Acromegaly and the long-term fracture risk of the vertebra and hip: a national cohort study.

In this national cohort study, the patients with acromegaly had significantly higher risks of clinical vertebral (HR 2.09 [1.58-2.78]) and hip (HR 2.52 [1.61-3.95]) fractures than the controls. The increased fracture risk in patients with acromegaly was time-dependent and was observed even during the early period of follow-up. PURPOSE: Acromegaly is characterized by the overproduction of growth hormone (GH) and insulin-like growth factor-1 (IGF-1), both play important roles in regulating bone metabolism. We investigated the risk of vertebral and hip fractures in patients with acromegaly compared to age- and sex-matched controls. METHODS: This nationwide population-based cohort study included 1,777 patients with acromegaly aged 40 years or older in 2006-2016 and 8,885 age- and sex-matched controls. A Cox proportional hazards model was used to estimate the adjusted hazard ratio (HR) [95% confidence interval]. RESULTS: The mean age was 54.3 years and 58.9% were female. For approximately 8.5 years of follow-up, the patients with acromegaly had significantly higher risks of clinical vertebral (HR 2.09 [1.58-2.78]) and hip (HR 2.52 [1.61-3.95]) fractures than the controls in the multivariate analyses. There were significant differences in the risks of clinical vertebral (P < 0.0001) and hip (P < 0.0001) fractures between the patients with acromegaly and the controls in the Kaplan-Meier survival analysis. The multivariable-adjusted HRs for clinical vertebral fractures comparing the patients with acromegaly with controls during and excluding the first 7 years of observation were 1.69 [1.15-2.49] and 2.70 [1.75-4.17], respectively. The HRs for hip fractures during and excluding the first 7 years of observation were 2.29 [1.25-4.18] and 3.36 [1.63-6.92], respectively. CONCLUSIONS: The patients with acromegaly had a higher risk of hip fractures as well as clinical vertebral fractures than the controls. The increased fracture risk in patients with acromegaly was time-dependent and was observed even during the early period of follow-up.

Bromophenols from Symphyocladia latiuscula (Harvey) Yamada as Novel Cholecystokinin 2 Receptor Antagonists.

BACKGROUND: Cholecystokinin (CCK) is one of the most abundant peptides in the central nervous system and is believed to function as a neurotransmitter as well as a gut hormone with an inverse correlation of its level to anxiety and depression. Therefore, CCK receptors (CCKRs) could be a relevant target for novel antidepressant therapy. METHODS: In silico target prediction was first employed to predict the probability of the bromophenols interacting with key protein targets based on a model trained on known bioactivity data and chemical similarity considerations. Next, we tested the functional effect of natural bromophenols from Symphyocladia latiuscula on the CCK2 receptor followed by a molecular docking simulation to predict interactions between a compound and the binding site of the target protein. RESULTS: Results of cell-based functional G-protein coupled receptor (GPCR) assays demonstrate that bromophenols 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol (1), 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (2), and bis-(2,3,6-tribromo-4,5-dihydroxybenzyl) ether (3) are full CCK2 antagonists. Molecular docking simulation of 1‒3 with CCK2 demonstrated strong binding by means of interaction with prime interacting residues: Arg356, Asn353, Val349, His376, Phe227, and Pro210. Simulation results predicted good binding scores and interactions with prime residues, such as the reference antagonist YM022. CONCLUSIONS: The results of this study suggest bromophenols 1-3 are CCK2R antagonists that could be novel therapeutic agents for CCK2R-related diseases, especially anxiety and depression.

In Vitro Human Monoamine Oxidase Inhibition and Human Dopamine D4 Receptor Antagonist Effect of Natural Flavonoids for Neuroprotection.

