Zoonotic Source Attribution of Salmonella enterica Serotype Typhimurium Using Genomic Surveillance Data, United States.

Increasingly, routine surveillance and monitoring of foodborne pathogens using whole-genome sequencing is creating opportunities to study foodborne illness epidemiology beyond routine outbreak investigations and case-control studies. Using a global phylogeny of Salmonella enterica serotype Typhimurium, we found that major livestock sources of the pathogen in the United States can be predicted through whole-genome sequencing data. Relatively steady rates of sequence divergence in livestock lineages enabled the inference of their recent origins. Elevated accumulation of lineage-specific pseudogenes after divergence from generalist populations and possible metabolic acclimation in a representative swine isolate indicates possible emergence of host adaptation. We developed and retrospectively applied a machine learning Random Forest classifier for genomic source prediction of Salmonella Typhimurium that correctly attributed 7 of 8 major zoonotic outbreaks in the United States during 1998-2013. We further identified 50 key genetic features that were sufficient for robust livestock source prediction.

SeqSero2: Rapid and Improved Salmonella Serotype Determination Using Whole-Genome Sequencing Data.

SeqSero, launched in 2015, is a software tool for Salmonella serotype determination from whole-genome sequencing (WGS) data. Despite its routine use in public health and food safety laboratories in the United States and other countries, the original SeqSero pipeline is relatively slow (minutes per genome using sequencing reads), is not optimized for draft genome assemblies, and may assign multiple serotypes for a strain. Here, we present SeqSero2 (github.com/denglab/SeqSero2; denglab.info/SeqSero2), an algorithmic transformation and functional update of the original SeqSero. Major improvements include (i) additional sequence markers for identification of Salmonella species and subspecies and certain serotypes, (ii) a k-mer based algorithm for rapid serotype prediction from raw reads (seconds per genome) and improved serotype prediction from assemblies, and (iii) a targeted assembly approach for specific retrieval of serotype determinants from WGS for serotype prediction, new allele discovery, and prediction troubleshooting. Evaluated using 5,794 genomes representing 364 common U.S. serotypes, including 2,280 human isolates of 117 serotypes from the National Antimicrobial Resistance Monitoring System, SeqSero2 is up to 50 times faster than the original SeqSero while maintaining equivalent accuracy for raw reads and substantially improving accuracy for assemblies. SeqSero2 further suggested that 3% of the tested genomes contained reads from multiple serotypes, indicating a use for contamination detection. In addition to short reads, SeqSero2 demonstrated potential for accurate and rapid serotype prediction directly from long nanopore reads despite base call errors. Testing of 40 nanopore-sequenced genomes of 17 serotypes yielded a single H antigen misidentification.IMPORTANCE Serotyping is the basis of public health surveillance of Salmonella It remains a first-line subtyping method even as surveillance continues to be transformed by whole-genome sequencing. SeqSero allows the integration of Salmonella serotyping into a whole-genome-sequencing-based laboratory workflow while maintaining continuity with the classic serotyping scheme. SeqSero2, informed by extensive testing and application of SeqSero in the United States and other countries, incorporates important improvements and updates that further strengthen its application in routine and large-scale surveillance of Salmonella by whole-genome sequencing.

Antimicrobial Resistance in Salmonella in the United States from 1948 to 1995.

We conducted a retrospective study of 2,149 clinicalSalmonellastrains to help document the historical emergence of antimicrobial resistance. There were significant increases in resistance to older drugs, including ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline, which were most common inSalmonella entericaserotype Typhimurium. An increase in multidrug resistance was observed for each decade since the 1950s. These data help show howSalmonellaevolved over the past 6 decades, after the introduction of new antimicrobial agents.

Salmonella serotype determination utilizing high-throughput genome sequencing data.

