Epidemiology and clinical presentation of community-acquired Staphylococcus aureus bacteraemia in children under 5 years of age admitted to the Manhiça District Hospital, Mozambique, 2001-2019.

Staphylococcus aureus bacteraemia (SAB) is one of the most common bloodstream infections globally. Data on the burden and epidemiology of community-acquired SAB in low-income countries are scarce but needed to define preventive and management strategies. Blood samples were collected from children < 5 years of age with fever or severe disease admitted to the Manhiça District Hospital for bacterial isolation, including S. aureus. Between 2001 and 2019, 7.6% (3,197/41,891) of children had bacteraemia, of which 12.3% corresponded to SAB. The overall incidence of SAB was 56.1 episodes/100,000 children-years at risk (CYAR), being highest among neonates (589.8 episodes/100,000 CYAR). SAB declined significantly between 2001 and 2019 (322.1 to 12.5 episodes/100,000 CYAR). In-hospital mortality by SAB was 9.3% (31/332), and significantly associated with infections by multidrug-resistant (MDR) strains (14.7%, 11/75 vs. 6.9%, 14/204 among non-MDR, p = 0.043) and methicillin-resistant S. aureus (33.3%, 5/15 vs. 7.6%, 20/264 among methicillin-susceptible S. aureus, p = 0.006). Despite the declining rates of SAB, this disease remains an important cause of death among children admitted to MDH, possibly in relation to the resistance to the first line of empirical treatment in use in our setting, suggesting an urgent need to review current policy recommendations.

Klebsiella spp. cause severe and fatal disease in Mozambican children: antimicrobial resistance profile and molecular characterization.

BACKGROUND: Klebsiella spp. are important pathogens associated with bacteremia among admitted children and is among the leading cause of death in children < 5 years in postmortem studies, supporting a larger role than previously considered in childhood mortality. Herein, we compared the antimicrobial susceptibility, mechanisms of resistance, and the virulence profile of Klebsiella spp. from admitted and postmortem children. METHODS: Antimicrobial susceptibility and virulence factors of Klebsiella spp. recovered from blood samples collected upon admission to the hospital (n = 88) and postmortem blood (n = 23) from children < 5 years were assessed by disk diffusion and multiplex PCR. RESULTS: Klebsiella isolates from postmortem blood were likely to be ceftriaxone resistant (69.6%, 16/23 vs. 48.9%, 43/88, p = 0.045) or extended-spectrum ß-lactamase (ESBL) producers (60.9%, 14/23 vs. 25%, 22/88, p = 0.001) compared to those from admitted children. blaCTX-M-15 was the most frequent ESBL gene: 65.3%, 9/14 in postmortem isolates and 22.7% (5/22) from admitted children. We found higher frequency of genes associated with hypermucoviscosity phenotype and invasin in postmortem isolates than those from admitted children: rmpA (30.4%; 7/23 vs. 9.1%, 8/88, p = 0.011), wzi-K1 (34.7%; 8/23 vs. 8%; 7/88, p = 0.002) and traT (60.8%; 14/23 vs. 10.2%; 9/88, p < 0.0001), respectively. Additionally, serine protease auto-transporters of Enterobacteriaceae were detected from 1.8% (pic) to 12.6% (pet) among all isolates. Klebsiella case fatality rate was 30.7% (23/75). CONCLUSION: Multidrug resistant Klebsiella spp. harboring genes associated with hypermucoviscosity phenotype has emerged in Mozambique causing invasive fatal disease in children; highlighting the urgent need for prompt diagnosis, appropriate treatment and effective preventive measures for infection control.

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

BACKGROUND: 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). METHODS: We assessed the contribution of EHEC, EIEC and DAEC isolated from stool samples from a case-control study conducted in children aged < 5 years 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. RESULTS: 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 = 0.243]) than in MSD cases (0%, [0/89] of cases vs. 0.4%, [1/222] of controls; p = 1.000). 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 months (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 = 0.383, respectively). Among these, DAEC predominated, being detected in 7.7% of cases vs. 17.6% of controls aged 24-59 months, 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. CONCLUSIONS: 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.

Detecting Staphylococcus aureus Virulence and Resistance Genes: a Comparison of Whole-Genome Sequencing and DNA Microarray Technology.

