Group A Streptococcal Infections Research Articles

Current epidemiological aspects of streptococcal and poststreptococcal diseases in the Russian Federation

Epidemiological situation related to the spread of streptococcal infection and post-streptococcal diseases in the world remains tense in recent decades. According to the World Health Organization, more than 616 million cases of streptococcal pharyngitis occur annually in the world. Group A Streptococcus (GAS) is the most common cause of morbidity and mortality in infectious diseases and one of the main human pathogens. The prevalence of severe cases of GAS infections amounted to 18.1 million people worldwide. At the same time, in recent years outbreaks of scarlet fever have been recorded in some countries. In 2011, scarlet fever rate was 10 times higher than the average rate of the previous decade. In the past 3 years, epidemics of scarlet fever have been registered in the UK, with an increased incidence every year. Although there are significant achievements in the fight against rheumatic fever (RF), it keeps reminding of itself in the form of new outbreaks. Currently, acute rheumatic fever (ARF) is not a mass disease, but it is a serious problem associated with heart damage. In the article, we analyzed incidence and prevalence of RF, rheumatic heart disease (RHD), chronic pharyngitis, nasopharyngitis, sinusitis and rhinitis. We present the comparative characteristic among children, adolescents and adults. We also describe high epidemiological and social significance of the problem and possible increase of RF and RHD, which cause significant social and economic damage, remaining the urgent problem of public health in Russia. It was revealed that the situation with ARF and RHD in Russia remains unstable. Between 1997 and 2016, epidemiological indicators of streptococcal infection tend to increase moderately. ARF incidence significantly increases with an average growth rate (AGR) of 2.5 %, while its prevalence has the opposite tendency to decrease. AGR was –29.2 %. RFD incidence showed a significant indicators increase with AGR of +1.1 %. At the same time, the prevalence tended to decrease and STP was –2 %. The situation makes it necessary to strengthen the epidemiological control of streptococcal infection and increase the alertness of doctors regarding the new cases of ARF and RHD.

Opsonic Activity of Conservative Versus Variable Regions of the Group A Streptococcus M Protein.

Group A Streptococcus (GAS) and GAS-associated infections are a global challenge, with no licensed GAS vaccine on the market. The GAS M protein is a critical virulence factor in the fight against GAS infection, and it has been a primary target for GAS vaccine development. Measuring functional opsonic antibodies against GAS is an important component in the clinical development path for effective vaccines. In this study, we compared the opsonic activity of two synthetic, self-adjuvanting subunit vaccines containing either the J8- or 88/30-epitope in Swiss outbred mice using intranasal administration. Following primary immunization and three boosts, sera were assessed for IgG activity using ELISA, and opsonization activity against seven randomly selected clinical isolates of GAS was measured. Vaccine constructs containing the conservative J8-epitope showed significant opsonic activity against six out of the seven GAS clinical isolates, while the vaccine containing the variable 88/30-epitope did not show any significant opsonic activity.

Emm Types and clusters and macrolide resistance of pediatric group A streptococcal isolates in Central Greece during 2011-2017.

BackgroundThe surveillance of emm types and macrolide susceptibility of group A streptococcus (GAS) in various areas and time periods enhances the understanding of the epidemiology of GAS infections and may guide treatment strategies and the formulation of type-specific vaccines. Greece has emerged as a country with high macrolide use. However, studies suggest a gradual reduction in macrolide consumption after 2007.MethodsDuring a 7-year period (2011–2017), 604 GAS isolates were recovered from consecutive children presenting with pharyngeal or nonpharyngeal infections in Central Greece; 517 viable isolates underwent molecular analysis, including emm typing.ResultsIsolates belonged to 20 different emm types (in decreasing order of prevalence: 1, 89, 4, 12, 28, 3, 75 and 6, accounting for 88.2% of total isolates). The emm types comprised 10 emm clusters (five most common clusters: E4, A-C3, E1, A-C4 and A-C5). The emm89 isolates were acapsular (‘new clade‘). Overall macrolide resistance rate was 15.4%, and cMLSB emerged as the predominant resistance phenotype (56.4%). The lowest annual resistance rates occurred in 2014 (13.1%), 2016 (5.5%) and 2017(8.0%) (P for trend = 0.002). Consumption of macrolide/lincosamide/streptogramin B declined by 22.6% during 2011–2017. Macrolide resistance and emm28 and emm77 types were associated (both P<0.001). The most frequently identified genetic lineages of macrolide-resistant GAS included emm28/ST52, emm77/ST63, emm12/ST36, emm89/ST101 and emm4/ST39. We estimated that 98.8% of the isolates belonged to emm types incorporated into a novel 30-valent M protein vaccine.ConclusionsIn Central Greece during 2011–2017, the acapsular emm89 isolates comprised the second most prevalent type. Susceptibility testing and molecular analyses revealed decreasing GAS macrolide resistance rates, which may be attributed to the reduction in the consumption of macrolides and/or the reduced circulation of macrolide-resistant clones in recent years. Such data may provide valuable baseline information in targeting therapeutic intervention and the formulation of type-specific GAS vaccines.

