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The last decade has seen an explosion of advanced assays for the diagnosis of infectious diseases, yet evidence-based recommendations to inform their optimal use in the care of transplant recipients are lacking. A consensus conference sponsored by the American Society of Transplantation (AST) was convened on December 7, 2021, to define the utility of novel infectious disease diagnostics in organ transplant recipients. The conference represented a collaborative effort by experts in transplant infectious diseases, diagnostic stewardship, and clinical microbiology from centers across North America to evaluate current uses, unmet needs, and future directions for assays in 5 categories including (1) multiplex molecular assays, (2) rapid antimicrobial resistance detection methods, (3) pathogen-specific T-cell reactivity assays, (4) next-generation sequencing assays, and (5) mass spectrometry-based assays. Participants reviewed and appraised available literature, determined assay advantages and limitations, developed best practice guidance largely based on expert opinion for clinical use, and identified areas of future investigation in the setting of transplantation. In addition, attendees emphasized the need for well-designed studies to generate high-quality evidence needed to guide care, identified regulatory and financial barriers, and discussed the role of regulatory agencies in facilitating research and implementation of these assays. Findings and consensus statements are presented.

Sore throat is one of the most common complaints encountered in the ambulatory clinical setting. Rapid, culture-independent diagnostic techniques that do not rely on pharyngeal swabs would be highly valuable as a point-of-care strategy to guide outpatient antibiotic treatment. Despite the promise of this approach, efforts to detect volatiles during oropharyngeal infection have yet been limited. In our research study, we sought to evaluate for specific bacterial volatile organic compounds (VOC) biomarkers in isolated culturesin vitro, in order to establish proof-of-concept prior to initial clinical studies of breath biomarkers. A particular challenge for diagnosis of pharyngitis due toStreptococcus pyogenesis the likelihood that many metabolites may be shared byS. pyogenesand other related oropharyngeal colonizing bacterial species. Therefore, we evaluated whether sufficient metabolic differences are present that distinguish the volatile metabolome of Group A streptococci from other streptococcal species that also colonize the respiratory mucosa, such asS. pneumoniaeandS. intermedius. In this work, we identify candidate biomarkers that distinguishS. pyogenesfrom other species, and establish highly produced VOCs that indicate presence ofS. pyogenes in vitro, supporting future breath-based diagnostic testing for streptococcal pharyngitis.IMPORTANCEAcute pharyngitis accounts for approximately 15 million ambulatory care visits in the USA. The most common and important bacterial cause of pharyngitis isStreptococcus pyogenesis, accounting for 15% to 30% of pediatric pharyngitis. Distinguishing between bacterial and viral pharyngitis is key to management in US practice. Culture of a specimen obtained by throat swab is the standard laboratory procedure for the microbiologic confirmation of pharyngitis, however this method is time consuming which delays appropriate treatment. If left untreated,S. pyogenespharyngitis may lead to local and distant complications. In this study, we characterized the volatile metabolomes ofS. pyogenesand other related oropharyngeal colonizing bacterial species. We identify candidate biomarkers that distinguishS. pyogenesfrom other species and provides evidence to support future breath-based diagnostic testing for streptococcal pharyngitis.

Objective:To describe the cumulative seroprevalence of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) antibodies during the coronavirus disease 2019 (COVID-19) pandemic among employees of a large pediatric healthcare system.Design, setting, and participants:Prospective observational cohort study open to adult employees at the Children’s Hospital of Philadelphia, conducted April 20–December 17, 2020.Methods:Employees were recruited starting with high-risk exposure groups, utilizing e-mails, flyers, and announcements at virtual town hall meetings. At baseline, 1 month, 2 months, and 6 months, participants reported occupational and community exposures and gave a blood sample for SARS-CoV-2 antibody measurement by enzyme-linked immunosorbent assays (ELISAs). A post hoc Cox proportional hazards regression model was performed to identify factors associated with increased risk for seropositivity.Results:In total, 1,740 employees were enrolled. At 6 months, the cumulative seroprevalence was 5.3%, which was below estimated community point seroprevalence. Seroprevalence was 5.8% among employees who provided direct care and was 3.4% among employees who did not perform direct patient care. Most participants who were seropositive at baseline remained positive at follow-up assessments. In a post hoc analysis, direct patient care (hazard ratio [HR], 1.95; 95% confidence interval [CI], 1.03–3.68), Black race (HR, 2.70; 95% CI, 1.24–5.87), and exposure to a confirmed case in a nonhealthcare setting (HR, 4.32; 95% CI, 2.71–6.88) were associated with statistically significant increased risk for seropositivity.Conclusions:Employee SARS-CoV-2 seroprevalence rates remained below the point-prevalence rates of the surrounding community. Provision of direct patient care, Black race, and exposure to a confirmed case in a nonhealthcare setting conferred increased risk. These data can inform occupational protection measures to maximize protection of employees within the workplace during future COVID-19 waves or other epidemics.

