Soluble Urokinase-Type Plasminogen Activator Receptor as a Prognostic Marker of Ugandan Children at Risk of Severe and Fatal Malaria.

BACKGROUND: Current malaria diagnostic tests do not reliably identify children at risk of severe and fatal infection. Host immune and endothelial activation contribute to malaria pathogenesis. Soluble urokinase-type plasminogen activator receptor (suPAR) is a marker of these pathways. We hypothesized that measuring suPAR at presentation could risk-stratify children with malaria. METHODS: Plasma suPAR levels were determined in consecutive febrile children with malaria at presentation to hospital in Jinja, Uganda. We evaluated the accuracy of suPAR in predicting in-hospital mortality, and whether suPAR could improve a validated clinical scoring system (Lambaréné Organ Dysfunction Score [LODS]). RESULTS: Of the 1226 children with malaria, 39 (3.2%) died. suPAR concentrations at presentation were significantly higher in children who went on to die than in those who survived (P < .0001). suPAR levels were associated with disease severity (LODS: 0 vs 1, P = .001; 1 vs 2, P < .001; 2 vs 3, 0 vs 2, 1 vs 3, and 0 vs 3, P < .0001). suPAR concentrations were excellent predictors of in-hospital mortality (area under the receiver operating characteristic curve [AUROC], 0.92 [95% confidence interval {CI}, .91-.94]). The prognostic accuracy of LODS (AUROC, 0.93 [95% CI, .91-.94]) was improved when suPAR was added (AUROC, 0.97 [95% CI, .96-.98]; P < .0001). CONCLUSIONS: Measuring suPAR at presentation can identify children at risk of severe and fatal malaria. Adding suPAR to clinical scores could improve the recognition and triage of children at risk of death. suPAR can be detected with a point-of-care test and can now be evaluated in prospective trials.

Risk Factors for Community Colonization With Extended-Spectrum Cephalosporin-Resistant Enterobacterales (ESCrE) in Botswana: An Antibiotic Resistance in Communities and Hospitals (ARCH) Study.

BACKGROUND: The epidemiology of extended-spectrum cephalosporin-resistant Enterobacterales (ESCrE) in low- and middle-income countries (LMICs) is poorly described. Identifying risk factors for ESCrE colonization is critical to inform antibiotic resistance reduction strategies because colonization is typically a precursor to infection. METHODS: From 15 January 2020 to 4 September 2020, we surveyed a random sample of clinic patients at 6 sites in Botswana. We also invited each enrolled participant to refer up to 3 adults and children. All participants had rectal swabs collected that were inoculated onto chromogenic media followed by confirmatory testing. Data were collected on demographics, comorbidities, antibiotic use, healthcare exposures, travel, and farm and animal contact. Participants with ESCrE colonization (cases) were compared with noncolonized participants (controls) to identify risk factors for ESCrE colonization using bivariable, stratified, and multivariable analyses. RESULTS: A total of 2000 participants were enrolled. There were 959 (48.0%) clinic participants, 477 (23.9%) adult community participants, and 564 (28.2%) child community participants. The median (interquartile range) age was 30 (12-41) and 1463 (73%) were women. There were 555 cases and 1445 controls (ie, 27.8% of participants were ESCrE colonized). Independent risk factors (adjusted odds ratio [95% confidence interval]) for ESCrE included healthcare exposure (1.37 [1.08-1.73]), foreign travel [1.98 (1.04-3.77]), tending livestock (1.34 [1.03-1.73]), and presence of an ESCrE-colonized household member (1.57 [1.08-2.27]). CONCLUSIONS: Our results suggest healthcare exposure may be important in driving ESCrE. The strong links to livestock exposure and household member ESCrE colonization highlight the potential role of common exposure or household transmission. These findings are critical to inform strategies to curb further emergence of ESCrE in LMICs.

Pulmonary Nontuberculous Mycobacteria, Ontario, Canada, 2020.

