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PubMed2002

[Four serotypes highly positive for pathogenic factor-related genes found among Escherichia coli isolates from sporadic diarrheal cases in Fukui Prefecture].

To evaluate the prevalence of pathogenic factor-related genes in 964 O-serotype Escherichia coli isolated from sporadic diarrhea cases at 2 hospitals in Fukui during 1997 to 2001, we checked all of these strains for H-serotype and examined them for 4 pathogenic factor-related genes (LT, ST, stx and invE) by PCR. Of these strains, 409 except for most of 9 serotypes which are usually low prevalence of pathogenic factor-related genes, and additional 16 strains isolated from other hospitals in Fukui Prefecture were also examined for 3 pathogenic factor-related genes (eaeA, astA and aggR) by PCR. It was found that 4 serotypes, O6:H16, O25:HNM, O111:H21 and O126:H27, were highly positive for pathogenic factor-related genes; O6:H16 (11/12 strains) positive for LT, ST, astA, O 25:HNM (10/14 strains) positive for ST and astA, O111:H21 (22/22 strains) positive for astA or aggR, and O126:H27 (8/9) positive for astA and aggR genes. According to the drug susceptibility test, these serotypes as O6:H16, O25:HNM, O111:H21 and O126:H27 showed a significant resistance to some drugs in 7/12, 4/14, 21/22 and 9/9 strains, respectively. Such results indicate that both O111 and O126-serotypes are highly positive for pathogenic factor-related genes and also drug-resistant, namely this fact suggests that the O-serotyping as laboratory screening is one of the useful measures of clinical management. Additionally, the pulsedfied gel electrophoresis patterns found in each of the 4 serotypes mentioned revealed that a part of E. coli in Fukui might be derived from the same source of infection.

Diarrhea↗
PubMed1997

DNA fragments from regions involved in surface antigen expression specifically identify Listeria monocytogenes serovar 4 and a subset thereof: cluster IIB (serotypes 4b, 4d, and 4e).

Listeria monocytogenes serotype 4b has frequently been implicated in sporadic as well as epidemic listeriosis. On the basis of pulsed-field fingerprinting, serotype 4b strains, along with strains of serotypes 4d and 4e, constitute one genomic cluster (IIB). We have identified two genomic regions essential for the expression of surface antigens which previously were shown to be specific to cluster IIB strains. A DNA probe of 1.1 kb derived from one of the regions (probe 1) hybridized only with strains of serotypes 4b, 4d, and 4e in Southern blots and dot blots. A different DNA probe of 0.3 kb (probe 2), derived from the other region, hybridized with all serovar 4 strains (serotypes 4b, 4a, 4c, 4d, and 4e). All other L. monocytogenes serotypes were negative with probe 1 or 2. Use of probe 1 in Southern blots of EcoRI-digested genomic DNA revealed a restriction fragment length polymorphism in serotype 4b strains, with the hybridizing EcoRI fragments being 4.5 kb (strains of the epidemic clone) and either 4.5 or 5.0 kb (all other serotype 4b strains). Although the probes hybridized with a special group of Listeria innocua strains which also expressed the surface antigens, the latter could be readily distinguished by the size of the hybridizing EcoRI fragment with probe 1 (ca. 2.2 kb). These data suggest that the combined use of these probes with L. monocytogenes can readily and specifically identify cluster IIB strains as well as the entire serovar 4 complex.

Antibodies, Monoclonal↗
PubMed2002

Restriction fragment length polymorphisms detected with novel DNA probes differentiate among diverse lineages of serogroup 4 Listeria monocytogenes and identify four distinct lineages in serotype 4b.

Listeria monocytogenes of serotype 4b has been implicated in numerous outbreaks of food-borne listeriosis and in ca. 40% of sporadic cases. Strains of this serotype appear to be relatively homogeneous genetically, and molecular markers specific for distinct serotype 4b lineages have not been frequently identified. Here we show that DNA fragments derived from the putative mannitol permease locus of Listeria monocytogenes had an unexpectedly high potential to differentiate among different strains of serotype 4b when used as probes in Southern blotting of EcoRI-digested genomic DNA, yielding four distinct restriction fragment length polymorphism (RFLP) patterns. Strains of two epidemic-associated lineages, including the major epidemic clone implicated in several outbreaks in Europe and North America, had distinct RFLPs which differed from those of all other serotype 4b strains that we screened but which were encountered among strains of serotypes 1/2b and 3b. In addition, three serogroup 4 lineages were found to have unique RFLPs that were not encountered among any other L. monocytogenes strains. One was an unusual lineage of serotype 4b, and the other two were members of the serotype 4a and 4c group. The observed polymorphisms may reflect evolutionary relationships among lineages of L. monocytogenes and may facilitate detection and population genetic analysis of specific lineages.