Diagnosis of acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection relies on detection of viral antigens or amplified viral nucleic acids. Serology, although invaluable for epidemiology, is not routinely needed clinically. However, in some settings, serologic data may have direct clinical utility: for example, in evaluation of persistent symptoms in patients without a prior diagnosis of acute infection. In contrast, SARS-CoV-2 serologic testing is sometimes used or requested in situations in which existing data do not support it, such as determination of need for vaccination. In this study, we describe available methods of serologic testing and provide cases supported by clinical vignettes of where such tests can be helpful, as well as examples where they are not. These examples may help clarify clinical decision making in this rapidly evolving area.

COVID-19 control efforts have been hampered by transmission from pre-symptomatic individuals infected with SARS-CoV2. Prolonged asymptomatic respiratory viral shedding in children has been described and may be another important reservoir for ongoing transmission. The primary diagnostic approach to identify SARS-CoV2 infection relies on qPCR of specific viral sequences from respiratory samples, which is expensive, uncomfortable, relatively slow, and susceptible to false-negative results. A rapid non-invasive method to detect mild or asymptomatic infection would have a major impact on public health campaigns to control COVID-19. We hypothesize that candidate biomarkers characterize the exhaled breath of children with SARS-CoV2 infection. To test this hypothesis, we enrolled SARS-CoV-2-infected and -uninfected children admitted to a major pediatric academic medical center and analyzed their breath volatile composition. Targeted volatiles analysis revealed that six volatile organic compounds increased significantly in SARS-CoV-2-infected children. Three aldehydes (octanal, nonanal, and heptanal) drew special attention as candidate biomarkers, because viral infections have previously been shown to induce aldehyde production. Together, these biomarkers demonstrated 100% sensitivity and 66.6% specificity. Our work provides a solid framework upon which to build a future "breathalyzer" test for SARS-CoV-2 infection in children.

Tackling the ancient infectious foe of malaria, Xie et al. (2022) uncover a novel class of nucleoside analogs that selectively hijack and inhibit the tyrosine tRNA synthase of the parasite. With high potency, good oral bioavailability, and minimal host cell toxicity, these inhibitors show promise as next-generation antimalarials.

Key Points sC5b9 plasma levels are elevated in children with SARS-CoV-2 infection, even if they have minimal symptoms of COVID-19. A high proportion of children with SARS-CoV-2 infection met clinical criteria for TMA., Most children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have mild or minimal disease, with a small proportion developing severe disease or multisystem inflammatory syndrome in children (MIS-C). Complement-mediated thrombotic microangiopathy (TMA) has been associated with SARS-CoV-2 infection in adults but has not been studied in the pediatric population. We hypothesized that complement activation plays an important role in SARS-CoV-2 infection in children and sought to understand if TMA was present in these patients. We enrolled 50 hospitalized pediatric patients with acute SARS-CoV-2 infection (n = 21, minimal coronavirus disease 2019 [COVID-19]; n = 11, severe COVID-19) or MIS-C (n = 18). As a biomarker of complement activation and TMA, soluble C5b9 (sC5b9, normal 247 ng/mL) was measured in plasma, and elevations were found in patients with minimal disease (median, 392 ng/mL; interquartile range [IQR], 244-622 ng/mL), severe disease (median, 646 ng/mL; IQR, 203-728 ng/mL), and MIS-C (median, 630 ng/mL; IQR, 359-932 ng/mL) compared with 26 healthy control subjects (median, 57 ng/mL; IQR, 9-163 ng/mL; P < .001). Higher sC5b9 levels were associated with higher serum creatinine (P = .01) but not age. Of the 19 patients for whom complete clinical criteria were available, 17 (89%) met criteria for TMA. A high proportion of tested children with SARS-CoV-2 infection had evidence of complement activation and met clinical and diagnostic criteria for TMA. Future studies are needed to determine if hospitalized children with SARS-CoV-2 should be screened for TMA, if TMA-directed management is helpful, and if there are any short- or long-term clinical consequences of complement activation and endothelial damage in children with COVID-19 or MIS-C., Visual Abstract

