Precision symptom phenotyping identifies early clinical and proteomic predictors of distinct COVID-19 sequelae.

BACKGROUND: Post-COVID conditions (PCC) are difficult to characterize, diagnose, predict, and treat due to overlapping symptoms and poorly understood pathology. Identifying inflammatory profiles may improve clinical prognostication and trial endpoints. METHODS: 1,988 SARS-CoV-2 positive U.S. Military Health System beneficiaries with quantitative post-COVID symptom scores were included in this analysis. Among participants who reported moderate-to-severe symptoms on surveys collected 6-months post-SARS-CoV-2 infection, principal component analysis (PCA) followed by K-means clustering identified distinct clusters of symptoms. RESULTS: Three symptom-based clusters were identified: a sensory cluster (loss of smell and/or taste), a fatigue/difficulty thinking cluster, and a difficulty breathing/exercise intolerance cluster. Individuals within the sensory cluster were all outpatients during their initial COVID-19 presentation. The difficulty breathing cluster had a higher likelihood of obesity and COVID-19 hospitalization compared to those with no/mild symptoms at 6-months post-infection. Multinomial regression linked early post-infection D-dimer and IL-1RA elevation to fatigue/difficulty thinking, and elevated ICAM-1 concentrations to sensory symptoms. CONCLUSIONS: We identified three distinct symptom-based PCC phenotypes with specific clinical risk factors and early post-infection inflammatory predictors. With further validation and characterization, this framework may allow more precise classification of PCC cases and potentially improve the diagnosis, prognostication, and treatment of PCC.

Understanding "Hybrid Immunity": Comparison and Predictors of Humoral Immune Responses to Severe Acute Respiratory Syndrome Coronavirus 2 Infection (SARS-CoV-2) and Coronavirus Disease 2019 (COVID-19) Vaccines.

BACKGROUND: Comparison of humoral responses in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccinees, those with SARS-CoV-2 infection, or combinations of vaccine/ infection ("hybrid immunity") may clarify predictors of vaccine immunogenicity. METHODS: We studied 2660 US Military Health System beneficiaries with a history of SARS-CoV-2 infection-alone (n = 705), vaccination-alone (n = 932), vaccine-after-infection (n = 869), and vaccine-breakthrough-infection (n = 154). Peak anti-spike-immunoglobulin G (IgG) responses through 183 days were compared, with adjustment for vaccine product, demography, and comorbidities. We excluded those with evidence of clinical or subclinical SARS-CoV-2 reinfection from all groups. RESULTS: Multivariable regression results indicated that vaccine-after-infection anti-spike-IgG responses were higher than infection-alone (P < .01), regardless of prior infection severity. An increased time between infection and vaccination was associated with greater post-vaccination IgG response (P < .01). Vaccination-alone elicited a greater IgG response but more rapid waning of IgG (P < .01) compared with infection-alone (P < .01). BNT162b2 and mRNA-1273 vaccine-receipt was associated with greater IgG responses compared with JNJ-78436735 vaccine-receipt (P < .01), regardless of infection history. Those with vaccine-after-infection or vaccine-breakthrough-infection had a more durable anti-spike-IgG response compared to infection-alone (P < .01). CONCLUSIONS: Vaccine-receipt elicited higher anti-spike-IgG responses than infection-alone, although IgG levels waned faster in those vaccinated (compared to infection-alone). Vaccine-after-infection elicits a greater humoral response compared with vaccine or infection alone; and the timing, but not disease severity, of prior infection predicted these post-vaccination IgG responses. While differences between groups were small in magnitude, these results offer insights into vaccine immunogenicity variations that may help inform vaccination timing strategies.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mRNA Vaccine-Breakthrough Infection Phenotype Includes Significant Symptoms, Live Virus Shedding, and Viral Genetic Diversity.

Little is known about severe acute respiratory syndrome coronavirus 2 "vaccine-breakthrough" infections (VBIs). Here we characterize 24 VBIs in predominantly young healthy persons. While none required hospitalization, a proportion endorsed severe symptoms and shed live virus as high as 4.13 × 103 plaque-forming units/mL. Infecting genotypes included both variant-of-concern (VOC) and non-VOC strains.