Natural flavone and isoflavone analogs such as 3',4',7-trihydroxyflavone (1), 3',4',7-trihydroxyisoflavone (2), and calycosin (3) possess significant neuroprotective activity in Alzheimer's and Parkinson's disease. This study highlights the in vitro human monoamine oxidase (hMAO) inhibitory potential and functional effect of those natural flavonoids at dopamine and serotonin receptors for their possible role in neuroprotection. In vitro hMAO inhibition and enzyme kinetics studies were performed using a chemiluminescent assay. The functional effect of three natural flavonoids on dopamine and serotonin receptors was tested via cell-based functional assays followed by a molecular docking simulation to predict interactions between a compound and the binding site of the target protein. A forced swimming test was performed in the male C57BL/6 mouse model. Results of in vitro chemiluminescent assays and enzyme kinetics depicted 1 as a competitive inhibitor of hMAO-A with promising potency (IC50 value: 7.57 ± 0.14 µM) and 3 as a competitive inhibitor of hMAO-B with an IC50 value of 7.19 ± 0.32 µM. Likewise, GPCR functional assays in transfected cells showed 1 as a good hD4R antagonist. In docking analysis, these active flavonoids interacted with a determinant-interacting residue via hydrophilic and hydrophobic interactions, with low docking scores comparable to reference ligands. The post-oral administration of 1 to male C57BL/6 mice did not reduce the immobility time in the forced swimming test. The results of this study suggest that 1 and 3 may serve as effective regulators of the aminergic system via hMAO inhibition and the hD4R antagonist effect, respectively, for neuroprotection. The route of administration should be considered.

Detection of Salmonella Typhi nucleic acid by RT-PCR and anti-HlyE, -CdtB, -PilL, and -Vi IgM by ELISA at sites in Ghana, Madagascar and Ethiopia.

BACKGROUND: We aimed to assess the prevalence of Salmonella Typhi through DNA and IgM-antibody detection methods as a prelude to extended surveillance activities at sites in Ghana, Madagascar, and Ethiopia. METHODS: We performed species-specific real-time polymerase reaction (RT-PCR) to identify bacterial nucleic acid, and enzyme-linked immunosorbent assay (ELISA) for detecting HlyE/STY1498-, CdtB/STY1886-, pilL/STY4539- and Vi-antigens in blood and biopsy specimens of febrile and non-febrile subjects. We generated antigen-specific ELISA proxy cut-offs by change-point analyses, and utilized cumulative sum as detection method coupled with 1000 repetitive bootstrap analyses. We computed prevalence rates in addition to odds ratios to assess correlations between ELISA outcomes and participant characteristics. RESULTS: Definitive positive RT-PCR results were obtained from samples of febrile subjects originating from Adama Zuria/Ethiopia (1.9%, 2/104), Wolayita Sodo/Ethiopia (1.0%, 1/100), Diego/Madagascar (1.0%, 1/100), and Kintampo/Ghana (1.0%, 1/100), and from samples of non-febrile subjects from Wolayita Sodo/Ethiopia (1%, 2/201). While IgM antibodies against all antigens were identified across all sites, prevalence rates were highest at all Ethiopian sites, albeit in non-febrile populations. Significant correlations in febrile subjects aged < 15 years versus ≥ 15 years were detected for Vi (Odds Ratio (OR): 8.00, p = 0.034) in Adama Zuria/Ethiopia, STY1498 (OR: 3.21, p = 0.008), STY1886 (OR: 2.31, p = 0.054) and STY4539 (OR: 2.82, p = 0.022) in Diego/Madagascar, and STY1498 (OR: 2.45, p = 0.034) in Kintampo/Ghana. We found statistical significance in non-febrile male versus female subjects for STY1498 (OR: 1.96, p = 0.020) in Adama Zuria/Ethiopia, Vi (OR: 2.84, p = 0.048) in Diego/Madagascar, and STY4539 (OR: 0.46, p = 0.009) in Kintampo/Ghana. CONCLUSIONS: Findings indicate non-discriminatory stages of acute infections, though with site-specific differences. Immune responses among non-febrile, presumably healthy participants may mask recall and/or reporting bias leading to misclassification, or asymptomatic, subclinical infection signs induced by suppression of inflammatory responses. As most Ethiopian participants were ≥ 15 years of age and not at high-risk, the true S. Typhi burden was likely missed. Change-point analyses for generating ELISA proxy cut-offs appeared robust, though misclassification is possible. Our findings provided important information that may be useful to assess sites prior to implementing surveillance for febrile illness including Salmonella disease.

A retrospective review of vaccine wastage and associated risk factors in the Littoral region of Cameroon during 2016-2017.

