Disease manifestations and pathogenic mechanisms of Group A Streptococcus.

Streptococcus pyogenes, also known as group A Streptococcus (GAS), causes mild human infections such as pharyngitis and impetigo and serious infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. Furthermore, repeated GAS infections may trigger autoimmune diseases, including acute poststreptococcal glomerulonephritis, acute rheumatic fever, and rheumatic heart disease. Combined, these diseases account for over half a million deaths per year globally. Genomic and molecular analyses have now characterized a large number of GAS virulence determinants, many of which exhibit overlap and redundancy in the processes of adhesion and colonization, innate immune resistance, and the capacity to facilitate tissue barrier degradation and spread within the human host. This improved understanding of the contribution of individual virulence determinants to the disease process has led to the formulation of models of GAS disease progression, which may lead to better treatment and intervention strategies. While GAS remains sensitive to all penicillins and cephalosporins, rising resistance to other antibiotics used in disease treatment is an increasing worldwide concern. Several GAS vaccine formulations that elicit protective immunity in animal models have shown promise in nonhuman primate and early-stage human trials. The development of a safe and efficacious commercial human vaccine for the prophylaxis of GAS disease remains a high priority.

Post-fever retinitis: a single center experience from south India.

Various retinal manifestations can occur following a febrile illness due to viral, bacterial or protozoal etiology. As there are limited data in the literature, we undertook this study to analyse the clinical presentation of post-fever retinitis due to various etiologies, as well as its course and management. This was a retrospective study of 14 consecutive cases who presented to the Vitreo Retina Department of our hospital over a 1-year period between January 2010 and December 2010. All patients underwent detailed ophthalmic examination and relevant investigations including fundus fluorescein angiography and optical coherence tomography (OCT). Basic and specific investigations were performed as necessary. All patients were given systemic steroids which were tapered based on clinical response. Twenty-one eyes of 14 patients (7 bilateral, 7 unilateral) were studied. Onset of ocular symptoms was approximately 3 weeks after fever. Four patients had specific etiology-one each of chikungunya, enteric fever, malaria and abdominal abscess with pneumococcal pneumonia. The presenting visual acuity of the affected eyes averaged 2/60. Six eyes had relative afferent pupillary defect. All patients had solitary or multiple patches of retinitis at the posterior pole and exudation at the macula. OCT through the lesions revealed inner retinal hyperreflectivity and thickening with after-shadowing. All patients showed improvement in vision with unilateral cases improving to an average of 6/12 and bilateral cases improving to an average of 6/24. Patients also showed resolution of retinitis, macular edema and serous detachment. Post-fever retinitis as a condition manifested approximately 3 weeks after onset of fever. Irrespective of the cause of the fever, clinical presentation of cases was similar with inner retinitis at the posterior pole and a favourable response to steroids, suggesting a possible immunological basis for this condition.

Molecular investigation of bacterial communities on the inner and outer surfaces of peripheral venous catheters.

Peripheral venous catheters (PVCs) are some of the most widely used medical devices in hospitals worldwide. PVC-related infections increase morbidity and treatment costs. The inner surfaces of PVCs are rarely examined for the population structure of bacteria, as it is generally believed that bacteria at this niche are similar to those on the external surface of PVCs. We primarily test this hypothesis and also study the effect of antibiotic treatment on bacterial communities from PVC surfaces. The inner and outer surfaces of PVCs from 15 patients were examined by 454 GS FLX Titanium 16S rRNA sequencing and the culture method. None of the PVCs were colonised according to the culture method and none of the patients had a bacteraemia. From a total of 127,536 high-quality sequence reads, 14 bacterial phyla and 268 diverse bacterial genera were detected. The number of operational taxonomic units for each sample was in the range of 86-157, even though 60 % of patients had received antibiotic treatment. Stenotrophomonas maltophilia was the predominant bacterial species in all the examined PVC samples. There were noticeable but not statistically significant differences between the inner and outer surfaces of PVCs in terms of the distribution of the taxonomic groups. In addition, the bacterial communities on PVCs from antibiotic-treated patients were significantly different from untreated patients. In conclusion, the surfaces of PVCs display complex bacterial communities. Although their significance has yet to be determined, these findings alter our perception of PVC-related infections.

Rabies in a blind patient: confusion after corneal transplantation.

Two blind persons received corneal transplants from a single donor who showed no signs of rabies before he died. One of the recipients, a young girl, died 16 days later of rabies and the other recipient survived. We discuss the possible mode of transmission of rabies to the first recipient and the management of the second recipient.

New multi-determinant strategy for a group A streptococcal vaccine designed for the Australian Aboriginal population.

