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Background: Invasive non-typhoidal Salmonella (iNTS) serovars S. Typhimurium and S. Enteritidis are major etiologic agents of invasive bacterial disease among infants and young children in sub-Saharan Africa, including in Mali. Early studies of iNTS serovars in several countries indicated that S. Typhimurium was more prevalent than S. Enteritidis, including in Mali before 2008. We investigated genomic and associated phenotypic changes associated with an increase in the relative proportion of iNTS caused by S. Enteritidis versus S. Typhimurium in Bamako, Mali, during the period 2002–2012. Methodology/Principal findings: Comparative genomics studies identified homologs of tetracycline resistance and arsenic utilization genes that were associated with the temporal shift of serovars causing iNTS shift, along with several hypothetical proteins. These findings, validated through PCR screening and phenotypic assays, provide initial steps towards characterizing the genomic changes consequent to unknown evolutionary pressures associated with the shift in serovar prevalence. Conclusions/Significance: This work identified a shift to S. Enteritidis from the more classic S. Typhimurium, associated with iNTS in Bamako, Mali, during the period 2002–2012. This type of shift in underlying iNTS pathogens are of great importance to pediatric public health in endemic regions of sub-Saharan Africa. Additionally, this work demonstrates the utility of combining epidemiologic data, whole genome sequencing, and functional characterization in the laboratory to identify and characterize genomic changes in the isolates that may be involved with the observed shift in circulating iNTS agents. [ABSTRACT FROM AUTHOR]

Among more than 2500 nontyphoidal Salmonella enterica (NTS) serovars, S. enterica serovar Typhimurium and S. enterica serovar Enteritidis account for approximately fifty percent of all human isolates of NTS reported globally. The global incidence of NTS gastroenteritis in 2010 was estimated to be 93 million cases, approximately 80 million of which were contracted via food-borne transmission. It is estimated that 155,000 deaths resulted from NTS in 2010. NTS also causes severe, extra-intestinal, invasive bacteremia, referred to as invasive nontyphoidal Salmonella (iNTS) disease. iNTS disease usually presents as a febrile illness, frequently without gastrointestinal symptoms, in both adults and children. Symptoms of iNTS are similar to malaria, often including fever (>90%) and splenomegaly (>40%). The underlying reasons for the high rates of iNTS disease in Africa are still being elucidated. Evidence from animal and human studies supports the feasibility of developing a safe and effective vaccine against iNTS. Both antibodies and complement can kill Salmonella species in vitro . Proof-of-principle studies in animal models have demonstrated efficacy for live attenuated and subunit vaccines that target the O-antigens, flagellin proteins, and other outer membrane proteins of serovars Typhimurium and Enteritidis. More recently, a novel delivery strategy for NTS vaccines has been developed: the Generalized Modules for Membrane Antigens (GMMA) technology which presents surface polysaccharides and outer membrane proteins in their native conformation. GMMA technology is self-adjuvanting, as it delivers multiple pathogen-associated molecular pattern molecules. GMMA may be particularly relevant for low- and middle-income countries as it has the potential for high immunologic potency at a low cost and involves a relatively simple production process without the need for complex conjugation. Several vaccines for the predominant NTS serovars Typhimurium and Enteritidis, are currently under development. [ABSTRACT FROM AUTHOR]

Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity. [ABSTRACT FROM AUTHOR]

Background. In 2002, following establishment of a clinical microbiology laboratory in the government hospital that admits children with severe illnesses in Bamako, Mali, surveillance to identify pathogens causing invasive bacterial infections (septicemia, bacteremia, meningitis, etc) was initiated. Methods. Parents/guardians of children aged <16 years admitted to l’Hôpital Gabriel Touré with high fever or clinical syndromes compatible with focal invasive bacterial disease were asked for consent to culture their child’s blood/body fluid. Standard bacteriologic techniques speciated isolates; Salmonella serovars were determined. Results. From July 2002 through June 2014, 687 nontyphoidal Salmonella (NTS) isolates were obtained from 667 children; 667 yielded a single serovar and 20 grew 2 Salmonella serovars, 1 being NTS. Four serovars accounted for 87% of the 687 NTS isolates, including Salmonella Enteritidis (n = 244 [35.5%]), Salmonella Typhimurium (n = 221 [32.2%]), I:4,[5],12:i:- (n = 42 [6.1%]), and Salmonella Dublin (n = 89 [13.0%]). Of 553 patients with invasive NTS from whom 1 of the 4 predominant serovars was isolated in pure culture, 448 (81.0%) were aged <5 years and case fatality was 20.3%; Salmonella Enteritidis case fatality (27.8%) was higher than for other serovars (P = .0009). NTS disease showed a seasonal peak following the rainy season and into the cool, dry season. Since 2010, Salmonella Enteritidis cases have risen and Salmonella Typhimurium fallen. Conclusions. NTS has become the predominant invasive pathogen as Haemophilus influenzae type b and pneumococcal vaccine use in Mali has diminished invasive disease due to those pathogens. The age distribution and limited serovars involved make control of NTS disease by vaccines epidemiologically feasible, if products under development prove safe and efficacious. [ABSTRACT FROM AUTHOR]

Background. The gold standard for diagnosis of enteric fever caused by Salmonella Typhi or Salmonella Paratyphi A or B is bone marrow culture. However, because bone marrow aspiration is highly invasive, many hospitals and large health centers perform blood culture instead. As blood culture has several limitations, there is a need for novel typhoid diagnostics with improved sensitivity and more rapid time to detection. Methods. We developed a clyA-based real-time polymerase chain reaction (qPCR) method to detect Salmonella Typhi and Salmonella Paratyphi A simultaneously in blood. The sensitivity and specificity of this probeset was first evaluated in vitro in the laboratory and then in a typhoid-endemic population, in Karachi, Pakistan, and in healthy US volunteers. Results. We optimized a DNA extraction and real-time PCR-based method that could reliably detect 1 colony-forming unit/mL of Salmonella Typhi. The probe set was able to detect clinical Salmonella Typhi and Salmonella Paratyphi A strains and also diarrheagenic Escherichia coli, but not invasive E. coli or other invasive bacteria. In the field, the clyA qPCR diagnostic was 40% as sensitive as blood culture. However, when qPCR-positive specimens were considered to be true positives, blood culture only exhibited 28.57% sensitivity. Specificity was ≥90% for all comparisons and in the healthy US volunteers. qPCR was significantly faster than blood culture in terms of detection of typhoid and paratyphoid. Conclusions. Based on lessons learned, we recommend that future field trials of this and other novel diagnostics that detect typhoidal and nontyphoidal Salmonella employ multiple methodologies to define a “positive” sample. [ABSTRACT FROM AUTHOR]

Highlights: [ • ] An attenuated nontyphoidal Salmonella live oral vaccine is designed by deleting genes guaBA and clpP. [ • ] We test the safety of this vaccine in monkeys infected with simian immunodeficiency virus. [ • ] The vaccine was safe and well-tolerated, establishing basis for efficacy and protection studies. [Copyright &y& Elsevier]

Abstract: Non-typhoidal Salmonella enterica (NTS) serovars Typhimurium and Enteritidis are important causes of bacterial gastroenteritis in the USA and worldwide. In sub-Saharan Africa these two serovars are emerging as agents associated with lethal invasive disease (e.g., bacteremia, meningitis). The development of NTS vaccines, based on mucosally administered live attenuated strains and parenteral non-living antigens, could diminish the NTS disease burden globally. Mouse models of S. Typhimurium and S. Enteritidis invasive disease can accelerate the development of NTS vaccines. Live attenuated NTS vaccines elicit both cellular and humoral immunity in mice and their efficacy is well established. In contrast, non-living vaccines that primarily elicit humoral immunity have demonstrated variable efficacy. An analysis of the reported studies with non-living vaccines against S. Typhimurium and S. Enteritidis reveals that efficacy is influenced by two important independent variables: (1) the innate susceptibility to NTS infection that differs dramatically between commonly used mouse strains and (2) the virulence of the NTS strain used for challenge. Protection by non-living vaccines has generally been seen only in host–pathogen interactions where a sub-lethal infection results, such as challenging resistant mice with either highly virulent or weakly virulent strains or susceptible mice with weakly virulent strains. The immunologic basis of this discrepancy and the implications for human NTS vaccine development are reviewed herein. [Copyright &y& Elsevier]