BACKGROUND: Immunization is an effective preventive health intervention. In Cameroon, the Expanded Program on Immunization (EPI) aims to vaccinate children under 5 years of age for free, but vaccination coverage has consistently remained below the national target. Vaccines are distributed based on the target population size, factoring in wastage norms. However, the vaccine wastage rate (VWR) may differ among various settings. Our study aimed to assess vaccine wastage for different site settings, seasonality, and vaccine types in comparison to vaccination coverage in order to provide comprehensive insights on vaccine wastage. METHODS: A retrospective data collection and analysis were conducted on immunization and vaccine wastage data in the Littoral Region of Cameroon during 2016 and 2017. Health districts were classified as urban or rural, seasonality was categorized as rainy or dry season, and vaccine types were grouped into liquid, lyophilized, oral, and injectable vaccines. VWRs and vaccination coverage rates (VCRs) were calculated, and the vaccine waste factor was investigated. RESULTS: The VWR of Bacillus Calmette-Guérin (BCG; 32.19%) was the highest, followed by measles and rubella (MR; 19.05%) and yellow fever (YF; 18.34%) among all EPI vaccines in the Littoral Region of Cameroon during 2016 and 2017. Single-dose vaccine vials exhibited lower VWRs than multi-dose vials. Dry season was associated with higher VWRs for most vaccines, although more lyophilized vaccines (BCG, MR, YF vaccines) were wasted in rainy season in 2016. The VWR was persistently higher in rural than urban health districts. The months of February and November saw a decrease in VCRs. The study found an overall negative correlation between VCR and VWR. CONCLUSIONS: Multiple factors may cause wastage of EPI vaccines in Cameroon. Vaccination area characteristics, seasonality, types of vaccines such as multi- or single-dose, lyophilized or injectable vaccines are related to VWRs in Littoral Region. Further research on vaccine wastage and vaccination coverage across Cameroon is needed to better understand the socio-behavioral aspect of vaccine in-take that may affect the level of vaccination and vaccine wastage. Public health system strengthening is warranted to adapt more real-time monitoring of the VWR and VCR for each vaccine in the government's immunization programs.

In Vitro and In Silico Characterization of G-Protein Coupled Receptor (GPCR) Targets of Phlorofucofuroeckol-A and Dieckol.

Phlorotannins are polyphenolic compounds in marine alga, especially the brown algae. Among numerous phlorotannins, dieckol and phlorofucofuroeckol-A (PFF-A) are the major ones and despite a wider biological activity profile, knowledge of the G protein-coupled receptor (GPCR) targets of these phlorotannins is lacking. This study explores prime GPCR targets of the two phlorotannins. In silico proteocheminformatics modeling predicted twenty major protein targets and in vitro functional assays showed a good agonist effect at the α2C adrenergic receptor (α2CAR) and an antagonist effect at the adenosine 2A receptor (A2AR), δ-opioid receptor (δ-OPR), glucagon-like peptide-1 receptor (GLP-1R), and 5-hydroxytryptamine 1A receptor (5-TH1AR) of both phlorotannins. Besides, dieckol showed an antagonist effect at the vasopressin 1A receptor (V1AR) and PFF-A showed a promising agonist effect at the cannabinoid 1 receptor and an antagonist effect at V1AR. In silico molecular docking simulation enabled us to investigate and identify distinct binding features of these phlorotannins to the target proteins. The docking results suggested that dieckol and PFF-A bind to the crystal structures of the proteins with good affinity involving key interacting amino acid residues comparable to reference ligands. Overall, the present study suggests α2CAR, A2AR, δ-OPR, GLP-1R, 5-TH1AR, CB1R, and V1AR as prime receptor targets of dieckol and PFF-A.

Neuroprotective Effect of Aurantio-Obtusin, a Putative Vasopressin V1A Receptor Antagonist, on Transient Forebrain Ischemia Mice Model.

