Infectious Disease

Gaiha GD, Rossin EJ, Urbach J, Landeros C, Collins DR, Nwonu C, Muzhingi I, Anahtar MN, Waring OM, Piechocka-Trocha A, Waring M, Worrall DP, Ghebremichael MS, Newman RM, Power KA, Allen TM, Chodosh J, Walker BD. Structural topology defines protective CD8 T cell epitopes in the HIV proteome. Science 2019;364(6439):480-484.Abstract
Mutationally constrained epitopes of variable pathogens represent promising targets for vaccine design but are not reliably identified by sequence conservation. In this study, we employed structure-based network analysis, which applies network theory to HIV protein structure data to quantitate the topological importance of individual amino acid residues. Mutation of residues at important network positions disproportionately impaired viral replication and occurred with high frequency in epitopes presented by protective human leukocyte antigen () class I alleles. Moreover, CD8 T cell targeting of highly networked epitopes distinguished individuals who naturally control HIV, even in the absence of protective alleles. This approach thereby provides a mechanistic basis for immune control and a means to identify CD8 T cell epitopes of topological importance for rational immunogen design, including a T cell-based HIV vaccine.
Deiner MS, McLeod SD, Wong J, Chodosh J, Lietman TM, Porco TC. Google Searches and Detection of Conjunctivitis Epidemics Worldwide. Ophthalmology 2019;126(9):1219-1229.Abstract
PURPOSE: Epidemic and seasonal infectious conjunctivitis outbreaks can impact education, workforce, and economy adversely. Yet conjunctivitis typically is not a reportable disease, potentially delaying mitigating intervention. Our study objective was to determine if conjunctivitis epidemics could be identified using Google Trends search data. DESIGN: Search data for conjunctivitis-related and control search terms from 5 years and countries worldwide were obtained. Country and term were masked. Temporal scan statistics were applied to identify candidate epidemics. Candidates then were assessed for geotemporal concordance with an a priori defined collection of known reported conjunctivitis outbreaks, as a measure of sensitivity. PARTICIPANTS: Populations by country that searched Google's search engine using our study terms. MAIN OUTCOME MEASURES: Percent of known conjunctivitis outbreaks also found in the same country and period by our candidate epidemics, identified from conjunctivitis-related searches. RESULTS: We identified 135 candidate conjunctivitis epidemic periods from 77 countries. Compared with our a priori defined collection of known reported outbreaks, candidate conjunctivitis epidemics identified 18 of 26 (69% sensitivity) of the reported country-wide or island nationwide outbreaks, or both; 9 of 20 (45% sensitivity) of the reported region or district-wide outbreaks, or both; but far fewer nosocomial and reported smaller outbreaks. Similar overall and individual sensitivity, as well as specificity, were found on a country-level basis. We also found that 83% of our candidate epidemics had start dates before (of those, 20% were more than 12 weeks before) their concurrent reported outbreak's report issuance date. Permutation tests provided evidence that on average, conjunctivitis candidate epidemics occurred geotemporally closer to outbreak reports than chance alone suggests (P < 0.001) unlike control term candidates (P = 0.40). CONCLUSIONS: Conjunctivitis outbreaks can be detected using temporal scan analysis of Google search data alone, with more than 80% detected before an outbreak report's issuance date, some as early as the reported outbreak's start date. Future approaches using data from smaller regions, social media, and more search terms may improve sensitivity further and cross-validate detected candidates, allowing identification of candidate conjunctivitis epidemics from Internet search data potentially to complementarily benefit traditional reporting and detection systems to improve epidemic awareness.
