Azithromycin for Bacterial Watery Diarrhea: A Reanalysis of the AntiBiotics for Children With Severe Diarrhea (ABCD) Trial Incorporating Molecular Diagnostics.

BACKGROUND: Bacterial pathogens cause substantial diarrhea morbidity and mortality among children living in endemic settings, yet antimicrobial treatment is only recommended for dysentery or suspected cholera. METHODS: AntiBiotics for Children with severe Diarrhea was a 7-country, placebo-controlled, double-blind efficacy trial of azithromycin in children 2-23 months of age with watery diarrhea accompanied by dehydration or malnutrition. We tested fecal samples for enteric pathogens utilizing quantitative polymerase chain reaction to identify likely and possible bacterial etiologies and employed pathogen-specific cutoffs based on genomic target quantity in previous case-control diarrhea etiology studies to identify likely and possible bacterial etiologies. RESULTS: Among 6692 children, the leading likely etiologies were rotavirus (21.1%), enterotoxigenic Escherichia coli encoding heat-stable toxin (13.3%), Shigella (12.6%), and Cryptosporidium (9.6%). More than one-quarter (1894 [28.3%]) had a likely and 1153 (17.3%) a possible bacterial etiology. Day 3 diarrhea was less common in those randomized to azithromycin versus placebo among children with a likely bacterial etiology (risk difference [RD]likely, -11.6 [95% confidence interval {CI}, -15.6 to -7.6]) and possible bacterial etiology (RDpossible, -8.7 [95% CI, -13.0 to -4.4]) but not in other children (RDunlikely, -0.3% [95% CI, -2.9% to 2.3%]). A similar association was observed for 90-day hospitalization or death (RDlikely, -3.1 [95% CI, -5.3 to -1.0]; RDpossible, -2.3 [95% CI, -4.5 to -.01]; RDunlikely, -0.6 [95% CI, -1.9 to .6]). The magnitude of risk differences was similar among specific likely bacterial etiologies, including Shigella. CONCLUSIONS: Acute watery diarrhea confirmed or presumed to be of bacterial etiology may benefit from azithromycin treatment. CLINICAL TRIALS REGISTRATION: NCT03130114.

The Etiology of Childhood Pneumonia in Mali: Findings From the Pneumonia Etiology Research for Child Health (PERCH) Study.

BACKGROUND: We present findings from the Pneumonia Etiology Research for Child Health (PERCH) site in Bamako, Mali. METHODS: Cases were patients 28 days to 59 months of age, admitted to hospital with severe or very severe pneumonia (2005 World Health Organization definition). Community controls were frequency matched by age. Both provided nasopharyngeal and oropharyngeal swabs for multiplex polymerase chain reaction and Streptococcus pneumoniae culture. Cases underwent blood culture and induced sputum culture for Mycobacterium tuberculosis. A subset had pleural fluid and lung aspirates collected for culture and polymerase chain reaction. Primary analyses included participants with negative or unknown HIV status (HIV-) and cases with abnormal chest radiographs (CXR+). Cases and controls were compared using logistic regression adjusting for age. Etiologic fractions were calculated by a Bayesian nested partially latent class analysis, the PERCH integrated analysis. RESULTS: Between January 1, 2012, and January 14, 2014, we enrolled 241 CXR+/HIV- cases and 725 HIV- controls. Compared with controls, cases were more likely to have moderate-to-severe wasting (43.1% vs. 14.1%, P < 0.001) and stunting (26.6% vs. 9.4%, P < 0.001). Predominant etiologies were respiratory syncytial virus [24.0%; 95% credible interval (CrI): 18.3%-31.1%], S. pneumoniae (15.2%; 95% CrI: 9.5-21.6), human metapneumovirus (11.8%; 95% CrI: 8.3%-16.2%) and parainfluenza virus type 3 (9.0%; 95% CrI: 5.8%-13.3%). Case fatality was 13.3%, with Staphylococcus aureus, Pneumocystis jirovecii and Haemophilus influenzae type b predominating (40% of fatal cases). CONCLUSIONS: PERCH uncovered high case fatality among children with severe pneumonia in Mali, highlighting a role for new interventions (eg, respiratory syncytial virus vaccines) and a need to improve vaccine coverage and strengthen healthcare delivery.