Certain serovars of Salmonella enterica subsp. enterica cause invasive disease (e.g., enteric fever, bacteremia, septicemia, meningitis, etc.) in humans and constitute a global public health problem. A rapid, sensitive diagnostic test is needed to allow prompt initiation of therapy in individual patients and for measuring disease burden at the population level. An innovative and promising new rapid diagnostic technique is microwave-accelerated metal-enhanced fluorescence (MAMEF). We have adapted this assay platform to detect the chromosomal oriC locus common to all Salmonella enterica subsp. enterica serovars. We have shown efficient lysis of biologically relevant concentrations of Salmonella spp. suspended in bacteriological media using microwave-induced lysis. Following lysis and DNA release, as little as 1 CFU of Salmonella in 1 ml of medium can be detected in ,30 seconds. Furthermore the assay is sensitive and specific: it can detect oriC from Salmonella serovars Typhi, Paratyphi A, Paratyphi B, Paratyphi C, Typhimurium, Enteritidis and Choleraesuis but does not detect Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae or Acinetobacter baumanii. We have also performed preliminary experiments using a synthetic Salmonella oriC oligonucleotide suspended in whole human blood and observed rapid detection when the sample was diluted 1:1 with PBS. These pre-clinical data encourage progress to the next step to detect Salmonella in blood (and other ordinarily sterile, clinically relevant body fluids). [ABSTRACT FROM AUTHOR]

Background: In sub-Saharan Africa, non-typhoidal Salmonella (NTS) are emerging as a prominent cause of invasive disease (bacteremia and focal infections such as meningitis) in infants and young children. Importantly, including data from Mali, three serovars, Salmonella enterica serovar Typhimurium, Salmonella Enteritidis and Salmonella Dublin, account for the majority of non-typhoidal Salmonella isolated from these patients. Methods: We have extended a previously developed series of polymerase chain reactions (PCRs) based on O serogrouping and H typing to identify Salmonella Typhimurium and variants (mostly I 4,[5],12:i:-), Salmonella Enteritidis and Salmonella Dublin. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR was used to differentiate diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium from other O serogroup B, H:i serovars. We used these PCRs to blind-test 327 Salmonella serogroup B and D isolates that were obtained from the blood cultures of febrile patients in Bamako, Mali. Principal Findings: We have shown that when used in conjunction with our previously described O-serogrouping PCR, our PCRs are 100% sensitive and specific in identifying Salmonella Typhimurium and variants, Salmonella Enteritidis, Salmonella Dublin and Salmonella Stanleyville. When we attempted to differentiate 171 Salmonella Typhimurium (I 4,[ 5],12:i:1,2) strains from 52 monophasic Salmonella Typhimurium (I 4,[5],12:i:-) strains, we were able to correctly identify 170 of the Salmonella Typhimurium and 51 of the Salmonella I 4,[5],12:i:- strains. Conclusion: We have described a simple yet effective PCR method to support surveillance of the incidence of invasive disease caused by NTS in developing countries. Author Summary: The genus Salmonella has more than 2500 serological variants (serovars), such as Salmonella enterica serovar Typhi and Salmonella Paratyphi A and B, that cause, respectively, typhoid and paratyphoid fevers (enteric fevers), and a large number of non-typhoidal Salmonella (NTS) serovars that cause gastroenteritis in healthy hosts. In young infants, the elderly and immunocompromised hosts, NTS can cause severe, fatal invasive disease. Multiple studies of pediatric patients in sub-Saharan Africa have documented the important role of NTS, in particular Salmonella Typhimurium and Salmonella Enteritidis (and to a lesser degree Salmonella Dublin), as invasive bacterial pathogens. Salmonella spp. are isolated from blood and identified by standard microbiological techniques and the serovar is ascertained by agglutination with commercial antisera. PCR-based typing techniques are becoming increasingly popular in developing countries, in part because high quality typing sera are difficult to obtain and expensive and H serotyping is technically difficult. We have developed a series of polymerase chain reactions (PCRs) to identify Salmonella Typhimurium and variants, Salmonella Enteritidis and Salmonella Dublin. We successfully identified 327 Salmonella isolates using our multiplex PCR. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR generally differentiated diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium variant strains from other closely related strains. The PCRs described here will enable more laboratories in developing countries to serotype NTS that have been isolated from blood. [ABSTRACT FROM AUTHOR]

Background: In sub-Saharan Africa, non-typhoidal Salmonella (NTS) are emerging as a prominent cause of invasive disease (bacteremia and focal infections such as meningitis) in infants and young children. Importantly, including data from Mali, three serovars, Salmonella enterica serovar Typhimurium, Salmonella Enteritidis and Salmonella Dublin, account for the majority of non-typhoidal Salmonella isolated from these patients. Methods: We have extended a previously developed series of polymerase chain reactions (PCRs) based on O serogrouping and H typing to identify Salmonella Typhimurium and variants (mostly I 4,[5],12:i:-), Salmonella Enteritidis and Salmonella Dublin. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR was used to differentiate diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium from other O serogroup B, H:i serovars. We used these PCRs to blind-test 327 Salmonella serogroup B and D isolates that were obtained from the blood cultures of febrile patients in Bamako, Mali. Principal Findings: We have shown that when used in conjunction with our previously described O-serogrouping PCR, our PCRs are 100% sensitive and specific in identifying Salmonella Typhimurium and variants, Salmonella Enteritidis, Salmonella Dublin and Salmonella Stanleyville. When we attempted to differentiate 171 Salmonella Typhimurium (I 4,[ ],12:i:1,2) strains from 52 monophasic Salmonella Typhimurium (I 4,[5],12:i:-) strains, we were able to correctly identify 170 of the Salmonella Typhimurium and 51 of the Salmonella I 4,[5],12:i:- strains. Conclusion: We have described a simple yet effective PCR method to support surveillance of the incidence of invasive disease caused by NTS in developing countries. [ABSTRACT FROM AUTHOR]

Yersinia enterocolitica strains of biotype 1A lack the known virulence determinants of strains in other categories, including the Yersinia virulence plasmid (pYV), and several chromosomal markers of pathogenicity. For this reason, and also because Y. enterocolitica strains of biotype 1A are frequently isolated from the environment or asymptomatic individuals, these bacteria are often assumed to be avirulent. On the other hand, there is a considerable body of clinical, epidemiological and experimental evidence to indicate that at least some strains of Y. enterocolitica biotype 1A are able to cause gastrointestinal symptoms which resemble those caused by pYV-bearing strains. The availability of a number of experimental systems, including cell culture and animal models of infection, provides an opportunity to identify and characterise the essential virulence determinants of biotype 1A strains. [Copyright &y& Elsevier]

Background Salmonella enterica bloodstream infections are an important cause of childhood morbidity and mortality, including in Mali. We report 17 years of surveillance for nontyphoidal and typhoidal S. enterica infections among inpatients and outpatients at l'Hôpital Gabriel Touré, the main source of pediatric tertiary care in Bamako, Mali. Methods Between June 2002 and December 2018, a blood culture was collected from 54 748 children aged ≤15 years with fever and/or suspected invasive bacterial infection who provided consent (38 152 inpatients, 16 596 outpatients). Bacterial pathogens were identified using standard microbiological techniques and serovars of S. enterica were determined by PCR and/or agglutination with antisera. Results Nontyphoidal Salmonella (NTS) was identified in 671 enrolled inpatients (1.8% of all enrolled inpatients, 13.8% of enrolled inpatients with a positive culture). S. Enteritidis, the most common NTS serovar, accounted for 38.5% of all NTS isolates (n = 258), followed by S. Typhimurium (31.7%, n = 213). The median (SD) age of children with a culture positive for NTS was 1.8 (3) years. Overall case fatality was 20.9%. An additional 138 inpatients (0.4%) had a positive culture for typhoidal Salmonella. NTS was identified in 11 outpatients (0.07%), while typhoidal Salmonella was found in 49 outpatients (0.3%). The annual incidence of invasive NTS disease decreased over the study period, but case fatality remained high. Conclusions Although incidence decreased, NTS remained a major cause of invasive bacterial infection and mortality among hospitalized children in Bamako, while typhoidal Salmonella was uncommon. Because 87% of NTS belonged to only 4 serovars, a multivalent vaccine may be an effective strategy to reduce the burden and mortality of invasive NTS. [ABSTRACT FROM AUTHOR]