Van Tyne D, Manson AL, Huycke MM, Karanicolas J, Earl AM, Gilmore MS. Impact of antibiotic treatment and host innate immune pressure on enterococcal adaptation in the human bloodstream. Sci Transl Med 2019;11(487)Abstract
Multidrug-resistant enterococcal strains emerged in the early 1980s and are now among the leading causes of drug-resistant bacterial infection worldwide. We used functional genomics to study an early bacterial outbreak in patients in a Wisconsin hospital between 1984 and 1988 that was caused by multidrug-resistant The goal was to determine how a clonal lineage of became adapted to growth and survival in the human bloodstream. Genome sequence analysis revealed a progression of increasingly fixed mutations and repeated independent occurrences of mutations in a relatively small set of genes. Repeated independent mutations suggested selection within the host during the course of infection in response to pressures such as host immunity and antibiotic treatment. We observed repeated independent mutations in a small number of loci, including a little studied polysaccharide utilization pathway and the locus. Functional studies showed that mutating these loci rendered better able to withstand antibiotic pressure and innate immune defenses in the human bloodstream. We also observed a shift in mutation pattern that corresponded to the introduction of carbapenem antibiotics in 1987. This work identifies pathways that allow enterococci to survive the transition from the human gut into the bloodstream, enabling them to cause severe bacteremia associated with high mortality.
Busch C, Iglicki M, Okada M, Gabrielle P-H, Cohen S, Mariussi M, Amphornphruet A, Cebeci Z, Chaikitmongkol V, Couturier A, Fraser-Bell S, Fung AT, Iannetta D, Radecka L, Laíns I, Rodrigues TM, Lupidi M, Ozimek M, Sala-Puigdollers A, Rehak M, Loewenstein A, Zur D, Zur D. Causative Pathogens of Endophthalmitis after Intravitreal Anti-VEGF Injection: An International Multicenter Study. Ophthalmologica 2019;241(4):211-219.Abstract
PURPOSE: The main objective of this study was to investigate the microbiological spectrum of endophthalmitis after anti-VEGF injections and to compare streptococcal with non-streptococcus-associated cases with regard to baseline characteristics and injection procedure. METHODS: Retrospective, international multicenter study of patients with culture-positive endophthalmitis after intravitreal anti-VEGF injection at 17 different retina referral centers. RESULTS: Eighty-three cases with 87 identified pathogens were included. Coagulase-negative staphylococci (59%) and viridans streptococci (15%) were the most frequent pathogens found. The use of postoperative antibiotics and performance of injections in an operating room setting significantly reduced the rate of streptococcus-induced endophthalmitis cases (p = 0.01 for both). CONCLUSION: We found a statistically significant lower rate of postinjectional local antibiotic therapy and operating room-based procedures among the streptococcus-induced cases compared to cases caused by other organisms.
Ramos Y, Rocha J, Hael AL, van Gestel J, Vlamakis H, Cywes-Bentley C, Cubillos-Ruiz JR, Pier GB, Gilmore MS, Kolter R, Morales DK. PolyGlcNAc-containing exopolymers enable surface penetration by non-motile Enterococcus faecalis. PLoS Pathog 2019;15(2):e1007571.Abstract
Bacterial pathogens have evolved strategies that enable them to invade tissues and spread within the host. Enterococcus faecalis is a leading cause of local and disseminated multidrug-resistant hospital infections, but the molecular mechanisms used by this non-motile bacterium to penetrate surfaces and translocate through tissues remain largely unexplored. Here we present experimental evidence indicating that E. faecalis generates exopolysaccharides containing β-1,6-linked poly-N-acetylglucosamine (polyGlcNAc) as a mechanism to successfully penetrate semisolid surfaces and translocate through human epithelial cell monolayers. Genetic screening and molecular analyses of mutant strains identified glnA, rpiA and epaX as genes critically required for optimal E. faecalis penetration and translocation. Mechanistically, GlnA and RpiA cooperated to generate uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) that was utilized by EpaX to synthesize polyGlcNAc-containing polymers. Notably, exogenous supplementation with polymeric N-acetylglucosamine (PNAG) restored surface penetration by E. faecalis mutants devoid of EpaX. Our study uncovers an unexpected mechanism whereby the RpiA-GlnA-EpaX metabolic axis enables production of polyGlcNAc-containing polysaccharides that endow E. faecalis with the ability to penetrate surfaces. Hence, targeting carbohydrate metabolism or inhibiting biosynthesis of polyGlcNAc-containing exopolymers may represent a new strategy to more effectively confront enterococcal infections in the clinic.