Infection with group A streptococci can result in acute and post-infectious pathology, including rheumatic fever and rheumatic heart disease. These diseases are associated with poverty and are increasing in incidence, particularly in developing countries and amongst indigenous populations, such as Australia's Aboriginal population, who suffer the highest incidence worldwide. Immunity to group A streptococci is mediated by antibodies against the M protein, a coiled-coil alpha helical surface protein of the bacterium. Vaccine development faces two substantial obstacles. Although opsonic antibodies directed against the N terminus of the protein are mostly responsible for serotypic immunity, more than 100 serotypes exist. Furthermore, whereas the pathogenesis of rheumatic fever is not well understood, increasing evidence indicates an autoimmune process. To develop a suitable vaccine candidate, we first identified a minimum, helical, non-host-cross-reactive peptide from the conserved C-terminal half of the protein and displayed this within a non-M-protein peptide sequence designed to maintain helical folding and antigenicity, J14 (refs. 8,9). As this region of the M protein is identical in only 70% of group A streptococci isolates, the optimal candidate might consist of the conserved determinant with common N-terminal sequences found in communities with endemic group A streptococci. We linked seven serotypic peptides with J14 using a new chemistry technique that enables the immunogen to display all the individual peptides pendant from an alkane backbone. This construct demonstrated excellent immunogenicity and protection in mice.

Molecular markers for discriminating Streptococcus pyogenes and S. dysgalactiae subspecies equisimilis.

Given the increasing aetiological importance of Streptococcus dysgalactiae subspecies equisimilis in diseases which are primarily attributed to S. pyogenes, molecular markers are essential to distinguish these species and delineate their epidemiology more precisely. Many clinical microbiology laboratories rely on agglutination reactivity and biochemical tests to distinguish them. These methods have limitations which are particularly exacerbated when isolates with mixed properties are encountered. In order to provide additional distinguishing parameters that could be used to unequivocally discriminate these two common pathogens, we assess here three molecular targets: the speB gene, intergenic region upstream of the scpG gene (IRSG) and virPCR. Of these, the former two respectively gave positive and negative results for S. pyogenes, and negative and positive results for S. dysgalactiae subsp. equisimilis. Thus, a concerted use of these nucleic acid-based methods is particularly helpful in epidemiological surveillance to accurately assess the relative contribution of these species to streptococcal infections and diseases.

Genes for the majority of group a streptococcal virulence factors and extracellular surface proteins do not confer an increased propensity to cause invasive disease.

BACKGROUND: The factors behind the reemergence of severe, invasive group A streptococcal (GAS) diseases are unclear, but it could be caused by altered genetic endowment in these organisms. However, data from previous studies assessing the association between single genetic factors and invasive disease are often conflicting, suggesting that other, as-yet unidentified factors are necessary for the development of this class of disease. METHODS: In this study, we used a targeted GAS virulence microarray containing 226 GAS genes to determine the virulence gene repertoires of 68 GAS isolates (42 associated with invasive disease and 28 associated with noninvasive disease) collected in a defined geographic location during a contiguous time period. We then employed 3 advanced machine learning methods (genetic algorithm neural network, support vector machines, and classification trees) to identify genes with an increased association with invasive disease. RESULTS: Virulence gene profiles of individual GAS isolates varied extensively among these geographically and temporally related strains. Using genetic algorithm neural network analysis, we identified 3 genes with a marginal overrepresentation in invasive disease isolates. Significantly, 2 of these genes, ssa and mf4, encoded superantigens but were only present in a restricted set of GAS M-types. The third gene, spa, was found in variable distributions in all M-types in the study. CONCLUSIONS: Our comprehensive analysis of GAS virulence profiles provides strong evidence for the incongruent relationships among any of the 226 genes represented on the array and the overall propensity of GAS to cause invasive disease, underscoring the pathogenic complexity of these diseases, as well as the importance of multiple bacteria and/or host factors.

Antigenic diversity within a family of M proteins from group A streptococci: evidence for the role of frameshift and compensatory mutations.

The genes (emm) encoding M proteins, from isolates of group-A streptococci (GAS) serotyped as M52, M53, M80 and M nontypeable (MNT; serologically related to M53 and M80), were examined. Characterization of emm from these GAS revealed some discrepancies with serotyping, illustrating the difficulty in serotype determination when cross-reactions occur. DNA sequences corresponding to the N-terminal region of M proteins from the isolates showed considerable similarity both in the hypervariable region and the repeat regions. We propose that these serotypes form a family of closely related M types. Frameshift mutations in the hypervariable region followed by a corrective (compensatory) frameshift were observed. This may be an effective mechanism for generating antigenic diversity in the M protein.

Vaccine development for group A streptococcus infections and associated disease.

