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Genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have repeatedly altered the course of the coronavirus disease 2019 (COVID-19) pandemic. Delta variants are now the focus of intense international attention because they are causing widespread COVID-19 globally and are associated with vaccine breakthrough cases. We sequenced 16,965 SARS-CoV-2 genomes from samples acquired March 15, 2021, through September 20, 2021, in the Houston Methodist hospital system. This sample represents 91% of all Methodist system COVID-19 patients during the study period. Delta variants increased rapidly from late April onward to cause 99.9% of all COVID-19 cases and spread throughout the Houston metroplex. Compared with all other variants combined, Delta caused a significantly higher rate of vaccine breakthrough cases (23.7% for Delta compared with 6.6% for all other variants combined). Importantly, significantly fewer fully vaccinated individuals required hospitalization. Vaccine breakthrough cases caused by Delta had a low median PCR cycle threshold value (a proxy for high virus load). This value was similar to the median cycle threshold value for unvaccinated patients with COVID-19 caused by Delta variants, suggesting that fully vaccinated individuals can transmit SARS-CoV-2 to others. Patients infected with Alpha and Delta variants had several significant differences. The integrated analysis indicates that vaccines used in the United States are highly effective in decreasing severe COVID-19, hospitalizations, and deaths.

Certain genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of substantial concern because they may be more transmissible or detrimentally alter the pandemic course and disease features in individual patients. SARS-CoV-2 genome sequences from 12,476 patients in the Houston Methodist health care system diagnosed from January 1 through May 31, 2021 are reported here. Prevalence of the B.1.1.7 (Alpha) variant increased rapidly and caused 63% to 90% of new cases in the latter half of May. Eleven B.1.1.7 genomes had an E484K replacement in spike protein, a change also identified in other SARS-CoV-2 lineages. Compared with non–B.1.1.7-infected patients, individuals with B.1.1.7 had a significantly lower cycle threshold (a proxy for higher virus load) and significantly higher hospitalization rate. Other variants [eg, B.1.429 and B.1.427 (Epsilon), P.1 (Gamma), P.2 (Zeta), and R.1] also increased rapidly, although the magnitude was less than that in B.1.1.7. Twenty-two patients infected with B.1.617.1 (Kappa) or B.1.617.2 (Delta) variants had a high rate of hospitalization. Breakthrough cases (n = 207) in fully vaccinated patients were caused by a heterogeneous array of virus genotypes, including many not currently designated variants of interest or concern. In the aggregate, this study delineates the trajectory of SARS-CoV-2 variants circulating in a major metropolitan area, documents B.1.1.7 as the major cause of new cases in Houston, TX, and heralds the arrival of B.1.617 variants in the metroplex.