Clinical, Immunological, and Virological SARS-CoV-2 Phenotypes in Obese and Nonobese Military Health System Beneficiaries.

BACKGROUND: The mechanisms underlying the association between obesity and coronavirus disease 2019 (COVID-19) severity remain unclear. After verifying that obesity was a correlate of severe COVID-19 in US Military Health System (MHS) beneficiaries, we compared immunological and virological phenotypes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in both obese and nonobese participants. METHODS: COVID-19-infected MHS beneficiaries were enrolled, and anthropometric, clinical, and demographic data were collected. We compared the SARS-CoV-2 peak IgG humoral response and reverse-transcription polymerase chain reaction viral load in obese and nonobese patients, stratified by hospitalization, utilizing logistic regression models. RESULTS: Data from 511 COVID-19 patients were analyzed, among whom 24% were obese and 14% severely obese. Obesity was independently associated with hospitalization (adjusted odds ratio [aOR], 1.91; 95% confidence interval [CI], 1.15-3.18) and need for oxygen therapy (aOR, 3.39; 95% CI, 1.61-7.11). In outpatients, severely obese had a log10 (1.89) higher nucleocapsid (N1) genome equivalents (GE)/reaction and log10 (2.62) higher N2 GE/reaction than nonobese (P = 0.03 and P < .001, respectively). We noted a correlation between body mass index and peak anti-spike protein IgG in inpatients and outpatients (coefficient = 5.48, P < .001). CONCLUSIONS: Obesity is a strong correlate of COVID-19 severity in MHS beneficiaries. These findings offer new pathophysiological insights into the relationship between obesity and COVID-19 severity.

Durability of SARS-CoV-2-Specific T-Cell Responses at 12 Months Postinfection.

BACKGROUND: Characterizing the longevity and quality of cellular immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enhances understanding of coronavirus disease 2019 (COVID-19) immunity that influences clinical outcomes. Prior studies suggest SARS-CoV-2-specific T cells are present in peripheral blood 10 months after infection. Analysis of the function, durability, and diversity of cellular response long after natural infection, over a range of ages and disease phenotypes, is needed to identify preventative and therapeutic interventions. METHODS: We identified participants in our multisite longitudinal, prospective cohort study 12 months after SARS-CoV-2 infection representing a range of disease severity. We investigated function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry, and compared magnitude of SARS-CoV-2-specific antibodies. RESULTS: SARS-CoV-2-specific antibodies and T cells were detected 12 months postinfection. Severe acute illness was associated with higher frequencies of SARS-CoV-2-specific CD4 T cells and antibodies at 12 months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity. CONCLUSIONS: SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12 months postinfection, with higher frequency noted in those who experienced severe disease.

Comparison of A(H3N2) Neutralizing Antibody Responses Elicited by 2018-2019 Season Quadrivalent Influenza Vaccines Derived from Eggs, Cells, and Recombinant Hemagglutinin.

BACKGROUND: Low vaccine effectiveness against A(H3N2) influenza in seasons with little antigenic drift has been attributed to substitutions in hemagglutinin (HA) acquired during vaccine virus propagation in eggs. Clinical trials comparing recombinant HA vaccine (rHA) and cell-derived inactivated influenza vaccine (IIV) to egg-derived IIVs provide opportunities to assess how egg-adaptive substitutions influence HA immunogenicity. METHODS: Neutralization titers in pre- and postimmunization sera from 133 adults immunized with 1 of 3 types of influenza vaccines in a randomized, open-label trial during the 2018-2019 influenza season were measured against egg- and cell-derived A/Singapore/INFIMH-16-0019/2016-like and circulating A(H3N2) influenza viruses using HA pseudoviruses. RESULTS: All vaccines elicited neutralizing antibodies to all H3 vaccine antigens, but the rHA vaccine elicited the highest titers and seroconversion rates against all strains tested. Egg- and cell-derived IIVs elicited responses similar to each other. Preimmunization titers against H3 HA pseudoviruses containing egg-adaptive substitutions T160K and L194P were high, but lower against H3 HA pseudoviruses without those substitutions. All vaccines boosted neutralization titers against HA pseudoviruses with egg-adaptive substitutions, but poorly neutralized wild-type 2019-2020 A/Kansas/14/2017 (H3N2) HA pseudoviruses. CONCLUSION: Egg- and cell-derived 2018-2019 season influenza vaccines elicited similar neutralization titers and response rates, indicating that the cell-derived vaccine did not improve immunogenicity against the A(H3N2) viruses. The higher responses after rHA vaccination may be due to its higher HA content. All vaccines boosted titers to HA with egg-adaptive substitutions, suggesting boosting from past antigens or better exposure of HA epitopes. Studies comparing immunogenicity and effectiveness of different influenza vaccines across many seasons are needed.