Traditional Chinese medicines (TCMs) have been a rich source of novel drug discovery, and Cassia seed is one of the common TCMs with numerous biological effects. Based on the existing reports on neuroprotection by Cassia seed extract, the present study aims to search possible pharmacological targets behind the neuroprotective effects of the Cassia seeds by evaluating the functional effect of specific Cassia compounds on various G-protein-coupled receptors. Among the four test compounds (cassiaside, rubrofusarin gentiobioside, aurantio-obtusin, and 2-hydroxyemodin 1-methylether), only aurantio-obtusin demonstrated a specific V1AR antagonist effect (71.80 ± 6.0% inhibition at 100 µM) and yielded an IC50 value of 67.70 ± 2.41 µM. A molecular docking study predicted an additional interaction of the hydroxyl group at C6 and a methoxy group at C7 of aurantio-obtusin with the Ser341 residue as functional for the observed antagonist effect. In the transient brain ischemia/reperfusion injury C57BL/6 mice model, aurantio-obtusin attenuated the latency time that was reduced in the bilateral common carotid artery occlusion (BCCAO) groups. Likewise, compared to neuronal damage in the BCCAO groups, treatment with aurantio-obtusin (10 mg/kg, p.o.) significantly reduced the severity of damage in medial cornu ammonis 1 (mCA1), dorsal CA1, and cortex regions. Overall, the findings of this study highlight V1AR as a possible target of aurantio-obtusin for neuroprotection.

The Surveillance for Enteric Fever in Asia Project (SEAP), Severe Typhoid Fever Surveillance in Africa (SETA), Surveillance of Enteric Fever in India (SEFI), and Strategic Typhoid Alliance Across Africa and Asia (STRATAA) Population-based Enteric Fever Studies: A Review of Methodological Similarities and Differences.

Building on previous multicountry surveillance studies of typhoid and others salmonelloses such as the Diseases of the Most Impoverished program and the Typhoid Surveillance in Africa Project, several ongoing blood culture surveillance studies are generating important data about incidence, severity, transmission, and clinical features of invasive Salmonella infections in sub-Saharan Africa and South Asia. These studies are also characterizing drug resistance patterns in their respective study sites. Each study answers a different set of research questions and employs slightly different methodologies, and the geographies under surveillance differ in size, population density, physician practices, access to healthcare facilities, and access to microbiologically safe water and improved sanitation. These differences in part reflect the heterogeneity of the epidemiology of invasive salmonellosis globally, and thus enable generation of data that are useful to policymakers in decision-making for the introduction of typhoid conjugate vaccines (TCVs). Moreover, each study is evaluating the large-scale deployment of TCVs, and may ultimately be used to assess post-introduction vaccine impact. The data generated by these studies will also be used to refine global disease burden estimates. It is important to ensure that lessons learned from these studies not only inform vaccination policy, but also are incorporated into sustainable, low-cost, integrated vaccine-preventable disease surveillance systems.

Metformin, resveratrol, and exendin-4 inhibit high phosphate-induced vascular calcification via AMPK-RANKL signaling.

Vascular calcification increases the risk of developing cardiovascular disease, and it is closely associated with metabolic disorders such as diabetes mellitus and non-alcoholic fatty liver disease. We investigated whether the activators of AMP-activated protein kinase (AMPK), metformin, resveratrol, and exendin-4, improved inorganic phosphate (Pi)-induced vascular calcification in rat vascular smooth muscle cells (VSMCs) and whether these effects were via AMPK. Pi increased calcium deposition in a dose-dependent manner, and metformin, resveratrol, and exendin-4 significantly decreased calcium deposition in the Pi-treated VSMCs. Moreover, metformin and exendin-4 increased the expression of a SMC marker gene, α-smooth muscle actin, and Ampk and reduced the receptor activator of nuclear factor kappa-Β ligand (Rankl)/osteoprotegerin ratio. Metformin, resveratrol, and exendin-4 reduced the expression of osteoblast differentiation-associated factors, such as runt-related transcription factor 2, bone morphogenic protein-2, p-small mothers against decapentaplegic 1/5/8, and Rankl. Inhibition of AMPK by siRNA adversely affected the anti-calcification effects of metformin, resveratrol, and exendin-4 and reversed the reduction of the expression of Rankl by metformin and exendin-4 in the Pi-treated VSMCs. These data suggest that metformin, resveratrol, and exendin-4 ameliorate Pi-induced vascular calcification by inhibiting osteoblast differentiation of VSMCs, which is mediated by AMPK.

Tetra-aryl cyclobutane and stilbenes from the rhizomes of Rheum undulatum and their α-glucosidase inhibitory activity: Biological evaluation, kinetic analysis, and molecular docking simulation.