Invasive Streptococcus pyogenes Infections in

Few data are available on invasive group A Streptococcus (GAS) infections (IGASIs) in infants. We described initial clinical and laboratory features and outcomes of <3-month-old infants hospitalized for an IGASI between 2007 and 2016 in France. Patients were identified from the French National Reference Centre for streptococci. IGASI was defined by the isolation of GAS from blood cultures or from other usually sterile sites. Data collection was performed by assessing the patients' hospitalization reports. Twenty-six patients (15 males; 57.7%) were included. Among 19 cases with available data, 14 (73.7%) were household contacts of a GAS infection, reaching 8/9 (88.9%) in neonates. The diagnoses were bacteremia (n = 18; 69.2%), pleural effusion or pneumonia (n = 6; 23.1%), meningitis with brain abscess (n = 1; 3.8%), and septic arthritis (n = 1; 3.8%). Fever (n = 10; 38.5%), hemodynamic disorders (n = 11; 42.3%), respiratory disorders (n = 7; 26.9%), thrombocytopenia (n = 7; 26.9%), and neutropenia (n = 5; 19.2%) were frequently observed. The main emm-genotype was emm-1 (n = 8; 30.8%). Thirteen (50.0%) infants have been admitted to the intensive care unit, and two (7.7%) died. Respiratory disorders, high C-reactive protein level, and the need for transfusion were significantly associated with severity. IGASI remains uncommon in <3-month-old children but leads to a high morbidity. Whether an antibiotic prophylaxis for contact neonates of a patient with GAS infection decreases the risk of infection remains to be determined.

The Contribution of Pulmonary Dendritic Cells to the Induction and Regulation of T Cell Responses During Respiratory Infection

Abstract The respiratory tract represents a major portal of entry for pathogens, including Group A Streptococci (GAS). GAS represents a significant burden in cases of primary infection as well as cases of secondary bacterial pneumonia following influenza virus infection. Control of infection and eventual resolution is dependent on the local adaptive immune response. This is facilitated by the uptake and subsequent presentation of antigen by dendritic cells (DC) to T cells. Furthermore, after GAS infection, DC drive T cell development towards a Th17 immune response that is associated with pathogen control. Our hypothesis is that pulmonary DC, both resident and recruited, contribute locally to the maintenance and regulation of an effector CD4 T cell response in the context of a GAS infection. Toward this end, our studies are defining the contribution of local DC: T cell interactions in shaping the phenotype and magnitude of Th17 and Tfh responses within the lungs during GAS infection. An improved understanding of the contribution of these DC to the development and regulation of these CD4 T cells responses during GAS infection should elaborate strategies to the development of better therapies.

P028: Antibiotic prescribing and use of corticosteroids for the emergency department management of acute pharyngitis