Staphylococcus aureusis a major bacterial pathogen causing a variety of diseases ranging from wound infections to severe bacteremia or intoxications. Besides host factors, the course and severity of disease is also widely dependent on the genotype of the bacterium. Whole-genome sequencing (WGS), followed by bioinformatic sequence analysis, is currently the most extensive genotyping method available. To identify clinically relevant staphylococcal virulence and resistance genes in WGS data, we developed anin silicotyping scheme for the software SeqSphere(+)(Ridom GmbH, Münster, Germany). The implemented target genes (n= 182) correspond to those queried by the IdentibacS. aureusGenotyping DNA microarray (Alere Technologies, Jena, Germany). Thein silicoscheme was evaluated by comparing the typing results of microarray and of WGS for 154 humanS. aureusisolates. A total of 96.8% (n= 27,119) of all typing results were equally identified with microarray and WGS (40.6% present and 56.2% absent). Discrepancies (3.2% in total) were caused by WGS errors (1.7%), microarray hybridization failures (1.3%), wrong prediction of ambiguous microarray results (0.1%), or unknown causes (0.1%). Superior to the microarray, WGS enabled the distinction of allelic variants, which may be essential for the prediction of bacterial virulence and resistance phenotypes. Multilocus sequence typing clonal complexes and staphylococcal cassette chromosomemecelement types inferred from microarray hybridization patterns were equally determined by WGS. In conclusion, WGS may substitute array-based methods due to its universal methodology, open and expandable nature, and rapid parallel analysis capacity for different characteristics in once-generated sequences.

Invasive Salmonella Infections Among Children From Rural Mozambique, 2001-2014.

BACKGROUND: Invasive nontyphoidal Salmonella (iNTS) has emerged as a cause of bacteremia in African children and HIV-infected adults, which is associated with high mortality. Epidemiological data and burden of iNTS infections in resource-constrained settings are needed to better define preventive and curative strategies. METHODS: Blood and, if appropriate, cerebrospinal fluid, were collected from children <15 years of age with fever or severe disease admitted to the Manhiça District Hospital and cultured for NTS; isolates were then characterized. RESULTS: From January 2001 to December 2014, 41,668 of the 51,878 admitted children had a blood culture performed. Invasive NTS was isolated from 670 (1.6%) specimens collected from 41,668 patients; 69 (10.3% died). Salmonella enterica subspecies enterica serovar Typhi or Salmonella enterica subspecies enterica serovar Paratyphi A or C were only isolated in 14 (0.03%) patients. A total of 460 of 620 (74.2%) NTS isolates serotyped were Salmonella enterica subspecies enterica serovar Typhimurium (45% [116/258] of which were multilocus sequence type 313). The incidence of iNTS was 61.8 (95% confidence interval, 55.4-68.9) cases per 100,000 child-years, being highest among infants (217.7 cases/100,000 child-years). The incidence of iNTS declined significantly (P < .0001) over time, but the case fatality ratio remained constant at approximately 10%. Antimicrobial resistance of iNTS against most available antimicrobials has steadily increased, with a predominance of multidrug-resistant strains. CONCLUSIONS: The decreasing but still high incidence of iNTS, its high associated case fatality ratio, and the common detection of multidrug-resistant strains call for a need to improve treatment and prevention strategies for iNTS.

The Challenge of Diagnosing and Treating Staphylococcus aureus Invasive Infections in a Resource-limited Sub-Saharan Africa Setting: A Case Report.

BACKGROUND: Community-acquired methicillin-sensitive Staphylococcus aureus (CA-MSSA) is responsible for the majority of skin and soft-tissue infections. CA-MSSA can also cause life-threatening infections, possibly in relation to particular virulence factors, including Panton-Valentine leukocidin (PVL). METHODS: We describe a severe CA-MSSA necrotizing pneumonia complicated with multifocal osteomyelitis, pericardial effusion and endocarditis in a 6-year-old boy admitted to a Mozambican hospital. Staphylococcus aureus isolation and antibiotic susceptibility testing were performed by conventional microbiology. Additionally, microarray assay was used for molecular characterization. RESULTS: Blood culture confirmed the presence of S. aureus susceptible to most antimicrobial agents, including methicillin. Molecular characterization confirmed the presence of PVL, together with alpha and beta haemolysin genes. CONCLUSIONS: To our knowledge, this is the first reported case of disseminated CA-MSSA disease with confirmed PVL exotoxin in sub-Saharan Africa. PVL-positive CA-MSSA should be considered in the differential diagnosis of community-acquired pneumonia, making laboratory testing a higher priority.