Serotyping forms the basis of national and international surveillance networks for Salmonella, one of the most prevalent foodborne pathogens worldwide (1-3). Public health microbiology is currently being transformed by whole-genome sequencing (WGS), which opens the door to serotype determination using WGS data. SeqSero (www.denglab.info/SeqSero) is a novel Web-based tool for determining Salmonella serotypes using high-throughput genome sequencing data. SeqSero is based on curated databases of Salmonella serotype determinants (rfb gene cluster, fliC and fljB alleles) and is predicted to determine serotype rapidly and accurately for nearly the full spectrum of Salmonella serotypes (more than 2,300 serotypes), from both raw sequencing reads and genome assemblies. The performance of SeqSero was evaluated by testing (i) raw reads from genomes of 308 Salmonella isolates of known serotype; (ii) raw reads from genomes of 3,306 Salmonella isolates sequenced and made publicly available by GenomeTrakr, a U.S. national monitoring network operated by the Food and Drug Administration; and (iii) 354 other publicly available draft or complete Salmonella genomes. We also demonstrated Salmonella serotype determination from raw sequencing reads of fecal metagenomes from mice orally infected with this pathogen. SeqSero can help to maintain the well-established utility of Salmonella serotyping when integrated into a platform of WGS-based pathogen subtyping and characterization.

Comparative analysis of subtyping methods against a whole-genome-sequencing standard for Salmonella enterica serotype Enteritidis.

A retrospective investigation was performed to evaluate whole-genome sequencing as a benchmark for comparing molecular subtyping methods for Salmonella enterica serotype Enteritidis and survey the population structure of commonly encountered S. enterica serotype Enteritidis outbreak isolates in the United States. A total of 52 S. enterica serotype Enteritidis isolates representing 16 major outbreaks and three sporadic cases collected between 2001 and 2012 were sequenced and subjected to subtyping by four different methods: (i) whole-genome single-nucleotide-polymorphism typing (WGST), (ii) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), (iii) clustered regularly interspaced short palindromic repeats combined with multi-virulence-locus sequence typing (CRISPR-MVLST), and (iv) pulsed-field gel electrophoresis (PFGE). WGST resolved all outbreak clusters and provided useful robust phylogenetic inference results with high epidemiological correlation. While both MLVA and CRISPR-MVLST yielded higher discriminatory power than PFGE, MLVA outperformed the other methods in delineating outbreak clusters whereas CRISPR-MVLST showed the potential to trace major lineages and ecological origins of S. enterica serotype Enteritidis. Our results suggested that whole-genome sequencing makes a viable platform for the evaluation and benchmarking of molecular subtyping methods.

Genomic epidemiology of Salmonella enterica serotype Enteritidis based on population structure of prevalent lineages.

Salmonella enterica serotype Enteritidis is one of the most commonly reported causes of human salmonellosis. Its low genetic diversity, measured by fingerprinting methods, has made subtyping a challenge. We used whole-genome sequencing to characterize 125 S. enterica Enteritidis and 3 S. enterica serotype Nitra strains. Single-nucleotide polymorphisms were filtered to identify 4,887 reliable loci that distinguished all isolates from each other. Our whole-genome single-nucleotide polymorphism typing approach was robust for S. enterica Enteritidis subtyping with combined data for different strains from 2 different sequencing platforms. Five major genetic lineages were recognized, which revealed possible patterns of geographic and epidemiologic distribution. Analyses on the population dynamics and evolutionary history estimated that major lineages emerged during the 17th-18th centuries and diversified during the 1920s and 1950s.

Multilocus sequence typing confirms wild birds as the source of a Campylobacter outbreak associated with the consumption of raw peas.

From August to September 2008, the Centers for Disease Control and Prevention (CDC) assisted the Alaska Division of Public Health with an outbreak investigation of campylobacteriosis occurring among the residents of Southcentral Alaska. During the investigation, pulsed-field gel electrophoresis (PFGE) of Campylobacter jejuni isolates from human, raw pea, and wild bird fecal samples confirmed the epidemiologic link between illness and the consumption of raw peas contaminated by sandhill cranes for 15 of 43 epidemiologically linked human isolates. However, an association between the remaining epidemiologically linked human infections and the pea and wild bird isolates was not established. To better understand the molecular epidemiology of the outbreak, C. jejuni isolates (n=130; 59 from humans, 40 from peas, and 31 from wild birds) were further characterized by multilocus sequence typing (MLST). Here we present the molecular evidence to demonstrate the association of many more human C.jejuni infections associated with the outbreak with raw peas and wild bird feces. Among all sequence types (STs) identified, 26 of 39 (67%) were novel and exclusive to the outbreak. Five clusters of overlapping STs (n=32 isolates; 17 from humans, 2 from peas, and 13 from wild birds) were identified. In particular, cluster E (n=7 isolates; ST-5049) consisted of isolates from humans,peas, and wild birds. Novel STs clustered closely with isolates typically associated with wild birds and the environment but distinct from lineages commonly seen in human infections. Novel STs and alleles recovered from human outbreak isolates allowed additional infections caused by these rare genotypes to be attributed to the contaminated raw peas.