Introduction: Acute pharyngitis is a common emergency department (ED) presentation. The Centor (Modified/McIsaac) score uses five criteria (age, tonsillar exudates, swollen tender anterior cervical nodes, absence of a cough, and history of fever) to predict Group A Streptococcus (GAS) infection. The recommendation is patients with a Centor score of 0-1 should not undergo testing and should not be given antibiotics, patients with a score of 2-3 may warrant throat cultures, and for patients with a score ≥ 4, empiric antibiotics may be appropriate. Associated pain is often first managed with acetaminophen or non-steroidal anti-inflammatory drugs, however recent evidence suggests a short course of low-to-moderate dose corticosteroids as adjunctive therapy may reduce inflammation and provide pain relief. The objective of this study was to describe the ED management of acute pharyngitis for adult patients presenting to an academic ED over a two-year study period. Methods: This was a retrospective chart review of all adult (&gt; 17 years) patients presenting to Mount Sinai Hospital ED with a discharge diagnosis of acute pharyngitis (ICD-10 code J02.9) from January 1st 2016 to December 31st 2018. Trained research personnel reviewed medical records and extracted data using a computerized, data abstraction form. Results: Of the 638 patients included in the study, 286 (44.8%) had a Centor score of 0-1, 328 (51.4%) had a score of 2-3, and 24 (3.8%) had a score of ≥ 4. Of those with a Centor score of 0-1, 83 (29.0%) had a throat culture, 88 (30.8%) were prescribed antibiotics, 15 (5.2%) were positive for GAS and 74 (25.9%) were given corticosteroids in the ED or at discharge. Of those with a Centor score of 2-3, 156 (47.6%) had a throat culture, 220 (67.1%) were prescribed antibiotics, 44 (13.4%) were positive for GAS, and 145 (44.2%) were given corticosteroids. Of those with a Centor score ≥ 4, 14 (58.3%) had a throat culture, 18 (75.0%) were prescribed antibiotics, 7 (29.2%) were positive for GAS and 12 (50.0%) were given corticosteroids. Conclusion: As predicted, a higher Centor score was associated with higher risk of GAS infection, increased antibiotic prescribing and use of corticosteroids. Many patients with low Centor scores were prescribed antibiotics and also had throat cultures. Further work is required to understand clinical decision making for the management of acute pharyngitis.

Structure–Activity Analysis of Cyclic Multicomponent Lipopeptide Self-Adjuvanting Vaccine Candidates Presenting Group A Streptococcus Antigens

Group A Streptococcus (GAS) infection causes a range of life-threatening diseases, including rheumatic heart disease. Cyclic peptides offer an attractive solution for presentation of short peptide antigens due to their stability and structurally constrained conformation. We investigated a cyclic carrier decapeptide incorporating a B cell GAS peptide epitope, a universal T helper epitope, and a synthetic toll-like receptor 2-targeting moiety as a possible self-adjuvanting GAS vaccine. A structure-activity relationship of the cyclic lipopeptide vaccine showed successful induction of J8-specific systemic immunoglobulin G (IgG) antibodies when administered subcutaneously without an additional adjuvant. Interestingly, the physical mixture control induced the highest titers of all vaccine compounds, with antibodies from mice immunized with this physical mixture control shown to effectively opsonize multiple strains of clinically isolated GAS bacteria. This study showed the capability for a self-adjuvanting cyclic delivery system to act as a vehicle for the delivery of GAS peptide antigens to treat GAS infections.

Immunization With a Secreted Esterase Protects Mice Against Multiple Serotypes (M1, M3, and M28) of Group A Streptococcus.

Streptococcal secreted esterase (Sse) is a platelet-activating factor acetylhydrolase that is critical for Group A Streptococcus (GAS) skin invasion and innate immune evasion. There are two Sse variant complexes that share >98% identity within each complex but display about 37% variation between the complexes in amino acid sequences. Sse immunization protects mice against lethal infection and skin invasion in subcutaneous infection with the hypervirulent CovRS mutant strain, MGAS5005. However, it is not known whether Sse immunization provides significant protection against infection of GAS with functional CovRS and whether immunization with Sse of one variant complex provides protection against infection of GAS that produces Sse of another variant complex. This study was designed to address these questions. Mice were immunized with recombinant Sse of M1 GAS (SseM1) and challenged with MGAS5005 (serotype M1, CovS mutant, and Sse of variant complex I), MGAS315 (M3, CovS mutant, and Sse of variant complex I), MGAS2221 (M1, wild-type CovRS, and Sse of variant complex I), and MGAS6180 (M28, wild-type CovRS, and Sse of variant complex II). SseM1 immunization significantly increased survival rates of mice in subcutaneous MGAS5005 and intraperitoneal MGAS6180 challenges and showed consistently higher or longer survival in the other challenges. Immunized mice had smaller skin lesion and higher neutrophil responses in subcutaneous infections and lower GAS burdens in spleen, liver, and kidney in most of the challenge experiments than control mice. SseM1 immunization enhanced proinflammatory responses. These data suggest that Sse immunization has a broad benefit against GAS infections that can vary in extent from strain to strain and that the benefit may be due to the immunization-enhanced proinflammatory responses. In particular, immunization with SseM1 can provide protection against M28 GAS infection even though its Sse and SseM1 have significant variations.