We measured annual prevalence of microbiologically defined nontuberculous mycobacterial lung disease in Ontario, Canada. Mycobacterium avium prevalence was 13 cases/100,000 persons in 2020, a 2.5-fold increase from 2010, indicating a large increase in true M. avium lung disease. During the same period, M. xenopi decreased nearly 50%, to 0.84 cases/100,000 persons.

Facilitating Safe Discharge Through Predicting Disease Progression in Moderate Coronavirus Disease 2019 (COVID-19): A Prospective Cohort Study to Develop and Validate a Clinical Prediction Model in Resource-Limited Settings.

BACKGROUND: In locations where few people have received coronavirus disease 2019 (COVID-19) vaccines, health systems remain vulnerable to surges in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Tools to identify patients suitable for community-based management are urgently needed. METHODS: We prospectively recruited adults presenting to 2 hospitals in India with moderate symptoms of laboratory-confirmed COVID-19 to develop and validate a clinical prediction model to rule out progression to supplemental oxygen requirement. The primary outcome was defined as any of the following: SpO2 < 94%; respiratory rate > 30 BPM; SpO2/FiO2 < 400; or death. We specified a priori that each model would contain three clinical parameters (age, sex, and SpO2) and 1 of 7 shortlisted biochemical biomarkers measurable using commercially available rapid tests (C-reactive protein [CRP], D-dimer, interleukin 6 [IL-6], neutrophil-to-lymphocyte ratio [NLR], procalcitonin [PCT], soluble triggering receptor expressed on myeloid cell-1 [sTREM-1], or soluble urokinase plasminogen activator receptor [suPAR]), to ensure the models would be suitable for resource-limited settings. We evaluated discrimination, calibration, and clinical utility of the models in a held-out temporal external validation cohort. RESULTS: In total, 426 participants were recruited, of whom 89 (21.0%) met the primary outcome; 257 participants comprised the development cohort, and 166 comprised the validation cohort. The 3 models containing NLR, suPAR, or IL-6 demonstrated promising discrimination (c-statistics: 0.72-0.74) and calibration (calibration slopes: 1.01-1.05) in the validation cohort and provided greater utility than a model containing the clinical parameters alone. CONCLUSIONS: We present 3 clinical prediction models that could help clinicians identify patients with moderate COVID-19 suitable for community-based management. The models are readily implementable and of particular relevance for locations with limited resources.

Monkeypox Virus Detection in Different Clinical Specimen Types.

A global monkeypox outbreak began in May 2022. Limited data exist on specimen type performance in associated molecular diagnostics. Consequently, a diverse range of specimen sources were collected in the initial weeks of the outbreak in Ontario, Canada. Our clinical evaluation identified skin lesions as the optimal diagnostic specimen source.

Immune and endothelial activation markers and risk stratification of childhood pneumonia in Uganda: A secondary analysis of a prospective cohort study.