Campylobacter fetus subsp. testudinum subsp. nov., isolated from humans and reptiles.

A polyphasic study was undertaken to determine the taxonomic position of 13 Campylobacter fetus-like strains from humans (n = 8) and reptiles (n = 5). The results of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS and genomic data from sap analysis, 16S rRNA gene and hsp60 sequence comparison, pulsed-field gel electrophoresis, amplified fragment length polymorphism analysis, DNA-DNA hybridization and whole genome sequencing demonstrated that these strains are closely related to C. fetus but clearly differentiated from recognized subspecies of C. fetus. Therefore, this unique cluster of 13 strains represents a novel subspecies within the species C. fetus, for which the name Campylobacter fetus subsp. testudinum subsp. nov. is proposed, with strain 03-427(T) ( = ATCC BAA-2539(T) = LMG 27499(T)) as the type strain. Although this novel taxon could not be differentiated from C. fetus subsp. fetus and C. fetus subsp. venerealis using conventional phenotypic tests, MALDI-TOF MS revealed the presence of multiple phenotypic biomarkers which distinguish Campylobacter fetus subsp. testudinum subsp. nov. from recognized subspecies of C. fetus.

Epidemiology of Salmonellosis Among Infants in the United States: 1968-2015.

OBJECTIVES: Describe characteristics of gastroenteritis, bacteremia, and meningitis caused by nontyphoidal Salmonella among US infants. METHODS: We analyze national surveillance data during 1968-2015 and active, sentinel surveillance data during 1996-2015 for culture-confirmed Salmonella infections by syndrome, year, serotype, age, and race. RESULTS: During 1968-2015, 190 627 culture-confirmed Salmonella infections among infants were reported, including 165 236 (86.7%) cases of gastroenteritis, 6767 (3.5%) bacteremia, 371 (0.2%) meningitis, and 18 253 (9.7%) with other or unknown specimen sources. Incidence increased during the late 1970s-1980s, declined during the 1990s-early 2000s, and has gradually increased since the mid-2000s. Infants' median age was 4 months for gastroenteritis and bacteremia and 2 months for meningitis. The most frequently reported serotypes were Typhimurium (35 468; 22%) for gastroenteritis and Heidelberg for bacteremia (1954; 29%) and meningitis (65; 18%). During 1996-2015 in sentinel site surveillance, median annual incidence of gastroenteritis was 120, bacteremia 6.2, and meningitis 0.25 per 100 000 infants. Boys had a higher incidence of each syndrome than girls in both surveillance systems, but most differences were not statistically significant. Overall, hospitalization and fatality rates were 26% and 0.1% for gastroenteritis, 70% and 1.6% for bacteremia, and 96% and 4% for meningitis. During 2004-2015, invasive salmonellosis incidence was higher for Black (incident rate ratio, 2.7; 95% confidence interval, 2.6-2.8) and Asian (incident rate ratio, 1.8; 95% confidence interval, 1.7-1.8) than white infants. CONCLUSIONS: Salmonellosis causes substantial infant morbidity and mortality; serotype heidelberg caused the most invasive infections. Infants with meningitis were younger than those with bacteremia or gastroenteritis. Research into risk factors for infection and invasive illness could inform prevention efforts.

A global genomic analysis of Salmonella Concord reveals lineages with high antimicrobial resistance in Ethiopia.