One achiral tetra-aryl cyclobutane [rheundulin A (1)] and three stilbene glycosides [rheundulins B-D (2-4)] were isolated from the methanol extract of Rheum undulatum L., along with eight known compounds (5-12). Structural determination of the new compounds (1-4) was accomplished using comprehensive spectroscopic methods. Compound 1 represents the first example of a dimeric stilbene linked via a cyclobutane ring from the Rheum genus. All isolates were screened for their inhibition against α-glucosidase. Among them, stilbene derivatives (5 and 6) showed strong inhibitory effects on α-glucosidase with IC50 values of 0.5 and 15.4 µM, respectively, which were significantly higher than that of the positive control, acarbose (IC50 = 126.8 µM). Rheundulin A (1) showed moderate α-glucosidase inhibition with an IC50 value of 80.1 µM. In addition, kinetic analysis and molecular docking simulation of the most active compound (5) with α-glucosidase were performed for the first time. Kinetic studies revealed that compound 5 competitively inhibited the active site of α-glucosidase (Ki = 0.40 µM), while 6 had a mixed-type inhibitory effect against α-glucosidase (Ki = 15.34 µM). Molecular docking simulations of 5 and 6 demonstrated negative-binding energies, indicating high proximity to the active site and tight binding to α-glucosidase enzyme.

Stilbenes with Potent Protein Tyrosine Phosphatase-1B Inhibitory Activity from the Roots of Polygonum multiflorum.

Seven new stilbene glycosides including three dimers (1-3) and four monomers (4-7) were isolated from the roots of Polygonum multiflorum along with nine previously identified stilbenes (8-16). In addition, two deglucosylated stilbenes, 2a and 3a, were also obtained as new dimeric stilbenes. The structures of the purified phytochemicals were elucidated by interpreting their spectroscopic data (NMR, HRMS, and ECD). To the best of our knowledge, this represents the first isolation of a phenylpropanoid (C6-C3) substituted with a stilbene unit (7) from the Polygonaceae family. In an in vitro enzyme assay with human recombinant protein tyrosine phosphatase-1B (PTP1B), compounds 2-5 showed weak PTP1B inhibition with an IC50 value range of 27.4-37.6 µM, while three deglucosylated stilbenes 2a, 3a, and 8a exhibited IC50 values of 2.1, 1.9, and 12.1 µM, respectively. The inhibition modes and binding mechanism of selected inhibitors (2a and 3a) were investigated using kinetic methods and molecular docking simulations.

CRISPR-Pass: Gene Rescue of Nonsense Mutations Using Adenine Base Editors.

A nonsense mutation is a substitutive mutation in a DNA sequence that causes a premature termination during translation and produces stalled proteins, resulting in dysfunction of a gene. Although it usually induces severe genetic disorders, there are no definite methods for inducing read through of premature termination codons (PTCs). Here, we present a targeted tool for bypassing PTCs, named CRISPR-pass, that uses CRISPR-mediated adenine base editors. CRISPR-pass, which should be applicable to 95.5% of clinically significant nonsense mutations in the ClinVar database, rescues protein synthesis in patient-derived fibroblasts, suggesting potential clinical utility.

Occurrence of Typhoid Fever Complications and Their Relation to Duration of Illness Preceding Hospitalization: A Systematic Literature Review and Meta-analysis.

BACKGROUND: Complications from typhoid fever disease have been estimated to occur in 10%-15% of hospitalized patients, with evidence of a higher risk in children and when delaying the implementation of effective antimicrobial treatment. We estimated the prevalence of complications in hospitalized patients with culture-confirmed typhoid fever and the effects of delaying the implementation of effective antimicrobial treatment and age on the prevalence and risk of complications. METHODS: A systematic review and meta-analysis were performed using studies in the PubMed database. We rated risk of bias and conducted random-effects meta-analyses. Days of disease at hospitalization (DDA) was used as a surrogate for delaying the implementation of effective antimicrobial treatment. Analyses were stratified by DDA (DDA <10 versus ≥10 mean/median days of disease) and by age (children versus adults). Differences in risk were assessed using odds ratios (ORs) and 95% confidence intervals (CIs). Heterogeneity and publication bias were evaluated with the I2 value and funnel plot analysis, respectively. RESULTS: The pooled prevalence of complications estimated among hospitalized typhoid fever patients was 27% (95% CI, 21%-32%; I2 = 90.9%, P < .0001). Patients with a DDA ≥ 10 days presented higher prevalence (36% [95% CI, 29%-43%]) and three times greater risk of severe disease (OR, 3.00 [95% CI, 2.14-4.17]; P < .0001) than patients arriving earlier (16% [95% CI, 13%- 18%]). Difference in prevalence and risk by age groups were not significant. CONCLUSIONS: This meta-analysis identified a higher overall prevalence of complications than previously reported and a strong association between duration of symptoms prior to hospitalization and risk of serious complications.