BACKGROUND: Despite the global burden of pneumonia, reliable triage tools to identify children in low-resource settings at risk of severe and fatal respiratory tract infection are lacking. This study assessed the ability of circulating host markers of immune and endothelial activation quantified at presentation, relative to currently used clinical measures of disease severity, to identify children with pneumonia who are at risk of death. METHODS AND FINDINGS: We conducted a secondary analysis of a prospective cohort study of children aged 2 to 59 months presenting to the Jinja Regional Hospital in Jinja, Uganda between February 2012 and August 2013, who met the Integrated Management of Childhood Illness (IMCI) diagnostic criteria for pneumonia. Circulating plasma markers of immune (IL-6, IL-8, CXCL-10/IP-10, CHI3L1, sTNFR1, and sTREM-1) and endothelial (sVCAM-1, sICAM-1, Angpt-1, Angpt-2, and sFlt-1) activation measured at hospital presentation were compared to lactate, respiratory rate, oxygen saturation, procalcitonin (PCT), and C-reactive protein (CRP) with a primary outcome of predicting 48-hour mortality. Of 805 children with IMCI pneumonia, 616 had severe pneumonia. Compared to 10 other immune and endothelial activation markers, sTREM-1 levels at presentation had the best predictive accuracy in identifying 48-hour mortality for children with pneumonia (AUROC 0.885, 95% CI 0.841 to 0.928; p = 0.03 to p < 0.001) and severe pneumonia (AUROC 0.870, 95% CI 0.824 to 0.916; p = 0.04 to p < 0.001). sTREM-1 was more strongly associated with 48-hour mortality than lactate (AUROC 0.745, 95% CI 0.664 to 0.826; p < 0.001), respiratory rate (AUROC 0.615, 95% CI 0.528 to 0.702; p < 0.001), oxygen saturation (AUROC 0.685, 95% CI 0.594 to 0.776; p = 0.002), PCT (AUROC 0.650, 95% CI 0.566 to 0.734; p < 0.001), and CRP (AUROC 0.562, 95% CI 0.472 to 0.653; p < 0.001) in cases of pneumonia and severe pneumonia. The main limitation of this study was the unavailability of radiographic imaging. CONCLUSIONS: In this cohort of Ugandan children, sTREM-1 measured at hospital presentation was a significantly better indicator of 48-hour mortality risk than other common approaches to risk stratify children with pneumonia. Measuring sTREM-1 at clinical presentation may improve the early triage, management, and outcome of children with pneumonia at risk of death. TRIAL REGISTRATION: The trial was registered at clinicaltrial.gov (NCT04726826).

Clinical sign and biomarker-based algorithm to identify bacterial pneumonia among outpatients with lower respiratory tract infection in Tanzania.

BACKGROUND: Inappropriate antibiotics use in lower respiratory tract infections (LRTI) is a major contributor to resistance. We aimed to design an algorithm based on clinical signs and host biomarkers to identify bacterial community-acquired pneumonia (CAP) among patients with LRTI. METHODS: Participants with LRTI were selected in a prospective cohort of febrile (≥ 38 °C) adults presenting to outpatient clinics in Dar es Salaam. Participants underwent chest X-ray, multiplex PCR for respiratory pathogens, and measurements of 13 biomarkers. We evaluated the predictive accuracy of clinical signs and biomarkers using logistic regression and classification and regression tree analysis. RESULTS: Of 110 patients with LRTI, 17 had bacterial CAP. Procalcitonin (PCT), interleukin-6 (IL-6) and soluble triggering receptor expressed by myeloid cells-1 (sTREM-1) showed an excellent predictive accuracy to identify bacterial CAP (AUROC 0.88, 95%CI 0.78-0.98; 0.84, 0.72-0.99; 0.83, 0.74-0.92, respectively). Combining respiratory rate with PCT or IL-6 significantly improved the model compared to respiratory rate alone (p = 0.006, p = 0.033, respectively). An algorithm with respiratory rate (≥ 32/min) and PCT (≥ 0.25 µg/L) had 94% sensitivity and 82% specificity. CONCLUSIONS: PCT, IL-6 and sTREM-1 had an excellent predictive accuracy in differentiating bacterial CAP from other LRTIs. An algorithm combining respiratory rate and PCT displayed even better performance in this sub-Sahara African setting.

Copan eNAT Transport System To Address Challenges in COVID-19 Diagnostics in Regions with Limited Testing Access.