Exploring the virulence potential of Staphylococcus aureus CC121 and CC152 lineages related to paediatric community-acquired bacteraemia in Manhiça, Mozambique.

Staphylococcus aureus is a frequent agent of bacteraemia. This bacterium has a variety of virulence traits that allow the establishment and maintenance of infection. This study explored the virulence profile of S. aureus strains causing paediatric bacteraemia (SAB) in Manhiça district, Mozambique. We analysed 336 S. aureus strains isolated from blood cultures of children younger than 5 years admitted to the Manhiça District Hospital between 2001 and 2019, previously characterized for antibiotic susceptibility and clonality. The strains virulence potential was evaluated by PCR detection of the Panton-Valentine leucocidin (PVL) encoding genes, lukS-PV/lukF-PV, assessment of the capacity for biofilm formation and pathogenicity assays in Galleria mellonella. The overall carriage of PVL-encoding genes was over 40%, although reaching ~ 70 to 100% in the last years (2014 to 2019), potentially linked to the emergence of CC152 lineage. Strong biofilm production was a frequent trait of CC152 strains. Representative CC152 and CC121 strains showed higher virulence potential in the G. mellonella model when compared to reference strains, with variations within and between CCs. Our results highlight the importance of monitoring the emergent CC152-MSSA-PVL+ and other lineages, as they display important virulence traits that may negatively impact the management of SAB paediatric patients in Manhiça district, Mozambique.

Detection of Enteric Viruses in Children under Five Years of Age before and after Rotavirus Vaccine Introduction in Manhiça District, Southern Mozambique, 2008-2019.

Enteric viruses are the leading cause of diarrhoea in children <5 years. Despite existing studies describing rotavirus diarrhoea in Mozambique, data on other enteric viruses remains scarce, especially after rotavirus vaccine introduction. We explored the prevalence of norovirus GI and GII, adenovirus 40/41, astrovirus, and sapovirus in children <5 years with moderate-to-severe (MSD), less severe (LSD) diarrhoea and community healthy controls, before (2008-2012) and after (2016-2019) rotavirus vaccine introduction in Manhiça District, Mozambique. The viruses were detected using ELISA and conventional reverse transcription PCR from stool samples. Overall, all of the viruses except norovirus GI were significantly more detected after rotavirus vaccine introduction compared to the period before vaccine introduction: norovirus GII in MSD (13/195, 6.7% vs. 24/886, 2.7%, respectively; p = 0.006) and LSD (25/268, 9.3% vs. 9/430, 2.1%, p < 0.001); adenovirus 40/41 in MSD (7.2% vs. 1.8%, p < 0.001); astrovirus in LSD (7.5% vs. 2.6%, p = 0.002); and sapovirus in MSD (7.1% vs. 1.4%, p = 0.047) and controls (21/475, 4.4% vs. 51/2380, 2.1%, p = 0.004). Norovirus GII, adenovirus 40/41, astrovirus, and sapovirus detection increased in MSD and LSD cases after rotavirus vaccine introduction, supporting the need for continued molecular surveillance for the implementation of appropriate control and prevention measures.

Antimicrobial resistance and clonality of Staphylococcus aureus causing bacteraemia in children admitted to the Manhiça District Hospital, Mozambique, over two decades.