Hypervirulent Streptococcus agalactiae septicemia in twin ex-premature infants transmitted by breast milk: report of source detection and isolate characterization using commonly available molecular diagnostic methods.

BACKGROUND: Group B Streptococcus (GBS) infections caused by Streptococcus agalactiae is a leading cause of meningitis and sepsis in neonates, with early-onset GBS symptoms emerging during the first week of life and late-onset occurring thereafter. Perinatal transmission of GBS to the neonate through the birth canal is the main factor associated with early-onset neonate infections, while less is understood about the source of late-onset infections. METHODS: In this report we describe a case of twin ex-premature infants who presented one month after birth with GBS septicemia. The mother had been appropriately screened at gestational age 35-37 weeks and laboratory methods failed to detect GBS colonization by culture or clinical molecular methods. In attempts to identify and isolate the source of GBS infection, additional surveillance swabs were collected from the mother at the time of neonate admission. Culture and a commercially available, FDA-cleared molecular PCR assay were performed. RESULTS: No GBS was detected from swabs collected from the perianal, thigh/groin or axillary areas. However, expressed breast milk and swabs from the breastmilk pump were positive by both methods. Since simultaneous culture and molecular methods which used breastmilk as a source were performed, investigators ascertained the limit of detection for GBS in breastmilk. The limit of detection was determined to be tenfold lower than that of LIM-broth enriched cultures-the FDA-approved source. Subsequent whole genome sequencing (WGS) analysis of isolates recovered from breastmilk and blood cultures from the infants demonstrated all strains were related and characterized as ST-452. Both infants responded very well to treatment and continued to have no related events or concerns at the two-year follow up appointment. CONCLUSIONS: Strain type 452 (capsular type IV) has recently emerged as a hypervirulent strain and has previously been documented as causing GBS infections in elderly populations. Antibiotic therapy resolved both mother and infant infections. Subsequent testing for the presence of GBS in breastmilk samples also showed an absence of bacteria. This is the first report of infant twins late-onset GBS infections caused by the hypervirulent S. agalactiae ST-452 with breastmilk as the source.

In Vitro activity of Manuka Honey and polyhexamethylene biguanide on filamentous fungi and toxicity to human cell lines.

Soft-tissue invasive fungal infections are increasingly recognized as significant entities directly contributing to morbidity and mortality. They complicate clinical care, requiring aggressive surgical debridement and systemic antifungal therapy. To evaluate new topical approaches to therapy, we examined the antifungal activity and cytotoxicity of Manuka Honey (MH) and polyhexamethylene biguanide (PHMB). The activities of multiple concentrations of MH (40%, 60%, 80%) and PHMB (0.01%, 0.04%, 0.1%) against 13 clinical mould isolates were evaluated using a time-kill assay between 5 min and 24 h. Concentrations were selected to represent current clinical use. Cell viability was examined in parallel for human epidermal keratinocytes, dermal fibroblasts and osteoblasts, allowing determination of the 50% viability (LD50) concentration. Antifungal activity of both agents correlated more closely with exposure time than concentration. Exophiala and Fusarium growth was completely suppressed at 5 min for all PHMB concentrations, and at 12 and 6 h, respectively, for all MH concentrations. Only Lichtheimia had persistent growth to both agents at 24 h. Viability assays displayed concentration-and time-dependent toxicity for PHMB. For MH, exposure time predicted cytotoxicity only when all cell types were analyzed in aggregate. This study demonstrates that MH and PHMB possess primarily time-dependent antifungal activity, but also exert in vitro toxicity on human cells which may limit clinical use. Further research is needed to determine ideal treatment strategies to optimize antifungal activity against moulds while limiting cytotoxicity against host tissues in vivo.

