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Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Darcy Rd, Westmead, New South Wales, 2145 Australia
Correspondence Gwendolyn L. Gilbert lyng{at}icpmr.wsahs.nsw.gov.au
Received April 5, 2003
Accepted August 28, 2003
Streptococcus pneumoniae polysaccharide and protein-conjugate vaccines are available against the most commonly isolated pneumococcal serotypes. Ongoing surveillance of invasive pneumococcal disease is needed in order to monitor changes in distribution of serotypes. Based on previously published sequences of capsular polysaccharide synthesis (cps) gene clusters of 16 pneumococcal serotypes, a molecular capsular typing (MCT) system has been developed, based on a combination of partial cpsA–cpsB sequencing and serotype- or serogroup-specific PCR, targeting the genes wzy and wzx (except for serotype 3). In this study, 151 S. pneumoniae isolates of known serotype (representing 51 serotypes) and 276 recent clinical isolates were used to develop MCT and compare it with conventional serotyping (CS) (total 427 isolates). On the basis of 376 heterogeneity sites in the cpsA–cpsB region, 89 sequence types (ST) were identified, of which 76 corresponded to a single serotype and 11 contained two serotypes. The correct serotypes in two of the latter (10A-23F-g and 23F-23A) were identified using serotype 23F-specific PCR. Limited CS was required for 92 (22 %) isolates to distinguish between the two serotypes in the nine other mixed ST (6A–6B-g, 6A–6B-q, 15B–22F, 33F–33A, 17F–35B, 18B–18C, 13–20, 25F–38, 31–42). MCT is a specific, objective and practical method that can predict the serotype of most S. pneumoniae isolates; it will facilitate epidemiological studies. Further study of the relationship between MCT and CS is needed in order to improve our understanding of serotype differentiation and to improve MCT methods further.
The GenBank/EMBL/DDBJ accession numbers for the new partial sequence data for cpsA–cpsB, wzy and wzx genes are respectively AF532632–AF532715, AY330713–AY330718 and AY163171–AY163232. The accession numbers for sequence data used in this study that had been previously reported by others, in addition to those listed in Tables 2 and 3, are U15171, U66846 and U66845 (partial cps gene cluster for serotype 3), AF246897 and AF298581 (cps gene cluster for serotype 6B), AF105113 (partial cps gene cluster for serotype 19A), AF105114 and AF106137 (partial cps gene clusters for serotype 19B) and AF105115 (partial cps gene cluster for serotype 19C).
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONConclusionACKNOWLEDGEMENTSREFERENCES |
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S. pneumoniae comprises at least 90 serotypes (Henrichsen, 1995), distinguished by capsular polysaccharide antigens (Garcia et al., 2000). Capsule production in S. pneumoniae is largely controlled by capsular polysaccharide synthesis (cps) gene clusters (Garcia et al., 2000). The cps gene clusters for at least 16 pneumococcal serotypes have been sequenced and serotype-specific genes identified (Jiang et al., 2001; van Selm et al., 2002). The cps gene cluster contains genes responsible for synthesis of the serotype-specific polysaccharide, including (except in serotype 3) wzy (polysaccharide polymerase gene) and wzx (polysaccharide flippase gene) (Garcia et al., 2000; Jiang et al., 2001). At the 5'-end of the cps gene cluster are four relatively conserved open reading frames, cpsA (wzg)–cpsB (wzh)–cpsC (wzd)–cpsD (wze) (Jiang et al., 2001; Morona et al., 2002). In the region between the 3'-end of cpsA and the 5'-end of cpsB, there are sites of heterogeneity between and within serotypes (Jiang et al., 2001; Lawrence et al., 2000). S. pneumoniae is characterized by high-frequency recombination within the cps gene cluster, leading to serotype ‘switching’ among isolates within genetic lineages defined by relationships between their more conserved housekeeping genes (Coffey et al., 1998; Jiang et al., 2001).
Pneumococcal serogroup and serotype identification is currently performed by using large panels of expensive antisera by various methods, including the capsular swelling (Quellung) reaction, the traditional ‘gold standard', latex agglutination and co-agglutination (Arai et al., 2001; Henrichsen, 1995; Lalitha et al., 1999). Cross-reactions between serotypes and discrepancies between methods can occur and some strains are non-serotypable (Barker et al., 1999; Heidelberger, 1983; Henrichsen, 1999; Kumar et al., 1985). Molecular typing has the potential to improve discrimination and provide additional information (Gillespie, 1999; Hall, 1998). Several molecular typing methods have been developed, including three designed to predict serotypes and serogroups. However, all require further evaluation and improvement (Brito et al., 2003; Lawrence et al., 2000, 2003).