Coronavirus disease 2019 (COVID-19) convalescent plasma has emerged as a promising therapy and has been granted Emergency Use Authorization by the US Food and Drug Administration for hospitalized COVID-19 patients. We recently reported results from interim analysis of a propensity score–matched study suggesting that early treatment of COVID-19 patients with convalescent plasma containing high-titer anti-spike protein receptor binding domain (RBD) IgG significantly decreases mortality. We herein present results from a 60-day follow-up of a cohort of 351 transfused hospitalized patients. Prospective determination of enzyme-linked immunosorbent assay anti-RBD IgG titer facilitated selection and transfusion of the highest titer units available. Retrospective analysis by the Ortho VITROS IgG assay revealed a median signal/cutoff ratio of 24.0 for transfused units, a value far exceeding the recent US Food and Drug Administration–required cutoff of 12.0 for designation of high-titer convalescent plasma. With respect to altering mortality, our analysis identified an optimal window of 44 hours after hospitalization for transfusing COVID-19 patients with high-titer convalescent plasma. In the aggregate, the analysis confirms and extends our previous preliminary finding that transfusion of COVID-19 patients soon after hospitalization with high-titer anti-spike protein RBD IgG present in convalescent plasma significantly reduces mortality.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and proven treatments are limited. Transfusion of convalescent plasma collected from donors who have recovered from COVID-19 is among many approaches being studied as potentially efficacious therapy. We are conducting a prospective, propensity score–matched study assessing the efficacy of COVID-19 convalescent plasma transfusion versus standard of care as treatment for severe and/or critical COVID-19. We present herein the results of an interim analysis of 316 patients enrolled at Houston Methodist hospitals from March 28 to July 6, 2020. Of the 316 transfused patients, 136 met a 28-day outcome and were matched to 251 non-transfused control COVID-19 patients. Matching criteria included age, sex, body mass index, comorbidities, and baseline ventilation requirement 48 hours from admission, and in a second matching analysis, ventilation status at day 0. Variability in the timing of transfusion relative to admission and titer of antibodies of plasma transfused allowed for analysis in specific matched cohorts. The analysis showed a significant reduction (P = 0.047) in mortality within 28 days, specifically in patients transfused within 72 hours of admission with plasma with an anti-spike protein receptor binding domain titer of ≥1:1350. These data suggest that treatment of COVID-19 with high anti–receptor binding domain IgG titer convalescent plasma is efficacious in early-disease patients.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for >100 years. Patients (n = 25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28, 2020, to April 14, 2020. Patients were transfused with convalescent plasma, obtained from donors with confirmed severe acute respiratory syndrome coronavirus 2 infection who had recovered. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 after transfusion. Clinical improvement was assessed on the basis of a modified World Health Organization six-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. At day 7 after transfusion with convalescent plasma, nine patients had at least a one-point improvement in clinical scale, and seven of those were discharged. By day 14 after transfusion, 19 (76%) patients had at least a one-point improvement in clinical status, and 11 were discharged. No adverse events as a result of plasma transfusion were observed. Whole genome sequencing data did not identify a strain genotype-disease severity correlation. The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease.

Invasive strains of Streptococcus pyogeneswith significantly reduced susceptibility to β-lactam antibiotics have been recently described. These reports have caused considerable concern in the international infectious disease, medical microbiology, and public health communities because S. pyogeneshas remained universally susceptible to β-lactam antibiotics for 70 years. Virtually all analyzed strains had single amino acid replacements in penicillin-binding protein 2X (PBP2X), a major target of β-lactam antibiotics in pathogenic bacteria. We used isogenic strains to test the hypothesis that a single amino acid replacement in PBP2X conferred a fitness advantage in a mouse model of necrotizing myositis. We determined that when mice were administered intermittent subtherapeutic dosing of benzylpenicillin, the strain with a Pro601Leu amino acid replacement in PBP2X that confers reduced β-lactam susceptibility in vitrowas more fit, as assessed by the magnitude of colony-forming units recovered from disease tissue. These data provide important pathogenesis information that bears on this emerging global infectious disease problem.

Group A streptococcus (GAS) is a major pathogen that impacts health and economic affairs worldwide. Although the oropharynx is the primary site of infection, GAS can colonize the female genital tract and cause severe diseases, such as puerperal sepsis, neonatal infections, and necrotizing myometritis. Our understanding of how GAS genes contribute to interaction with the primate female genital tract is limited by the lack of relevant animal models. Using two genome-wide transposon mutagenesis screens, we identified 69 GAS genes required for colonization of the primate vaginal mucosa in vivoand 96 genes required for infection of the uterine wall ex vivo. We discovered a common set of 39 genes important for GAS fitness in both environments. They include genes encoding transporters, surface proteins, transcriptional regulators, and metabolic pathways. Notably, the genes that encode the surface-exclusion protein (SpyAD) and the immunogenic secreted protein 2 (Isp2) were found to be crucial for GAS fitness in the female primate genital tract. Targeted gene deletion confirmed that isogenic mutant strains ΔspyADand Δisp2are significantly impaired in ability to colonize the primate genital tract and cause uterine wall pathologic findings. Our studies identified novel GAS genes that contribute to female reproductive tract interaction that warrant translational research investigation.