Activity of Norspermidine on Bacterial Biofilms of Multidrug-Resistant Clinical Isolates Associated with Persistent Extremity Wound Infections.

Biofilm formation is a major virulence factor for numerous pathogenic bacteria and is cited as a central event in the pathogenesis of chronic human infections, which is in large part due to excessive extracellular matrix secretion and metabolic changes that occur within the biofilm rendering them highly tolerant to antimicrobial treatments. Polyamines, including norspermidine, play central roles in bacterial biofilm development, but have also recently been shown to inhibit biofilm formation in select strains of various pathogenic bacteria. The aim of this study was to evaluate in vitro the biofilm dispersive and inhibitory activities of norspermidine against multidrug-resistant clinical isolates of Acinetobacter baumannii(n = 4), Klebsiella pneumoniae (n = 3), Pseudomonas aeruginosa (n = 5) and Staphylococcus aureus (n = 4) associated with chronic extremity wound infections using the semi-quantitative 96-well plate method and confocal laser microscopy. In addition to the antibiofilm activity, biocompatibility of norspermidine was also evaluated by measuring toxicity in vitro to human cell lines and whole porcine tissue explants using MTT viability assay and histological analysis. Norspermidine (5-20 mM) had variable dispersive and inhibitory activity on biofilms which was dependent on both the strain and species. Of the clinical bacterial species evaluated herein, A. baumannii isolates were the most sensitive to the effect of norspermidine, which was in part due to the inhibitory effects of norspermidine on bacterial motility and expression of genes involved in the production of homoserine lactones and quorum sensing molecules both essential for biofilm formation. Importantly, exposure of cell lines and whole tissues to norspermidine for prolonged periods of time (≥24 h) was observed to reduce viability and alter tissue histology in a time and concentration dependent manner, with 20 mM exposure having the greatest negative effects on both tissues and individual cell lines. Collectively our findings demonstrate that, similar to other polyamines, norspermidine displays both inhibitory and dispersive activities on biofilms of clinical multidrug-resistant bacterial isolates, in particular for strains of A. baumannii. Additionally our findings suggest that direct application may be considered on tissues, albeit for limited exposure times.

The use of PCR/Electrospray Ionization-Time-of-Flight-Mass Spectrometry (PCR/ESI-TOF-MS) to detect bacterial and fungal colonization in healthy military service members.

BACKGROUND: The role of microbial colonization in disease is complex. Novel molecular tools to detect colonization offer theoretical improvements over traditional methods. We evaluated PCR/Electrospray Ionization-Time-of-Flight-Mass Spectrometry (PCR/ESI-TOF-MS) as a screening tool to study colonization of healthy military service members. METHODS: We assessed 101 healthy Soldiers using PCR/ESI-TOF-MS on nares, oropharynx, and groin specimens for the presence of gram-positive and gram-negative bacteria (GNB), fungi, and antibiotic resistance genes. A second set of swabs was processed by traditional culture, followed by identification using the BD Phoenix automated system; comparison between PCR/ESI-TOF-MS and culture was carried out only for GNB. RESULTS: Using PCR/ESI-TOF-MS, at least one colonizing organism was found on each individual: mean (SD) number of organisms per subject of 11.8(2.8). The mean number of organisms in the nares, groin and oropharynx was 3.8(1.3), 3.8(1.4) and 4.2(2), respectively. The most commonly detected organisms were aerobic gram-positive bacteria: primarily coagulase-negative Staphylococcus (101 subjects: 341 organisms), Streptococcus pneumoniae (54 subjects: 57 organisms), Staphylococcus aureus (58 subjects: 80 organisms) and Nocardia asteroides (45 subjects: 50 organisms). The mecA gene was found in 96 subjects. The most commonly found GNB was Haemophilus influenzae (20 subjects: 21 organisms) and the most common anaerobe was Propionibacterium acnes (59 subjects). Saccharomyces species (30 subjects) were the most common fungi detected. Only one GNB (nares E. coli) was identified in the same subject by both diagnostic systems. CONCLUSION: PCR/ESI-TOF-MS detected common colonizing organisms and identified more typically-virulent bacteria in asymptomatic, healthy adults. PCR/ESI-TOF-MS appears to be a useful method for detecting bacterial and fungal organisms, but further clinical correlation and validation studies are needed.