In a previous study, we developed a molecular serotype identification assay for group B streptococcus based on sequencing and serotype-specific PCR, targeting the cps gene cluster (Kong et al., 2002). In this study, we aimed to use similar methods to develop a molecular capsular typing (MCT) method for S. pneumoniae.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONConclusionACKNOWLEDGEMENTSREFERENCES |
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Isolates were retrieved from storage by subculture on blood agar plates (Columbia II agar base supplemented with 5 % horse blood) and incubated overnight at 37 °C in 5 % CO2.
Conventional serotyping (CS).
CS was performed by the Quellung reaction using rabbit polyclonal antisera from the Statens Serum Institute, Copenhagen, Denmark (Sorensen, 1993; Henrichsen, 1995). Briefly, 2 µl of a suspension of isolate in 10 % formalin saline and 1 µl antiserum were mixed under a glass coverslip and examined for capsular swelling using a light microscope at 400x magnification. All were serotyped by the donor laboratory, except clinical isolates from CIDM, which were serotyped at Department of Microbiology, Children's Hospital at Westmead, Sydney, Australia. Selected New Zealand clinical isolates for which only serogroup results were available and some other selected isolates were retested at Children's Hospital at Westmead.
MCT
Oligonucleotide primers.
The oligonucleotide primers used in this study, their target sites, specificity, sequences and numbered base positions and melting temperatures (Tm) are shown in Table 2. Expected amplicon lengths of different primer pairs can be calculated from the 5'-end positions of the corresponding primers.
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DNA preparation, PCR and sequencing.
DNA extraction, PCR and sequencing were performed as described previously (Kong et al., 2002). Briefly, five individual S. pneumoniae colonies or a sweep of culture were sampled using a disposable loop and resuspended in 0.2–1 ml digestion buffer (10 mM Tris/HCl, pH 8.0, 0.45 % Triton X-100 and 0.45 % Tween 20) in 2 ml Eppendorf tubes. The tubes containing S. pneumoniae suspension were heated at 100 °C (dry block heater or water bath) for 10 min and then cooled on ice and centrifuged for 2 min at 14 000 r.p.m. to pellet the cell debris. An aliquot (5 µl) of each supernatant containing extracted DNA was used as template for PCR.
PCR systems were used as described previously (Kong et al., 1999). The denaturation, annealing and elongation temperatures and times used were 96 °C for 1 s, 60–70 °C (depending on the primer Tm values) for 1 s and 74 °C for 10–90 s (depending on the length of amplicons) for 35 cycles.
Aliquots (10 µl) of PCR products were analysed by electrophoresis on 1.5 % agarose gels, which were stained with 0.5 µg ethidium bromide ml-1. For detection and serotype prediction, the presence of PCR amplicons of the expected length, as shown by ultraviolet transillumination, was accepted as positive.
For sequencing, PCR products were further purified by polyethylene glycol precipitation (Ahmet et al., 1999). The PCR products were sequenced using Applied Biosystems Taq DyeDeoxy terminator cycle-sequencing kits according to standard protocols. The corresponding amplification primers or inner primers were used as sequencing primers.
Sequence management, comparison and multiple sequence alignments.
WebANGIS (http://www1.angis.org.au/pbin/WebANGIS/ wrapper.pl) in ANGIS (Australian National Genomic Information Service) provided all programs used in the study, in particular, for sequence file management (WebFM), two-sequence comparison (BESTFIT in the Comparison program group) and multiple sequence alignments (PILEUP and PRETTY in the Multiple Sequence Analysis program group).
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONConclusionACKNOWLEDGEMENTSREFERENCES |
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Pre-test by S. pneumoniae-specific PCR
All isolates were tested by PCR using primer pairs targeting the pneumoccocal surface antigen A (psaA; P1–P2) (Morrison et al., 2000) and pneumolysin (ply; IIa–IIb) genes (Salo et al., 1995) to confirm the identity of isolates. All 427 isolates included in this study produced amplicons of the expected size with both primer sets, which confirmed their identity as S. pneumoniae and demonstrated that the quality (non-inhibitory) and quantity of DNA extracts (Brito et al., 2003) were adequate for further analysis. This was particularly important for five non-serotypable isolates and eight that belonged to serotypes 25F and 38, which do not form amplicons with primers cpsS1–cpsA3 (see below).