Two-component systems (TCSs) are signal transduction proteins that enable bacteria to respond to external stimuli by altering the global transcriptome. Accessory proteins interact with TCSs to fine-tune their activity. In group A Streptococcus(GAS), regulator of Cov (RocA) is an accessory protein that functions with the control of virulence regulator/sensor TCS, which regulates approximately 15% of the GAS transcriptome. Whole-genome sequencing analysis of serotype M28 GAS strains collected from invasive infections in humans identified a higher number of missense (amino acid–altering) and nonsense (protein-truncating) polymorphisms in rocAthan expected. We hypothesized that polymorphisms in RocA alter the global transcriptome and virulence of serotype M28 GAS. We used naturally occurring clinical isolates with rocApolymorphisms (n = 48), an isogenic rocAdeletion mutant strain, and five isogenic rocApolymorphism mutant strains to perform genome-wide transcript analysis (RNA sequencing), in vitrovirulence factor assays, and mouse and nonhuman primate pathogenesis studies to test this hypothesis. Results demonstrated that polymorphisms in rocAresult in either a subtle transcriptome change, causing a wild-type–like virulence phenotype, or a substantial transcriptome change, leading to a significantly increased virulence phenotype. Each polymorphism had a unique effect on the global GAS transcriptome. Taken together, our data show that naturally occurring polymorphisms in one gene encoding an accessory protein can significantly alter the global transcriptome and virulence phenotype of GAS, an important human pathogen.

Streptococcus pyogenessecretes many toxins that facilitate human colonization, invasion, and dissemination. NADase (SPN) and streptolysin O (SLO) are two toxins that play important roles in pathogenesis. We previously showed that increased production of SPN and SLO in epidemic serotype M1 and M89 S. pyogenesstrains is associated with rapid intercontinental spread and enhanced virulence. The biological functions of SPN and SLO have been extensively studied using eukaryotic cell lines, but the relative contribution of each of these two toxins to pathogenesis of epidemic M1 or M89 strains remains unexplored. Herein, using a genetically representative epidemic M1 strain and a panel of isogenic mutant derivative strains, we evaluated the relative contributions of SPN and SLO toxins to virulence in mouse models of necrotizing myositis, bacteremia, and skin and soft tissue infection. We found that isogenic mutants lacking SPN, SLO, and both toxins are equally impaired in ability to cause necrotizing myositis. In addition, mutants lacking either SPN or SLO are significantly attenuated in the bacteremia and soft tissue infection models, and the mutant strain lacking production of both toxins is further attenuated. The mutant strain lacking both SPN and SLO production is severely attenuated in ability to resist killing by human polymorphonuclear leukocytes. We conclude that both SPN and SLO contribute significantly to S. pyogenespathogenesis in these virulence assays.

Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation within a species; however, few investigations demonstrate how naturally occurring SNPs may increase strain virulence. We recently used group A Streptococcus as a model pathogen to study bacteria strain genotype–patient disease phenotype relationships. Whole-genome sequencing of approximately 800 serotype M59 group A Streptococcus strains, recovered during an outbreak of severe invasive infections across North America, identified a disproportionate number of SNPs in the gene encoding multiple gene regulator of group A Streptococcus (mga). Herein, we report results of studies designed to test the hypothesis that the most commonly occurring SNP, encoding a replacement of arginine for histidine at codon 201 of Mga (H201R), significantly increases virulence. Whole transcriptome analysis revealed that the H201R replacement significantly increased expression of mgaand 54 other genes, including many proven virulence factors. Compared to the wild-type strain, a H201R isogenic mutant strain caused significantly larger skin lesions in mice. Serial quantitative bacterial culture and noninvasive magnetic resonance imaging also demonstrated that the isogenic H201R strain was significantly more virulent in a nonhuman primate model of joint infection. These findings show that the H201R replacement in Mga increases the virulence of M59 group A Streptococcus and provide new insight to how a naturally occurring SNP in bacteria contributes to human disease phenotypes.

Group A Streptococcus(GAS) causes an exceptionally broad range of infections in humans, from relatively mild pharyngitis and skin infections to life-threatening necrotizing fasciitis and toxic shock syndrome. An epidemic of severe invasive human infections caused by type emm59GAS, heretofore an exceedingly rare cause of disease, spread west to east across Canada over a 3-year period (2006 to 2008). By sequencing the genomes of 601 epidemic, historic, and other emm59organisms, we discovered that a recently emerged, genetically distinct emm59clone is responsible for the Canadian epidemic. Using near-real-time genome sequencing, we were able to show spread of the Canadian epidemic clone into the United States. The extensive genome data permitted us to identify patterns of geographic dissemination as well as links between emm59subclonal lineages that cause infections. Mouse and nonhuman primate models of infection demonstrated that the emerged clone is unusually virulent. Transmission of epidemic emm59strains may have occurred primarily by skin contact, as suggested by an experimental model of skin transmission. In addition, the emm59strains had a significantly impaired ability to persist in human saliva and to colonize the oropharynx of mice, and seldom caused human pharyngitis. Our study contributes new information to the rapidly emerging field of molecular pathogenomics of bacterial epidemics and illustrates how full-genome data can be used to precisely illuminate the landscape of strain dissemination during a bacterial epidemic.