Colistin pharmacokinetics in burn patients during continuous venovenous hemofiltration.

While colistin is considered a last resort for the treatment of multidrug-resistant Gram-negative bacterial infections, there has been an increase in its use due to the increasing prevalence of drug-resistant infections worldwide. The pharmacology of colistin is complex, and pharmacokinetic data are limited, especially in patients requiring renal replacement therapy. As a result, dosing for patients who require renal replacement remains a challenge. Here, we present pharmacokinetic data for colistin from two burn patients (37 and 68 years old) infected with colistin-susceptible isoclonal Acinetobacter baumannii and receiving continuous venovenous hemofiltration (CVVH). To our knowledge, we are the first to examine data from before and during CVVH (for one patient), allowing analysis of the effect of CVVH on colistin pharmacokinetics. Pharmacokinetic/pharmacodynamic analysis indicated that a dose increase from 1.5 to 2.2 mg/kg of body weight colistin base activity on CVVH was insufficient to satisfy the target parameter of an AUC24/MIC (area under the concentration-time curve over 24 h in the steady state divided by the MIC) of ≥ 60 at an MIC of ≥ 1 µg/ml in one patient with residual endogenous renal function. Plasma concentrations of colistin ranged from 0 to 15 µg/ml, with free colistin levels ranging from 0.4 to 2.2 µg/ml. While both patients resolved their clinical infections and survived to discharge, colistin-resistant colonizing isolates resulted from therapy in one patient. The variabilities observed in colistin concentrations and pharmacokinetic characteristics highlight the importance of pharmacokinetic monitoring of antibiotics in patients undergoing renal replacement therapy.

Simultaneous Identification of Multiple ß-Lactamases in Acinetobacter baumannii in Relation to Carbapenem and Ceftazidime Resistance, Using Liquid Chromatography-Tandem Mass Spectrometry.

Shotgun proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to detect ß-lactamases in clinical Acinetobacter baumannii isolates. The correlation of the detection of ß-lactamase proteins (rather than PCR detection of the corresponding genes) with the resistance phenotypes demonstrated an added value for LC-MS/MS in antimicrobial susceptibility testing.

Combat-Related Pythium aphanidermatum Invasive Wound Infection: Case Report and Discussion of Utility of Molecular Diagnostics.

We describe a 22-year-old soldier with 19% total body surface area burns, polytrauma, and sequence- and culture-confirmed Pythium aphanidermatum wound infection. Antemortem histopathology suggested disseminated Pythium infection, including brain involvement; however, postmortem PCR revealed Cunninghamella elegans, Lichtheimia corymbifera, and Saksenaea vasiformis coinfection. The utility of molecular diagnostics in invasive fungal infections is discussed.

In vitro activity of Melaleuca alternifolia (tea tree) oil on filamentous fungi and toxicity to human cells.

Invasive fungal wound infections (IFIs) are increasingly reported in trauma patients and cause considerable morbidity and mortality despite standard of care treatment in trauma centers by experienced medical personnel. Topical agents such as oil of melaleuca, also known as tea tree oil (TTO), have been proposed for adjunctive treatment of IFIs. We evaluated the activity of TTO against filamentous fungi associated with IFIs by testing 13 clinical isolates representing nine species via time-kill assay with seven concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, and 1%). To ascertain the safety of topical application to wounds, cell viability assays were performed in vitro using human fibroblasts, keratinocytes, osteoblasts, and umbilical vein endothelial cells with 10 concentrations of TTO (75%, 50%, 25%, 10%, 5%, and 10-fold serial dilutions from 1 to 0.0001%) at five time points (5, 15, 30, 60, and 180 min). Compatibility of TTO with explanted porcine tissues was also assessed with eight concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, 1%, and 0.1%) at the time points used for cellular assays and at 24 h. The time-kill studies showed that fungicidal activity was variable between isolates. The effect of TTO on cell viability was primarily concentration dependent with significant cytotoxicity at concentrations of ≥ 10% and ≥ 50% for cells lines and whole tissue, respectively. Our findings demonstrate that TTO possesses antifungal activity against filamentous fungi associated with IFIs; furthermore that negligible effects on whole tissues, in contrast to individual cells, were observed following exposure to TTO. Collectively, these findings indicate a potential use of TTO as topical treatment of IFIs.