Community-based health care clinics and hospital outreach services have the potential to expand coronavirus disease 2019 (COVID-19) diagnostics to rural areas. However, reduced specimen stability during extended transport, the absence of a cold chain to centralized laboratories, and biosafety concerns surrounding specimen handling have limited this expansion. In the following study, we evaluated eNAT (Copan Italia, Brescia, Italy) as an alternative transport system to address the biosafety and stability challenges associated with expanding COVID-19 diagnostics to rural and remote regions. In this study, we demonstrated that high-titer severe acute respiratory virus syndrome coronavirus 2 (SARS-CoV-2) lysate placed into eNAT medium cannot be propagated in cell culture, supporting viral inactivation. To account for off-site testing in these settings, we assessed the stability of contrived nasopharyngeal (NP) specimens stored for up to 14 days in various transport media (eNAT, eSwab, viral transport medium [VTM], saline, and phosphate-buffered saline [PBS]) at 4°C, 22 to 25°C, and 35°C. The molecular detection of SARS-CoV-2 was unaffected by sample storage temperature over the 2 weeks when stored in eNAT or PBS (change in cycle threshold, ≤1). In contrast, variable stability was observed across test conditions for other transport media. As eNAT can inactivate SARS-CoV-2, it may support COVID-19 diagnostics at the point of care. Evaluation of compatibility of eNAT with Cepheid Xpert Xpress SARS-CoV-2 assay demonstrated diagnostic accuracy and sensitivity equivalent to those of VTM. Taken together, these findings suggest that the implementation of eNAT as a collection device can expand COVID-19 testing to areas with limited health care access.

Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Tracking.

BACKGROUND: High-sensitivity severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations. METHODS: We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection of 0.5 pg/mL (10.6 fmol/L) nucleocapsid antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives and quantified and tracked antigen dynamics in intensive care unit coronavirus disease 2019 (COVID-19) inpatients and immunocompromised COVID-19 patients. RESULTS: Compared to qualitative reverse transcription-polymerase chain reaction methods, the MSAA demonstrated a positive percentage agreement of 97% (95% CI 92%-99%) and a negative percentage agreement of 97% (95% CI 94%-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with cycle threshold values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden. CONCLUSIONS: The MSAA enables sensitive and specific detection of acute infections and quantification and tracking of antigen burden and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.

Prognostic Accuracy of Soluble Triggering Receptor Expressed on Myeloid Cells (sTREM-1)-based Algorithms in Febrile Adults Presenting to Tanzanian Outpatient Clinics.

BACKGROUND: The inability to identify individuals with acute fever at risk of death is a barrier to effective triage and management of severe infections, especially in low-resource settings. Since endothelial and immune activation contribute to the pathogenesis of various distinct life-threatening infections, we hypothesized that measuring mediators of these pathways at clinical presentation would identify febrile adults at risk of death. METHODS: Plasma concentrations of markers of endothelial (angiopoetin-1/2, soluble fms-like tyrosine kinase-1, soluble vascular cell adhesion molecule-1, soluble intercellular adhesion molecule-1) and immune (soluble triggering receptor expressed on myeloid cells [sTREM-1], interleukin-6, interleukin-8, chitinase-3-like protein-1, soluble tumor necrosis factor receptor-1, procalcitonin [PCT], C-reactive protein [CRP]) activation pathways were determined in consecutive adults with acute fever (≥38°C) at presentation to outpatient clinics in Dar es Salaam, Tanzania. We evaluated the accuracy of these mediators in predicting all-cause mortality and examined whether markers could improve the prognostic accuracy of clinical scoring systems, including the quick sequential organ failure assessment (qSOFA) and Glasgow coma scale (GCS). RESULTS: Of 507 febrile adults, 32 died (6.3%) within 28 days of presentation. We found that sTREM-1 was the best prognostic marker for 28-day mortality (area under the receiver operating characteristic [AUROC] 0.87, 95% confidence interval [CI] 0.81-0.92) and was significantly better than CRP (P < .0001) and PCT (P = .0001). The prognostic accuracy of qSOFA and the GCS were significantly enhanced when sTREM-1 was added (0.80 [95% CI 0.76-0.83] to 0.91 [95% CI 0.88-0.94; P < .05] and 0.72 [95% CI 0.63-0.80] to 0.94 [95% CI 0.91-0.97; P < .05], respectively). CONCLUSIONS: Measuring sTREM-1 at clinical presentation can identify febrile individuals at risk of all-cause febrile mortality. Adding severity markers such as sTREM-1 to simple clinical scores could improve the recognition and triage of patients with life-threatening infections in resource-limited settings.

Evaluation of the FecalSwab for Stool Specimen Storage and Molecular Detection of Enteropathogens on the BD Max System.