Surveillance for Invasive Salmonella Disease in Bamako, Mali, From 2002 to 2018.

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.

Development of a PCR algorithm to detect and characterize Neisseria meningitidis carriage isolates in the African meningitis belt.

Improved methods for the detection and characterization of carried Neisseria meningitidis isolates are needed. We evaluated a multiplex PCR algorithm for the detection of a variety of carriage strains in the meningitis belt. To further improve the sensitivity and specificity of the existing PCR assays, primers for gel-based PCR assays (sodC, H, Z) and primers/probe for real-time quantitative PCR (qPCR) assays (porA, cnl, sodC, H, E, Z) were modified or created using Primer Express software. Optimized multiplex PCR assays were tested on 247 well-characterised carriage isolates from six countries of the African meningitis belt. The PCR algorithm developed enabled the detection of N. meningitidis species using gel-based and real-time multiplex PCR targeting porA, sodC, cnl and characterization of capsule genes through sequential multiplex PCR assays for genogroups (A, W, X, then B, C, Y and finally H, E and Z). Targeting both porA and sodC genes together allowed the detection of meningococci with a sensitivity of 96% and 89% and a specificity of 78% and 67%, for qPCR and gel-based PCR respectively. The sensitivity and specificity ranges for capsular genogrouping of N. meningitidis are 67% - 100% and 98%-100% respectively for gel-based PCR and 90%-100% and 99%-100% for qPCR. We developed a PCR algorithm that allows simple, rapid and systematic detection and characterisation of most major and minor N. meningitidis capsular groups, including uncommon capsular groups (H, E, Z).

Characterization of Invasive Salmonella Serogroup C1 Infections in Mali.

Nontyphoidal Salmonella (NTS) are the leading cause of foodborne infections worldwide and a major cause of bloodstream infections in infants and HIV-infected adults in sub-Saharan Africa (SSA). Salmonella Typhimurium (serogroup B) and Salmonella Enteritidis (serogroup D) are the most common serovars in this region. However, data describing rarer invasive NTS serovars, particularly those belonging to serogroups C1 and C2, circulating in SSA are lacking. We previously conducted systematic blood culture surveillance on pediatric patients in Bamako, Mali, from 2002 to 2014, and the results showed that serovars Typhimurium and Enteritidis accounted for 32% and 36% of isolates, respectively. Here, we present data on 27 Salmonella serogroup C1 strains that were isolated during this previous study. The strains were typed by serum agglutination and multilocus sequence typing (MLST). Sixteen strains were Salmonella Paratyphi C, four were Salmonella Colindale, and two were Salmonella Virchow. Interestingly, five strains were identified as the very rare Salmonella Brazzaville using a combination of serum agglutination and flagellin gene typing. Phenotypic characterization showed that Salmonella Brazzaville produced biofilm and exhibited catalase activity, which were not statistically different from the gastroenteritis-associated Salmonella Typhimurium sequence type (ST) 19. All tested Salmonella Paratyphi C strains were poor biofilm producers and showed significantly less catalase activity than Salmonella Typhimurium ST19. Overall, our study provides insight into the Salmonella serogroup C1 serovars that cause invasive disease in infants in Mali. In addition, we show that MLST and flagellin gene sequencing, in association with traditional serum agglutination, are invaluable tools to help identify rare Salmonella serovars.

Standardization of Laboratory Methods for the PERCH Study.