Infection with malarial parasites renders hosts more mosquito-attractive than their uninfected, healthy counterparts. One volatile organic compound, α-pinene, is associated with Plasmodium spp. infection in multiple studies and is a known mosquito attractant. However, how malarial infection results in elevated levels of host-associated α-pinene remains unclear. One study suggested that exposure of erythrocytes to the malarial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) results in increased levels of α-pinene. Here we establish that endogenous levels of α-pinene are present in human erythrocytes, that these levels vary widely by erythrocyte donor, and that α-pinene levels are not altered by HMBPP treatment.

We present a series of 6 critically ill children with multisystem inflammatory syndrome in children. Key findings of this syndrome include fever, diarrhea, shock, and variable presence of rash, conjunctivitis, extremity edema, and mucous membrane changes.

In this study, we identified three novel compound classes with potent activity against Plasmodium falciparum, the most dangerous human malarial parasite. Resistance of this pathogen to known drugs is increasing and compounds with different modes of action are urgently needed. One promising drug target is the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) of the methylerythritol 4-phosphate (MEP) pathway for which we have previously identified three active compound classes against Mycobacterium tuberculosis. The close structural similarities in the active sites of the DXPS enzymes of P. falciparum and M. tuberculosis prompted investigation of its antiparasitic action, displaying good cell-based activity for all classes. Through structure-activity relationship studies we increased their antimalarial potency, and two classes also show good metabolic stability and low toxicity against human liver cells. The most active compound 1 inhibits the growth of blood-stage P. falciparum with an IC50 of 600 nM. The results from three different methods for target validation of compound 1 suggest intracellular polypharmacy. Similarity-based searches revealed two other possible target enzymes for this compound, which were further analyzed by docking calculations. All inhibitor classes are active against chloroquine resistant strains, confirming a new mode of action.

Efforts to control the global malaria health crisis are undermined by antimalarial resistance. Iden-tifying mechanisms of resistance will uncover the underlying biology of the Plasmodium falciparum malaria parasites that allow evasion of our most promising therapeutics and may reveal new drug targets. We utilized fosmidomycin (FSM) as a chemical inhibitor of plastidial isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway. We have thus identified an unusual metabolic regulation scheme in the malaria parasite through the essential glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Two parallel genetic screens converged on independent but functionally analogous resistance alleles in GAPDH. Metabolic profiling of FSM-resistant gapdh mutant parasites indicates that neither of these mutations disrupt overall glycolytic output. While FSM-resistant GAPDH variant proteins are catalytically active, they have reduced assembly into the homotetrameric state favored by wild-type GAPDH. Disrupted oligomerization of FSM-resistant GAPDH variant proteins is accompanied by altered enzymatic cooperativity and reduced susceptibility to inhibition by free heme. Together, our data identifies a new genetic biomarker of FSM-resistance and reveals the central role of GAPDH cooperativity in MEP pathway control and antimalarial sensitivity.

Carboxy ester prodrugs are widely employed to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can mask problematic chemical features that prevent cellular uptake and may enable tissue-specific compound delivery. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, limiting their therapeutic potential. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes as microbial esterase-specific promoieties have not been described. Here we identify the bacterial esterases, GloB and FrmB, that activate carboxy ester prodrugs in

Red blood cells (RBCs) are essential for aerobic respiration through delivery of oxygen to distant tissues. However, RBCs are currently considered immunologically inert, and few, if any, secondary ...