Antimicrobial resistant Salmonella enterica serovar Concord (S. Concord) is known to cause severe gastrointestinal and bloodstream infections in patients from Ethiopia and Ethiopian adoptees, and occasional records exist of S. Concord linked to other countries. The evolution and geographical distribution of S. Concord remained unclear. Here, we provide a genomic overview of the population structure and antimicrobial resistance (AMR) of S. Concord by analysing genomes from 284 historical and contemporary isolates obtained between 1944 and 2022 across the globe. We demonstrate that S. Concord is a polyphyletic serovar distributed among three Salmonella super-lineages. Super-lineage A is composed of eight S. Concord lineages, of which four are associated with multiple countries and low levels of AMR. Other lineages are restricted to Ethiopia and horizontally acquired resistance to most antimicrobials used for treating invasive Salmonella infections in low- and middle-income countries. By reconstructing complete genomes for 10 representative strains, we demonstrate the presence of AMR markers integrated in structurally diverse IncHI2 and IncA/C2 plasmids, and/or the chromosome. Molecular surveillance of pathogens such as S. Concord supports the understanding of AMR and the multi-sector response to the global AMR threat. This study provides a comprehensive baseline data set essential for future molecular surveillance.

Molecular determination of H antigens of Salmonella by use of a microsphere-based liquid array.

Serotyping of Salmonella has been an invaluable subtyping method for epidemiologic studies for more than 70 years. The technical difficulties of serotyping, primarily in antiserum production and quality control, can be overcome with modern molecular methods. We developed a DNA-based assay targeting the genes encoding the flagellar antigens (fliC and fljB) of the Kauffmann-White serotyping scheme. Fifteen H antigens (H:a, -b, -c, -d, -d/j, -e,h, -i, -k, -r, -y, -z, -z(10), -z(29), -z(35), and -z(6)), 5 complex major antigens (H:G, -EN, -Z4, -1, and -L) and 16 complex secondary antigens (H:2, -5, -6, -7, -f, -m/g,m, -m/m,t, -p, -s, -t/m,t, -v, -x, -z(15), -z(24), -z(28), and -z(51)) were targeted in the assay. DNA probes targeting these antigens were designed and evaluated on 500 isolates tested in parallel with traditional serotyping methods. The assay correctly identified 461 (92.2%) isolates based on the 36 antigens detected in the assay. Among the isolates considered correctly identified, 47 (9.4%) were partially serotyped because probes corresponding to some antigens in the strains were not in the assay, and 13 (2.6%) were monophasic or nonmotile strains that possessed flagellar antigen genes that were not expressed but were detected in the assay. The 39 (7.8%) strains that were not correctly identified possessed an antigen that should have been detected by the assay but was not. Apparent false-negative results may be attributed to allelic divergence. The molecular assay provided results that paralleled traditional methods with a much greater throughput, while maintaining the integrity of the Kauffmann-White serotyping scheme, thus providing backwards-compatible epidemiologic data. This assay should greatly enhance the ability of clinical and public health laboratories to serotype Salmonella.

Exploration of risk factors for ceftriaxone resistance in invasive non-typhoidal Salmonella infections in western Kenya.

Multidrug-resistant non-typhoidal Salmonella (NTS) infection has emerged as a prominent cause of invasive infections in Africa. We investigated the prevalence of ceftriaxone-resistant invasive NTS infections, conducted exploratory analysis of risk factors for resistance, and described antimicrobial use in western Kenya. We conducted a secondary analysis of existing laboratory, epidemiology, and clinical data from three independent projects, a malaria vaccine trial, a central nervous system (CNS) study, and the International Emerging Infections Program morbidity surveillance (surveillance program) during 2009-2014. We calculated odds ratios (OR) with 95% confidence intervals (CI) for ceftriaxone-resistant NTS infections compared with ceftriaxone-susceptible infections. We surveyed hospitals, pharmacies, and animal drug retailers about the availability and use of antimicrobials. In total, 286 invasive NTS infections were identified in the three projects; 43 NTS isolates were ceftriaxone-resistant. The absolute prevalence of ceftriaxone resistance varied among these methodologically diverse projects, with 18% (16/90) of isolates resistant to ceftriaxone in the vaccine trial, 89% (16/18) in the CNS study, and 6% (11/178) in the surveillance program. Invasive ceftriaxone-resistant infections increased over time. Most ceftriaxone-resistant isolates were co-resistant to multiple other antimicrobials. Having an HIV-positive mother (OR = 3.7; CI = 1.2-11.4) and taking trimethoprim-sulfamethoxazole for the current illness (OR = 9.6, CI = 1.2-78.9) were significantly associated with acquiring ceftriaxone-resistant invasive NTS infection. Ceftriaxone and other antibiotics were widely prescribed; multiple issues related to prescription practices and misuse were identified. In summary, ceftriaxone-resistant invasive NTS infection is increasing and limiting treatment options for serious infections. Efforts are ongoing to address the urgent need for improved microbiologic diagnostic capacity and an antimicrobial surveillance system in Kenya.