Antifungal wound penetration of amphotericin and voriconazole in combat-related injuries: case report.

BACKGROUND: Survivors of combat trauma can have long and challenging recoveries, which may be complicated by infection. Invasive fungal infections are a rare but serious complication with limited treatment options. Currently, aggressive surgical debridement is the standard of care, with antifungal agents used adjunctively with uncertain efficacy. Anecdotal evidence suggests that antifungal agents may be ineffective in the absence of surgical debridement, and studies have yet to correlate antifungal concentrations in plasma and wounds. CASE PRESENTATION: Here we report the systemic pharmacokinetics and wound effluent antifungal concentrations of five wounds from two male patients, aged 28 and 30 years old who sustained combat-related blast injuries in southern Afghanistan, with proven or possible invasive fungal infection. Our data demonstrate that while voriconazole sufficiently penetrated the wound resulting in detectable effluent levels, free amphotericin B (unbound to plasma) was not present in wound effluent despite sufficient concentrations in circulating plasma. In addition, considerable between-patient and within-patient variability was observed in antifungal pharmacokinetic parameters. CONCLUSION: These data highlight the need for further studies evaluating wound penetration of commonly used antifungals and the role for therapeutic drug monitoring in providing optimal care for critically ill and injured war fighters.

Clinical infectious outcomes associated with biofilm-related bacterial infections: a retrospective chart review.

BACKGROUND: Biofilms are associated with persistent infection. Reports characterizing clinical infectious outcomes and patient risk factors for colonization or infection with biofilm forming isolates are scarce. Our institution recently published a study examining the biofilm forming ability of 205 randomly selected clinical isolates. This present study aims to identify potential risk factors associated with these isolates and assess clinical infectious outcomes. METHODS: 221 clinical isolates collected from 2005 to 2012 and previously characterized for biofilm formation were studied. Clinical information from the associated patients, including demographics, comorbidities, antibiotic usage, laboratory values, and clinical infectious outcomes, was determined retrospectively through chart review. Duplicate isolates and non-clinical isolates were excluded from analysis. Associations with biofilm forming isolates were determined by univariate analysis and multivariate analysis. RESULTS: 187 isolates in 144 patients were identified for analysis; 113 were biofilm producers and 74 were not biofilm producers. Patients were primarily male (78 %) military members (61 %) with combat trauma (52 %). On multivariate analysis, the presence of methicillin-resistant Staphylococcus aureus (p < 0.01, OR 5.09, 95 % CI 1.12, 23.1) and Pseudomonas aeruginosa (p = 0.02, OR 3.73, 95 % CI 1.46, 9.53) were the only characteristics more likely to be present in the biofilm producing isolate group. Infectious outcomes of patients with non-biofilm forming isolates, including cure, relapse/reinfection, and chronic infection, were similar to infectious outcomes of patients with biofilm-forming isolates. Mortality with initial infection was higher in the biofilm producing isolate group (16 % vs 5 %, p = 0.01) but attributable mortality was low (1 of 14). No characteristics examined in this study were found to be associated with relapse/reinfection or chronic infection on multivariate analysis. CONCLUSIONS: Bacteria species, but not clinical characteristics, were associated with biofilm formation on multivariate analysis. Biofilm forming isolates and non-biofilm forming isolates had similar infectious outcomes in this study.

Phenotypic and genotypic changes over time and across facilities of serial colonizing and infecting Escherichia coli isolates recovered from injured service members.

