Publications by authors named "Marcelino Garrine"

16 Publications

  • Page 1 of 1

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

BMC Infect Dis 2021 Jun 5;21(1):526. Epub 2021 Jun 5.

Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique.

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 β-lactamase (ESBL) producers (60.9%, 14/23 vs. 25%, 22/88, p = 0.001) compared to those from admitted children. bla 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.
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http://dx.doi.org/10.1186/s12879-021-06245-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178901PMC
June 2021

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

Pathogens 2021 Apr 9;10(4). Epub 2021 Apr 9.

Centro de Investigação em Saúde de Manhiça, Maputo 1929, Mozambique.

is a leading cause of childhood diarrhoea and associated physical and cognitive impairment in low-resource settings. -positive faecal samples ( = 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 or rRNA loci for species determination and genotyping. Three species including (72.6%, 138/190), (22.6%, 43/190), and (4.2%, 8/190) were detected. Children ≤ 23 months were more exposed to spp. infections than older children. Both and were more prevalent among children with diarrhoeal disease compared to those children without it (47.6% vs. 33.3%, = 0.007 and 23.7% vs. 11.8%, = 0.014, respectively). A high intra-species genetic variability was observed within (subtype families Ia, Ib, Id, Ie, and If) and (subtype families IIb, IIc, IIe, and IIi) but not within (subtype family IIIb). No association between species/genotypes and child's age was demonstrated. The predominance of and IIc suggests that most of the 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.
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http://dx.doi.org/10.3390/pathogens10040452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070020PMC
April 2021

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

PLoS One 2021 8;16(4):e0249714. Epub 2021 Apr 8.

Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0249714PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8031087PMC
April 2021

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.

PLoS Negl Trop Dis 2021 01 19;15(1):e0008987. Epub 2021 Jan 19.

Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique.

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.
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http://dx.doi.org/10.1371/journal.pntd.0008987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846004PMC
January 2021

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

BMC Infect Dis 2020 Sep 7;20(1):659. Epub 2020 Sep 7.

Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique.

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.
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http://dx.doi.org/10.1186/s12879-020-05380-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7475947PMC
September 2020

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

PLoS Negl Trop Dis 2020 06 30;14(6):e0008419. Epub 2020 Jun 30.

Parasitology Reference and Research Laboratory, National Centre for Microbiology, Health Institute Carlos III, Majadahonda, Madrid, Spain.

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.
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http://dx.doi.org/10.1371/journal.pntd.0008419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357779PMC
June 2020

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

PLoS Negl Trop Dis 2020 05 1;14(5):e0008274. Epub 2020 May 1.

Centro de Investigação em Saúde da Manhiça (CISM), Maputo, Mozambique.

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.
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http://dx.doi.org/10.1371/journal.pntd.0008274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7219792PMC
May 2020

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

Int J Antimicrob Agents 2018 Jun 12;51(6):881-887. Epub 2018 Feb 12.

ISGlobal, Barcelona Centre for International Health Research, Barcelona, Spain; Department of Clinical Microbiology, Centre for Biomedical Diagnosis, Hospital Clinic, Barcelona, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ijantimicag.2018.02.005DOI Listing
June 2018

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

PLoS Negl Trop Dis 2017 Jun 16;11(6):e0005671. Epub 2017 Jun 16.

Department of Epidemiology and Public Health, University of Maryland Baltimore, Baltimore, Maryland, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pntd.0005671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489214PMC
June 2017

Molecular Characterization of Community Acquired Bacteremia in Young Children in Southern Mozambique, 2001-2009.

Front Microbiol 2017 4;8:730. Epub 2017 May 4.

Centro de Investigação em Saúde de Manhiça (CISM)Maputo, Mozambique.