Group A streptococcus (GAS) is responsible for a number of diseases ranging from uncomplicated pharyngitis through to life-treating invasive and post-infectious diseases such as necrotizing fasciitis and rheumatic heart disease. GAS associated diseases occur globally and are serious problems in many developing nations and indigenous populations of many developed nations. This, and the resurgence in industrialized countries, and increased virulence of GAS in the 1980s highlight the need of cost-effective control strategies. Here we highlight the GAS diseases that are still a problem in many populations and discuss potentially useful strategies to combat GAS infections and disease.

Limited repertoire of the C-terminal region of the M protein in Streptococcus pyogenes.

We have amplified genomic sequences (emm) that may encode M protein from strains of Streptococcus pyogenes using the polymerase chain reaction (PCR). Genomic DNA from 22 isolates representing 14 M serotypes was selected for the study. Primers which corresponded to the observed N-terminal signal sequence and the variable C-terminal sequences of emm6, emm49 and ennX were used. PCR products using emm6 and emm49 oligonucleotides were classified into two mutually exclusive groups which correspond to the presence or absence of serum opacity factor. These findings support the concept of limited heterogeneity in the C-terminal sequences of the M protein.

Mapping of transcripts encoded by the plasmid in Chlamydia trachomatis.

Mapping of the plasmid-encoded RNA of the intracellular parasite, Chlamydia trachomatis revealed that the upstream control elements are different from those of other Gram-negative bacteria. A tetranucleotide, AYAA was found near the -10 position, in 5 out of 8 upstream sequences described so far. The plasmid also has a developmentally regulated promoter. The chlamydial upstream elements do not function as promoters in E. coli and vice versa. An E. coli promoter-like sequence has been found to occur fortuitously upstream from the plasmid-encoded dnaB gene. Such sequences may be evolutionary relics.

Antibodies to streptococcal inhibitor of complement function and M peptides in a post-streptococcal glomerulonephritis endemic region of Australia.

Post-streptococcal glomerulonephritis (PSGN) is an immune-mediated disease in which an immune complex containing a streptococcal antigen are deposited in affected glomeruli. Strains of only some M types are known to be associated with PSGN. A secretory protein called SIC inhibits complement function. Whereas all M1 and M57 strains express closely related SIC (CRS), all M12 and M55 strains express distantly related SIC (DRS) proteins. Strains belonging to these four M types are historically associated with PSGN. This study used ELISA to analyse 112 sera from individuals with a recorded history of PSGN and 86 sera from individuals who had no such recorded history, all from a PSGN endemic region in tropical Australia. Antibody reactions to CRS, DRS and peptides corresponding to the N-termini of M1, M5, M12, M49, M55 and M57 antigens were assessed. A large proportion of the population showed reactions to each of these antigens and there was no correlation between CRS seropositivity and antibodies to CRS-positive M types. Likewise there was no correlation between DRS seropositivity and antibodies to DRS-positive M types. Interestingly, in this community endemic for PSGN a significantly higher proportion of DRS seropositive subjects had a recorded history of PSGN than did DRS seronegative subjects. DRS may have a predictive value for PSGN diagnosis or a role in PSGN pathogenesis.

Oligonucleotide probes to identify three sibling species of the Anopheles farauti Laveran complex (Diptera: Culicidae).

We have sequenced recombinant plasmid probes for each of 3 Anopheles farauti complex species and identified internal oligonucleotides of 25-26 base pairs, specific for each member of the complex. Synthetic oligonucleotides oAf1, oAf2 and oAf3, when radiolabelled and hybridized with deoxyribonucleic acid from An. farauti, reacted as highly specific probes for An. farauti numbers 1, 2 and 3 respectively. These probes are effective on air-dried or alcohol-preserved larval, pupal or adult specimens.

An unexpected response to Torulopsis glabrata fusion products to X-irradiation.

Intra-species fusion products of Saccharomyces cerevisiae, Saccharomyces unisporus and Torulopsis glabrata have been isolated following polyethylene glycol-induced fusion of protoplasts and selection for prototrophic colonies. Staining with lomofungin showed that all fusion products were uninucleate. Measurement of DNA content mostly gave values between haploid and diploid levels indicating that the majority of fusion products were aneuploid, Nevertheless fusion products of S. cerevisiae and S. unisporus were, as expected, more resistant to X-irradiation than their haploid parents. By contrast, the X-ray dose-response curve of all T. glabrata fusion products was indistinguishable from their progenitors despite the fact that mitotic segregants could be recovered amongst the survivors to X-rays. A possible explanation for the behaviour towards X-rays of T. glabrata fusion products is that this species lacks a DNA repair pathway involving recombination between homologous chromosomes. We conclude from this study that the shape of the X-ray dose-response curve should not be taken to indicate the ploidy of new yeast isolates without supporting data.

