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.

High burden of invasive beta-haemolytic streptococcal infections in Fiji.

We undertook a 5-year retrospective study of group A streptococcal (GAS) bacteraemia in Fiji, supplemented by a 9-month detailed retrospective study of beta-haemolytic streptococcal (BHS) infections. The all-age incidence of GAS bacteraemia over 5 years was 11.6/100,000. Indigenous Fijians were 4.7 times more likely to present with invasive BHS disease than people of other ethnicities, and 6.4 times more likely than Indo-Fijians. The case-fatality rate for invasive BHS infections was 28%. emm-typing was performed on 23 isolates: 17 different emm-types were found, and the emm-type profile was different from that found in industrialized nations. These data support the contentions that elevated rates of invasive BHS and GAS infections are widespread in developing countries, and that the profile of invasive organisms in these settings reflects a wide diversity of emm-types and a paucity of types typically found in industrialized countries.

Streptococcus pyogenes strains containing emm12 and emm55 possess a novel gene coding for distantly related SIC protein.

Streptococcus pyogenes infection and acute glomerulonephritis (AGN), a non-suppurtave disease, are endemic in the Aboriginal people of the Northern Territory (NT) of Australia. Vir typing, a locus-specific polymerase chain reaction (PCR)-based typing method [Gardiner, Hartas, Currie et al PCR Meth Appl 1995 4: 288-93], has revealed high divergence among the NT streptococcal strains. A total of 76 Vir types (VTs) representing about 95% of the NT isolates were screened for sic, a gene for streptococcal inhibitor of complement function, by PCR and hybridization. This revealed that seven VTs are positive for sic, and there are two classes of the gene: those closely related to sic (CRS) originally described by Akesson, Sjoholm & Bjorck [ J. Biol. Chem. 1996 271: 1081-8] and those distantly related to sic (DRS). Among the CRS-positive VTs, VT16, VT78 and VT91 have emm (gene for M protein) encoding type 1 M protein or related specificity, and VT8 and VT101 contain emm57 or related alleles. Chromosomal location of CRS in emm57 is different from that in emm1 or related strains. The DRS-positive VT18 and VT52 contained emm55 and emm12 respectively, which are phylogenetically related. Strains of S. pyogenes types 1, 12, 55 and 57 are known to be associated with AGN. Restricted distribution of CRS and DRS among the M types historically associated with AGN suggests that these sic alleles may have a role in AGN pathogenesis.

Hairpin extension. A general method for the improvement of sensitivity of oligonucleotide probes.

A general and sensitive detection method of target DNA is described. The system is based on an oligonucleotide probe labeled to high specific activity. This involves a novel oligonucleotide design incorporating at the 3' end a hairpin structure, allowing extension by polymerase reaction.

Lateral genetic transfers between group A and G streptococci for M-like genes are ongoing.

Previously we described a long-polymerase chain reaction (PCR) method to amplify a 4-7 kb target containing most of the components of the vir regulon (mga, emm-like genes and scpA) in a number of group A streptococcus (GAS) isolates. In contrast to GAS, strains of human group G streptococcus (GGS) gave approximately 1.6 or 1.8 kb products. Sequence analysis of the amplified products issued from GGS templates revealed a mosaic consisting of upstream sequence from mga (the gene for positive regulator of vir regulon), an unidentified open reading frame, a short segment of emm (the gene for M protein, an antiphagocytic molecule) and an upstream sequence of scp (C5a-peptidase gene). A full length scpG is present immediately downstream from the mosaic segment in the human GGS genome. The GGS PCR fragment did not code for mga or full length emm. All human GGS isolates are known to code for emm but the gene is separated from scpG by at least 10 kb. Our data, obtained using long-PCR and unrelated strains of GGS, confirm this. We could not detect a homologue of mga in human GGS by hybridization analysis. The mosaic sequence suggests that enbloc transfer of the vir regulon from GAS to a GGS progenitor may have occurred, following which deletion and rearrangement events may have taken place. Partial nucleotide sequences of emm corresponding to the variable domain of M proteins from three local GGS isolates were determined. One sequence (emmGGS6) is 99% identical to emm from a geographically separated isolate of GGS recently described.3 emmGGS6 also has significant homology with emm from a GAS strain (STDONALD) isolated from the same geographical area as was GGS6. The two emm sequences (emmGGS6 and emmSTDONALD) revealed frameshift-compensatory frameshift mutations relative to each other, contributing to lower amino acid homology between the two predicted M proteins. Since emmSTDONALD has no known relatives within the 80 or so emm sequences in the database, we speculate that it could have been laterally acquired from GGS. Horizontal transfers between GGS and GAS may be ongoing.

Simplification of a locus-specific DNA typing method (Vir typing) for Streptococcus pyogenes.

We describe a simplification of a highly discriminatory molecular typing method, called Vir typing, for Streptococcus pyogenes (D. Gardiner, J. Hartas, B. Currie, J. D. Mathews, D. J. Kemp, and K. S. Sriprakash, PCR Methods Appl. 4:288-293, 1995). The procedure can be completed within a day, is reproducible, and can be applied directly to colonies growing on primary culture plates, allowing rapid establishment of strain identity in an outbreak.

Characterisation of group A streptococcal isolates from tropical Australia with high prevalence of rheumatic fever: probing for signature sequences to identify members of the family of serotype 5.

The prevalence of rheumatic heart disease (RHD) in Northern Territory Aboriginal communities is high, but there is a low isolation rate of historically rheumatic fever associated M types (such as M5) of group A streptococci (GAS). Many isolates are M non-typable (MNT). Serology suggests that the population is exposed to M5-like isolates; some RHD patients having high IgM or IgG titres to two M5 B-repeat region peptide epitopes, B1 (KQQESK) and B4 (EQKSKQ). To identify relatives of M5 in our collection of GAS, oligonucleotide probes to the B1 and B4-repeat regions shared by M5 and a local M5-like isolate, were used to screen 101 isolates for the presence of signature sequences. In all, 28% of the tropical Australian isolates contained the signature sequences, identifying members of the M5 family. The 5' region of the genes for M proteins from three members of the M5 family fell into two sequence types. Hybridisation to probes based on these sequences suggested that among tropical Australian isolates there are at least three distinct sequence types that contained the M5 signature sequences. These results suggest that a considerable number of M5 family GAS are circulating in tropical Australia.

Vir typing: a long-PCR typing method for group A streptococci.

We have developed a new procedure (Vir typing) for typing Streptococcus pyogenes, by amplifying the entire 5- to 7-kb variable vir regulon by long PCR. The amplified DNA is then cleaved with HaeIII and visualized by ethidium bromide fluorescence after agarose gel electrophoresis. A simple procedure for preparing DNA of sufficiently high quality from 96 samples was employed simultaneously. This DNA was also used to develop a random amplified polymorphic DNA (RAPD) procedure. The discriminatory power of the two DNA-based procedures was compared with previous methods, M typing, and multilocus enzyme electrophoresis. Both procedures were highly discriminatory, but the stoichiometric yield of restriction fragments in Vir typing allows unambiguous interpretation of results.