Singh G, Ismail AM, Lee JY, Ramke M, Lee JS, Dyer DW, Seto D, Rajaiya J, Chodosh J. Divergent Evolution of E1A CR3 in Human Adenovirus Species D. Viruses 2019;11(2)Abstract
Adenovirus E1A is the first viral protein expressed during infection. E1A controls critical aspects of downstream viral gene expression and cell cycle deregulation, and its function is thought to be highly conserved among adenoviruses. Various bioinformatics analyses of E1A from 38 human adenoviruses of species D (HAdV-D), including likelihood clade model partitioning, provided highly significant evidence of divergence of HAdV-Ds into two distinct groups for the conserved region 3 (CR3), present only in the E1A 13S isoform. This variance within E1A 13S of HAdV-Ds was not found in any other human adenovirus (HAdV) species. By protein sequence and structural analysis, the zinc finger motif of E1A CR3, previously shown as critical for transcriptional activation, showed the greatest differences. Subsequent codon usage bias analysis revealed substantial divergence in E1A 13S between the two groups of HAdV-Ds, suggesting that these two sub-groups of HAdV-D evolved under different cellular conditions. Hence, HAdV-D E1A embodies a previously unappreciated evolutionary divergence among HAdVs.
Dabul ANG, Avaca-Crusca JS, Navais RB, Merlo TP, Van Tyne D, Gilmore MS, da Camargo ILBC. Molecular basis for the emergence of a new hospital endemic tigecycline-resistant Enterococcus faecalis ST103 lineage. Infect Genet Evol 2019;67:23-32.Abstract
Enterococcus faecalis are a major cause of nosocomial infection worldwide, and the spread of vancomycin resistant strains (VRE) limits treatment options. Tigecycline-resistant VRE began to be isolated from inpatients at a Brazilian hospital within months following the addition of tigecycline to the hospital formulary. This was found to be the result of a spread of an ST103 E. faecalis clone. Our objective was to identify the basis for tigecycline resistance in this lineage. The genomes of two closely related tigecycline-susceptible (MIC = 0.06 mg/L), and three representative tigecycline-resistant (MIC = 1 mg/L) ST103 isolates were sequenced and compared. Further, efforts were undertaken to recapitulate the emergence of resistant strains in vitro. The specific mutations identified in clinical isolates in several cases were within the same genes identified in laboratory-evolved strains. The contribution of various polymorphisms to the resistance phenotype was assessed by trans-complementation of the wild type or mutant alleles, by testing for differences in mRNA abundance, and/or by examining the phenotype of transposon insertion mutants. Among tigecycline-resistant clinical isolates, five genes contained non-synonymous mutations, including two genes known to be related to enterococcal tigecycline resistance (tetM and rpsJ). Finally, within the in vitro-selected resistant variants, mutation in the gene for a MarR-family response regulator was associated with tigecycline resistance. This study shows that E. faecalis mutates to attain tigecycline resistance through the complex interplay of multiple mechanisms, along multiple evolutionary trajectories.
Lee JS, Ismail AM, Lee JY, Zhou X, Materne EC, Chodosh J, Rajaiya J. Impact of dynamin 2 on adenovirus nuclear entry. Virology 2019;529:43-56.Abstract
The large GTPase dynamin 2 controls both endosomal fission and microtubule acetylation. Here we report that dynamin 2 alters microtubules and regulates the trafficking of human adenovirus type 37. Dynamin 2 knockdown by siRNA in infected cells resulted in accumulation of acetylated tubulin, repositioning of microtubule organizing centers (MTOCs) closer to cell nuclei, increased virus in the cytosol (with a compensatory decrease in endosomal virus), reduced proinflammatory cytokine induction, and increased binding of virus to the nucleoporin, Nup358. These events led to increased viral DNA nuclear entry and viral replication. Overexpression of dynamin 2 generated opposite effects. Therefore, dynamin 2 inhibits adenovirus replication and promotes innate immune responses by the infected cell. MTOC transposition in dynamin 2 knockdown promotes a closer association with nuclear pore complexes to facilitate viral DNA delivery. Dynamin 2 plays a key role in adenoviral trafficking and influences host responses to infection.