Background Few studies describe the respiratory syncytial virus (RSV) burden in African populations, and most have utilized hospital-based surveillance. In Mali, no community-based studies exist of the incidence or epidemiology of RSV infection. This study provides the first estimates of RSV incidence in Mali. Methods In a cohort of infants enrolled in a clinical trial of maternal influenza vaccination, we estimate incidence of RSV-associated febrile illness in the first 6 months of life and identify risk factors for RSV infection and progression to severe disease. Infants (N = 1871) were followed from birth to 6 months of age and visited weekly to detect pneumonia and influenza-like illness. Baseline covariates were explored as risk factors for RSV febrile illness and RSV pneumonia or hospitalization. Results Incidence of RSV illness was estimated at 536.8 per 1000 person-years, and 86% (131/153) of RSV illness episodes were positive for RSV-B. RSV illness was most frequent in the fifth month of life and associated with having older mothers and with lower parity. The incidence of RSV-associated hospitalizations was 45.6 per 1000 person-years. Among infants with RSV illness, males were more likely to be hospitalized. The incidence of RSV pneumonia was 29 cases per 1000 person-years. Conclusions In the first 6 months of life, Malian infants have a high incidence of RSV illness, primarily caused by RSV-B. Prevention of early RSV will require passive protection via maternal immunization in pregnancy. Mali is the first country where RSV-B has been identified as the dominant subtype, with potential implications for vaccine development. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Typhi produces significant morbidity and mortality worldwide despite the fact that there are licensed Salmonella Typhi vaccines available. This is primarily due to the fact that these vaccines are not used in the countries that most need them. There is growing recognition that an effective invasive Salmonella vaccine formulation must also prevent infection due to other Salmonella serovars. We anticipate that a multivalent vaccine that targets the following serovars will be needed to control invasive Salmonella infections worldwide: Salmonella Typhi, Salmonella Paratyphi A, Salmonella Paratyphi B (currently uncommon but may become dominant again), Salmonella Typhimurium, Salmonella Enteritidis and Salmonella Choleraesuis (as well as other Group C Salmonella ). Live attenuated vaccines are an attractive vaccine formulation for use in developing as well as developed countries. Here, we describe the methods of attenuation that have been used to date to create live attenuated Salmonella vaccines and provide an update on the progress that has been made on these vaccines. [ABSTRACT FROM AUTHOR]

Background: As part of the Vaccine Impact on Diarrhea in Africa (VIDA) Study, we examined the prevalence, clinical presentation, and seasonality of Cryptosporidium in children to understand its relative burden after the introduction of rotavirus vaccine. Methods: VIDA was a 3-year, age-stratified, matched case-control study of medically attended acute moderate-to-severe diarrhea (MSD) in children aged 0–59 months residing in censused populations at sites in Kenya, Mali, and The Gambia. Clinical and epidemiologic data were collected at enrollment, and a stool sample was tested for enteropathogens by quantitative PCR. An algorithm was created based on the organism's cycle threshold (Ct) and association with MSD to identify the subset of Cryptosporidium PCR-positive (Ct <35) cases most likely to be attributed to MSD. Clinical outcomes were assessed at 2–3 months after enrollment. Results: One thousand one hundred six (22.9%) cases of MSD and 873 controls (18.1%) were PCR positive for Cryptosporidium; 465 cases (42.0%) were considered attributable to Cryptosporidium, mostly among children 6–23 months. Cryptosporidium infections peaked in The Gambia and Mali during the rainy season, while in Kenya they did not have clear seasonality. Compared with cases with watery MSD who had a negative PCR for Cryptosporidium, cases with watery MSD attributed to Cryptosporidium were less frequently dehydrated but appeared more severely ill using a modified Vesikari scale (38.1% vs 27.0%; P < 0.001), likely due to higher rates of hospitalization and intravenous fluid administration, higher prevalence of being wasted or very thin very thin (23.4% vs 14.7%; P < 0.001), and having severe acute malnutrition (midupper arm circumference <115 mm, 7.7% vs 2.5%; P < 0.001). On follow-up, Cryptosporidium-attributed cases had more prolonged and persistent episodes (43.2% vs 32.7%; P <0.001) and linear growth faltering (change in height-for-age z score between enrollment and follow-up: −0.29 vs −0.17; P < 0.001). Conclusions: The burden of Cryptosporidium remains high among young children in sub-Saharan Africa. Its propensity to cause illness and further impact children longer term by compromising nutritional status early in life calls for special attention to enable appropriate management of clinical and nutritional consequences. The burden of Cryptosporidium remains high among young children in sub-Saharan Africa. Its propensity to cause illness and further impact children longer term by compromising nutritional status early in life necessitates appropriate management of clinical and nutritional consequences. [ABSTRACT FROM AUTHOR]

Background: Pediatric exposures to unsafe sources of water, unsafely managed sanitation, and animals are prevalent in low- and middle-income countries. In the Vaccine Impact on Diarrhea in Africa case-control study, we examined associations between these risk factors and moderate-to-severe diarrhea (MSD) in children <5 years old in The Gambia, Kenya, and Mali. Methods: We enrolled children <5 years old seeking care for MSD at health centers; age-, sex-, and community-matched controls were enrolled at home. Conditional logistic regression models, adjusted for a priori confounders, were used to evaluate associations between MSD and survey-based assessments of water, sanitation, and animals living in the compound. Results: From 2015 to 2018, 4840 cases and 6213 controls were enrolled. In pan-site analyses, children with drinking water sources below "safely managed" (onsite, continuously accessible sources of good water quality) had 1.5–2.0-fold higher odds of MSD (95% confidence intervals [CIs] ranging from 1.0 to 2.5), driven by rural site results (The Gambia and Kenya). In the urban site (Mali), children whose drinking water source was less available (several hours/day vs all the time) had higher odds of MSD (matched odds ratio [mOR]: 1.4, 95% CI: 1.1, 1.7). Associations between MSD and sanitation were site-specific. Goats were associated with slightly increased odds of MSD in pan-site analyses, whereas associations with cows and fowl varied by site. Conclusions: Poorer types and availability of drinking water sources were consistently associated with MSD, whereas the impacts of sanitation and household animals were context-specific. The association between MSD and access to safely managed drinking water sources post-rotavirus introduction calls for transformational changes in drinking water services to prevent acute child morbidity from MSD. We assessed water, sanitation, and animal-associated risk factors for moderate-to-severe diarrhea in children in a matched case-control study. Less availability (hours/day) and access to quality drinking water were consistent risk factors; poor sanitation and animal ownership were context-specific risk factors. [ABSTRACT FROM AUTHOR]

Background: Diarrheal disease is heterogeneous, including watery diarrhea (WD) and dysentery, some cases of which become persistent diarrhea (PD). Changes in risk over time necessitate updated knowledge of these syndromes in sub-Saharan Africa. Methods: The Vaccine Impact on Diarrhea in Africa (VIDA) study was an age-stratified, case-control study of moderate-to-severe diarrhea among children <5 years old in The Gambia, Mali, and Kenya (2015–2018). We analyzed cases with follow-up of about 60 days after enrollment to detect PD (lasting ≥14 days), examined the features of WD and dysentery, and examined determinants for progression to and sequelae from PD. Data were compared with those from the Global Enteric Multicenter Study (GEMS) to detect temporal changes. Etiology was assessed from stool samples using pathogen attributable fractions (AFs), and predictors were assessed using χ2 tests or multivariate regression, where appropriate. Results: Among 4606 children with moderate-to-severe diarrhea, 3895 (84.6%) had WD and 711 (15.4%) had dysentery. PD was more frequent among infants (11.3%) than in children 12–23 months (9.9%) or 24–59 months (7.3%), P =.001 and higher in Kenya (15.5%) than in The Gambia (9.3%) or Mali (4.3%), P <.001; the frequencies were similar among children with WD (9.7%) and those with dysentery (9.4%). Compared to children not treated with antibiotics, those who received antibiotics had a lower frequency of PD overall (7.4% vs 10.1%, P =.01), and particularly among those with WD (6.3% vs 10.0%; P =.01) but not among children with dysentery (8.5% vs 11.0%; P =.27). For those with watery PD, Cryptosporidium and norovirus had the highest AFs among infants (0.16 and 0.12, respectively), while Shigella had the highest AF (0.25) in older children. The odds of PD decreased significantly over time in Mali and Kenya while increasing significantly in The Gambia. Conclusions: The burden of PD endures in sub-Saharan Africa, with nearly 10% of episodes of WD and dysentery becoming persistent. This report updates our knowledge of the risk factors and etiology associated with persistent diarrhea and dysentery. We demonstrate that both persistent and bloody diarrhea remain serious problems with distinct etiology, presentation, and outcomes among sub-Saharan African infants. [ABSTRACT FROM AUTHOR]