A single preliminary PCR is adequate for confirmation of species identity and DNA quality; primers P1–P2, targeting psaA, are preferred because the amplicons produced are of similar size and have similar sensitivity to those produced by primers cpsS1–cpsA3 (see below). After further evaluation of the specificity of cps-related primers and the DNA extraction method, it may be possible to omit this S. pneumoniae-specific PCR pre-test.
Development of serotype-/group-specific PCR
PCR primer target selection.
Sequence analysis of the cps gene clusters of 16 serotypes shows that wzy and wzx are highly variable (Jiang et al., 2001) and therefore suitable PCR targets for serotype/group identification (Table 2), except for serotype 3, which has no wzy and wzx. For this serotype, we used primers targeting orf2 (wze)–cap3A–cap3B (Arrecubieta et al., 1996).
Targeting wzx or wzy? Fifteen previously published wzx and wzy sequences were used to design primer pairs for each target for each of the following serotypes or serogroups: 1, 2, 4, 6, 8, 9V, 14, 18C, 19F, 19A, 19B, 19C, 23F and 33F/37 (Table 2). A set of 56 well-characterized isolates that included one representative of each of 51 serotypes and five non-serotypable isolates were tested using all serotype-/group-specific primer pairs targeting both wzx and wzy. The ‘unknown’ CIDM clinical isolates were initially examined by the most common serotype-/group-specific PCR primer pairs (all except 1, 2, 19B/19C, 33F/37). Finally, after sequencing and CS results were known, isolates that apparently belonged to relevant serotypes were tested by the corresponding serotype-/group-specific PCR to confirm the results or to resolve discrepancies between CS and ST (sequence typing) results, if necessary.
With few exceptions, both primer sets gave consistent identification of all isolates. The exceptions were that PCR targeting wzx but not wzy of serotype 23F amplified all four serotype 23A isolates and PCR targeting wzx but not wzy of serotypes 33F/37 amplified all three serotype 33B isolates. These exceptions help to identify serotypes 23A and 33B, respectively, by exclusion. Since the specificities of primer pairs targeting wzx and wzy were similar for most serotypes, one set targeting either wzx or wzy would be adequate for routine use, except for serotypes 23F/23A and 33F/33B/37. In addition, both sets of primers targeting orf2 (wze)–cap3A–cap3B of serotype 3 produced amplicons of the expected size from all serotype 3 isolates.
Serotype or serogroup specific? Two primer pairs designed to identify serotypes 6B and 6A amplified both, i.e. were serogroup 6-specific (Lawrence et al., 2003). Two primer pairs designed to identify serotype 18C amplified all serotypes in serogroup 18, i.e. were serogroup 18-specific. One primer pair targeting wzx of serotype 23F was neither entirely 23F-specific (see above) nor able to identify all serogroup 23 isolates: it failed to amplify four serotype 23B isolates. Two primer pairs targeting wzx and wzy of serotypes 33F and 37 (Llull et al., 1999) also amplified a 33A isolate, and those targeting wzx amplified three serotype 33B isolates (see above), i.e. they were neither serotype nor serogroup specific. Our findings relating to serogroups 6, 18 and 23 are consistent with those of two reports published since completion of our study, which described multiplex PCR-based methods for identification of up to nine pneumococcal serotypes/groups (Lawrence et al., 2003; Brito et al., 2003).
Sequencing partial wzx and wzy. In order to explore further the sequence heterogeneity and to improve serotype- and serogroup-specific PCR methods, we sequenced portions of wzy and wzx of isolates belonging to serogroups 6, 18, 23 and 33/37. The results showed that there is enough sequence heterogeneity to allow us to design serotype-specific PCRs for serotypes 33B, 18A and 18F. However, there are only minor differences and ‘cross-talk’ between wzy and wzx sequences of serotypes 6A and 6B, 18B and 18C and 33F and 33A and, hence, serotype-specific PCR assays targeting these genes are unlikely to be successful.
Development of a partial cpsA–cpsB sequence database
Sequencing and primer target selection.
It has been shown previously that PCR-RFLP based on the cpsA–cpsB region can predict S. pneumoniae serotypes (Lawrence et al., 2000). However, the method generates a long amplicon (1.8 kbp), requires the use of three restriction enzymes and special equipment and has limited discriminatory ability. We believed that sequencing would be a more straightforward way to improve the discriminatory ability. The region we chose contains the most variable sites in the cpsA–cpsB region (Jiang et al., 2001), corresponding to positions 1119–1917 of the S. pneumoniae serotype 19F cps gene cluster or position 951 of cpsA to position 302 of cpsB (GenBank accession no. U09239). We designed several primer pairs to amplify a 1001 bp segment within this region, corresponding to positions 1030–2030 or position 923 of cpsA to position 415 of cpsB (accession no. U09239).