Prospecting for new group A streptococcal vaccine candidates.

BACKGROUND & OBJECTIVES: Most group A streptococcal (GAS) vaccine strategies focused on the surface M protein of the GAS. However, vaccine based on M protein have some drawbacks. In the present study, we used two approaches to identify new proteins and peptides that may have utility as vaccine candidates. METHODS: A whole gel elution procedure was used to separate GAS surface antigens into 9 size fractionated pools. Mice were vaccinated with each pool and antibody titre, opsonic ability and protective capacity measured. In an alternative approach BioInformatics was used to identify putative GAS surface proteins. Peptides from within these proteins were then selected on the basis of predicted antigenicity or location. These peptides were conjugated to keyhole lymphocyanin (KLH) and immunogenicity measured in a mouse model. RESULTS: One pool of GAS surface proteins (approximately 29kDa) induced antibodies that were both opsonic and potentially protective. Immunoflourescent microscopy demonstrated that these antibodies bound to the surface of M1 GAS. Amino acid sequencing subsequently identified superoxide dismutase as the major antigen in this pool. A BioInformatic search of the M1 GAS genome and subsequent analysis identified several peptides that fulfilled criteria as potential vaccine candidates. Each peptide when conjugated to KLH was able to induce a strong antibody response. INTERPRETATION & CONCLUSION: Several new antigens were identified that may have potential as vaccine targets. A future GAS vaccine may have multiple peptide epitopes, providing protection against multiple GAS strains.

Immune responses of a liposome/ISCOM vaccine adjuvant against streptococcal fibronectin binding protein 1 (Sfb1) in mice.

BACKGROUND & OBJECTIVES: The fibronectin binding protein Sfb1 of Streptococcus pyogenes is a well characterised antigen which induces protection against lethal challenge with group A streptococcus (GAS) when adjuvanted with cholera toxin B-subunit (CTB). As an alternative to CTB adjuvanted intranasal immunisations we investigated the immune responses generated in mice using Sfb1 incorporated in to the skin and mucosal adjuvant SAMA4. METHODS: Mice (BALB/c) were vaccinated intradermally with 100 microl of either SAMA4 (adjuvant only group) or SAMA4/Sfb1 and were boosted 7 days later. Mice vaccinated with CTB based vaccines were immunised by intranasal inoculation with a mixture containing 30 microg Sfb1 and 10 microg CTB on days 1, 3, 5 and 15. At 14 days after the last booster immunisation the immune response was characterised and mice were challenged with 10(8) CFU of S. pyogenes. RESULTS: Mice vaccinated with SAMA4/Sfb1 elicited a Sfb1-specific IgG response in the sera that was significantly higher than that seen in control mice and mice immunised with the adjuvant only (P<0.05). No significant differences were seen for specific IgA antibodies in the sera in all groups examined. Compared with non-immunised and adjuvant only immunised controls, mice immunised with the Sfb1/SAMA4 vaccine exhibited a significant increase (P<0.05) in the number of Sfb1 reactive spleen cells in lymphoproliferation assays which were three fold higher than those seen for mice vaccinated with the Sfb1/CTB vaccine. Mice vaccinated with CTB/Sfb1 had the highest level of protection (80%) as where mice vaccinated with SAMA4 and SAMA4/Sfb1 displayed no protection (20% and 40%). INTERPRETATION & CONCLUSION: These data suggest that the SAMA4 adjuvant used in this study fails to elicit protective immunity in BALB/c mice when used to adjuvant the known protective antigen Sfb1.

Updated model of group A Streptococcus M proteins based on a comprehensive worldwide study.

Group A Streptococcus (GAS) M protein is an important virulence factor and potential vaccine antigen, and constitutes the basis for strain typing (emm-typing). Although >200 emm-types are characterized, structural data were obtained from only a limited number of emm-types. We aim to evaluate the sequence diversity of near-full-length M proteins from worldwide sources and analyse their structure, sequence conservation and classification. GAS isolates recovered from throughout the world during the last two decades underwent emm-typing and complete emm gene sequencing. Predicted amino acid sequence analyses, secondary structure predictions and vaccine epitope mapping were performed using MUSCLE and Geneious software. A total of 1086 isolates from 31 countries were analysed, representing 175 emm-types. emm-type is predictive of the whole protein structure, independent of geographical origin or clinical association. Findings of an emm-type paired with multiple, highly divergent central regions were not observed. M protein sequence length, the presence or absence of sequence repeats and predicted secondary structure were assessed in the context of the latest vaccine developments. Based on these global data, the M6 protein model is updated to a three representative M protein (M5, M80 and M77) model, to aid in epidemiological analysis, vaccine development and M protein-related pathogenesis studies.