P[8] were prevalent in moderate to severe diarrhoea (10%, 5/49 vs. 0, p = 0.0002; and 14%, 7/49 vs. 1%, 1/177, p < 0.0001; respectively), and in less severe diarrhoea (21%, 12/58 vs. 0, p = 0.003; and 24%, 14/58 vs. 0, p < 0.0001; respectively). Our surveillance demonstrated the circulation of similar genotypes contemporaneously among cases and controls, as well as switching from pre-to post-vaccine introduction. Continuous surveillance is needed to evaluate the dynamics of the changes in genotypes following vaccine introduction.publishersversionpublishe

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

Minimal genetic change in Vibrio cholerae in Mozambique over time: Multilocus variable number tandem repeat analysis and whole genome sequencing.

Although cholera is a major public health concern in Mozambique, its transmission patterns remain unknown. We surveyed the genetic relatedness of 75 Vibrio cholerae isolates from patients at Manhiça District Hospital between 2002-2012 and 3 isolates from river using multilocus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS). MLVA revealed 22 genotypes in two clonal complexes and four unrelated genotypes. WGS revealed i) the presence of recombination, ii) 67 isolates descended monophyletically from a single source connected to Wave 3 of the Seventh Pandemic, and iii) four clinical isolates lacking the cholera toxin gene. This Wave 3 strain persisted for at least eight years in either an environmental reservoir or circulating within the human population. Our data raises important questions related to where these isolates persist and how identical isolates can be collected years apart despite our understanding of high change rate of MLVA loci and the V. cholerae molecular clock