Escherichia coli is the most common colonizing and infecting organism isolated from U.S. service members injured during deployment. Our objective was to evaluate the phenotypic and genotypic changes of infecting and colonizing E. coli organisms over time and across facilities to better understand their transmission patterns. E. coli isolates were collected via surveillance cultures and infection workups from U.S. military personnel injured during deployment (June 2009 to May 2011). The isolates underwent antimicrobial susceptibility testing, pulsed-field gel electrophoresis, and multiplex PCR for phylotyping to determine their resistance profiles and clonality. A total of 343 colonizing and 136 infecting E. coli isolates were analyzed, of which 197 (57%) and 109 (80%) isolates, respectively, produced extended-spectrum ß-lactamases (ESBL). Phylogroup A was predominant among both colonizing (38%) and infecting isolates (43%). Although 188 unique pulsed-field types (PFTs) were identified from the colonizing isolates, and 54 PFTs were identified from the infecting isolates, there was a lack of PFT overlap between study years, combat zones, and military treatment facilities. On a per-subject basis, 26% and 32% of the patients with serial colonizing isolates and 10% and 21% with serial infecting isolates acquired changes in their phylogroup and PFT profiles, respectively, over time. The production of ESBL remained high over time and across facilities, with no substantial changes in antimicrobial susceptibilities. Overall, our results demonstrated an array of genotypic and phenotypic differences for the isolates without large clonal clusters; however, the same PFTs were occasionally observed in the colonizing and infecting isolates, suggesting that the source of infections may be endogenous host organisms.

Biofilms and persistent wound infections in United States military trauma patients: a case-control analysis.

BACKGROUND: Complex traumatic injuries sustained by military personnel, particularly when involving extremities, often result in infectious complications and substantial morbidity. One factor that may further impair patient recovery is the persistence of infections. Surface-attached microbial communities, known as biofilms, may play a role in hindering the management of infections; however, clinical data associating biofilm formation with persistent or chronic infections are lacking. Therefore, we evaluated the production of bacterial biofilms as a potential risk factor for persistent infections among wounded military personnel. METHODS: Bacterial isolates and clinical data from military personnel with deployment-related injuries were collected through the Trauma Infectious Disease Outcomes Study. The study population consisted of patients with diagnosed skin and soft-tissue infections. Cases (wounds with bacterial isolates of the same organism collected 14 days apart) were compared to controls (wounds with non-recurrent bacterial isolates), which were matched by organism and infectious disease syndrome. Potential risk factors for persistent infections, including biofilm formation, were examined in a univariate analysis. Data are expressed as odds ratios (OR; 95% confidence interval [CI]). RESULTS: On a per infected wound basis, 35 cases (representing 25 patients) and 69 controls (representing 60 patients) were identified. Eight patients with multiple wounds were utilized as both cases and controls. Overall, 235 bacterial isolates were tested for biofilm formation in the case-control analysis. Biofilm formation was significantly associated with infection persistence (OR: 29.49; CI: 6.24-infinity) in a univariate analysis. Multidrug resistance (OR: 5.62; CI: 1.02-56.92), packed red blood cell transfusion requirements within the first 24 hours (OR: 1.02; CI: 1.01-1.04), operating room visits prior to and on the date of infection diagnosis (OR: 2.05; CI: 1.09-4.28), anatomical location of infected wound (OR: 5.47; CI: 1.65-23.39), and occurrence of polymicrobial infections (OR: 69.71; CI: 15.39-infinity) were also significant risk factors for persistent infections. CONCLUSIONS: We found that biofilm production by clinical strains is significantly associated with the persistence of wound infections. However, the statistical power of the analysis was limited due to the small sample size, precluding a multivariate analysis. Further data are needed to confirm biofilm formation as a risk factor for persistent wound infections.

A Seminested PCR Method for the Diagnosis of Invasive Fungal Infections in Combat Injured.