Group A Streptococci Shift the Balance of Muscular Mitochondrial Dynamics

The most severe form of invasive group A Streptococcus (GAS) disease is necrotizing fasciitis described as rapidly muscle destruction. Muscles are responsible for energetic functions which are mostly provided by proper mitochondrial function. Mitochondria are the highly dynamic organelles, constantly undergoing fusion and fission. Pathogen aim at preserving the bacterial replication niche by targeting to mitochondria and inducing cell death. This prompted us to specifically analyze the effect on muscular mitochondria upon GAS infection. By using electron microscopic analysis of GAS‐infected muscle tissue, the muscle is strongly damaged and the mitochondria are fragmented and structurally abnormal. Further, we found GAS alters the morphology of cell mitochondria, inducing fragmentation of the mitochondria network which accompanied by decreasing the levels of fusion protein in mouse muscle cell, C2C12. Furthermore, our data demonstrate that mitochondrial function is not strictly dependent on morphology. Our results reveal that disruption of fusion induces more profound defects on lacking of fission during GAS infection, and imply that while fusion is required throughout life, fission is more important later in life likely to combat bacterial infection. Overall, our study provides important new insights into the central role of mitochondrial dynamics in innate immunity.Support or Funding InformationMOST 108‐2320‐B‐006‐013

Group A Streptococcus May Suppress V‐ATPase Activity To Inhibit THP‐1 Macrophage Phagolysosomal Acidification

Infection with Group A Streptococcus (GAS), a human‐specific bacterial pathogen, results in a multitude of diseases ranging from strep throat to toxic shock, and can lead to serious post‐infection sequelae such as rheumatic heart disease. During infection, macrophages engulf bacteria within the phagosome, which then fuses with the lysosome. Subsequent acidification of the phagolysosome allows for degradation of bacteria. Our lab and others have shown that during GAS infection, phagolysosomes are not acidified, allowing GAS to persist. The overall objective of this study was to determine how GAS inhibits phagolysosomal acidification. Our lab has previously shown that secretion of the pore‐forming toxin Streptolysin O (SLO) causes phagolysosomal leakage of relatively large proteins. In our current work, we demonstrate that during infection using a mutant strain lacking SLO or using heat‐killed bacteria that GAS‐infected phagosomes are still not acidified, indicating that lack of acidification is not due to mere leakage. Phagolysosomes undergo acidification using the enzyme complex vacuolar ATPase (VATPase), which needs to properly assemble cytosolic and membrane components to function. To assess whether V‐ATPase assembly was affected, infected cells were fractionated and recruitment of cytosolic V‐ATPase subunits to the phagosome membrane was monitored by Western blot. Our preliminary results indicate that V‐ATPase assembly is not inhibited, suggesting that GAS may instead prevent acidification by inhibiting V‐ATPase activity. We are currently conducting further research to investigate this potential inhibitory mechanism of GAS. Restoring the ability of lysosomes to become acidified and activate proteolytic enzymes may serve as a means to enhance macrophage function, both bactericidal capacity and antigen presentation capability, to elicit effective long‐lasting immunity.Support or Funding InformationAmerican Heart Association (17GRNT3410851), Occidental College Undergraduate Research Center

Isolating Group A Streptococcus Infected Phagosomes

The number of Group A Streptococcus (GAS) infections has dramatically risen in the last several decades and is an increasing public health concern worldwide. Normally, the body’s innate immune system responds to pathogens by phagocytosing and degrading the bacteria within the phagolysosome by a number of different molecular processes, including the oxidative burst response and lysosomal acidification. However, it has been shown by our lab and others that GAS has a number of different mechanisms to subvert immune defenses, including evasion of digestion in the phagolysosome. Because it is unusual for pathogens to reside in phagolysosomes, we want to develop a proteomic profile of GAS‐infected phagosomes. Because isolation of phagosomes is technically difficult, we set out to develop a method to isolate and purify GAS‐infected phagosomes using magnetic particles. We coupled magnetic particles to live GAS and demonstrated that we can effectively isolate viable coupled bacteria. Following infection of THP‐1 macrophages, we employ a magnetic separation protocol that yields a high concentration of GAS‐infected phagosomes largely absent of other organellar contamination. With these magnetically‐isolated GAS phagolysosomes, we aim to conduct a proteomics survey using mass spectrometry to characterize GAS phagosome modification to elucidate its mechanisms of survival. Our work will help identify novel therapeutic targets and strategies to treat GAS infections to enhance normal immune processes aimed at combating GAS infections, and thus limiting the use of antibiotics.Support or Funding InformationAmerican Heart Association (17GRNT3410851), Occidental College Undergraduate Research Center