Panton-Valentine leukocidin (PVL) is a two-component cytolytic toxin epidemiologically linked to community-associated methicillin-resistant Staphylococcus aureus(CA-MRSA) infections, including serious invasive infections caused by the epidemic clone referred to as strain USA300. Although PVL has long been known to be a S. aureusvirulence molecule in vitro, the relative contribution of this leukotoxin to invasive CA-MRSA infections such as pneumonia remains controversial. We developed a nonhuman primate model of CA-MRSA pneumonia and used it to test the hypothesis that PVL contributes to lower respiratory tract infections caused by S. aureusstrain USA300. The lower respiratory tract disease observed in this monkey model mimicked the clinical and pathological features of early mild to moderate S. aureuspneumonia in humans, including fine-structure histopathology. In this experiment using a large sample of monkeys and multiple time points of examination, no involvement of PVL in virulence could be detected. Compared with the wild-type parental USA300 strain, the isogenic PVL deletion-mutant strain caused equivalent lower respiratory tract pathology. We conclude that PVL does not contribute to lower respiratory tract infection in this nonhuman primate model of human CA-MRSA pneumonia.

Genetic variants of SARS-CoV-2 continue to dramatically alter the landscape of the COVID-19 pandemic. The recently described variant of concern designated Omicron (B.1.1.529) has rapidly spread worldwide and is now responsible for the majority of COVID-19 cases in many countries. Because Omicron was recognized very recently, many knowledge gaps exist about its epidemiology, clinical severity, and disease course. A genome sequencing study of SARS-CoV-2 in the Houston Methodist healthcare system identified 4,468 symptomatic patients with infections caused by Omicron from late November 2021 through January 5, 2022. Omicron very rapidly increased in only three weeks to cause 90% of all new COVID-19 cases, and at the end of the study period caused 98% of new cases. Compared to patients infected with either Alpha or Delta variants in our healthcare system, Omicron patients were significantly younger, had significantly increased vaccine breakthrough rates, and were significantly less likely to be hospitalized. Omicron patients required less intense respiratory support and had a shorter length of hospital stay, consistent with on average decreased disease severity. Two patients with Omicron “stealth” sublineage BA.2 also were identified. The data document the unusually rapid spread and increased occurrence of COVID-19 caused by the Omicron variant in metropolitan Houston, and address the lack of information about disease character among US patients.

Molecular mechanisms mediating group A Streptococcus(GAS)-host interactions remain poorly understood but are crucial for diagnostic, therapeutic, and vaccine development. An optimized high-density microarray was used to analyze the transcriptome of GAS during experimental mouse soft tissue infection. The transcriptome of a wild-type serotype M1 GAS strain and an isogenic transcriptional regulator knockout mutant (covR) also were compared. Array datasets were verified by quantitative real-time reverse transcriptase-polymerase chain reaction and in situimmunohistochemistry. The results unambiguously demonstrate that coordinated expression of proven and putative GAS virulence factors is directed toward overwhelming innate host defenses leading to severe cellular damage. We also identified adaptive metabolic responses triggered by nutrient signals and hypoxic/acidic conditions in the host, likely facilitating pathogen persistence and proliferation in soft tissues. Key discoveries included that oxidative stress genes, virulence genes, genes related to amino acid and maltodextrin utilization, and several two-component transcriptional regulators were highly expressed in vivo. This study is the first global analysis of the GAS transcriptome during invasive infection. Coupled with parallel analysis of the covRmutant strain, novel insights have been made into the regulation of GAS virulence in vivo, resulting in new avenues for targeted therapeutic and vaccine research.