Background: Studies conducted before rotavirus vaccine introduction found that moderate-to-severe diarrhea (MSD) in children aged <5 years was associated with stunting at follow-up. It is unknown whether the reduction in rotavirus-associated MSD following vaccine introduction decreased the risk of stunting. Methods: The Global Enteric Multicenter Study (GEMS) and the Vaccine Impact on Diarrhea in Africa (VIDA) study, two comparable matched case-control studies, were conducted during 2007–2011 and 2015–2018, respectively. We analyzed data from 3 African sites where rotavirus vaccine was introduced after GEMS and before starting VIDA. Children with acute MSD (<7 days onset) were enrolled from a health center and children without MSD (diarrhea-free for ≥7 days) were enrolled at home within 14 days of the index MSD case. The odds of being stunted at a follow-up visit 2–3 months after enrollment for an episode of MSD was compared between GEMS and VIDA using mixed-effects logistic regression models controlling for age, sex, study site, and socioeconomic status. Results: We analyzed data from 8808 children from GEMS and 10 579 from VIDA. Among those who were not stunted at enrollment in GEMS, 8.6% with MSD and 6.4% without MSD became stunted during the follow-up period. In VIDA, 8.0% with MSD and 5.5% children without MSD developed stunting. An episode of MSD was associated with higher odds of being stunted at follow-up compared with children without MSD in both studies (adjusted odds ratio [aOR], 1.31; 95% confidence interval [CI]: 1.04–1.64 in GEMS and aOR, 1.30; 95% CI: 1.04–1.61 in VIDA). However, the magnitude of association was not significantly different between GEMS and VIDA (P =.965). Conclusions: The association of MSD with subsequent stunting among children aged <5 years in sub-Saharan Africa did not change after rotavirus vaccine introduction. Focused strategies are needed for prevention of specific diarrheal pathogens that cause childhood stunting. Despite reductions in the attributable burden of rotavirus following vaccine introduction, the association between moderate to severe diarrhea and stunting at a 2–3 month follow-up did not change between studies conducted before and after rotavirus vaccine introduction at 3 sub-Saharan African sites. [ABSTRACT FROM AUTHOR]

Background: The magnitude of pediatric enteric pathogen exposures in low-income settings necessitates substantive water and sanitation interventions, including animal feces management. We assessed associations between pediatric enteric pathogen detection and survey-based water, sanitation, and animal characteristics within the Vaccine Impact on Diarrhea in Africa case-control study. Methods: In The Gambia, Kenya, and Mali, we assessed enteric pathogens in stool of children aged <5 years with moderate-to-severe diarrhea and their matched controls (diarrhea-free in prior 7 days) via the TaqMan Array Card and surveyed caregivers about household drinking water and sanitation conditions and animals living in the compound. Risk ratios (RRs) and 95% confidence intervals (CIs) were calculated using modified Poisson regression models, stratified for cases and controls and adjusted for age, sex, site, and demographics. Results: Bacterial (cases, 93%; controls, 72%), viral (63%, 56%), and protozoal (50%, 38%) pathogens were commonly detected (cycle threshold <35) in the 4840 cases and 6213 controls. In cases, unimproved sanitation (RR, 1.56; 95% CI, 1.12–2.17), as well as cows (RR, 1.61; 95% CI, 1.16–2.24) and sheep (RR, 1.48; 95% CI, 1.11–1.96) living in the compound, were associated with Shiga toxin–producing Escherichia coli. In controls, fowl (RR, 1.30; 95% CI, 1.15–1.47) were associated with Campylobacter spp. In controls, surface water sources were associated with Cryptosporidium spp., Shigella spp., heat-stable toxin-producing enterotoxigenic E. coli, and Giardia spp. Conclusions: Findings underscore the importance of enteric pathogen exposure risks from animals alongside more broadly recognized water and sanitation risk factors in children. We assessed survey-based water, sanitation, and animal factors associated with enteric pathogens in cases of moderate to severe diarrhea and controls. Animals, cows and sheep (cases) and fowl (controls), were associated with bacterial carriage; surface water was associated with bacteria and protozoa in controls. [ABSTRACT FROM AUTHOR]

Human cryptosporidiosis, caused primarily by Cryptosporidium hominis and a subset of Cryptosporidium parvum, is a major cause of moderate-to-severe diarrhea in children under 5 years of age in developing countries and can lead to nutritional stunting and death. Cryptosporidiosis is particularly severe and potentially lethal in immunocompromised hosts. Biological and technical challenges have impeded traditional vaccinology approaches to identify novel targets for the development of vaccines against C. hominis, the predominant species associated with human disease. We deemed that the existence of genomic resources for multiple species in the genus, including a muchimproved genome assembly and annotation for C. hominis, makes a reverse vaccinology approach feasible. To this end, we sought to generate a searchable online resource, termed C. hominis gene catalog, which registers all C. hominis genes and their properties relevant for the identification and prioritization of candidate vaccine antigens, including physical attributes, properties related to antigenic potential and expression data. Using bioinformatic approaches, we identified ~400 C. hominis genes containing properties typical of surface-exposed antigens, such as predicted glycosylphosphatidylinositol (GPI)-anchor motifs, multiple transmembrane motifs and/or signal peptides targeting the encoded protein to the secretory pathway. This set can be narrowed further, e.g. by focusing on potential GPI-anchored proteins lacking homologs in the human genome, but with homologs in the other Cryptosporidium species for which genomic data are available, and with low amino acid polymorphism. Additional selection criteria related to recombinant expression and purification include minimizing predicted post-translation modifications and potential disulfide bonds. Forty proteins satisfying these criteria were selected from 3745 proteins in the updated C. hominis annotation. The immunogenic potential of a few of these is currently being tested. [ABSTRACT FROM AUTHOR]

Innate immune inflammatory responses are subject to complex layers of negative regulation at intestinal mucosal surfaces. Although the type I IFN system is critical for amplifying antiviral immunity, it has been shown to play a homeostatic role in some models of autoimmune inflammation. Type I IFN is triggered in the gut by select bacterial pathogens, but whether and how the type I IFN might regulate innate immunity in the intestinal environment have not been investigated in the context of Salmonella enterica serovar Typhimurium (ST). ST infection of human or murine macrophages reveals that IFN-β selectively restricts the transcriptional responses mediated by both the TLRs and the NOD-Iike receptors. Specifically, IFN-β potently represses ST-dependent innate induction of IL-1 family cytokines and neutrophil chemokines. This IFN-β-mediated transcriptional repression was independent of the effects of IFN-β on .Si-induced macrophage cell death, but significantly dependent on IL-10 regulation. We further evaluated ST pathogenesis in vivo following oral inoculation of mice lacking IFN-β. We show that IFN-β-/- mice exhibit greater resistance to oral ST infection and a slower spread of ST to distal sterile sites. This Work provides mechanistic insight into the relationship between ST and type I IFN, and demonstrates an additional mechanism by which IFN-β may promote spread of enteric pathogens. [ABSTRACT FROM AUTHOR]

Typhoid fever epidemiology was investigated rigorously in Santiago, Chile during the 1980s, when Salmonella enterica serovar Typhi (S. Typhi) caused seasonal, hyperendemic disease. Targeted interventions reduced the annual typhoid incidence rates from 128–220 cases/105 population occurring between 1977–1984 to <8 cases/105 from 1992 onwards. As such, Santiago represents a contemporary example of the epidemiologic transition of an industrialized city from amplified hyperendemic typhoid fever to a period when typhoid is no longer endemic. We used whole genome sequencing (WGS) and phylogenetic analysis to compare the genotypes of S. Typhi cultured from acute cases of typhoid fever occurring in Santiago during the hyperendemic period of the 1980s (n = 74) versus the nonendemic 2010s (n = 80) when typhoid fever was rare. The genotype distribution between "historical" (1980s) isolates and "modern" (2011–2016) isolates was similar, with genotypes 3.5 and 2 comprising the majority of isolations, and 73/80 (91.3%) of modern isolates matching a genotype detected in the 1980s. Additionally, phylogenomically 'ancient' genotypes 1.1 and 1.2.1, uncommon in the global collections, were also detected in both eras, with a notable rise amongst the modern isolates. Thus, genotypes of S. Typhi causing acute illness in the modern nonendemic era match the genotypes circulating during the hyperendemic 1980s. The persistence of historical genotypes may be explained by chronic typhoid carriers originally infected during or before the 1980s. Author summary: Studies of Salmonella Typhi (the cause of typhoid fever) rarely include isolates collected both before and after the interruption of hyperendemic transmission because this typically occurred decades before modern bacteria preservation methods. After interruption, it was assumed that sporadic cases and infrequent outbreaks were due to either chronic biliary carriers or importations, but this was difficult to characterize with low resolution bacterial typing methods. In Santiago, Chile, typhoid fever persisted at hyperendemic levels through the 1980s until organized control efforts in the 1980s and changes to wastewater policy in 1991 caused annual typhoid incidence to plummet. In this study, we used whole genome sequencing (WGS) to investigate whether recent sporadic cases occurring in Santiago in the 2010s were genomically similar to S. Typhi circulating in the 1980s, or dissimilar, possibly representing importations of S. Typhi from outside of Chile. We found concordance amongst S. Typhi genotypes between the 1980s and 2010s, and differences from genotypes circulating in Southeast Asia and Africa where typhoid remains hyperendemic. Our findings suggest that a proportion of modern, rare typhoid cases in Santiago are autochthonous, and that chronic carriers or another unknown reservoir likely contribute. Broadly, our findings corroborate the epidemiologic importance of long-term reservoirs of typhoid fever decades after typhoid elimination. [ABSTRACT FROM AUTHOR]

