Streptococcus suis infection: a prospective study in northern Thailand.

A prospective study of all cases of Streptococcus suis blood or CSF culture positive admitted to Lamphun Provincial Hospital in northern Thailand was carried out. Fifty-three cases of S. suis were identified, which comprised 70% of all viridans streptococci cases. The majority of cases (88.6%) were contracted orally and 83.0% had an underlying disease present. Five clinical syndromes were identified: acute meningitis (37.2%), septicemia (27.9%), toxic shock syndrome (TSS) (23.3%), subacute bacterial endocarditis(SBE) (9.3%) and spondylitis (2.3%). The patients with TSS had a lower mean age than those without TSS. S. suis IgG and IgM antibody levels in the TSS group were lower than those without TSS which is important when considering the clinical syndrome and severity of the infection.

Antibody levels to the class I and II epitopes of the M protein and myosin are related to group A streptococcal exposure in endemic populations.

Rheumatic fever (RF)/rheumatic heart disease (RHD) and post-streptococcal glomerulonephritis are thought to be autoimmune diseases, and follow group A streptococcal (GAS) infection. Different GAS M types have been associated with rheumatogenicity or nephritogenicity and categorized into either of two distinct classes (I or II) based on amino acid sequences present within the repeat region ('C' repeats) of the M protein. Sera from ARF patients have previously been shown to contain elevated levels of antibodies to the class I-specific epitope and myosin with the class I-specific antibodies also being cross-reactive to myosin, suggesting a disease association. This study shows that immunoreactivity of the class I-specific peptide and myosin does not differ between controls and acute RF (ARF)/RHD in populations that are highly endemic for GAS, raising the possibility that the association is related to GAS exposure, not the presence of ARF/RHD. Peptide inhibition studies suggest that the class I epitope may be conformational and residue 10 of the peptide is critical for antibody binding. We demonstrate that correlation of antibody levels between the class I and II epitope is due to class II-specific antibodies recognizing a common epitope with class I which is contained within the sequence RDL-ASRE. Our results suggest that antibody prevalence to class I and II epitopes and myosin is associated with GAS exposure, and that antibodies to these epitopes are not an indicator of disease nor a pathogenic factor in endemic populations.

Construction of infectious dengue 2 virus cDNA clones using high copy number plasmid.

Procedures for cloning entire dengue serotype 2 virus genome in the multiple cloning site of a commercially available high copy number plasmid are described. The 10.7 kb viral RNA genome was reverse transcribed, amplified as three overlapping DNA fragments and successively ligated into pBluescript II KS, which contains the colE1 origin of replication. When propagated at room temperature (20-25 degrees C) under low level of antibiotic selection, the full-length recombinant plasmid was stable upon serial passages in two common Escherichia coli strains employed. Under the same culture conditions the whole dengue cDNA sequence was transferred successfully to another high copy number plasmid, pGem 3Z. Following in vitro transcription and lipofectin-mediated transfection, capped RNA transcripts derived from the plasmid initiated virus replication in C6/36 mosquito cells and BHK-21 cells within 3-4 days of transfection. Upon subsequent expansion in C6/36 cells, dengue viruses derived from the first- and eighth-plasmid passages achieved similar titers as the parent virus. They were also indistinguishable from the parent virus by the criteria of replication kinetics in mosquito and mammalian cell lines, and size and reactivity of selected viral proteins as detected with polyclonal and monoclonal antibodies. The cloning scheme and resultant recombinant plasmids based on high copy number cloning vectors allows greater flexibility in manipulation of dengue viral genome when compared with previous attempts employing low-copy number counterparts.

Streptococcus suis infection in northern Thailand.

A ten-case report of Streptococcus suis infection was reported in Lamphun, northern Thailand from 1999 to 2000. Ten patients were admitted to Lampoon Provincial Hospital with a history of high fever, watery diarrhea, severe myalgia and ecchymosis rashes. The disease progressed rapidly and all patients died within 24-48 hours after admission from complications such as disseminated intravascular coagulation (DIC), acute renal failure (ARF) or acute respiratory distress syndrome (ARDS). Epidemiological data revealed that all cases were healthy men aged between 40-49, residing in the same geographical area and had a history of raw pork or uncooked pig's blood consumption prior to their illnesses. Blood culture and genetic investigation (16 s rRNA polymerase chain reaction with restriction enzyme PstII) confirmed diagnoses of the same species of Streptococcus suis infections.

Epidemiological analysis of non-M-typeable group A Streptococcus isolates from a Thai population in northern Thailand.