Genome-wide analysis of microbial pathogens and molecular pathogenesis processes has become an area of considerable activity in the last 5 years. These studies have been made possible by several advances, including completion of the human genome sequence, publication of genome sequences for many human pathogens, development of microarray technology and high-throughput proteomics, and maturation of bioinformatics. Despite these advances, relatively little effort has been expended in the bacterial pathogenesis arena to develop and use integrated research platforms in a systems biology approach to enhance our understanding of disease processes. This review discusses progress made in exploiting an integrated genome-wide research platform to gain new knowledge about how the human bacterial pathogen group A Streptococcuscauses disease. Results of these studies have provided many new avenues for basic pathogenesis research and translational research focused on development of an efficacious human vaccine and novel therapeutics. One goal in summarizing this line of study is to bring exciting new findings to the attention of the investigative pathology community. In addition, we hope the review will stimulate investigators to consider using analogous approaches for analysis of the molecular pathogenesis of other microbes.

The molecular basis for bacterial responses to host signals during natural infections is poorly understood. The gram-positive bacterial pathogen group A Streptococcus(GAS) causes human mucosal, skin, and life-threatening systemic infections. During the transition from a throat or skin infection to an invasive infection, GAS must adapt to changing environments and host factors. To better understand how GAS adapts, we used transcript profiling and functional analysis to investigate the transcriptome of a wild-type serotype M1 GAS strain in human blood. Global changes in GAS gene expression occur rapidly in response to human blood exposure. Increased transcription was observed for many genes that likely enhance bacterial survival, including those encoding superantigens and host-evasion proteins regulated by a multiple gene activator called Mga. GAS also coordinately expressed genes involved in proteolysis, transport, and catabolism of oligopeptides to obtain amino acids in this protein-rich host environment. Comparison of the transcriptome of the wild-type strain to that of an isogenic deletion mutant (ΔcovR) mutated in the two-component regulatory system designated CovR-CovS reinforced the hypothesis that CovR-CovS has an important role linking key biosynthetic, catabolic, and virulence functions during transcriptome restructuring. Taken together, the data provide crucial insights into strategies used by pathogenic bacteria for thwarting host defenses and surviving in human blood.

This Guest Editorial introduces this month's special Infectious Disease Theme Issue, a series of reviews focusing on the molecular pathogenic processes of four representative pathogens, including two bacteria (brucellae and Staphylococcus aureus), a virus (influenza), and a parasite (Trypanosoma cruzi).

Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there has been international concern about the emergence of virus variants with mutations that increase transmissibility, enhance escape from the human immune response, or otherwise alter biologically important phenotypes. In late 2020, several variants of concern emerged globally, including the UK variant (B.1.1.7), the South Africa variant (B.1.351), Brazil variants (P.1 and P.2), and two related California variants of interest (B.1.429 and B.1.427). These variants are believed to have enhanced transmissibility. For the South Africa and Brazil variants, there is evidence that mutations in spike protein permit it to escape from some vaccines and therapeutic monoclonal antibodies. On the basis of our extensive genome sequencing program involving 20,453 coronavirus disease 2019 patient samples collected from March 2020 to February 2021, we report identification of all six of these SARS-CoV-2 variants among Houston Methodist Hospital (Houston, TX) patients residing in the greater metropolitan area. Although these variants are currently at relatively low frequency (aggregate of 1.1%) in the population, they are geographically widespread. Houston is the first city in the United States in which active circulation of all six current variants of concern has been documented by genome sequencing. As vaccine deployment accelerates, increased genomic surveillance of SARS-CoV-2 is essential to understanding the presence, frequency, and medical impact of consequential variants and their patterns and trajectory of dissemination.

In a series of four articles published between 1916 and 1919 in The Journal of Medical Research, precursor to The American Journal of Pathology, the investigative pathologist S. Burt Wolbach unambiguously showed that Rocky Mountain spotted fever has a tick-borne mode of transmission, the causative agent replicates intracellularly, and the disease is fundamentally a vasculitis. Although underappreciated, Wolbach’s tour-de-force work epitomized investigative pathology. These four articles should be mandatory reading for young investigators and are recommended also to seasoned investigators who seek reinvigoration in the beauty in their craft.