Cholera remains a major cause of infectious diarrhea globally. Despite the increased availability of cholera vaccines, there is still an urgent need for other effective interventions to reduce morbidity and mortality. Furthermore, increased prevalence of antibiotic-resistant Vibrio cholerae threatens the use of many drugs commonly used to treat cholera. We developed iOWH032, a synthetic small molecule inhibitor of the cystic fibrosis transmembrane conductance regulator chloride channel, as an antisecretory, host-directed therapeutic for cholera. In the study reported here, we tested iOWH032 in a Phase 2a cholera controlled human infection model. Forty-seven subjects were experimentally infected with V. cholerae El Tor Inaba strain N16961 in an inpatient setting and randomized to receive 500 mg iOWH032 or placebo by mouth every 8 hours for 3 days to determine the safety and efficacy of the compound as a potential treatment for cholera. We found that iOWH032 was generally safe and achieved a mean (± standard deviation) plasma level of 4,270 ng/mL (±2,170) after 3 days of oral dosing. However, the median (95% confidence interval) diarrheal stool output rate for the iOWH032 group was 25.4 mL/hour (8.9, 58.3), compared to 32.6 mL/hour (15.8, 48.2) for the placebo group, a reduction of 23%, which was not statistically significant. There was also no significant decrease in diarrhea severity and number or frequency of stools associated with iOWH032 treatment. We conclude that iOWH032 does not merit future development for treatment of cholera and offer lessons learned for others developing antisecretory therapeutic candidates that seek to demonstrate proof of principle in a cholera controlled human infection model study. Trial registration: This study is registered with ClinicalTrials.gov as NCT04150250. Author summary: Cholera, a disease caused by infection with the bacterium Vibrio cholerae, remains a major cause of diarrheal illness and death, particularly in settings with poor sanitation and hygiene. We developed a synthetic chemical, named "iOWH032," as a potential treatment for cholera, which is administered as oral tablets. The chemical acts by blocking secretions from cells in the intestine, and thereby was expected to prevent fluid loss and dehydration caused by cholera illness. We tested iOWH032 in a clinical study using a cholera human challenge model. Study volunteers were intentionally infected with V. cholerae in an inpatient clinic setting to better study the effects of iOWH032 on infected individuals. This challenge model had been used previously to test cholera vaccine candidates, but this study represents the first test of a potential cholera treatment using the model. We found that treatment of individuals with iOWH032 was safe, but did not result in a significant reduction of cholera illness, based on several different measurements of diarrheal symptoms and severity. This study demonstrates how human challenge models incorporating a relatively small number of subjects can help support decision-making about potential new therapeutics and other interventions for infectious diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Older adults are more susceptible to viral and bacterial infection, and experience higher incidence and severity of infectious diseases. Although vaccination is the most logical solution in preventing infectious diseases, primary vaccine responses in individuals aged ≥65 years-old fail to generate complete protection. This is presumably attributed to immunosenescence, a term that describes functional differences associated with the immune system and natural age advancement. Both the innate and adaptive immune systems experience age-related impairments that contribute to insufficient protection following vaccination. This review addresses current knowledge of age-related changes that affect vaccine responsiveness; including the deficits in innate cell functions, dampened humoral and cell-mediated immune responses, current vaccination schedules for older adults, and concludes with potential strategies for improving vaccine efficacy specifically for this age group. Due to an age-related decline in immunity and poor vaccine responses, infectious diseases remain a burden among the aged population. [ABSTRACT FROM AUTHOR]

With the emergence of multidrug resistant Salmonella strains, the development of anti‐Salmonella vaccines is an important task. Currently there are no approved vaccines against Salmonella Paratyphi A, the leading cause of paratyphoid fever. To fill this gap, oligosaccharides corresponding to the O‐polysaccharide repeating units from the surface of Salmonella Paratyphi A have been synthesized through convergent stereoselective glycosylations. The synthetic glycan antigen was conjugated with a powerful immunogenic carrier system, the bacteriophage Qβ. The resulting construct was able to elicit strong and long‐lasting anti‐glycan IgG antibody responses, which were highly selective toward Salmonella Paratyphi A associated glycans. The availability of well‐defined glycan antigen enabled the determination that one repeating unit of the polysaccharide is sufficient to induce protective antibodies, and the paratose residue and/or the O‐acetyl modifications on the backbone are important for recognition by antibodies elicited by a Qβ‐tetrasaccharide conjugate. Immune sera provided excellent protection to mice from lethal challenge with Salmonella Paratyphi A, highlighting the potential of the synthetic glycan‐based vaccine. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Typhi H58, an antimicrobial-resistant lineage, is globally disseminated but has not been reported in Latin America. Genomic analysis revealed 3 independent introductions of Salmonella Typhi H58 with reduced fluoroquinolone susceptibility into Chile. Our findings highlight the utility of enhanced genomic surveillance for typhoid fever in this region. [ABSTRACT FROM AUTHOR]

Background Typhoid fever has been endemic on the island nation of Samoa (2016 population, 195 979) since the 1960s and has persisted through 2019, despite economic development and improvements in water supply and sanitation. Methods Salmonella enterica serovar Typhi isolates from the 2 hospitals with blood culture capability and matched patient demographic and clinical data from January 2008 through December 2019 were analyzed. Denominators to calculate incidence by island, region, and district came from 2011 and 2016 censuses and from 2017–2019 projections from Samoa's Bureau of Statistics. Data were analyzed to describe typhoid case burden and incidence from 2008 to 2019 by time, place, and person. Results In sum, 53–193 blood culture-confirmed typhoid cases occurred annually from 2008 to 2019, without apparent seasonality. Typhoid incidence was low among children age < 48 months (17.6–27.8/105), rose progressively in ages 5–9 years (54.0/105), 10–19 years (60.7–63.4/105), and 20–34 years (61.0–79.3/105), and then tapered off; 93.6% of cases occurred among Samoans < 50 years of age. Most typhoid cases and the highest incidence occurred in Northwest Upolu, but Apia Urban Area (served by treated water supplies) also exhibited moderate incidence. The proportion of cases from short-cycle versus long-cycle transmission is unknown. Samoan S. Typhi are pansusceptible to traditional first-line antibiotics. Nevertheless, enhanced surveillance in 2019 detected 4 (2.9%) deaths among 140 cases. Conclusions Typhoid has been endemic in Samoa in the period 2008–2019. Interventions, including mass vaccination with a Vi-conjugate vaccine coadministered with measles vaccine are planned. [ABSTRACT FROM AUTHOR]

Background: Enterotoxigenic Escherichia coli (ETEC) encoding heat-stable enterotoxin (ST) alone or with heat-labile enterotoxin (LT) cause moderate-to-severe diarrhea (MSD) in developing country children. The Global Enteric Multicenter Study (GEMS) identified ETEC encoding ST among the top four enteropathogens. Since the GEMS objective was to provide evidence to guide development and implementation of enteric vaccines and other interventions to diminish diarrheal disease morbidity and mortality, we examined colonization factor (CF) prevalence among ETEC isolates from children age <5 years with MSD and from matched controls in four African and three Asian sites. We also assessed strength of association of specific CFs with MSD. Methodology/Principal findings: MSD cases enrolled at healthcare facilities over three years and matched controls were tested in a standardized manner for many enteropathogens. To identify ETEC, three E. coli colonies per child were tested by polymerase chain reaction (PCR) to detect genes encoding LT, ST; confirmed ETEC were examined by PCR for major CFs (Colonization Factor Antigen I [CFA/I] or Coli Surface [CS] antigens CS1-CS6) and minor CFs (CS7, CS12, CS13, CS14, CS17, CS18, CS19, CS20, CS21, CS30). ETEC from 806 cases had a single toxin/CF profile in three tested strains per child. Major CFs, components of multiple ETEC vaccine candidates, were detected in 66.0% of LT/ST and ST-only cases and were associated with MSD versus matched controls by conditional logistic regression (p≤0.006); major CFs detected in only 25.0% of LT-only cases weren’t associated with MSD. ETEC encoding exclusively CS14, identified among 19.9% of 291 ST-only and 1.5% of 259 LT/ST strains, were associated with MSD (p = 0.0011). No other minor CF exhibited prevalence ≥5% and significant association with MSD. Conclusions/Significance: Major CF-based efficacious ETEC vaccines could potentially prevent up to 66% of pediatric MSD cases due to ST-encoding ETEC in developing countries; adding CS14 extends coverage to ~77%. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae (KP) and Pseudomonas aeruginosa (PA) are important human pathogens that are associated with a range of infection types, including wound and disseminated infections. Treatment has been complicated by rising rates of antimicrobial resistance. Immunoprophylactic strategies are not constrained by antimicrobial resistance mechanisms. Vaccines against these organisms would be important public health tools, yet they are not available. KP surface O polysaccharides (OPS) are protective antigens in animal models of infection. Similarly, PA flagellin (Fla), the major subunit of the flagellar filament, is required for virulence and is a target of protective antibodies in animal models. We report herein the development of a combined KP and PA glycoconjugate vaccine comprised of the four most common KP OPS types associated with human infections (O1, O2, O3, O5), chemically linked to the two Fla types of PA (FlaA, FlaB). Conjugation of KP OPS to PA Fla enhanced anti-polysaccharide immune responses and produced a formulation that generated antibody titers to the four KP OPS types and both PA Fla antigens in rabbits. Passive transfer of vaccine-induced rabbit antisera reduced the bacterial burden and protected mice against fatal intravenous KP infection. Mice passively transferred with conjugate-induced antisera were also protected against PA infection after thermal injury with a FlaB-expressing isolate, but not a FlaA isolate. Taken together, these promising preclinical results provide important proof-of-concept for a broad spectrum human vaccine to prevent KP and PA infections. [ABSTRACT FROM AUTHOR]