Group A Streptococcus Prevents NOX‐2 Complex Formation to Limit Oxidative Burst in Macrophages

Group A Streptococcus (GAS) is a Gram positive bacterial pathogen responsible for about 500,000 deaths a year, causing diseases ranging from superficial pharyngitis to acute rheumatic fever. Immune cells such as macrophages and neutrophils generate reactive oxygen species (ROS) to aid in bacterial destruction. Unlike other bacterial species, GAS lacks enzymes that can detoxify ROS. We thus sought to determine whether GAS can prevent ROS production altogether. We found that THP‐1 macrophages infected with GAS indeed produce less ROS as measured by DCF‐DA assays. We next sought to determine whether GAS affect the formation of the NOX‐2 enzyme complex that produces ROS. The NOX‐2 complex is made up of cytosolic and membrane bound subunits that come together when activated by the phagocytosis of a pathogen. Analysis of whole cell lysates by Western blot indicate decreased NOX‐2 complex formation in GAS‐infected cells compared with controls. Furthermore, we analyzed recruitment of the cytosolic components to the phagosome membrane by analyzing crude cell fractions. These results confirmed that GAS infection prevents NOX‐2 complex assembly. Interestingly, our data indicates that GAS can not only prevent the formation of the NOX‐2 complex, but can disassemble the complex in stimulated cells. Our findings suggest that one way GAS evades defenses of our immune system is by preventing the formation of toxic ROS. Inducing ROS production in GAS‐infected cells would therefore provide an alternative to antibiotic treatment for GAS infections.Support or Funding InformationAmerican Heart Association (17GRNT3410851), Occidental College Undergraduate Research Center

Th17 lymphocytes drive vascular and neuronal deficits in a mouse model of postinfectious autoimmune encephalitis

Antibodies against neuronal receptors and synaptic proteins are associated with a group of ill-defined central nervous system (CNS) autoimmune diseases termed autoimmune encephalitides (AE), which are characterized by abrupt onset of seizures and/or movement and psychiatric symptoms. Basal ganglia encephalitis (BGE), representing a subset of AE syndromes, is triggered in children by repeated group A Streptococcus (GAS) infections that lead to neuropsychiatric symptoms. We have previously shown that multiple GAS infections of mice induce migration of Th17 lymphocytes from the nose into the brain, causing blood-brain barrier (BBB) breakdown, extravasation of autoantibodies into the CNS, and loss of excitatory synapses within the olfactory bulb (OB). Whether these pathologies induce functional olfactory deficits, and the mechanistic role of Th17 lymphocytes, is unknown. Here, we demonstrate that, whereas loss of excitatory synapses in the OB is transient after multiple GAS infections, functional deficits in odor processing persist. Moreover, mice lacking Th17 lymphocytes have reduced BBB leakage, microglial activation, and antibody infiltration into the CNS, and have their olfactory function partially restored. Th17 lymphocytes are therefore critical for selective CNS entry of autoantibodies, microglial activation, and neural circuit impairment during postinfectious BGE.

Group A streptococcal bacteremias in Southwest Finland 2007\u20132018: epidemiology and role of infectious diseases consultation in antibiotic treatment selection