How Can the Typhoid Fever Surveillance in Africa and the Severe Typhoid Fever in Africa Programs Contribute to the Introduction of Typhoid Conjugate Vaccines?

BACKGROUND: The World Health Organization now recommends the use of typhoid conjugate vaccines (TCVs) in typhoid-endemic countries, and Gavi, the Vaccine Alliance, added TCVs into the portfolio of subsidized vaccines. Data from the Severe Typhoid Fever in Africa (SETA) program were used to contribute to TCV introduction decision-making processes, exemplified for Ghana and Madagascar. METHODS: Data collected from both countries were evaluated, and barriers to and benefits of introduction scenarios are discussed. No standardized methodological framework was applied. RESULTS: The Ghanaian healthcare system differs from its Malagasy counterpart: Ghana features a functioning insurance system, antimicrobials are available nationwide, and several sites in Ghana deploy blood culture-based typhoid diagnosis. A higher incidence of antimicrobial-resistant Salmonella Typhi is reported in Ghana, which has not been identified as an issue in Madagascar. The Malagasy people have a low expectation of provided healthcare and experience frequent unavailability of medicines, resulting in limited healthcare-seeking behavior and extended consequences of untreated disease. CONCLUSIONS: For Ghana, high typhoid fever incidence coupled with spatiotemporal heterogeneity was observed. A phased TCV introduction through an initial mass campaign in high-risk areas followed by inclusion into routine national immunizations prior to expansion to other areas of the country can be considered. For Madagascar, a national mass campaign followed by routine introduction would be the introduction scenario of choice as it would protect the population, reduce transmission, and prevent an often-deadly disease in a setting characterized by lack of access to healthcare infrastructure. New, easy-to-use diagnostic tools, potentially including environmental surveillance, should be explored and improved to facilitate identification of high-risk areas.

Bacteremia Among Febrile Patients Attending Selected Healthcare Facilities in Ibadan, Nigeria.

BACKGROUND: The relative contribution of bacterial infections to febrile disease is poorly understood in many African countries due to diagnostic limitations. This study screened pediatric and adult patients attending 4 healthcare facilities in Ibadan, Nigeria, for bacteremia and malaria parasitemia. METHODS: Febrile patients underwent clinical diagnosis, malaria parasite testing, and blood culture. Bacteria from positive blood cultures were isolated and speciated using biochemical and serological methods, and Salmonella subtyping was performed by polymerase chain reaction. Antimicrobial susceptibility was tested by disk diffusion. RESULTS: A total of 682 patients were recruited between 16 June and 16 October 2017; 467 (68.5%) were <18 years of age. Bacterial pathogens were cultured from the blood of 117 (17.2%) patients, with Staphylococcus aureus (69 [59.0%]) and Salmonella enterica (34 [29.1%]) being the most common species recovered. Twenty-seven (79.4%) of the Salmonella isolates were serovar Typhi and the other 7 belonged to nontyphoidal Salmonella serovarieties. Thirty-four individuals were found to be coinfected with Plasmodium falciparum and bacteria. Five (14.7%) of these coinfections were with Salmonella, all in children aged <5 years. Antimicrobial susceptibility testing revealed that most of the Salmonella and Staphylococcus isolates were multidrug resistant. CONCLUSIONS: The study demonstrates that bacteria were commonly recovered from febrile patients with or without malaria in this location. Focused and extended epidemiological studies are needed for the introduction of typhoid conjugate vaccines that have the potential to prevent a major cause of severe community-acquired febrile diseases in our locality.

Acute Febrile Illness Among Children in Butajira, South-Central Ethiopia During the Typhoid Fever Surveillance in Africa Program.