The FecalSwab system (Copan Italia, Brescia, Italy) is a convenient alternative to bulk stool for the diagnosis of enteric pathogens. Although the U.S. Food and Drug Administration (FDA) approved for transport and culture of enteric bacterial pathogens, the FecalSwab has not been well assessed for its suitability with molecular platforms. In this study, we evaluated the FecalSwab as a specimen type for the BD Max system using the viral and bacterial enteric panels (BD Diagnostics, Baltimore, MD, USA). A total of 186 unpreserved stool specimens were collected and used to prepare matched bulk stool and FecalSwab samples. Performance was equivalent (P > 0.48) to bulk stool for all targets when 50 µl of FecalSwab specimen was loaded onto the BD Max assays. As stool specimens are often collected off-site from the clinical microbiology laboratory and require transport, we assessed the stability of stool specimens stored for up to 14 days at 4°C, 22°C, or 35°C to account for varying transportation conditions. Molecular detection for the majority of viral targets (excluding astrovirus) was unaffected (change in cycle threshold [ΔCT ] ≤ 1) by sample storage temperature over the 2-week period; however, detection of enteric bacteria was variable if specimens were not refrigerated (22°C or 35°C). By demonstrating equivalent performance to matched bulk stool and maintaining molecular detection sensitivity when stored at 4°C, we suggest that the FecalSwab is a suitable specimen type for enteropathogen diagnostics on the BD Max system.

The prevalence and clinical characteristics of pertussis-associated pneumonia among infants in Botswana.

BACKGROUND: There are scant data on the prevalence and clinical course of pertussis disease among infants with pneumonia in low- and middle-income countries. While pertussis vaccination coverage is high (≥90%) among infants in Botswana, human immunodeficiency virus (HIV) infection affects nearly one-third of pregnancies. We aimed to evaluate the prevalence and clinical course of pertussis disease in a cohort of HIV-unexposed uninfected (HUU), HIV-exposed uninfected (HEU), and HIV-infected infants with pneumonia in Botswana. METHODS: We recruited children 1-23 months of age with clinical pneumonia at a tertiary care hospital in Gaborone, Botswana between April 2012 and June 2016. We obtained nasopharyngeal swab specimens at enrollment and tested these samples using a previously validated in-house real-time PCR assay that detects a unique sequence of the porin gene of Bordetella pertussis. RESULTS: B. pertussis was identified in 1/248 (0.4%) HUU, 3/110 (2.7%) HEU, and 0/33 (0.0%) HIV-infected children. All pertussis-associated pneumonia cases occurred in infants 1-5 months of age (prevalence, 1.0% [1/103] in HUU and 4.8% [3/62] in HEU infants). No HEU infants with pertussis-associated pneumonia were taking cotrimoxazole prophylaxis at the time of hospital presentation. One HUU infant with pertussis-associated pneumonia required intensive care unit admission for mechanical ventilation, but there were no deaths. CONCLUSIONS: The prevalence of pertussis was low among infants and young children with pneumonia in Botswana. Although vaccination against pertussis in pregnancy is designed to prevent classical pertussis disease, reduction of pertussis-associated pneumonia might be an important additional benefit.

Integrated fever management: disease severity markers to triage children with malaria and non-malarial febrile illness.

Febrile symptoms in children are a leading cause of health-care seeking behaviour worldwide. The majority of febrile illnesses are uncomplicated and self-limited, without the need for referral or hospital admission. However, current diagnostic tools are unable to identify which febrile children have self-limited infection and which children are at risk of progressing to life-threatening infections, such as severe malaria. This paper describes the need for a simple community-based tool that can improve the early recognition and triage of febrile children, with either malarial or non-malarial illness, at risk of critical illness. The integration of a disease severity marker into existing malaria rapid diagnostic tests (RDT) could enable detection of children at risk of severe infection in the hospital and community, irrespective of aetiology. Incorporation of a disease severity marker could inform individualized management and early triage of children at risk of life-threatening infection. A child positive for both malaria and a disease severity marker could be prioritized for urgent referral/admission and parenteral therapy. A child positive for malaria and negative for a disease severity marker could be managed conservatively, as an out-patient, with oral anti-malarial therapy. An RDT with a disease severity marker could facilitate an integrated community-based approach to fever syndromes and improve early recognition, risk stratification, and prompt treatment of severe malaria and other life-threatening infections.