The incidence of invasive group A streptococcal (GAS) infections has shown a fluctuating but increasing trend in Finland. The impact of infectious diseases specialist consultation (IDSC) on the antimicrobial therapy of GAS bacteremia has not been studied earlier. A retrospective study on adult GAS bacteremia in The Hospital District of Southwest Finland (HDSWF) was conducted from 2007 to 2018. Data on incidence of bacteremic GAS cases were gathered from the National Infectious Disease Register. Clinical data were obtained by reviewing the electronic patient records. The overall incidence of GAS bacteremia in HDSWF was 3.52/100,000, but year-to-year variation was observed with the highest incidence of 7.93/100,000 in 2018. A total of 212 adult GAS bacteremia cases were included. A record of IDSC was found (+) in 117 (55.2%) cases, not found (−) in 71 (33.5%) cases and data were not available in 24 (11.3%) cases. Among IDSC+ cases, 57.3% were on penicillin G treatment whereas in the group IDSC− only 22.5%, respectively (OR = 4.61, 95% CI 2.37–8.97; p < 0.001). The use of clindamycin as adjunctive antibiotic was more common among IDSC+ (54.7%) than IDSC− (21.7%) (OR = 4.51, 95% CI 2.29–8.87; p < 0.001). There was an increasing trend in incidence of GAS bacteremia during the study period. Narrow-spectrum beta-lactam antibiotics were chosen, and adjunctive clindamycin was more commonly used, if IDSC took place. This highlights the importance of availability of IDSC but calls for improved practice among infectious diseases specialists by avoiding combination therapy with clindamycin in non-severe invasive GAS infections.

Nicotinamide Increases Intracellular NAD+ Content to Enhance Autophagy-Mediated Group A Streptococcal Clearance in Endothelial Cells.

Group A streptococcus (GAS) is a versatile pathogen that causes a wide spectrum of diseases in humans. Invading host cells is a known strategy for GAS to avoid antibiotic killing and immune recognition. However, the underlying mechanisms of GAS resistance to intracellular killing need to be explored. Endothelial HMEC-1 cells were infected with GAS, methicillin-resistant Staphylococcus aureus (MRSA) and Salmonella Typhimurium under nicotinamide (NAM)-supplemented conditions. The intracellular NAD+ level and cell viability were respectively measured by NAD+ quantification kit and protease-based cytotoxicity assay. Moreover, the intracellular bacteria were analyzed by colony-forming assay, transmission electron microscopy, and confocal microscopy. We found that supplementation with exogenous nicotinamide during infection significantly inhibited the growth of intracellular GAS in endothelial cells. Moreover, the NAD+ content and NAD+/NADH ratio of GAS-infected endothelial cells were dramatically increased, whereas the cell cytotoxicity was decreased by exogenous nicotinamide treatment. After knockdown of the autophagy-related ATG9A, the intracellular bacterial load was increased in nicotinamide-treated endothelial cells. The results of Western blot and transmission electron microscopy also revealed that cells treated with nicotinamide can increase autophagy-associated LC3 conversion and double-membrane formation during GAS infection. Confocal microscopy images further showed that more GAS-containing vacuoles were colocalized with lysosome under nicotinamide-supplemented conditions than without nicotinamide treatment. In contrast to GAS, supplementation with exogenous nicotinamide did not effectively inhibit the growth of MRSA or S. Typhimurium in endothelial cells. These results indicate that intracellular NAD+ homeostasis is crucial for controlling intracellular GAS infection in endothelial cells. In addition, nicotinamide may be a potential new therapeutic agent to overcome persistent infections of GAS.

Polyacrylate-Peptide Antigen Conjugate as a Single-Dose Oral Vaccine against Group A Streptococcus.

Group A Streptococcus (GAS)-associated rheumatic heart disease is a leading cause of death caused by GAS infection. While antibiotics can treat the infection in most cases, growing antibiotic resistance, late medical intervention, and recurrent infection are major obstacles to the effective treatment of GAS-associated diseases. As GAS infection typically originates from the bacterial colonization of mucosal tissue in the throat, an oral vaccine that can generate both systemic and mucosal immune responses would solve problems associated with traditional medical interventions. Moreover, orally delivered vaccines are more easily administered and less expensive for mass immunization. In this study, the B-cell epitope J8, derived from GAS M protein, and universal T-helper Pan HLA-DR-binding epitope peptide (PADRE), were conjugated to poly (methyl acrylate) (PMA) to form a self-assembled nanoparticle vaccine candidate (PMA-P-J8). Strong systemic and mucosal immune responses were induced upon single oral immunization of mice with the conjugate. The antibodies generated were opsonic against GAS clinical isolates as measured after boost immunization. Thus, we developed a simple conjugate as an effective, adjuvant-free oral peptide-based vaccine.

Isolation of Monoclonal Antibodies to Group A Streptococcus Antigens Using Phage Display.