BACKGROUND: Clearly differentiating causes of fever is challenging where diagnostic capacities are limited, resulting in poor patient management. We investigated acute febrile illness in children aged ≤15 years enrolled at healthcare facilities in Butajira, Ethiopia, during January 2012 to January 2014 for the Typhoid Fever Surveillance in Africa Program. METHODS: Blood culture, malaria microscopy, and blood analyses followed by microbiological, biochemical, and antimicrobial susceptibility testing of isolates were performed. We applied a retrospectively developed scheme to classify children as malaria or acute respiratory, gastrointestinal or urinary tract infection, or other febrile infections and syndromes. Incidence rates per 100 000 population derived from the classification scheme and multivariate logistic regression to determine fever predictors were performed. RESULTS: We rarely observed stunting (4/513, 0.8%), underweight (1/513, 0.2%), wasting (1/513, 0.2%), and hospitalization (21/513, 4.1%) among 513 children with mild transient fever and a mean disease severity score of 12 (95% confidence interval [CI], 11-13). Blood cultures yielded 1.6% (8/513) growth of pathogenic agents; microscopy detected 13.5% (69/513) malaria with 20 611/µL blood (95% CI, 15 352-25 870) mean parasite density. Incidences were generally higher in children aged ≤5 years than >5 to ≤15 years; annual incidences in young children were 301.3 (95% CI, 269.2-337.2) for malaria and 1860.1 (95% CI, 1778.0-1946.0) for acute respiratory and 379.9 (95% CI, 343.6-420.0) for gastrointestinal tract infections. CONCLUSIONS: We could not detect the etiological agents in all febrile children. Our findings may prompt further investigations and the reconsideration of policies and frameworks for the management of acute febrile illness.

The Typhoid Fever Surveillance in Africa Program: Geospatial Sampling Frames for Household-based Studies: Lessons Learned From a Multicountry Surveillance Network in Senegal, South Africa, and Sudan.

BACKGROUND: Robust household sampling, commonly applied for population-based investigations, requires sampling frames or household lists to minimize selection bias. We have applied Google Earth Pro satellite imagery to constitute structure-based sampling frames at sites in Pikine, Senegal; Pietermaritzburg, South Africa; and Wad-Medani, Sudan. Here we present our experiences in using this approach and findings from assessing its applicability by determining positional accuracy. METHODS: Printouts of satellite imagery combined with Global Positioning System receivers were used to locate and to verify the locations of sample structures (simple random selection; weighted-stratified sampling). Positional accuracy was assessed by study site and administrative subareas by calculating normalized distances (meters) between coordinates taken from the sampling frame and on the ground using receivers. A higher accuracy in conjunction with smaller distances was assumed. Kruskal-Wallis and Dunn multiple pairwise comparisons were performed to evaluate positional accuracy by setting and by individual surveyor in Pietermaritzburg. RESULTS: The median normalized distances and interquartile ranges were 0.05 and 0.03-0.08 in Pikine, 0.09 and 0.05-0.19 in Pietermaritzburg, and 0.05 and 0.00-0.10 in Wad-Medani, respectively. Root mean square errors were 0.08 in Pikine, 0.42 in Pietermaritzburg, and 0.17 in Wad-Medani. Kruskal-Wallis and Dunn comparisons indicated significant differences by low- and high-density setting and interviewers who performed the presented approach with high accuracy compared to interviewers with poor accuracy. CONCLUSIONS: The geospatial approach presented minimizes systematic errors and increases robustness and representativeness of a sample. However, the findings imply that this approach may not be applicable at all sites and settings; its success also depends on skills of surveyors working with aerial data. Methodological modifications are required, especially for resource-challenged sites that may be affected by constraints in data availability and area size.

The Monitoring and Evaluation of a Multicountry Surveillance Study, the Severe Typhoid Fever in Africa Program.

BACKGROUND: There is limited information on the best practices for monitoring multicountry epidemiological studies. Here, we describe the monitoring and evaluation procedures created for the multicountry Severe Typhoid Fever in Africa (SETA) study. METHODS: Elements from the US Food and Drug Administration (FDA) and European Centre for Disease Prevention and Control (ECDC) recommendations on monitoring clinical trials and data quality, respectively were applied in the development of the SETA monitoring plan. The SETA core activities as well as the key data and activities required for the delivery of SETA outcomes were identified. With this information, a list of key monitorable indicators was developed using on-site and centralized monitoring methods, and a dedicated monitoring team was formed. The core activities were monitored on-site in each country at least twice per year and the SETA databases were monitored centrally as a collaborative effort between the International Vaccine Institute and study sites. Monthly reports were generated for key indicators and used to guide risk-based monitoring specific for each country. RESULTS: Preliminary results show that monitoring activities have increased compliance with protocol and standard operating procedures. A reduction in blood culture contamination following monitoring field visits in two of the SETA countries are preliminary results of the impact of monitoring activities. CONCLUSIONS: Current monitoring recommendations applicable to clinical trials and routine surveillance systems can be adapted for monitoring epidemiological studies. Continued monitoring efforts ensure that the procedures are harmonized across sites. Flexibility, ongoing feedback, and team participation yield sustainable solutions.