Do Salmonella carry spare tyres?

Salmonellae are enterobacteria that have the unique ability to change their flagellar composition by switching expression among two loci that encode the major flagellin protein. This property is not available to all Salmonella, but is species, subspecies and serotype specific. Curiously, the subsequent loss of the second locus in some lineages of Salmonella has apparently been tolerated and, indeed, has led to considerable success for some lineages. We discuss here an evolutionary model for maintenance of this unique function and the possible evolutionary advantages of loss or preservation of this mechanism. We hypothesize that the second flagellin locus is a genetic 'spare tyre' used in particular environmental circumstances.

Laboratory-based surveillance of paratyphoid fever in the United States: travel and antimicrobial resistance.

BACKGROUND: The incidence of paratyphoid fever, including paratyphoid fever caused by antimicrobial-resistant strains, is increasing globally. However, the epidemiologic and laboratory characteristics of paratyphoid fever in the United States have never been studied. METHODS: We attempted to interview all patients who had been infected with laboratory-confirmed Salmonella serotypes Paratyphi A, Paratyphi B, or Paratyphi C in the United States with specimens collected from 1 April 2005 through 31 March 2006. At the Centers for Disease Control and Prevention (CDC), isolates underwent serotype confirmation, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis typing. RESULTS: Of 149 patients infected with Salmonella Paratyphi A, we obtained epidemiologic information for 89 (60%); 55 (62%) of 86 were hospitalized. Eighty-five patients (96%) reported having travel internationally, and 80 (90%) had traveled to South Asia. Of the 146 isolates received at the CDC, 127 (87%) were nalidixic acid resistant; nalidixic acid resistance was associated with travel to South Asia (odds ratio, 17.0; 95% confidence interval, 3.8-75.9). All nalidixic acid-resistant isolates showed decreased susceptibility to ciprofloxacin (minimum inhibitory concentration, > or = 0.12 microg/mL). Of 49 patients infected with Salmonella Paratyphi B, only 12 (24%) were confirmed to have Paratyphi B when tested at the CDC. Four (67%) of 6 patients were hospitalized, and 5 (83%) reported travel (4 to the Andean region of South America). One case of Salmonella Paratyphi C infection was reported in a traveler to West Africa with a urinary tract infection. CONCLUSIONS: Physicians should be aware of the increasing incidence of infection due to Salmonella Paratyphi A and treatment options given its widespread antimicrobial resistance. A paratyphoid fever vaccine is urgently needed. Continued surveillance for paratyphoid fever will help guide future prevention and treatment recommendations.

PCR method to identify Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B among Salmonella Isolates from the blood of patients with clinical enteric fever.

PCR methodology was developed to identify Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B. One multiplex PCR identifies serogroup D, A, and B and Vi-positive strains; another confirms flagellar antigen "d," "a," or "b." Blinded testing of 664 Malian and Chilean Salmonella blood isolates demonstrated 100% sensitivity and specificity.

Comparative genomics of Salmonella enterica serovar Montevideo reveals lineage-specific gene differences that may influence ecological niche association.

Salmonella enterica serovar Montevideo has been linked to recent foodborne illness outbreaks resulting from contamination of products such as fruits, vegetables, seeds and spices. Studies have shown that Montevideo also is frequently associated with healthy cattle and can be isolated from ground beef, yet human salmonellosis outbreaks of Montevideo associated with ground beef contamination are rare. This disparity fuelled our interest in characterizing the genomic differences between Montevideo strains isolated from healthy cattle and beef products, and those isolated from human patients and outbreak sources. To that end, we sequenced 13 Montevideo strains to completion, producing high-quality genome assemblies of isolates from human patients (n=8) or from healthy cattle at slaughter (n=5). Comparative analysis of sequence data from this study and publicly available sequences (n=72) shows that Montevideo falls into four previously established clades, differentially occupied by cattle and human strains. The results of these analyses reveal differences in metabolic islands, environmental adhesion determinants and virulence factors within each clade, and suggest explanations for the infrequent association between bovine isolates and human illnesses.