High-affinity monoclonal antibodies are valuable tools for studying the humoral immune response to Group A Streptococcus (GAS) antigens. This protocol describes a method for the selection of monoclonal antibody fragments that bind to GAS antigens using either naïve or immune repertoires displayed on the surface of M13 bacteriophage. Clones that specifically bind to GAS antigens are enriched for during the biopanning process, in which antibody-phage clones bind to an immobilized GAS antigen and are then washed, eluted, and amplified for subsequent rounds of selection. After the final round of biopanning, individual clones are screened by phage enzyme-linked immunosorbent assay (ELISA), and unique clones are identified by DNA fingerprinting and sequencing. The isolated monoclonal antibodies can be used to explore antibody-antigen interactions in molecular detail and provide insight into the protective mechanisms from GAS infection.

Lancefield Whole Blood Killing Assay to Evaluate Vaccine Efficacy.

While the Lancefield whole blood killing assay is named after the renowned streptococcal researcher Rebecca Lancefield, the protocol was first described by Todd in 1927 (Br J Exp Pathol 8:1-5, 1927). Initially, the assay was used to identify novel Group A Streptococcal (GAS) serotypes through the supplementation of non-immune human blood (often from infants) with type-specific antisera prepared in rabbits (Lancefield, J Exp Med 106:525-544, 1957; Maxted, Br J Exp Pathol 37:415-422, 1956) and to demonstrate the impressive longevity of type-specific immunity in patients following invasive GAS infection (Lancefield, J Exp Med 110:271-292, 1959). The modern assay is routinely used to screen defined GAS mutants (Wessels, Bronze, Proc Natl Acad Sci U S A 91:12238-12242, 1994; Zinkernagel et al., Cell Host Microbe 4:170-178, 2008) or transposon libraries (Le Breton et al., Infect Immun 81:862-875, 2013) for enhanced susceptibility to opsonophagocytic killing or to screen vaccine antisera (Salehi et al., mSphere 3:e00617-e00618, 2018) or other serological preparations (Reglinski et al., Sci Rep 5:15825, 2015) for anti-streptococcal activity.

Prevalence of Group A Streptococcus in Primary Care Patients and the Utility of C-Reactive Protein and Clinical Scores for Its Identification in Thailand.

.Pharyngitis is usually caused by a viral infection for which antibiotics are often unnecessarily prescribed, adding to the burden of antimicrobial resistance. Identifying who needs antibiotics is challenging; microbiological confirmation and clinical scores are used but have limitations. In a cross-sectional study nested within a randomized controlled trial, we estimated the prevalence and antibiotic susceptibility profiles of group A Streptococcus (GAS) in patients presenting to primary care with a sore throat and fever in northern Thailand. We then evaluated the use of C-reactive protein (CRP) and clinical scores (Centor and FeverPAIN) to identify the presence of GAS. One hundred sixty-nine patients were enrolled, of whom 35 (20.7%) had β-hemolytic Streptococci (BHS) isolated from throat swab culture, and 11 (6.5%) had GAS. All GAS isolates were sensitive to penicillin G. The median CRP of those without BHS isolation was 10 mg/L (interquartile range [IQR] ≤ 8–18), compared with 18 mg/L (IQR 9–71, P = 0.0302) for those with GAS and 14 mg/L (IQR ≤ 8–38, P = 0.0516) for those with any BHS isolated. However, there were no significant relationships between CRP > 8 mg/L (P = 0.112), Centor ≥ 3 (P = 0.212), and FeverPAIN ≥ 4 (P = 1.000), and the diagnosis of GAS compared with no BHS isolation. Identifying who requires antibiotics for pharyngitis remains challenging and necessitates further larger studies. C-reactive protein testing alone, although imperfect, can reduce prescribing compared with routine care. Targeted CRP testing through clinical scoring may be the most cost-effective approach to ruling out GAS infection.

Erratum for Barth et al., "Molecular Epidemiology of Noninvasive and Invasive Group A Streptococcal Infections in Cape Town".

Volume 4, no. 5, e00421-19, 2019, [https://doi.org/10.1128/mSphere.00421-19][1]. Table 2: in the last column, row 7, “2222 (8)” should read “22 (8).” [1]: /lookup/doi/10.1128/mSphere.00421-19