In sub-Saharan Africa, invasive nontyphoidal Salmonella (iNTS) infections with serovars S. Enteritidis, S. Typhimurium and I 4,[],12:i:- are widespread in children < 5 years old. Development of an efficacious vaccine would provide an important public health tool to prevent iNTS disease in this population. Glycoconjugates of S. Enteritidis core and O-polysaccharide (COPS) coupled to the homologous serovar phase 1 flagellin protein (FliC) were previously shown to be immunogenic and protected adult mice against death following challenge with a virulent Malian S. Enteritidis blood isolate. This study extends these observations to immunization of mice in early life and also assesses protection with partial and full regimens. Anti-COPS and anti-FliC serum IgG titers were assessed in infant and adult mice after immunization with 1, 2 or 3 doses of S. Enteritidis COPS:FliC alone or co-formulated with aluminum hydroxide or monophosphoryl lipid A (MPL) adjuvants. S. Enteritidis COPS:FliC was immunogenic in both age groups, although the immune responses were quantitatively lower in infants. Kinetics of antibody production were similar for the native and adjuvanted formulations after three doses; conjugates formulated with MPL elicited significantly increased anti-COPS IgG titers in adult but not infant mice. Nevertheless, robust protection against S. Enteritidis challenge was seen for all three formulations when three doses were given either during infancy or as adults. We further found that significant protection could be achieved with two COPS:FliC doses, despite elicitation of modest serum anti-COPS IgG antibody titers. These findings guide potential immunization strategies that may be translated to develop a human pediatric iNTS vaccine for sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Salmonella Typhimurium sequence type (ST) 313 produces septicemia in infants in sub-Saharan Africa. Although there are known genetic and phenotypic differences between ST313 strains and gastroenteritis-associated ST19 strains, conflicting data about the in vivo virulence of ST313 strains have been reported. To resolve these differences, we tested clinical Salmonella Typhimurium ST313 and ST19 strains in murine and rhesus macaque infection models. The 50% lethal dose (LD50) was determined for three Salmonella Typhimurium ST19 and ST313 strains in mice. For dissemination studies, bacterial burden in organs was determined at various time-points post-challenge. Indian rhesus macaques were infected with one ST19 and one ST313 strain. Animals were monitored for clinical signs and bacterial burden and pathology were determined. The LD50 values for ST19 and ST313 infected mice were not significantly different. However, ST313-infected BALB/c mice had significantly higher bacterial numbers in blood at 24 h than ST19-infected mice. ST19-infected rhesus macaques exhibited moderate-to-severe diarrhea while ST313-infected monkeys showed no-to-mild diarrhea. ST19-infected monkeys had higher bacterial burden and increased inflammation in tissues. Our data suggest that Salmonella Typhimurium ST313 invasiveness may be investigated using mice. The non-human primate results are consistent with clinical data, suggesting that ST313 strains do not cause diarrhea. [ABSTRACT FROM AUTHOR]

Invasive infections associated with non-typhoidal Salmonella (NTS) serovars Enteritidis (SE), Typhimurium (STm) and monophasic variant 1,4,[5],12:i:- are a major health problem in infants and young children in sub-Saharan Africa, and currently, there are no approved human NTS vaccines. NTS O-polysaccharides and flagellin proteins are protective antigens in animal models of invasive NTS infection. Conjugates of SE core and O-polysaccharide (COPS) chemically linked to SE flagellin have enhanced the anti-COPS immune response and protected mice against fatal challenge with a Malian SE blood isolate. We report herein the development of a STm glycoconjugate vaccine comprised of STm COPS conjugated to the homologous serovar phase 1 flagellin protein (FliC) with assessment of the role of COPS O-acetyls for functional immunity. Sun-type COPS conjugates linked through the polysaccharide reducing end to FliC were more immunogenic and protective in mice challenged with a Malian STm blood isolate than multipoint lattice conjugates (>95% vaccine efficacy [VE] versus 30–43% VE). Immunization with de-O-acetylated STm-COPS conjugated to CRM197 provided significant but reduced protection against STm challenge compared to mice immunized with native STm-COPS:CRM197 (63–74% VE versus 100% VE). Although OPS O-acetyls were highly immunogenic, post-vaccination sera that contained various O-acetyl epitope-specific antibody profiles displayed similar in vitro bactericidal activity when equivalent titers of anti-COPS IgG were assayed. In-silico molecular modeling further indicated that STm OPS forms a single dominant conformation, irrespective of O-acetylation, in which O-acetyls extend outward and are highly solvent exposed. These preclinical results establish important quality attributes for an STm vaccine that could be co-formulated with an SE-COPS:FliC glycoconjugate as a bivalent NTS vaccine for use in sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Background: In sub-Saharan Africa, systematic surveillance of young children with suspected invasive bacterial disease (e.g., septicemia, meningitis) has revealed non-typhoidal Salmonella (NTS) to be a major pathogen exhibiting high case fatality (~20%). Where infant vaccination against Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae has been introduced to prevent invasive disease caused by these pathogens, as in Bamako, Mali, their burden has decreased markedly. In parallel, NTS has become the predominant invasive bacterial pathogen in children aged <5 years. While NTS is believed to be acquired orally via contaminated food/water, epidemiologic studies have failed to identify the reservoir of infection or vehicles of transmission. This has precluded targeting food chain interventions to diminish disease transmission but conversely has fostered the development of vaccines to prevent invasive NTS (iNTS) disease. We developed a mathematical model to estimate the potential impact of NTS vaccination programs in Bamako. Methodology/Principal Findings: A Markov chain transmission model was developed utilizing age-specific Bamako demographic data and hospital surveillance data for iNTS disease in children aged <5 years and assuming vaccine coverage and efficacy similar to the existing, successfully implemented, Hib vaccine. Annual iNTS hospitalizations and deaths in children <5 years, with and without a Salmonella Enteritidis/Salmonella Typhimurium vaccine, were the model’s outcomes of interest. Per the model, high coverage/high efficacy iNTS vaccination programs would drastically diminish iNTS disease except among infants age <8 weeks. Conclusions/Significance: The public health impact of NTS vaccination shifts as disease burden, vaccine coverage, and serovar distribution vary. Our model shows that implementing an iNTS vaccine through an analogous strategy to the Hib vaccination program in Bamako would markedly reduce cases and deaths due to iNTS among the pediatric population. The model can be adjusted for use elsewhere in Africa where NTS epidemiologic patterns, serovar prevalence, and immunization schedules differ from Bamako. [ABSTRACT FROM AUTHOR]