Methodologies towards the development of an oligonucleotide microarray for determination of Salmonella serotypes.

A DNA-based microarray designed to detect somatic (O) and flagellar (H) antigens present in the five most commonly isolated Salmonella serovars within Canada was developed as an alternative to the traditional Kauffmann-White serotyping scheme currently used to serotype salmonellae. Short oligonucleotide probes were designed based on publicly available sequence data of selected genes responsible for O and H antigen biosynthesis. These targets included: antigen-specific sequences within the flagella (H) antigen phase 1 (fliC) and phase 2 (fljB) genes and somatic (O) antigen biosynthesis genes within the rfb cluster (Groups B--rfbJ, C1--wbaA, C2--rfbJ, D1--rfbS). A prototype microarray with 117 O and H antigen-specific probes and controls was used to assess probe performance against two pools of gene target PCR amplicons. A set of 31 of these antigen-specific probes (8 O and 23 H) with high specific signal and low non-specific signal were selected based on t-test (p-value <0.01) and log(2) ratio distribution analysis to create a prototype microarray. The microarray was tested against 16 Salmonella strains of known serotype. Based on the strains tested in this study, these probes successfully identified and differentiated 11 of the 12 antigens targeted. The prototype DNA-based typing microarray described here has the potential to be an automated alternative to the traditional antigen-antibody serotyping scheme currently used for Salmonella.

Surveillance for human Salmonella infections in the United States.

Surveillance for human Salmonella infections plays a critical role in understanding and controlling foodborne illness due to Salmonella. Along with its public health partners, the Centers for Disease Control and Prevention (CDC) has several surveillance systems that collect information on Salmonella infections in the United States. The National Salmonella Surveillance System, begun in 1962, receives reports of laboratory-confirmed Salmonella infections through state public health laboratories. Salmonella outbreaks are reported by state and local health departments through the Foodborne Disease Outbreak Reporting System, which became a Web-based, electronic system (eFORS) in 2001. PulseNet facilitates the detection of clusters of Salmonella infections through standardized molecular subtyping (DNA "fingerprinting") of isolates and maintenance of "fingerprint" databases. The National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS) monitors antimicrobial resistance in Salmonella by susceptibility testing of every 20th Salmonella isolate received by state and local public health laboratories. FootNet is an active surveillance system that monitors Salmonella infections in sentinel areas, providing population-based estimates of infection rates. Efforts are underway to electronically link all of the Salmonella surveillance systems at CDC to facilitate optimum use of available data and minimize duplication.

Invasive Infections with Nontyphoidal Salmonella in Sub-Saharan Africa.

Invasive nontyphoidal Salmonella (NTS) infections in Africa cause an enormous burden of illness. These infections are often devastating, with mortality estimated at 20%, even with appropriate antimicrobial therapy. Two major groups-young children and HIV-infected adults-suffer the great majority of these infections. In children, younger age itself, as well as malaria, malnutrition, and HIV infection, are prominent risk factors. In adults, HIV infection is by far the most important risk factor. The most common serotypes in invasive infections are Salmonella enterica serotypes Typhimurium and Enteritidis. In recent years, a specific strain of Salmonella Typhimurium, multilocus sequence type 313, has caused epidemics of invasive disease. Little is known about risk factors for exposure to NTS, making the design of rational interventions to decrease exposure difficult. Antimicrobial therapy is critically important for treatment of invasive NTS infections. Thus, the emergence and spread of resistance to agents commonly used for treatment of invasive NTS infection, now including third-generation cephalosporins, is an ominous development. Already, many invasive NTS infections are essentially untreatable in many health care facilities in sub-Saharan Africa. Several candidate vaccines are in early development and, if safe and effective, could be promising. Interventions to prevent exposure to NTS (e.g., improved sanitation), to prevent the occurrence of disease if exposure does occur (e.g., vaccination, malaria control), and to prevent severe disease and death in those who become ill (e.g., preserving antimicrobial effectiveness) are all important in reducing the toll of invasive NTS disease in sub-Saharan Africa.