Background Starkly highlighted by the current COVID-19 pandemic, infectious diseases continue to have an outsized impact on human health worldwide. Diagnostic testing for infection can be challenging due to resource limitations, time constraints, or shortcomings in the accuracy of existing diagnostics. Rapid, simple diagnostics are highly desirable. There is increasing interest in the development of diagnostics that use exhaled breath analysis as a convenient and safe diagnostic method, as breath sampling is noninvasive, secure, and easy to perform. Volatile organic compounds (VOCs) present in exhaled breath reflect the fingerprint of the underlying metabolic and biophysical processes during disease. Content In this review, we overview the major biomarkers present in exhaled breath in infectious diseases. We outline the promising recent advances in breath-based diagnosis of respiratory infections, including those caused by influenza virus, SARS-CoV-2, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Aspergillus fumigatus. In addition, we review the current landscape of diagnosis of 2 other globally important infections: Helicobacter pylori gastrointestinal infection and malaria. Summary Characteristic and reproducible breath VOCs are associated with several infectious diseases, suggesting breath analysis as a promising strategy for diagnostic development. Ongoing challenges include poor standardization of breath collection and analysis and lack of validation studies. Further research is required to expand the applicability of breath analysis to clinical settings.

Summary Widespread Plasmodium falciparum resistance to first-line antimalarials underscores the vital need to develop compounds with novel modes of action and identify new druggable targets. Here, we profile five compounds that potently inhibit P. falciparum asexual blood stages. Resistance selection studies with three carboxamide-containing compounds, confirmed by gene editing and conditional knockdowns, identify point mutations in the parasite transporter ABCI3 as the primary mediator of resistance. Selection studies with imidazopyridine or quinoline-carboxamide compounds also yield changes in ABCI3, this time through gene amplification. Imidazopyridine mode of action is attributed to inhibition of heme detoxification, as evidenced by cellular accumulation and heme fractionation assays. For the copy-number variation-selecting imidazopyridine and quinoline-carboxamide compounds, we find that resistance, manifesting as a biphasic concentration-response curve, can independently be mediated by mutations in the chloroquine resistance transporter PfCRT. These studies reveal the interconnectedness of P. falciparum transporters in overcoming drug pressure in different parasite strains.

The malaria parasite, Plasmodium falciparum, proliferates rapidly in human erythrocytes by actively scavenging multiple carbon sources and essential nutrients from its host cell. However, a global overview of the metabolic capacity of intraerythrocytic stages is missing. Using multiplex 13C‐labelling coupled with untargeted mass spectrometry and unsupervised isotopologue grouping, we have generated a draft metabolome of P. falciparum and its host erythrocyte consisting of 911 and 577 metabolites, respectively, corresponding to 41% of metabolites and over 70% of the metabolic reaction predicted from the parasite genome. An additional 89 metabolites and 92 reactions were identified that were not predicted from genomic reconstructions, with the largest group being associated with metabolite damage‐repair systems. Validation of the draft metabolome revealed four previously uncharacterised enzymes which impact isoprenoid biosynthesis, lipid homeostasis and mitochondrial metabolism and are necessary for parasite development and proliferation. This study defines the metabolic fate of multiple carbon sources in P. falciparum, and highlights the activity of metabolite repair pathways in these rapidly growing parasite stages, opening new avenues for drug discovery., Multiplex stable‐isotope labelling defines the observable metabolome of red blood cell stages of the malaria parasite Plasmodium falciparum and indicates potential roles for metabolic enzymes of unknown function.

* Abbreviations: CHOP — : Children’s Hospital of Philadelphia COVID-19 — : coronavirus disease 2019 RT-PCR — : reverse transcriptase polymerase chain reaction SARS-CoV-2 — : severe acute respiratory syndrome coronavirus 2 Surgery in patients with coronavirus disease 2019 (COVID-19) is associated with increased mortality and complications.1 Procedures may generate aerosols or require endotracheal intubation, increasing risk of occupational exposure of health care workers. As medical centers navigate aerosol-generating procedures, preprocedure testing is important for patient and provider safety. Testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immediately before the procedure would yield the most accurate results. However, scheduling logistics and inadequate turnaround times often necessitate preprocedure testing several days in advance. Thus, patients may have negative preprocedure testing results, yet have active viral replication at the time of surgery. Although patients are instructed to monitor for symptoms after testing, symptom reporting is likely to be insensitive in children because most have mild or no … Address correspondence to Audrey R. Odom John, MD, PhD, 907C ARC/Children’s Hospital of Philadelphia, 3615 Civic Center Blvd, Philadelphia, PA 19104-4318. E-mail: johna3{at}email.chop.edu