Background: Staphylococcus aureus is one of the main causes of bacteraemia, associated with high mortality, mainly due to the occurrence of multidrug resistant (MDR) strains. Data on antibiotic susceptibility and genetic lineages of bacteraemic S. aureus are still scarce in Mozambique. The study aims to describe the antibiotic susceptibility and clonality of S. aureus isolated from blood cultures of children admitted to the Manhiça District Hospital over two decades (2001-2019). Methods: A total of 336 S. aureus isolates detected in blood cultures of children aged <5 years were analyzed for antibiotic susceptibility by disk diffusion or minimal inhibitory concentration, and for the presence of resistance determinants by PCR. The clonality was evaluated by SmaI-PFGE, spa typing, and MLST. The SCCmec element was characterized by SCCmec typing. Results: Most S. aureus (94%, 317/336) were resistant to at least one class of antibiotics, and one quarter (25%) showed a MDR phenotype. High rates of resistance were detected to penicillin (90%) and tetracycline (48%); followed by erythromycin/clindamycin (25%/23%), and co-trimoxazole (11%), while resistance to methicillin (MRSA strains) or gentamicin was less frequent (≤5%). The phenotypic resistance to distinct antibiotics correlated well with the corresponding resistance determinants (Cohen's κ test: 0.7-1.0). Molecular typing revealed highly diverse clones with predominance of CC5 (17%, 58/336) and CC8 (16%), followed by CC15 (11%) and CC1 (11%). The CC152, initially detected in 2001, re-emerged in 2010 and became predominant throughout the remaining surveillance period, while other CCs (CC1, CC5, CC8, CC15, CC25, CC80, and CC88) decreased over time. The 16 MRSA strains detected belonged to clones t064-ST612/CC8-SCCmecIVd (69%, 11/16), t008-ST8/CC8-SCCmecNT (25%, 4/16) and t5351-ST88/CC88-SCCmecIVa (6%, 1/16). Specific clonal lineages were associated with extended length of stay and high in-hospital mortality. Conclusion: We document the circulation of diverse MDR S. aureus causing paediatric bacteraemia in Manhiça district, Mozambique, requiring a prompt recognition of S. aureus bacteraemia by drug resistant clones to allow more targeted clinical management of patients.

Genomic characterization of the rotavirus G3P[8] strain in vaccinated children, reveals possible reassortment events between human and animal strains in Manhiça District, Mozambique.

Mozambique introduced the rotavirus vaccine (Rotarix®; GlaxoSmithKline Biologicals, Rixensart, Belgium) in 2015, and since then, the Centro de Investigação em Saúde de Manhiça has been monitoring its impact on rotavirus-associated diarrhea and the trend of circulating strains, where G3P[8] was reported as the predominant strain after the vaccine introduction. Genotype G3 is among the most commonly detected Rotavirus strains in humans and animals, and herein, we report on the whole genome constellation of G3P[8] detected in two children (aged 18 months old) hospitalized with moderate-to-severe diarrhea at the Manhiça District Hospital. The two strains had a typical Wa-like genome constellation (I1-R1-C1-M1-A1-N1-T1-E1-H1) and shared 100% nucleotide (nt) and amino acid (aa) identities in 10 gene segments, except for VP6. Phylogenetic analysis demonstrated that genome segments encoding VP7, VP6, VP1, NSP3, and NSP4 of the two strains clustered most closely with porcine, bovine, and equine strains with identities ranging from 86.9-99.9% nt and 97.2-100% aa. Moreover, they consistently formed distinct clusters with some G1P[8], G3P[8], G9P[8], G12P[6], and G12P[8] strains circulating from 2012 to 2019 in Africa (Mozambique, Kenya, Rwanda, and Malawi) and Asia (Japan, China, and India) in genome segments encoding six proteins (VP2, VP3, NSP1-NSP2, NSP5/6). The identification of segments exhibiting the closest relationships with animal strains shows significant diversity of rotavirus and suggests the possible occurrence of reassortment events between human and animal strains. This demonstrates the importance of applying next-generation sequencing to monitor and understand the evolutionary changes of strains and evaluate the impact of vaccines on strain diversity.

Child Health and Mortality Prevention Surveillance (CHAMPS): Manhiça site description, Mozambique.

The Manhiça Health Research Centre (Manhiça HDSS) was established in 1996 in Manhiça, a rural district at Maputo Province in the southern part of Mozambique with approximately 49,000 inhabited households, a total population of 209.000 individuals, and an annual estimated birth cohort of about 5000 babies. Since 2016, Manhiça HDSS is implementing the Child Health and Mortality Prevention Surveillance (CHAMPS) program aiming to investigate causes of death (CoD) in stillbirths and children under the age of 5 years using an innovative post-mortem technique known as Minimally Invasive Tissue sampling (MITS), comprehensive pathogen screening using molecular methods, clinical record abstraction and verbal autopsy. Both in-hospital and community pediatric deaths are investigated using MITS. For this, community-wide socio-demographic approaches (notification of community deaths by key informants, formative research involving several segments of the community, availability of free phone lines for notification of medical emergencies and deaths, etc.) are conducted alongside to foster community awareness, involvement and adherence as well as to compute mortality estimates and collect relevant information of health and mortality determinants. The main objective of this paper is to describe the Manhiça Health and Demographic Surveillance System (HDSS) site and the CHAMPS research environment in place including the local capacities among its reference hospital, laboratories, data center and other relevant areas involved in this ambitious surveillance and research project, whose ultimate aim is to improve child survival through public health actions derived from credible estimates and understanding of the major causes of childhood mortality in Mozambique.