The Severe Typhoid Fever in Africa Program: Study Design and Methodology to Assess Disease Severity, Host Immunity, and Carriage Associated With Invasive Salmonellosis.

BACKGROUND: Invasive salmonellosis is a common community-acquired bacteremia in persons residing in sub-Saharan Africa. However, there is a paucity of data on severe typhoid fever and its associated acute and chronic host immune response and carriage. The Severe Typhoid Fever in Africa (SETA) program, a multicountry surveillance study, aimed to address these research gaps and contribute to the control and prevention of invasive salmonellosis. METHODS: A prospective healthcare facility-based surveillance with active screening of enteric fever and clinically suspected severe typhoid fever with complications was performed using a standardized protocol across the study sites in Burkina Faso, the Democratic Republic of Congo (DRC), Ethiopia, Ghana, Madagascar, and Nigeria. Defined inclusion criteria were used for screening of eligible patients for enrollment into the study. Enrolled patients with confirmed invasive salmonellosis by blood culture or patients with clinically suspected severe typhoid fever with perforation were eligible for clinical follow-up. Asymptomatic neighborhood controls and immediate household contacts of each case were enrolled as a comparison group to assess the level of Salmonella-specific antibodies and shedding patterns. Healthcare utilization surveys were performed to permit adjustment of incidence estimations. Postmortem questionnaires were conducted in medically underserved areas to assess death attributed to invasive Salmonella infections in selected sites. RESULTS: Research data generated through SETA aimed to address scientific knowledge gaps concerning the severe typhoid fever and mortality, long-term host immune responses, and bacterial shedding and carriage associated with natural infection by invasive salmonellae. CONCLUSIONS: SETA supports public health policy on typhoid immunization strategy in Africa.

Multicountry Distribution and Characterization of Extended-spectrum ß-Lactamase-associated Gram-negative Bacteria From Bloodstream Infections in Sub-Saharan Africa.

BACKGROUND: Antimicrobial resistance (AMR) is a major global health concern, yet, there are noticeable gaps in AMR surveillance data in regions such as sub-Saharan Africa. We aimed to measure the prevalence of extended-spectrum ß-lactamase (ESBL) producing Gram-negative bacteria in bloodstream infections from 12 sentinel sites in sub-Saharan Africa. METHODS: Data were generated during the Typhoid Fever Surveillance in Africa Program (TSAP), in which standardized blood cultures were performed on febrile patients attending 12 health facilities in 9 sub-Saharan African countries between 2010 and 2014. Pathogenic bloodstream isolates were identified at the sites and then subsequently confirmed at a central reference laboratory. Antimicrobial susceptibility testing, detection of ESBL production, and conventional multiplex polymerase chain reaction (PCR) testing for genes encoding for ß-lactamase were performed on all pathogens. RESULTS: Five hundred and five pathogenic Gram-negative bloodstream isolates were isolated during the study period and available for further characterization. This included 423 Enterobacteriaceae. Phenotypically, 61 (12.1%) isolates exhibited ESBL activity, and genotypically, 47 (9.3%) yielded a PCR amplicon for at least one of the screened ESBL genes. Among specific Gram-negative isolates, 40 (45.5%) of 88 Klebsiella spp., 7 (5.7%) of 122 Escherichia coli, 6 (16.2%) of 37 Acinetobacter spp., and 2 (1.3%) of 159 of nontyphoidal Salmonella (NTS) showed phenotypic ESBL activity. CONCLUSIONS: Our findings confirm the presence of ESBL production among pathogens causing bloodstream infections in sub-Saharan Africa. With few alternatives for managing ESBL-producing pathogens in the African setting, measures to control the development and proliferation of AMR organisms are urgently needed.