Storey PP, Tauqeer Z, Yonekawa Y, Todorich B, Wolfe JD, Shah SP, Shah AR, Koto T, Abbey AM, Morizane Y, Sharma P, Wood EH, Morizane-Hosokawa M, Pendri P, Pancholy M, Harkey S, Jeng-Miller KW, Obeid A, Borkar DS, Chen E, Williams P, Okada AA, Inoue M, Shiraga F, Hirakata A, Shah CP, Prenner J, Garg S, Garg S. The Impact of Prefilled Syringes on Endophthalmitis Following Intravitreal Injection of Ranibizumab. Am J Ophthalmol 2019;199:200-208.Abstract
PURPOSE: To compare the rates of infectious endophthalmitis following intravitreal injection of ranibizumab using prefilled syringes vs conventional preparation. DESIGN: Multicenter retrospective cohort study. METHODS: All eyes receiving intravitreal injection of 0.5 mg ranibizumab for retinal vascular diseases at 10 retina practices across the United States (2016 to 2017) and Japan (2009 to 2017) were included. The total numbers of eyes and injections were determined from billing codes. Endophthalmitis cases were determined from billing records and evaluated with chart review. Primary outcome was the rate of postinjection acute endophthalmitis. Secondary outcomes were visual acuity and microbial spectrum. RESULTS: A total of 243 754 intravitreal 0.5 mg ranibizumab injections (165 347 conventional and 78 407 prefilled) were administered to 43 132 unique patients during the study period. In the conventional ranibizumab group, a total of 43 cases of suspected endophthalmitis occurred (0.026%; 1 in 3845 injections) and 22 cases of culture-positive endophthalmitis occurred (0.013%; 1 in 7516 injections). In the prefilled ranibizumab group, 12 cases of suspected endophthalmitis occurred (0.015%; 1 in 6534 injections) and 2 cases of culture-positive endophthalmitis occurred (0.0026%; 1 in 39 204 injections). Prefilled syringes were associated with a trend toward decreased risk of suspected endophthalmitis (odds ratio 0.59; 95% confidence interval 0.31-1.12; P = .10) and a statistically significant decreased risk of culture-positive endophthalmitis (odds ratio 0.19; 95% confidence interval 0.045-0.82; P = .025). Average logMAR vision loss at final follow-up was significantly worse for eyes that developed endophthalmitis from the conventional ranibizumab preparation compared to the prefilled syringe group (4.45 lines lost from baseline acuity vs 0.38 lines lost; P = .0062). Oral-associated flora was found in 27.3% (6/22) of conventional ranibizumab culture-positive endophthalmitis cases (3 cases of Streptococcus viridans, 3 cases of Enterococcus faecalis) compared to 0 cases in the prefilled ranibizumab group. CONCLUSION: In a large, multicenter, retrospective study the use of prefilled syringes during intravitreal injection of ranibizumab was associated with a reduced rate of culture-positive endophthalmitis, including from oral flora, as well as with improved visual acuity outcomes.
Barros EM, Martin MJ, Selleck EM, Lebreton F, Sampaio JLM, Gilmore MS. Daptomycin Resistance and Tolerance Due to Loss of Function in Staphylococcus aureus dsp1 and asp23. Antimicrob Agents Chemother 2019;63(1)Abstract
Lipopeptide daptomycin is a last-line cell-membrane-targeting antibiotic to treat multidrug-resistant Alarmingly, daptomycin-resistant isolates have emerged. The mechanisms underlying daptomycin resistance are diverse and share similarities with resistances to cationic antimicrobial peptides and other lipopeptides, but they remain to be fully elucidated. We selected mutants with increased resistance to daptomycin from a library of transposon insertions in sequent type 8 (ST8) HG003. Insertions conferring increased daptomycin resistance were localized to two genes, one coding for a hypothetical lipoprotein (SAOUHSC_00362, Dsp1), and the other for an alkaline shock protein (SAOUHSC_02441, Asp23). Markerless loss-of-function mutants were then generated for comparison. All transposon mutants and knockout strains exhibited increased daptomycin resistance compared to those of wild-type and complemented strains. Null and transposon insertion mutants also exhibited increased resistance to cationic antimicrobial peptides. Interestingly, the mutant also showed increased resistance to vancomycin, a cell-wall-targeting drug with a different mode of action. Null mutations in both and resulted in increased tolerance as reflected by reduced killing to both daptomycin and vancomycin, as well as an increased tolerance to surfactant (Triton X-100). Neither mutant exhibited increased resistance to lysostaphin, a cell-wall-targeting endopeptidase. These findings identified two genes core to the species that make previously uncharacterized contributions to antimicrobial resistance and tolerance in .