Background: Despite the heightened risk of serious influenza during infancy, vaccination is not recommended in infants younger than 6 months. We aimed to assess the safety, immunogenicity, and efficacy of maternal immunisation with trivalent inactivated influenza vaccine for protection of infants against a first episode of laboratory-confirmed influenza.Methods: We did this prospective, active-controlled, observer-blind, randomised phase 4 trial at six referral centres and community health centres in Bamako, Mali. Third-trimester pregnant women (≥28 weeks' gestation) were randomly assigned (1:1), via a computer-generated, centre-specific list with alternate block sizes of six or 12, to receive either trivalent inactivated influenza vaccine or quadrivalent meningococcal vaccine. Study personnel administering vaccines were not masked to treatment allocation, but allocation was concealed from clinicians, laboratory personnel, and participants. Infants were visited weekly until age 6 months to detect influenza-like illness; laboratory-confirmed influenza diagnosed with RT-PCR. We assessed two coprimary objectives: vaccine efficacy against laboratory-confirmed influenza in infants born to women immunised any time prepartum (intention-to-treat population), and vaccine efficacy in infants born to women immunised at least 14 days prepartum (per-protocol population). The primary outcome was the occurrence of a first case of laboratory-confirmed influenza by age 6 months. This trial is registered with ClinicalTrials.gov, number NCT01430689.Findings: We did this trial from Sept 12, 2011, to Jan 28, 2014. Between Sept 12, 2011, and April 18, 2013, we randomly assigned 4193 women to receive trivalent inactivated influenza vaccine (n=2108) or quadrivalent meningococcal vaccine (n=2085). There were 4105 livebirths; 1797 (87%) of 2064 infants in the trivalent inactivated influenza vaccine group and 1793 (88%) of 2041 infants in the quadrivalent meningococcal vaccine group were followed up until age 6 months. We recorded 5279 influenza-like illness episodes in 2789 (68%) infants, of which 131 (2%) episodes were laboratory-confirmed influenza. 129 (98%) cases of laboratory-confirmed influenza were first episodes (n=77 in the quadrivalent meningococcal vaccine group vs n=52 in the trivalent inactivated influenza vaccine group). In the intention-to-treat population, overall infant vaccine efficacy was 33·1% (95% CI 3·7-53·9); in the per-protocol population, vaccine efficacy was 37·3% (7·6-57·8). Vaccine efficacy remained robust during the first 4 months of follow-up (67·9% [95% CI 35·1-85·3] by intention to treat and 70·2% [35·7-87·6] by per protocol), before diminishing during the fifth month (57·3% [30·6-74·4] and 60·7 [33·8-77·5], respectively). Adverse event rates in women and infants were similar among groups. Pain at the injection site was more common in women given quadrivalent meningococcal vaccine than in those given trivalent inactivated influenza vaccine (n=253 vs n=132; p<0·0001), although 354 [92%] reactions were mild. Obstetrical and non-obstetrical serious adverse events were reported in 60 (3%) women in the quadrivalent meningococcal vaccine group and 61 (3%) women in the trivalent inactivated influenza vaccine group. Presumed neonatal infection was more common in infants in the trivalent inactivated influenza vaccine group than in those in the quadrivalent meningococcal vaccine group (n=60 vs n=37; p=0·02). No serious adverse events were related to vaccination.Interpretation: Vaccination of pregnant women with trivalent inactivated influenza vaccine in Mali-a poorly resourced country with high infant mortality-was technically and logistically feasible and protected infants from laboratory-confirmed influenza for 4 months. With adequate financing to procure the vaccine, implementation will parallel the access to antenatal care and immunisation coverage of pregnant women with tetanus toxoid.Funding: Bill & Melinda Gates Foundation. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Typhimurium, an enteric pathogen that causes a self-limiting gastroenteritis, forms biofilms on different surfaces. In sub-Saharan Africa, Salmonella Typhimurium of a novel sequence type (ST) 313 was identified and produces septicemia in the absence of gastroenteritis. No animal reservoir has been identified, and it is hypothesized that transmission occurs via human to human. In this study, we show that invasive Salmonella Typhimurium ST313 strains from Mali are poor biofilm producers compared to Salmonella Typhimurium ST19 strains, which are found worldwide and are known to be associated with gastroenteritis. We evaluated biofilms using crystal violet staining, examination of the red, dry and rough morphotype, pellicle formation and a continuous flow system. One month-old Salmonella Typhimurium ST19 colonies survived in the absence of exogenous nutrients and were highly resistant to sodium hypochlorite treatment compared to Salmonella Typhimurium ST313. This study for the first time demonstrates the comparative biofilm-forming ability and long-term survival of clinical Salmonella Typhimurium ST19 and ST313 isolates. Salmonella Typhimurium ST19 strains are strong biofilm producers and can survive desiccation compared to Salmonella Typhimurium ST313 that form weak biofilms and survive poorly following desiccation. Our data suggest that like Salmonella Typhi, Salmonella Typhimurium ST313 lack mechanisms that allow it to persist in the environment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Invasive non-typhoidal Salmonella (iNTS) are an important cause of septicemia in children under the age of five years in sub-Saharan Africa. A novel genotype of Salmonella enterica subsp. enterica serovar Typhimurium (multi-locus sequence type [ST] 313) circulating in this geographic region is genetically different to from S. Typhimurium ST19 strains that are common throughout the rest of the world. S. Typhimurium ST313 strains have acquired pseudogenes and genetic deletions and appear to be evolving to become more like the typhoidal serovars S. Typhi and S. Paratyphi A. Epidemiological and clinical data show that S. Typhimurium ST313 strains are clinically associated with invasive systemic disease (bacteremia, septicemia, meningitis) rather than with gastroenteritis. The current work summarizes investigations of the broad hypothesis that S. Typhimurium ST313 isolates from Mali, West Africa, will behave differently from ST19 isolates in various in vitro assays. Here, we show that strains of the ST313 genotype are phagocytosed more efficiently and are highly resistant to killing by macrophage cell lines and primary mouse and human macrophages compared to ST19 strains. S. Typhimurium ST313 strains survived and replicated within different macrophages. Infection of macrophages with S. Typhimurium ST19 strains resulted in increased apoptosis and higher production of proinflammatory cytokines, as measured by gene expression and protein production, compared to S. Typhimurium ST313 strains. This difference in proinflammatory cytokine production and cell death between S. Typhimurium ST19 and ST313 strains could be explained, in part, by an increased production of flagellin by ST19 strains. These observations provide further evidence that S. Typhimurium ST313 strains are phenotypically different to ST19 strains and instead share similar pathogenic characteristics with typhoidal Salmonella serovars. Author Summary: Non-typhoidal Salmonella, such as Salmonella Typhimurium, generally cause self-limiting gastroenteritis. However, in sub-Saharan Africa, a novel genotype called sequence type (ST) 313 is circulating and causes bloodstream infections in infants and HIV-infected individuals. In contrast, the most common genotype found throughout the rest of the world is ST19, which has been highly studied. Currently, the pathogenesis of S. Typhimurium ST313 is not well understood. In our study, we present evidence that S. Typhimurium ST313 strains from Mali, West Africa, survive and replicate better within a diverse set of primary human and murine macrophages and cell lines, inducing significantly less host-cell death as compared to S. Typhimurium ST19. Flagellin expression in the S. Typhimurium ST313 strains was found to be attenuated compared to S. Typhimurium ST19, thereby causing decreased inflammation. We conclude that S. Typhimurium ST313 have evolved phenotypically to be an intermediate between S. Typhimurium ST19 and the highly invasive typhoidal Salmonella serovars S. Typhi and S. Paratyphi A. [ABSTRACT FROM AUTHOR]

Invasive non-typhoidal Salmonella (iNTS) are an important cause of septicemia in children under the age of five years in sub-Saharan Africa. A novel genotype of Salmonella enterica subsp. enterica serovar Typhimurium (multi-locus sequence type [ST] 313) circulating in this geographic region is genetically different to from S. Typhimurium ST19 strains that are common throughout the rest of the world. S. Typhimurium ST313 strains have acquired pseudogenes and genetic deletions and appear to be evolving to become more like the typhoidal serovars S. Typhi and S. Paratyphi A. Epidemiological and clinical data show that S. Typhimurium ST313 strains are clinically associated with invasive systemic disease (bacteremia, septicemia, meningitis) rather than with gastroenteritis. The current work summarizes investigations of the broad hypothesis that S. Typhimurium ST313 isolates from Mali, West Africa, will behave differently from ST19 isolates in various in vitro assays. Here, we show that strains of the ST313 genotype are phagocytosed more efficiently and are highly resistant to killing by macrophage cell lines and primary mouse and human macrophages compared to ST19 strains. S. Typhimurium ST313 strains survived and replicated within different macrophages. Infection of macrophages with S. Typhimurium ST19 strains resulted in increased apoptosis and higher production of proinflammatory cytokines, as measured by gene expression and protein production, compared to S. Typhimurium ST313 strains. This difference in proinflammatory cytokine production and cell death between S. Typhimurium ST19 and ST313 strains could be explained, in part, by an increased production of flagellin by ST19 strains. These observations provide further evidence that S. Typhimurium ST313 strains are phenotypically different to ST19 strains and instead share similar pathogenic characteristics with typhoidal Salmonella serovars. [ABSTRACT FROM AUTHOR]