While the world awaits a widely available COVID-19 vaccine, availability of testing is limited in many regions and can be further compounded by shortages of reagents, prolonged processing time and delayed results. One approach to rapid testing is to leverage the volatile organic compound (VOC) signature of SARS-CoV-2 infection. Detection dogs, a biological sensor of VOCs, were utilized to investigate whether SARS-CoV-2 positive urine and saliva patient samples had a unique odor signature. The virus was inactivated in all training samples with either detergent or heat treatment. Using detergent-inactivated urine samples, dogs were initially trained to find samples collected from hospitalized patients confirmed with SARS-CoV-2 infection, while ignoring samples collected from controls. Dogs were then tested on their ability to spontaneously recognize heat-treated urine samples as well as heat-treated saliva from hospitalized SARS-CoV-2 positive patients. Dogs successfully discriminated between infected and uninfected urine samples, regardless of the inactivation protocol, as well as heat-treated saliva samples. Generalization to novel samples was limited, particularly after intensive training with a restricted sample set. A unique odor associated with SARS-CoV-2 infection present in human urine as well as saliva, provides impetus for the development of odor-based screening, either by electronic, chemical, or biological sensing methods. The use of dogs for screening in an operational setting will require training with a large number of novel SARS-CoV-2 positive and confirmed negative samples.

Rash is a common feature of multisystem inflammatory syndrome in children (MIS-C), a postinfectious hyperinflammatory disease associated with prior severe acute respiratory syndrome coronavirus 2 infection. Because the differential diagnosis of fever and rash in children is broad, understanding clinical characteristics of MIS-C may assist with diagnosis. Here we describe the cutaneous findings observed in a series of children with MIS-C-associated rash.

Malaria, caused by Plasmodium parasites, continues to be a devastating global health issue, causing 405,000 deaths and 228 million cases in 2018. Understanding key metabolic processes in malaria parasites is critical to the development of new drugs to combat this major infectious disease. The Plasmodium glycolytic pathway is essential to the malaria parasite, providing energy for growth and replication and supplying important biomolecules for other essential Plasmodium anabolic pathways. Despite this overreliance on glycolysis, no current drugs target glycolysis, and there is a paucity of information on critical glycolysis targets. Our work addresses this unmet need, providing new mechanistic insights into this key pathway., Plasmodium parasites rely heavily on glycolysis for ATP production and for precursors for essential anabolic pathways, such as the methylerythritol phosphate (MEP) pathway. Here, we show that mutations in the Plasmodium falciparum glycolytic enzyme, phosphofructokinase (PfPFK9), are associated with in vitro resistance to a primary sulfonamide glycoside (PS-3). Flux through the upper glycolysis pathway was significantly reduced in PS-3-resistant parasites, which was associated with reduced ATP levels but increased flux into the pentose phosphate pathway. PS-3 may directly or indirectly target enzymes in these pathways, as PS-3-treated parasites had elevated levels of glycolytic and tricarboxylic acid (TCA) cycle intermediates. PS-3 resistance also led to reduced MEP pathway intermediates, and PS-3-resistant parasites were hypersensitive to the MEP pathway inhibitor, fosmidomycin. Overall, this study suggests that PS-3 disrupts core pathways in central carbon metabolism, which is compensated for by mutations in PfPFK9, highlighting a novel metabolic drug resistance mechanism in P. falciparum.

Carboxy ester prodrugs have been widely employed as a means to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can successfully mask problematic chemical features that prevent cellular uptake and can be used to target delivery of compounds to specific tissues. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, curbing their potential therapeutic applications. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes due to a paucity of information about the selectivity of microbial esterases. Here we identify the bacterial esterases, GloB and FrmB, that are required for carboxy ester prodrug activation in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, which revealed several promoieties likely to be serum esterase-resistant while still being microbially labile. These studies lay the groundwork for structure-guided design of anti-staphyloccal promoieties and expand the range of molecules to target staphyloccal pathogens.