Infection with group A streptococci (GAS) can lead to the development of severe postinfectious sequelae such as rheumatic fever (RF). In Thailand, RF and rheumatic heart disease (RHD) remain important health problems. More than 80% of GAS circulating in this population are non-M antigen typeable by conventional M serotyping methods. In this study, we determine the M protein sequence types of GAS isolates found in northern Thailand. The emm genes from 53 GAS isolates, collected between 1985 and 1995 from individuals with pharyngitis, impetigo, acute RF (ARF), RHD, or meningitis as well as from individuals without infections, were amplified by PCR and sequenced. Thirteen new sequence types that did not show homology to previously published sequences were characterized. Six of these sequence types could be isolated from both skin and throat sites of impetigo and pharyngitis/ARF patients, respectively. In many cases we could not specifically differentiate skin strains or throat strains that could be associated with ARF or acute glomerulonephritis. Antigenic variations in the emm gene of the isolates investigated, compared to published M protein sequences, were predominantly due to point mutations, small deletions, and insertions in the hypervariable region. One group of isolates with homology to M44 exhibited corrected frameshift mutations. A new M type isolated from an RHD patient exhibited nucleotide sequence corresponding to the N terminus of M58 and the C terminus of M25, suggesting that recombination between the two types may have occurred. This study provided epidemiological data relating to GAS endemic to northern Thailand which could be useful for identification of vaccine candidates in a specific region of endemicity.

Human antibodies to the conserved region of the M protein: opsonization of heterologous strains of group A streptococci.

A 20-mer peptide (p145) in the carboxyl-terminal region of the M protein of group A streptococci (GAS) has previously been defined as the target of bactericidal antibodies. Sequence analysis of seven field isolates from indigenous Australians living in an area highly endemic for GAS and five laboratory reference strains (encompassing nine unique serotypes plus three nontypeables) demonstrates that this region is highly conserved (sequence identity ranging from 65 to 95%) with six of the 12 sequences being identical to p145. Most of the sequence dissimilarity is contained within the last seven amino acids of p145. Competitive ELISA demonstrates that human antibodies specific for p145 cannot discriminate between p145 and synthetic peptides representing four from four of the variant sequences tested. Ig purified from endemic sera was able to opsonize each of the GAS isolates and free p145 as well as a peptide expressing a minimal conformational epitope within p145 (requiring amino acids between positions 2 and 13 of p145), but not an irrelevant peptide, were able to partially or completely inhibit opsonization of all isolates and reference strains. Thus adult endemic sera contain antibodies which are bactericidal for multiple GAS serotypes and which are specific for a sequence of 12 amino acids contained within the p145 region of the M protein.

Mapping a conserved conformational epitope from the M protein of group A streptococci.

The carboxyl terminus of the M protein of group A streptococci (GAS) is highly conserved and contains epitopes that have been shown to induce opsonic antibodies and protection against GAS infection. This region of the protein can also stimulate T cells, which can react in vitro with heart antigens. Since different segments of the carboxyl terminus may be involved in immunity to GAS and in the pathogenesis of autoimmune disease (rheumatic heart disease), it is important to precisely define critical epitopes. However, the M protein is known to be a coiled coil, and a critical immunodominant antibody-binding epitope within this region (peptide 145, a 20-mer with the sequence LRRDLDASREAKK-QVEKALE) is shown here to be conformational. Thus, small synthetic overlapping peptides of 8-12 amino acids in length that span peptide 145 (p145) were unable to capture antibodies present in p145-immune mouse sera or in endemic human sera, even though antibodies raised to these small peptides coupled to diphtheria toxoid could bind the smaller peptides and, in some cases, p145. A series of mutated peptides in which every residue of p145 was sequentially altered also failed to identify critical residues for antibody binding. We thus devised a strategy to produce chimeric peptides in which small peptides copying the M protein sequence were displayed within a larger 28-mer peptide derived from the sequence of the GCN4 leucine zipper DNA binding protein of yeast. A 12-amino-acid window of the p145 sequence was inserted into the GCN4 peptide in such a way as to preserve any potential helical structure. The window was moved along one residue at a time to give a series of peptides representing p145. Circular dichroism demonstrated that these larger chimeric peptides and p145, but not a shorter 12-mer peptide, displayed alpha-helical potential in 50% trifluoroethanol. Certain chimeric peptides efficiently captured antibodies specific for p145 and thus enabled us to map the minimal antibody-binding sequence. RRDLDASREAKK, referred to as J(1)2. The chimeric peptide containing this sequence, referred to as J2, was able to inhibit opsonization of GAS by human antisera containing anti-peptide 145 antibodies. The T-cell response from p145-immunized responder B10.BR mice to J2 and J(I)2 was much lower than the response to p145 and mapped to a different peptide.

Towards a vaccine for rheumatic fever: identification of a conserved target epitope on M protein of group A streptococci.