Kempen JH, Tekle-Haimanot R, Hunduma L, Alemayehu M, Pistilli M, Abashawl A, Lawrence SD, Alemayehu W. Fluorometholone 0.1% as Ancillary Therapy for Trachomatous Trichiasis Surgery: Randomized Clinical Trial. Am J Ophthalmol 2019;197:145-155.Abstract
PURPOSE: To assess the hypothesis that fluorometholone 0.1% eye drops are safe and effective as adjunctive therapy for trachomatous trichiasis (TT) surgery; determining the most promising dose. DESIGN: Randomized, placebo-controlled, double-masked parallel dose-ranging clinical trial. METHODS: Patients undergoing upper lid TT surgery at a rural Ethiopian hospital were randomized to fluorometholone 0.1% twice daily for 4 weeks, 4 times daily for 4 weeks, 4 times daily for 8 weeks, or matching frequency placebo in a 3:1:3:1:3:1 ratio for 1 eye. Randomization was stratified by TT severity (1-4 vs ≥5 lashes touching the globe). Safety outcomes (intraocular pressure [IOP] elevation, cataract, and other dose-limiting toxicities) and postoperative TT incidence were assessed over 1 year. RESULTS: Subjects randomized were 39:13:39:13:38:13 in the respective groups, and 1 subject in the 8-weeks fluorometholone group was withdrawn. Of 154 subjects, 148 (96.1%) completed 1 year's follow-up. Among 76 eyes receiving fluorometholone 4 times daily, 1 developed IOP elevation ≥ 30 mm Hg (to 37 mm Hg) and 1 had an allergic reaction attributed to the study drug; each resolved upon drug cessation without sequelae. No cataract or other dose-limiting toxicity events occurred. Postoperative TT within 1 year occurred in 29.3% of placebo eyes vs 17.7%, 19.6%, and 23.2% among the respective fluorometholone groups (P = .29 comparing placebo vs all active treatments combined). CONCLUSIONS: The results suggest fluorometholone 0.1% is likely to be safe and efficacious to reduce postoperative TT following TT surgery, and 1 drop twice daily for 4 weeks is the most promising dose. Confirmation in a full-scale clinical trial is needed before programmatic implementation.
Ung L, Bispo PJM, Shanbhag SS, Gilmore MS, Chodosh J. The Persistent Dilemma of Microbial Keratitis: Global Burden, Diagnosis, and Antimicrobial Resistance. Surv Ophthalmol 2018;Abstract
Microbial keratitis (MK) is a potentially blinding condition which must be treated emergently to preserve vision. Although long recognized as a significant cause of corneal blindness, our understanding of its true global scale, associated burden of disease, and etiological patterns remains somewhat limited. Current epidemiological data suggests that MK may be epidemic in parts of the world--particularly within South, South-East and East Asia--and may exceed two million cases per year world-wide. Etiological patterns vary between economically developed and developing countries, with bacterial predominance in the former and fungal predominance in the latter. The key to effective management lies in timely diagnosis; however, the current gold standard of stain and culture remains time consuming and often yields no clinically useful results. For this reason, there are attempts to develop highly sensitive and accurate molecular diagnostic tools to provide rapid diagnosis, inform treatment decision making, and minimize the threat of antimicrobial resistance. We provide an overview of these key areas and of avenues for further research toward the goal of more effectively addressing the problem of MK on both an individual and public health level.
Ismail AM, Cui T, Dommaraju K, Singh G, Dehghan S, Seto J, Shrivastava S, Fedorova NB, Gupta N, Stockwell TB, Halpin R, Madupu R, Heim A, Kajon AE, Romanowski EG, Kowalski RP, Malathi J, Therese KL, Madhavan HN, Zhang Q, Ferreyra LJ, Jones MS, Rajaiya J, Dyer DW, Chodosh J, Seto D. Author Correction: Genomic analysis of a large set of currently-and historically-important human adenovirus pathogens. Emerg Microbes Infect 2018;7(1):208.Abstract
The original publication of this article [1] was missing the below author that made contributions to the research and the published article.