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

BACKGROUND: 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.

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

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 genotypes circulating among symptomatic and asymptomatic children in Manhiça District, Mozambique, pre- and post-vaccine introduction. Stool was collected from enrolled children and screened for rotavirus 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.

Effectiveness of Monovalent Rotavirus Vaccine in Mozambique, a Country with a High Burden of Chronic Malnutrition.

Mozambique introduced monovalent rotavirus vaccine (Rotarix®) in September 2015. We evaluated the effectiveness of Rotarix® under conditions of routine use in Mozambican children hospitalized with acute gastroenteritis (AGE). A test negative case-control analysis was performed on data collected during 2017−2019 from children <5 years old, admitted with AGE in seven sentinel hospital sites in Mozambique. Adjusted VE was calculated for ≥1 dose of vaccine vs. zero doses using unconditional logistic regression, where VE = (1 − aOR) × 100%. VE estimates were stratified by age group, AGE severity, malnutrition, and genotype. Among 689 children eligible for analysis, 23.7% were rotavirus positive (cases) and 76.3% were negative (controls). The adjusted VE of ≥1 dose in children aged 6−11 months was 52.0% (95% CI, −11, 79), and −24.0% (95% CI, −459, 62) among children aged 12−23 months. Estimated VE was lower in stunted than non-stunted children (14% (95% CI, −138, 66) vs. 59% (95% CI, −125, 91)). Rotavirus vaccination appeared moderately effective against rotavirus gastroenteritis hospitalization in young Mozambican children. VE point estimates were lower in older and stunted children, although confidence intervals were wide and overlapped across strata. These findings provide additional evidence for other high-mortality countries considering rotavirus vaccine introduction.

Molecular Characterization of Staphylococcus aureus Isolated from Raw Milk Samples of Dairy Cows in Manhiça District, Southern Mozambique.

Staphylococcal infections are among the most common foodborne diseases. We performed the antibiotic susceptibility and molecular characterization of S. aureus from milk samples of dairy cows in Manhiça District. We observed a high frequency of S. aureus (41%, 58/143), in which 71% (41/58) were from commercial farms and 29% (17/58) from smallholder farms. Half of the isolates (50%, 29/58) were resistant to at least one antibiotic, with higher rates of resistance to penicillin (43%, 25/58), followed by tetracycline (16%, 9/58). Multidrug-resistant and methicillin-resistant S. aureus isolates were rare (5%, 3/58 and 3%, 2/58, respectively). The genetic diversity was low, with predominance of human-adapted strains being: ST1/CC1-t5388 (78%) and ST152-t1299 (10%), followed by ST8/CC8-t1476 (5%) and ST5/CC5-t002 (3%) and lastly, ST508/CC45-t331 and ST152-t355, with 2% each. The Panton-Valentine leukocidin (PVL) gene was detected among 14% (8/58) of the isolates, while genes encoding staphylococcal enterotoxins were scarce (3%, 2/58). Our findings revealed a high frequency of S. aureus, with high rates of resistance to the antibiotics commonly used in veterinary and human medicine. Further investigations focusing on the molecular epidemiology of S. aureus from cattle and farmers will provide detailed insights on the genetic relatedness between the strains.

Rotavirus disease burden pre-vaccine introduction in young children in Rural Southern Mozambique, an area of high HIV prevalence.