Background: Among combat injured, invasive fungal infections (IFIs) result in significant morbidity. Cultures and histopathology are the primary diagnostic methods for IFIs, but they have limitations. We previously evaluated a panfungal polymerase chain reaction assay, which was 83% sensitive and 99% specific for angioinvasive IFIs. Here, we evaluated 3 less resource-intensive seminested assays targeting clinically relevant fungi in the order Mucorales and genera Aspergillus and Fusarium. Methods: Formalin-fixed paraffin-embedded tissue specimens from a multicenter trauma IFI cohort (2009-2014) were used. Cases were US military personnel injured in Afghanistan with histopathologic IFI evidence. Controls were patients with similar injury patterns and no laboratory IFI evidence (negative culture and histopathology). Seminested assays specific to Mucorales (V4/V5 regions of 18S rDNA), Aspergillus (mitochondrial tRNA), and Fusarium (internal transcribed spacer [ITS]/28A regions of DNA) were compared with a panfungal assay amplifying the internal transcribed spacer 2 region of rDNA and to histopathology. Results: Specimens from 92 injury sites (62 subjects) were compared with control specimens from 117 injuries (101 subjects). We observed substantial agreement between the seminested and panfungal assays overall, especially for the order Mucorales. Moderate agreement was observed at the genus level for Aspergillus and Fusarium. When compared with histopathology, sensitivity and specificity of seminested assays were 67.4% and 96.6%, respectively (sensitivity increased to 91.7% when restricted to sites with angioinvasion). Conclusions: Prior studies of seminested molecular diagnostics have focused on culture-negative samples from immunocompromised patients. Our findings underscore the utility of the seminested approach in diagnosing soft-tissue IFIs using formalin-fixed paraffin-embedded tissue samples, especially with angioinvasion.

SARS-CoV-2 infection is associated with self-reported post-acute neuropsychological symptoms within six months of follow-up.

BACKGROUND: Chronic neuropsychological sequelae following SARS-CoV-2 infection, including depression, anxiety, fatigue, and general cognitive difficulties, are a major public health concern. Given the potential impact of long-term neuropsychological impairment, it is important to characterize the frequency and predictors of this post-infection phenotype. METHODS: The Epidemiology, Immunology, and Clinical Characteristics of Emerging Infectious Diseases with Pandemic Potential (EPICC) study is a longitudinal study assessing the impact of SARS-CoV-2 infection in U.S. Military Healthcare System (MHS) beneficiaries, i.e. those eligible for care in the MHS including active duty servicemembers, dependents, and retirees. Four broad areas of neuropsychological symptoms were assessed cross-sectionally among subjects 1-6 months post-infection/enrollment, including: depression (Patient Health Questionnaire-9), anxiety (General Anxiety Disorder-7), fatigue (PROMIS® Fatigue 7a), and cognitive function (PROMIS® Cognitive Function 8a and PROMIS® Cognitive Function abilities 8a). Multivariable Poisson regression models compared participants with and without SARS-CoV-2 infection history on these measures, adjusting for sex, ethnicity, active-duty status, age, and months post-first positive or enrollment of questionnaire completion (MPFP/E); models for fatigue and cognitive function were also adjusted for depression and anxiety scores. RESULTS: The study population included 2383 participants who completed all five instruments within six MPFP/E, of whom 687 (28.8%) had at least one positive SARS-CoV-2 test. Compared to those who had never tested positive for SARS-CoV-2, the positive group was more likely to meet instrument-based criteria for depression (15.4% vs 10.3%, p<0.001), fatigue (20.1% vs 8.0%, p<0.001), impaired cognitive function (15.7% vs 8.6%, p<0.001), and impaired cognitive function abilities (24.3% vs 16.3%, p<0.001). In multivariable models, SARS-CoV-2 positive participants, assessed at an average of 2.7 months after infection, had increased risk of moderate to severe depression (RR: 1.44, 95% CI 1.12-1.84), fatigue (RR: 2.07, 95% CI 1.62-2.65), impaired cognitive function (RR: 1.64, 95% CI 1.27-2.11), and impaired cognitive function abilities (RR: 1.41, 95% CI 1.15-1.71); MPFP/E was not significant. CONCLUSIONS: Participants with a history of SARS-CoV-2 infection were up to twice as likely to report cognitive impairment and fatigue as the group without prior SARS-CoV-2 infection. These findings underscore the continued importance of preventing SARS-CoV-2 infection and while time since infection/enrollment was not significant through 6 months of follow-up, this highlights the need for additional research into the long-term impacts of COVID-19 to mitigate and reverse these neuropsychological outcomes.