The Pneumonia Etiology Research for Child Health study was conducted across 7 diverse research sites and relied on standardized clinical and laboratory methods for the accurate and meaningful interpretation of pneumonia etiology data. Blood, respiratory specimens, and urine were collected from children aged 1-59 months hospitalized with severe or very severe pneumonia and community controls of the same age without severe pneumonia and were tested with an extensive array of laboratory diagnostic tests. A standardized testing algorithm and standard operating procedures were applied across all study sites. Site laboratories received uniform training, equipment, and reagents for core testing methods. Standardization was further assured by routine teleconferences, in-person meetings, site monitoring visits, and internal and external quality assurance testing. Targeted confirmatory testing and testing by specialized assays were done at a central reference laboratory.

Microscopic Analysis and Quality Assessment of Induced Sputum From Children With Pneumonia in the PERCH Study.

BACKGROUND.: It is standard practice for laboratories to assess the cellular quality of expectorated sputum specimens to check that they originated from the lower respiratory tract. The presence of low numbers of squamous epithelial cells (SECs) and high numbers of polymorphonuclear (PMN) cells are regarded as indicative of a lower respiratory tract specimen. However, these quality ratings have never been evaluated for induced sputum specimens from children with suspected pneumonia. METHODS.: We evaluated induced sputum Gram stain smears and cultures from hospitalized children aged 1-59 months enrolled in a large study of community-acquired pneumonia. We hypothesized that a specimen representative of the lower respiratory tract will contain smaller quantities of oropharyngeal flora and be more likely to have a predominance of potential pathogens compared to a specimen containing mainly saliva. The prevalence of potential pathogens cultured from induced sputum specimens and quantity of oropharyngeal flora were compared for different quantities of SECs and PMNs. RESULTS.: Of 3772 induced sputum specimens, 2608 (69%) had <10 SECs per low-power field (LPF) and 2350 (62%) had >25 PMNs per LPF, measures traditionally associated with specimens from the lower respiratory tract in adults. Using isolation of low quantities of oropharyngeal flora and higher prevalence of potential pathogens as markers of higher quality, <10 SECs per LPF (but not >25 PMNs per LPF) was the microscopic variable most associated with high quality of induced sputum. CONCLUSIONS.: Quantity of SECs may be a useful quality measure of induced sputum from young children with pneumonia.

Invasive Nontyphoidal Salmonella Infections Among Children in Mali, 2002-2014: Microbiological and Epidemiologic Features Guide Vaccine Development.

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.

Quantitative PCR for detection of Shigella improves ascertainment of Shigella burden in children with moderate-to-severe diarrhea in low-income countries.

Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 10(4) ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 10(4) ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ≥ 2.9 × 10(4) ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 10(4) ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture in both samples. Among a subset (n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n = 129) for culture to 17.6% (n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 10(4) ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella.

Genomic characterization of enteroaggregative Escherichia coli from children in Mali.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is a cause of epidemic and sporadic diarrhea, yet its role as an enteric pathogen is not fully understood. METHODS: We characterized 121 EAEC strains isolated in 2008 as part of a case-control study of moderate to severe acute diarrhea among children 0-59 months of age in Bamako, Mali. We applied multiplex polymerase chain reaction and comparative genome hybridization to identify potential virulence factors among the EAEC strains, coupled with classification and regression tree modeling to reveal combinations of factors most strongly associated with illness. RESULTS: The gene encoding the autotransporter protease SepA, originally described in Shigella species, was most strongly associated with diarrhea among the EAEC strains tested (odds ratio, 5.6 [95% confidence interval, 1.92-16.17]; P = .0006). In addition, we identified 3 gene combinations correlated with diarrhea: (1) a clonal group positive for sepA and a putative hemolysin; (2) a group harboring the EAST-1 enterotoxin and the flagellar type H33 but no other previously identified EAEC virulence factor; and (3) a group carrying several of the typical EAEC virulence genes. CONCLUSION: Our data suggest that only a subset of EAEC strains are pathogenic in Mali and suggest that sepA may serve as a valuable marker for the most virulent isolates.