The sequencing primers cpsS1–cpsA3 (Table 2) produced amplicons from all but 13 of the 427 isolates, of which eight belonged to two rare serotypes, 25F and 38, and five were non-serotypable. Two additional primer pairs, cpsS1–cpsA1 and cpsS3–cpsA2, were designed to test these 13 isolates further; three non-serotypable isolates were still not amplified using these primers.
Sequence heterogeneity analysis. Based on the raw sequencing data, we analysed the 798–800 bp fragments (several serotypes had a 1 bp insertion or deletion) of the most heterogeneous region between the 3'-end of cpsA (starting at cpsA position 951) and the 5'-end of cpsB (ending at cpsB position 302). Among all isolates of known CS studied, there were 424 sites that were identical for all 51 serotypes represented. They included 24 isolates with sequences that corresponded to cpsA–cpsB sequences previously available in GenBank. Therefore, there were 376 (47 %) heterogeneity sites (of 798–800 bp) in this partial cpsA–cpsB region, some of which were specific for individual serotypes, while others were shared between several (see below).
ST nomenclature. Based on sequence heterogeneity at one or more sites for all isolates, there were 89 different sequence types (ST) or 87 if the ST of two non-serotypable isolates were excluded. Forty-three of 51 CS and 53 of 89 ST studied were represented by more than one isolate. Eighteen of these 43 CS (4, 7F, 7C, 8, 9N, 9V, 13 and 20, 18B and 18C, 22F, 22A, 31, 33B, 34, 35F, 35B and 38) corresponded to a single ST and 25 included two to seven different ST. Eleven ST were shared by isolates belonging to two serotypes (Table 3).
ST were named according to the corresponding CS, with a suffix representing the source of the isolate for which the ST was first identified: -g for ST sequences previously available in GenBank; -c for CIDM isolates; -n, New South Wales; -q, Queensland; -w, Western Australia; -v, Victoria; -ca, Canada; -nz, New Zealand. When sequences characteristic of two serotypes were identified, the ST name included both, with the lower number serotype first (e.g. 13–20, 15B–22F, etc.) (Henrichsen, 1995). Representative sequences of all ST were deposited in GenBank (see Table 3 for ST name and GenBank accession numbers).
Ongoing nature of our ST database. Database development will continue until it includes all 90 known CS and all possible ST. In future, larger numbers of isolates of all serotypes will be examined as they become available and the results added to our database. It may be necessary to modify the database if additional ST or discrepancies are identified. Progress so far demonstrates that it is possible to generate an accessible cpsA–cpsB sequence database for practical use by S. pneumoniae serotyping reference laboratories. In general, the more CS, ST and isolates of each are included in the database, the greater the accuracy with which serotypes will be predicted by sequence data.
Five of 427 isolates studied were non-typable by both CS and ST. Three were not amplified by any of three sets of primers targeting the 
800 bp variable region of cpsA–cpsB; amplicon sequences of the other two did not correspond to any of the 51 CS represented among other isolates. Isolates that are non-serotypable may have decreased type-specific-antigen synthesis or non-encapsulated phase variation; failure to type them by ST may result from insertion or mutation of genes in the cps cluster (Brito et al., 2003) or may simply reflect the fact that our database is still incomplete.
Advantages and disadvantages of serotype-/group- specific PCR. Serotype- or serogroup-specific PCR was able to resolve many ST results that could not distinguish between different CS. There were 24 isolates for which sequencing could not distinguish between serotypes 10A and 23F; negative 23F-specific PCR results confirmed that six were serotype 10A and positive 23F-specific PCR confirmed that 18 were serotype 23F. Similarly, when sequencing could not distinguish between serotypes 23A and 23F, 23F-specific PCR (targeting both wzx and wzy) confirmed that one was serotype 23A and one was 23F. One clinical isolate gave positive results in serotype 9V- and serotype 14-specific PCR (targeting both wzx and wzy), but was identified by sequencing as 9V. Subsequent investigation of multiple colonies showed that it was a mixture of serotypes 9V and 14, predominantly serotype 9V.