Rheumatic fever and rheumatic heart disease remain very common in developing countries, and a vaccine to protect against these disorders would have a great impact on public health. A vaccine must target the M protein of group A streptococci (Streptococcus pyogenes), but until lately immunity was thought to be strain-specific and dependent on antibodies to the variable serotype-specific regions of the protein. Experiments in animals have suggested the conserved region of the M protein as a possible alternative target for protective antibodies. We constructed a 20-aminoacid peptide (peptide 145) within the conserved region of the carboxyl terminus of the protein. In mice the peptide induced serum antibodies that could opsonise reference type 5 streptococci. By enzyme-linked immunosorbent assay, positive responses to peptide 145 were obtained with serum from 77 (90%) of 86 Aboriginal subjects and 135 (81%) of 167 Thai subjects living in areas with high exposure to streptococci. Only 10 (14%) of 71 Caucasian subjects with low exposure to streptococci showed positive responses. There was no difference in the proportion positive between subjects with rheumatic heart disease and control groups (other or no heart disease). Antibodies to peptide 145 were able to opsonise isolates of streptococci from Aboriginal and Thai subjects with acute rheumatic fever as well as reference strains. This highly conserved part of the M protein may be a suitable target for vaccines to prevent streptococcal infections and their sequelae.

Identification of T cell autoepitopes that cross-react with the C-terminal segment of the M protein of group A streptococci.

Rheumatic fever (RF) follows a throat infection with different M-serotypes of beta-hemolytic group A streptococci (GAS) and can affect different tissues, predominantly the heart. It is thought to be an autoimmune illness. Although histological examination of affected heart shows an infiltrate consisting mainly of T cells, antigens or epitopes that could be putative targets of autoimmune T cells have not been identified. We have examined the T cell response to the conserved C-terminal region of the M protein--a streptococcal surface coiled-coil protein which is the target of opsonic antibodies and antibodies which cross-react with human heart tissue. Australian Aborigine, Caucasian and Thai patients, controls and mice were studied to define regions of the protein immunogenic for T cells, and T cell lines and clones were tested for cross-reactivity to myosin as well as an extract of RF-diseased mitral heart valve. Murine (B10, B10.D2, B10.BR) M peptide-specific T cells were often cross-reactive for other M peptides but did not cross-react with human heart antigens. Patients with RF or other heart diseases, or control subjects exposed more commonly to GAS were more likely to have T cell responses to the M protein, with many regions of the C-terminus being recognized. T cell lines and a clone specific for different M peptides were generated from five donors. Cross-reactivity could be shown between different M peptides, but unlike murine M peptide-specific T cells three of the human T cell lines reacted strongly to peptides representing homologous regions of cardiac and skeletal muscle myosins, and two of these lines also responded to porcine myosin and an extract of human rheumatic mitral valve. However, these last two lines were derived from a normal donor without history of RF or other heart disease. Our data demonstrate that regions of the M protein, including regions that are being considered as subunit vaccines, have the potential to stimulate pre-existing heart cross-reactive T cells, but that the ability of such T cells to cross-react (as measured in vitro) is not in itself sufficient to lead to disease.

Impetigo:an assessment of etiology and appropriate therapy in infants and children.

We found that mixed organisms of S.aureus and GABHS were the most common cause of impetigo in children in our study; that, of the two regimens evaluated, cloxacillin is the most effective treatment; that penicillin is equally effective in cases of mild to moderate forms and may be preferred on the basis of cost-effectiveness.

Conserved T and B cell epitopes on the M protein of group A streptococci. Induction of bactericidal antibodies.

To identify conserved T and B cell epitopes on the M protein of group A beta-hemolytic streptococci, overlapping synthetic peptides that span the conserved carboxyl-terminal segment of the M-5 protein were constructed and used to immunize a panel of H-2 congenic mice. Proliferative T cell epitopes were identified and, in many cases, mice immunized with these peptides produced high titer antibodies to the same peptides indicating that these proliferative epitopes could also stimulate Th cells. Peptide-specific T cells and antisera were tested for their reactivity with porcine myosin, tropomyosin, human heart myosin synthetic peptides, and extracts of human pericardial and atrial heart tissue. Although there was minimal response of M peptide-specific T cells to any of these Ag, certain M peptide-specific antisera reacted to immunoblotted porcine myosin and to an immunoblotted extract of human atrial heart tissue. However, two conserved peptides, LRRDLDASREAKKQVEKALE and KLTEKEKAELQAKLEAEAKA, stimulated peptide-specific antibodies in B10.BR and B10.D2 mice respectively, which reacted minimally if at all with human atrial heart tissue extract. Furthermore, antisera to the former peptide, in a bactericidal assay involving human monocytes, could mediate killing of streptococci (82% of bacteria). Although this level of killing is less than that produced by antisera to the highly polymorphic type-specific aminoterminus (up to 100% killing), it provides evidence that conserved epitopes can be the targets of bactericidal antibodies. These conserved epitopes may be useful in a vaccine because they also stimulate T cells, thus allowing development of immunologic memory and natural boosting of an immune response after natural exposure.