Flagellins are the main structural proteins of bacterial flagella and potent stimulators of innate and adaptive immunity in mammals. The flagellins of Salmonella are virulence factors and protective antigens, and form the basis of promising vaccines. Despite broad interest in flagellins as antigens and adjuvants in vaccine formulations, there have been few advances towards the development of scalable and economical purification methods for these proteins. We report here a simple and robust strategy to purify flagellin monomers from the supernatants of liquid growth culture. Phase 1 flagellins from Salmonella enterica serovars Typhimurium (i epitope) and Enteritidis (g,m epitopes) were purified directly from conditioned fermentation growth media using sequential cation- and anion-exchange chromatography coupled with a final tangential flow-filtration step. Conventional porous chromatography resin was markedly less efficient than membrane chromatography for flagellin purification. Recovery after each process step was robust, with endotoxin, nucleic acid and residual host–cell protein effectively removed. The final yield was 200–300 mg/L fermentation culture supernatant, with ∼45–50% overall recovery. A final pH 2 treatment step was instituted to ensure uniformity of flagellin in the monomeric form. Flagellins purified by this method were recognized by monoclonal anti-flagellin antibodies and maintained capacity to activate Toll-like Receptor 5. The process described is simple, readily scalable, uses standard bioprocess methods, and requires only a few steps to obtain highly purified material. [ABSTRACT FROM AUTHOR]

Shigella case isolates from the Global Enteric Multicenter Study were serotyped to guide vaccine development. A quadrivalent vaccine that includes O antigens from S. sonnei, S. flexneri 2a, S. flexneri 3a, and S. flexneri 6 should provide broad protection.Background. Shigella, a major diarrheal disease pathogen worldwide, is the target of vaccine development. The Global Enteric Multicenter Study (GEMS) investigated burden and etiology of moderate-to-severe diarrheal disease in children aged <60 months and matched controls without diarrhea during 3 years at 4 sites in Africa and 3 in Asia. Shigella was 1 of the 4 most common pathogens across sites and age strata. GEMS Shigella serotypes are reviewed to guide vaccine development.Methods. Subjects' stool specimens/rectal swabs were transported to site laboratories in transport media and plated onto xylose lysine desoxycholate and MacConkey agar. Suspect Shigella colonies were identified by biochemical tests and agglutination with antisera. Shigella isolates were shipped to the GEMS Reference Laboratory (Baltimore, MD) for confirmation and serotyping of S. flexneri; one-third of isolates were sent to the Centers for Disease Control and Prevention for quality control.Results. Shigella dysenteriae and S. boydii accounted for 5.0% and 5.4%, respectively, of 1130 Shigella case isolates; S. flexneri comprised 65.9% and S. sonnei 23.7%. Five serotypes/subserotypes comprised 89.4% of S. flexneri, including S. flexneri 2a, S. flexneri 6, S. flexneri 3a, S. flexneri 2b, and S. flexneri 1b.Conclusions. A broad-spectrum Shigella vaccine must protect against S. sonnei and 15 S. flexneri serotypes/subserotypes. A quadrivalent vaccine with O antigens from S. sonnei, S. flexneri 2a, S. flexneri 3a, and S. flexneri 6 can provide broad direct coverage against these most common serotypes and indirect coverage against all but 1 (rare) remaining subserotype through shared S. flexneri group antigens. [ABSTRACT FROM AUTHOR]

Non-typhoidal Salmonella (NTS) serovars S. Enteritidis and S. Typhimurium are a major cause of invasive bacterial disease (e.g., bacteremia, meningitis) in infants and young children in sub-Saharan Africa and also occasionally cause invasive disease in highly susceptible hosts (young infants, the elderly, and immunocompromised subjects) in industrialized countries. No licensed vaccines exist against human NTS infections. NTS core and O polysaccharide (COPS) and FliC (Phase 1 flagellin subunits) each constitute protective antigens in murine models. S. Enteritidis COPS conjugated to FliC represents a promising vaccine approach that elicits binding and opsonophagocytic antibodies and protects mice against lethal challenge with virulent S. Enteritidis. We examined the protective efficacy of fractional dosages of S. Enteritidis COPS:FliC conjugate vaccines in mice, and also established that protection can be passively transferred to naïve mice by administering sera from mice immunized with conjugate. Mice were immunized with three doses of either 10 µg, 2.5 µg (full dose), 0.25 µg, or 0.025 µg S. Enteritidis COPS:FliC conjugate at 28 day intervals. Antibody titers to COPS and FliC measured by ELISA fell consonant with progressively smaller vaccine dosage levels; anti-FliC IgG responses remained robust at fractional dosages for which anti-COPS serum IgG titers were decreased. Nevertheless, >90% protection against intraperitoneal challenge was observed in mice immunized with fractional dosages of conjugate that elicited diminished titers to both FliC and COPS. Passive transfer of immune sera from mice immunized with the highest dose of COPS:FliC to naïve mice was also protective, demonstrating the role of antibodies in mediating protection. These results provide important insights regarding the potency of Salmonella glycoconjugate vaccines that use flagellin as a carrier protein. [ABSTRACT FROM AUTHOR]

A highly invasive form of non-typhoidal Salmonella (iNTS) disease has recently been documented in many countries in sub-Saharan Africa. The most common Salmonella enterica serovar causing this disease is Typhimurium (Salmonella Typhimurium). We applied whole-genome sequence-based phylogenetic methods to define the population structure of sub-Saharan African invasive Salmonella Typhimurium isolates and compared these to global Salmonella Typhimurium populations. Notably, the vast majority of sub-Saharan invasive Salmonella Typhimurium isolates fell within two closely related, highly clustered phylogenetic lineages that we estimate emerged independently ?52 and ?35 years ago in close temporal association with the current HIV pandemic. Clonal replacement of isolates from lineage I by those from lineage II was potentially influenced by the use of chloramphenicol for the treatment of iNTS disease. Our analysis suggests that iNTS disease is in part an epidemic in sub-Saharan Africa caused by highly related Salmonella Typhimurium lineages that may have occupied new niches associated with a compromised human population and antibiotic treatment. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovars Typhi and Paratyphi ( S . Paratyphi) A and B cause enteric fever in humans. Of the paratyphoid group, S . Paratyphi A is the most common serovar. In 2000, there were an estimated 5.4 million cases of S . Paratyphi A worldwide. More recently paratyphoid fever has accounted for an increasing fraction of all cases of enteric fever. Although vaccines for typhoid fever have been developed and in use for decades, vaccines for paratyphoid fever have not yet been licensed. Several S . Paratyphi A vaccines, however, are in development and based on either whole cell live-attenuated strains or repeating units of the lipopolysaccharide O-antigen (O:2) conjugated to different protein carriers. An O-specific polysaccharide (O:2) of S . Paratyphi A conjugated to tetanus toxoid (O:2-TT), for example, has been determined to be safe and immunogenic after one dose in Phase I and Phase II trials. Two other conjugated vaccine candidates linked to diphtheria toxin and a live-attenuated oral vaccine candidate are currently in preclinical development. As promising vaccine candidates are advanced along the development pipeline, an adequate supply of vaccines will need to be ensured to meet growing demand, particularly in the most affected countries. [ABSTRACT FROM AUTHOR]

Background: Shiga toxin-producing Escherichia coli (STEC) is an important cause of bloody diarrhoea (BD), non-bloody diarrhoea (NBD) and the haemolytic uraemic syndrome (HUS). In Argentina and New Zealand, the most prevalent STEC serotype is O157:H7, which is responsible for the majority of HUS cases. In Australia, on the other hand, STEC O157:H7 is associated with a minority of HUS cases. The main aims of this study were to compare the phenotypic and genotypic characteristics of STEC O157 strains isolated between 1993 and 1996 from humans in Argentina, Australia and New Zealand, and to establish their clonal relatedness. Results: Seventy-three O157 STEC strains, isolated from HUS (n = 36), BD (n = 20), NBD (n = 10), or unspecified conditions (n = 7) in Argentina, Australia and New Zealand, were analysed. The strains were confirmed to be E. coli O157 by biochemical tests and serotyping. A multiplex polymerase chain reaction (PCR) was used to amplify the stx1, stx2 and rfbO157 genes and a genotyping method based on PCR-RFLP was used to determine stx1 and stx2 variants. This analysis revealed that the most frequent stx genotypes were stx2/stx2c (vh-a) (91%) in Argentina, stx2 (89%) in New Zealand, and stx1/stx2 (30%) in Australia. No stx1-postive strains were identified in Argentina or New Zealand. All strains harboured the eae gene and 72 strains produced enterohaemolysin (EHEC-Hly). The clonal relatedness of strains was investigated by phage typing and pulsed-field gel electrophoresis (PFGE). The most frequent phage types (PT) identified in Argentinian, Australian, and New Zealand strains were PT49 (n = 12), PT14 (n = 9), and PT2 (n = 15), respectively. Forty-six different patterns were obtained by XbaI-PFGE; 37 strains were grouped in 10 clusters and 36 strains showed unique patterns. Most clusters could be further subdivided by BlnI-PFGE. Conclusion: STEC O157 strains isolated in Argentina, Australia, and New Zealand differed from each other in terms of stx-genotype and phage type. Additionally, no common PFGE patterns were found in strains isolated in the three countries. International collaborative studies of the type reported here are needed to detect and monitor potentially hypervirulent STEC clones. [ABSTRACT FROM AUTHOR]