BACKGROUND: Rotavirus vaccines have been adopted in African countries since 2009, including Mozambique (2015). Disease burden data are needed to evaluate the impact of rotavirus vaccine. We report the burden of rotavirus-associated diarrhea in Mozambique from the Global Enteric Multicenter Study (GEMS) before vaccine introduction. METHODS: A case-control study (GEMS), was conducted in Manhiça district, recruiting children aged 0-59 months with moderate-to-severe diarrhea (MSD) and less-severe-diarrhea (LSD) between December 2007 and November 2012; including 1-3 matched (age, sex and neighborhood) healthy community controls. Clinical and epidemiological data and stool samples (for laboratory investigation) were collected. Association of rotavirus with MSD or LSD was determined by conditional logistic regression and adjusted attributable fractions (AF) calculated, and risk factors for rotavirus diarrhea assessed. RESULTS: Overall 915 cases and 1,977 controls for MSD, and 431 cases and 430 controls for LSD were enrolled. Rotavirus positivity was 44% (217/495) for cases and 15% (160/1046) of controls, with AF = 34.9% (95% CI: 32.85-37.06) and adjusted Odds Ratio (aOR) of 6.4 p< 0.0001 in infants with MSD compared to 30% (46/155) in cases and 14% (22/154) in controls yielding AF = 18.7%, (95% CI: 12.02-25.39) and aOR = 2.8, p = 0.0011 in infants with LSD. The proportion of children with rotavirus was 32% (21/66) among HIV-positive children and 23% (128/566) among HIV-negative ones for MSD. Presence of animals in the compound (OR = 1.9; p = 0.0151) and giving stored water to the child (OR = 2.0, p = 0.0483) were risk factors for MSD; while animals in the compound (OR = 2.37, p = 0.007); not having routine access to water on a daily basis (OR = 1.53, p = 0.015) and washing hands before cooking (OR = 1.76, p = 0.0197) were risk factors for LSD. CONCLUSION: The implementation of vaccination against rotavirus may likely result in a significant reduction of rotavirus-associated diarrhea, suggesting the need for monitoring of vaccine impact.

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.

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.

First identification of genotypes of Enterocytozoon bieneusi (Microsporidia) among symptomatic and asymptomatic children in Mozambique.

Enterocytozoon bieneusi is a human pathogen with a broad range of animal hosts. Initially, E. bieneusi was considered an emerging opportunistic pathogen in immunocompromised, mainly HIV-infected patients, but it has been increasingly reported in apparently healthy individuals globally. As in other African countries, the molecular epidemiology of E. bieneusi in Mozambique remains completely unknown. Therefore, we undertook a study to investigate the occurrence and genetic diversity of E. bieneusi infections in children with gastrointestinal symptoms as well as in asymptomatic children in Mozambique. Individual stool specimens were collected from 1,247 children aged between 0 and 14 years-old living in urban and rural settings in Zambézia (n = 1,097) and Maputo (n = 150) provinces between 2016 and 2019. Samples were analysed for E. bieneusi by nested-PCR targeting the internal transcribed spacer (ITS) region of the rRNA gene. All positive amplicons were confirmed and genotyped. Penalised logistic regression (Firth) was used to evaluate risk associations. The overall prevalence of E. bieneusi in this children population was 0.7% (9/1,247). A 10-fold higher prevalence was found in Maputo (4.0%; 6/150) than in Zambézia (0.3%; 3/1,097). All E. bieneusi-positive samples were from children older than 1-year of age, and most (8/9) from asymptomatic children. Nucleotide sequence analysis of the ITS region revealed the presence of four genotypes, three previously reported (Peru11, n = 1; Type IV, n = 2, and S2, n = 2) and a novel genotype (named HhMzEb1, n = 4). Novel genotype HhMzEb1 was identified in both asymptomatic (75%, 3/4) and symptomatic (25%, 1/4) children from a rural area in Maputo province in southern Mozambique. Genotypes HhMzEb1, Peru11, S2, and Type IV belonged to the Group 1 that includes genotypes with low host specificity and the potential for zoonotic and cross-species transmission. Being infected by enteric protozoan parasites and no handwashing were identified as risk associations for E. bieneusi infection. This study reports the first investigation of E. bieneusi genotypes in Mozambique with the identification of three previously reported genotypes in humans as well as a novel genotype (HhMzEb1). Findings highlight the need to conduct additional research to elucidate the epidemiology of E. bieneusi in the country, especially in rural areas where poor hygiene conditions still prevail. Special attention should be paid to the identification of suitable animal and environmental reservoirs of this parasite and to the characterization of transmission pathways.

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.