Wurster JI, Bispo PJM, Van Tyne D, Cadorette JJ, Boody R, Gilmore MS. Staphylococcus aureus from ocular and otolaryngology infections are frequently resistant to clinically important antibiotics and are associated with lineages of community and hospital origins. PLoS One 2018;13(12):e0208518.Abstract
Staphylococcus aureus is an important human pathogen that causes serious antibiotic-resistant infections. Its population structure is marked by the appearance and dissemination of successful lineages across different settings. To begin understanding the population structure of S. aureus causing ocular and otolaryngology infections, we characterized 262 isolates by antimicrobial sensitivity testing and multilocus sequence typing (MLST). Methicillin-resistant S. aureus were subjected to SCCmec typing and Panton-Valentine leukocidin (PVL) screening. Although we detected a high level of genetic diversity among methicillin-sensitive (MSSA) isolates, (63 sequence types-STs), the population was dominated by five lineages: ST30, ST5, ST8, ST15 and ST97. Resistance to penicillin, erythromycin and clindamycin was common among the major MSSA lineages, with fluctuations markedly impacted by genetic background. Isolates belonging to the predominant lineage, ST30, displayed high rates of resistance to penicillin (100%), erythromycin (71%), and clindamycin (63%). Overall, 21% of the isolates were methicillin-resistant (MRSA), with an apparent enrichment among otitis and orbital cellulitis isolates (>40%). MRSA isolates belonged to 14 STs grouped in 5 clonal complexes (CC), however, CC5 (56.1%) and CC8 (38.6%) dominated the population. Most CC5 strains were SCCmec type II, and resembled the hospital-adapted USA100 clone. CC8 strains were SCCmec type IV, and 86% were positive for the PVL toxin, common features of the community-acquired clone USA300. CC5 strains harboring a SCCmec type IV, typical for the USA800 clone, comprised 15.5% of the population. USA100 strains were highly resistant to clindamycin, erythromycin and levofloxacin (100%), while USA300 strains were frequently resistant to erythromycin (89%) but displayed lower rates of resistance to levofloxacin (39%) and clindamycin (17%). Our data demonstrate that the ocular and otolaryngology S. aureus populations are composed of strains that are commonly resistant to clinically relevant antibiotics, and are associated with the major epidemic clonal complexes of both community and hospital origins.
Duarte MJ, Kozin ED, Bispo PJM, Mitchell AH, Gilmore MS, Remenschneider AK. Methicillin-resistant Staphylococcus aureus in acute otitis externa. World J Otorhinolaryngol Head Neck Surg 2018;4(4):246-252.Abstract
Objective: Otologic methicillin-resistant (MRSA) infection has historically been rare, but given the rise in community-acquired MRSA carriage and infection at other body sites, prevalence rates may be changing. The goal of this study was to determine the prevalence of MRSA in recent otologic cultures from patients with acute otitis externa (AOE). Study design: Retrospective review of an institutional microbiologic database. Methods: A retrospective analysis was performed on serial culture isolates taken from the ear at a quaternary care hospital from January 2014 to April 2016. The causative pathogen and antibiotic sensitivity was determined by culture isolation and end point mean inhibitory concentration (MIC) testing. Medical records were reviewed to document patient characteristics, chronicity of infection, symptomatology, and previous treatments. Results: Over the study period, 173 patients were diagnosed with AOE and underwent otologic cultures of the ear. Fifty-three (30.6%) of cultures grew (SA). Of SA infections, 15 (28.3%) were identified as MRSA. MRSA patients were typically older than patients with methicillin-sensitive SA (MSSA) (mean age 46.7 ± 17.9 29 ± 19.4,  = 0.003) and had more medical comorbidities (4 1.7,  = 0.001). Compared to patients with MSSA, patients with MRSA were significantly more likely to have had prior ototopical antibiotic exposure (37% 73%,  = 0.019). Conclusion: Contemporary ear culture isolates at quaternary care center show higher rates of MRSA compared to historical reports in the literature. Clinicians should consider ear cultures to identify MRSA AOE. Level of Evidence: IV.