Synthesis, antimycobacterial activity and influence on mycobacterial InhA and PknB of 12-membered cyclodepsipeptides.

In recent years, several small natural cyclopeptides and cyclodepsipeptides were reported to have antimycobacterial activity. Following this lead, a synthetic pathway was developed for a small series of 12-membered ring compounds with one amide and two ester bonds (cyclotridepsipeptides). Within the series, the ring system proved to be necessary for growth inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis in the low micromolar range. Open-chain precursors and analogues were inactive. The compounds modulated autophosphorylation of the mycobacterial protein kinase B (PknB). PknB inhibitors were active at µM concentration against mycobacteria while inducers were inactive. PknB regulates the activity of the mycobacterial reductase InhA, the target of isoniazid. The activity of the series against Mycobacterium bovis BCG InhA overexpressing strains was indistinguishable from that of the parental strain suggesting that they do not inhibit InhA. All substances were not cytotoxic (HeLa > 5 µg/ml) and did not show any significant antiproliferative effect (HUVEC > 5 µg/ml; K-562 > 5 µg/ml). Within the scope of this study, the molecular target of this new type of small cyclodepsipeptide was not identified, but the data suggest interaction with PknB or other kinases may partly cause the activity.

Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium tuberculosis Growth and Infectivity.

Ergothioneine (EGT) is synthesized in mycobacteria, but limited knowledge exists regarding its synthesis, physiological role, and regulation. We have identified Rv3701c from Mycobacterium tuberculosis to encode for EgtD, a required histidine methyltransferase that catalyzes first biosynthesis step in EGT biosynthesis. EgtD was found to be phosphorylated by the serine/threonine protein kinase PknD. PknD phosphorylates EgtD both in vitro and in a cell-based system on Thr(213). The phosphomimetic (T213E) but not the phosphoablative (T213A) mutant of EgtD failed to restore EGT synthesis in a ΔegtD mutant. The findings together with observed elevated levels of EGT in a pknD transposon mutant during in vitro growth suggests that EgtD phosphorylation by PknD negatively regulates EGT biosynthesis. We further showed that EGT is required in a nutrient-starved model of persistence and is needed for long term infection of murine macrophages.

Maternal and neonatal IgG against Klebsiella pneumoniae are associated with broad protection from neonatal sepsis: a case-control study of hospitalized neonates in Botswana.

Sepsis is the leading postnatal cause of neonatal mortality worldwide. Globally Klebsiella pneumoniae is the leading cause of sepsis in hospitalized neonates. This study reports development and evaluation of ELISA for anti-Klebsiella IgG using dried blood spot samples and evaluates the association of anti-Klebsiella IgG (anti-Kleb IgG) antibodies in maternal and neonatal samples and the risk of neonatal sepsis. Neonates and their mothers were enrolled at 0-96 hours of life in the neonatal unit of a tertiary referral hospital in Gaborone, Botswana and followed until death or discharge to assess for episodes of blood culture-confirmed neonatal sepsis. Neonates with sepsis had significantly lower levels of Kleb-IgG compared to neonates who did not develop sepsis (Mann-Whitney U, p=0.012). Similarly, samples from mothers of neonates who developed sepsis tended to have less Kleb-IgG compared to mothers of controls (p=0.06). The inverse correlation between Kleb-IgG levels and all-cause bacteremia suggests that maternal Kleb-IgG is broadly protective through cross-reactivity with common bacterial epitopes. These data support the continued use of immunoglobulin assays using DBS samples to explore the role of passive immunity on neonatal sepsis risk and reaffirm the critical need for research supporting the development of maternal vaccines for neonatal sepsis.