This PCR method can be extended to other serotypes and serogroups when additional wzx and wzy or other specific sequences are available (Brito et al., 2003; Lawrence et al., 2003). However, the large number of pneumococcal serotypes would make it impractical for serotyping all of them. The use of multiplex PCR provides a partial solution (Brito et al., 2003; Lawrence et al., 2003), but is also limited to a relatively small number of serotypes. In future, the use of genechip technology and microarrays will provide a more practicable platform (Magee et al., 2001) but, in the meantime, serotype prediction based on partial cpsA–cpsB sequencing is more practical.
Advantages and disadvantages of partial cpsA–cpsB sequence typing.
For most serotypes (except rare serotypes 25F and 38 and non-serotypable isolates), a single amplification using primers cpsS1–cpsA3 will be needed. With future improvements in DNA sequencing facilities and chemistry, it will be possible to sequence the 800 bp region in a single sequencing reaction. A small proportion of isolates belonging to ST shared by two serotypes will be incompletely assigned by this method alone. CS or serotype-/serogroup-specific PCR will still be needed to identify these serotypes correctly (Table 3).
Algorithm for predicting S. pneumoniae serotype by MCT
Based on our study, we have developed an algorithm for practical use of our MCT method to predict S. pneumoniae serotypes.
Step 1. Download our partial cpsA–cpsB database to a personal computer. Our sequence files are directly located in WebFM (a sequence file manager program group) from WebANGIS, which makes sequence analysis easier. The 798–800 bp cpsA–cpsB sequences of 89 ST and their GenBank accession numbers, as shown in Table 3, can be downloaded to your own computer.
Step 2. Select the most appropriate multiple sequence analysis software. Many different multiple sequence analysis software programs are available from the Internet and elsewhere for installation on a personal computer. We selected programs PILEUP and PRETTY from among many related programs in WebANGIS.
Step 3. Sequencing and sequence editing. Using primer pair cpsS1–cpsA3, generate and sequence amplicons from extracted DNA of confirmed S. pneumoniae isolates (cpsS2 could be used as an inner sequencing primer if necessary). A few isolates will not form amplicons using primer pair cpsS1–cpsA3; most will be amplified using cpsS1–cpsA1 and/or cpsS3–cpsA2. Edit the raw sequencing data so they have the same start and end points as the sequences in our database (and so contain 798–800 bp). We used the program BESTFIT (in Comparison program group) and WebFM from WebANGIS to simplify sequence editing.
Step 4. ST analysis and interpretation. Use multiple sequence analysis software programs to compare test isolate sequences against the 89 sequences in our database. After identifying an identical match, the corresponding serotype(s) corresponding to the ST can be inferred. For the 11 ST shared by two serotypes, CS (mainly) and/or serotype-/group-specific PCR (for those available) are needed to identify individual serotypes (Table 3).
Step 5. New ST nomenclature. Test isolate sequences for which there is no match in our database may represent new or rare serotypes or ST. We recommend:
(i) use CS to decide their serotype;
(ii) to name the new ST, use our suggested nomenclature (CS-source code);
(iii) submit the new ST to GenBank and append the ST to the existing ST database.
Advantages and disadvantages of MCT. The main advantage of using MCT to predict pneumococcal serotype is that it does not require the use of large panels of expensive antisera (Brito et al., 2003; Henrichsen, 1995). It also has the potential to improve discrimination and provide additional information (Gillespie, 1999; Hall, 1998).
There are potential pitfalls in applying an indirect test to predict capsular antigen serotype (Lawrence et al., 2000). The genes examined (cpsA–cpsB, wzx and wzy) do not entirely determine serotype (Jiang et al., 2001; Lawrence et al., 2000; Llull et al., 1999). It is well documented that serotype can become dislocated through mutation or recombination (Coffey et al., 1998; Jiang et al., 2001). Thus, MCT is a useful adjunct to, but cannot yet replace, CS for all pneumococcal serotypes.
Comprehensive MCT results. The final MCT results (Table 3) show that cpsA–cpsB sequence typing alone correctly predicted the serotypes of more isolates (309) than serotype-/serogroup-specific PCR (186) alone. The combination of cpsA–cpsB sequence typing and 23F-specific PCR correctly predicted the serotypes of 335 isolates, leaving 92, belonging to nine ST that shared sequences between two serotypes, for which CS was needed to differentiate the serotypes.
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Conclusion |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONConclusionACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONConclusionACKNOWLEDGEMENTSREFERENCES |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONConclusionACKNOWLEDGEMENTSREFERENCES |
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