The emergence of community-acquired infections is increasingly recognized as life threating problem worldwide. In Manhiça district, southern Mozambique, is the leading cause of community-acquired bacteremia in neonates. Eighty-four isolates from children less than 5 years admitted to Manhiça District Hospital from 2001 to 2009 were randomly selected and genetically characterized by DNA microarray and typing. Antimicrobial susceptibility was determined by VITEK 2. Thirty-eight different types and 14 clonal complexes (CC) were identified. -type t084 ( = 10; 12%) was the most predominant while CC8 ( = 18; 21%) and CC15 ( = 14; 16%) were the most frequent CCs. Mortality tended to be higher among children infected with CC45 (33.3%, 1/3) and CC8 (27.8%, 5/18). The majority of isolates possessed the accessory gene regulator I (45%) and belonged to either capsule type 8 (52%) or 5 (47%). Panton valentine leukocidin (PVL) encoding genes were detected in 30%. Antibiotic resistance was high for penicillin (89%), tetracycline (59%) and Trimethoprim Sulfamethoxazole (36%) while MRSA was uncommon (8%). Although MRSA were uncommon, we found high genetic diversity of methicillin susceptible causing bacteremia in Mozambican children, associated with high resistance to the most available antibiotics in this community. Some CCs are likely to be more lethal indicating the need for prompt recognition and appropriate treatment.
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http://dx.doi.org/10.3389/fmicb.2017.00730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415612PMC
May 2017

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

J Clin Microbiol 2016 Apr 27;54(4):1008-16. Epub 2016 Jan 27.

Institute of Hygiene, Westphalian Wilhelms-University, Münster, Germany

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.
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http://dx.doi.org/10.1128/JCM.03022-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4809937PMC
April 2016

Evaluation in Cameroon of a Novel, Simplified Methodology to Assist Molecular Microbiological Analysis of V. cholerae in Resource-Limited Settings.

PLoS Negl Trop Dis 2016 Jan 6;10(1):e0004307. Epub 2016 Jan 6.

Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, United States of America.

Background: Vibrio cholerae is endemic in South Asia and Africa where outbreaks of cholera occur widely and are particularly associated with poverty and poor sanitation. Knowledge of the genetic diversity of toxigenic V. cholerae isolates, particularly in Africa, remains scarce. The constraints in improving this understanding is not only the lack of regular cholera disease surveillance, but also the lack of laboratory capabilities in endemic countries to preserve, store and ship isolates in a timely manner. We evaluated the use of simplified sample preservation methods for molecular characterization using multi-locus variable-number tandem-repeat analysis (MLVA) for differentiation of Vibrio cholerae genotypes.

Methods And Findings: Forty-seven V. cholerae isolates and 18 enriched clinical specimens (e.g. stool specimens after enrichment in broth) from cholera outbreaks in Cameroon were preserved on Whatman filter paper for DNA extraction. The samples were collected from two geographically distinct outbreaks in the Far North of Cameroon (FNC) in June 2014 and October 2014. In addition, a convenience sample of 14 isolates from the Philippines and 8 from Mozambique were analyzed. All 87 DNAs were successfully analyzed including 16 paired samples, one a cultured isolate and the other the enriched specimen from which the isolate was collected. Genotypic results were identical between 15 enriched specimens and their culture isolates and the other pair differed at single locus. Two closely related, but distinct clonal complexes were identified among the Cameroonian specimens from 2014.

Conclusions: Collecting V. cholerae using simplified laboratory methods in remote and low-resource settings allows for subsequent advanced molecular characterization of V. cholerae O1. These simplified DNA preservation methods identify V. cholerae and make possible timely information regarding the genetic diversity of V. cholerae; our results set the stage for continued molecular epidemiological research to better understand the transmission and dissemination of V. cholerae in Africa and elsewhere worldwide.
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http://dx.doi.org/10.1371/journal.pntd.0004307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703203PMC
January 2016

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

Clin Infect Dis 2015 Nov;61 Suppl 4:S339-45

Centro de Investigação em Saúde da Manhiça ISGlobal, Barcelona Ctr. Int. Health Res. (CRESIB), Hospital Clínic - Universitat de Barcelona, Spain.

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.
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http://dx.doi.org/10.1093/cid/civ712DOI Listing
November 2015

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

J Trop Pediatr 2015 Oct 17;61(5):397-402. Epub 2015 Jul 17.

Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique, CP1929 ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic - Universitat de Barcelona, Barcelona, 08036, Spain

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.
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http://dx.doi.org/10.1093/tropej/fmv045DOI Listing
October 2015
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