Bacterial Typing Techniques↗
PubMed2005

Assignment of weight-based immunoglobulin G1 (IgG1) and IgG2 units in antipneumococcal reference serum lot 89-S(F) for pneumococcal polysaccharide serotypes 1, 4, 5, 7F, 9V, and 18C.

Weight-based assignments for immunoglobulin G1 (IgG1) and IgG2 subclass antibodies to Streptococcus pneumoniae capsular polysaccharides (PnPs) in antipneumococcal standard reference serum lot 89-S (lot 89-S), also known as lot 89-SF, have been determined for serotypes 1, 4, 5, 7F, 9V, and 18C. This extends the usefulness of lot 89-S beyond the IgG1 and IgG2 subclass assignments for serotypes 3, 6B, 14, 19F, and 23F made previously (A. Soininen, H. Kayhty, I. Seppala, and T. Wuorimaa, Clin. Diagn. Lab. Immunol. 5:561-566, 1998) to cover 11 major serotypes associated with the highest percentage of pneumococcal disease worldwide. A method of equivalence of absorbances in enzyme immunosorbent assays was used to determine the IgG1 and IgG2 antibody concentrations for the additional serotypes in lot 89-S, based on the subclass values previously assigned for PnPs serotypes 6B, 14, and 23F. This cross-standardization method assures consistency with previous antibody assignments in that reference serum. The newly assigned subclass values for serotype 9V, and previously assigned values for serotype 14, were used to quantitate PnPs antibodies in sera from adult and pediatric subjects immunized with a pneumococcal conjugate vaccine. There was a predominance of IgG1 anti-PnPs antibodies in pediatric sera and IgG2 anti-PnPs antibodies in the adult sera. The IgG1 and IgG2 subclass assignments for the 11 PnPs serotypes in antipneumococcal standard reference serum lot 89-S are useful for quantitating and characterizing immune responses to pneumococcal infection and vaccination regimens.

Adult↗
PubMed1998

Correlation between serological and sequencing analyses of the PorB outer membrane protein in the Neisseria meningitidis serotyping system.

The current serological typing scheme for Neisseria meningitidis is not comprehensive; a proportion of isolates are not serotypeable. DNA sequence analysis and predicted amino acid sequences were used to characterize the structures of variable-region (VR) epitopes on N. meningitidis PorB proteins (PorB VR typing). Twenty-six porB gene sequences were obtained from GenBank and aligned with 41 new sequences. Primary amino acid structures predicted from those genes were grouped into 30 VR families of related variants that displayed at least 60% similarity. We correlated VR families with monoclonal antibody (MAb) reactivities, establishing a relationship between VR families and epitope locations for 15 serotype-defining MAbs. The current panel of serotype-defining MAbs underestimates by at least 50% the PorB VR variability because reagents for several major VR families are lacking or because a number of VR variants within some families are not recognized by serotype-defining MAbs. These difficulties, also reported for serosubtyping based on the PorA protein, are shown as inconsistent results between serological and sequence analyses, leading to inaccurate strain identification and incomplete epidemiological data. The information from this study enabled the expansion of the panel of MAbs currently available for serotyping, by including MAbs of previously undetermined specificities. Use of the expanded serotype panel enabled us to improve the sensitivity of serotyping by resolving a number of formerly nonserotypeable strains. In most cases, this information can be used to predict the VR family placement of unknown PorB proteins without sequencing the entire porB gene. PorB VR typing complements serotyping, and a combination of both techniques may be used for full characterization of meningococcal strains. The present work represents the most complete and integrated data set of PorB VR sequences and MAb reactivities of serogroup B and C meningococci produced to date.

Amino Acid Sequence↗
PubMed1998

Development of a fluorescent focus identification assay using serotype-specific monoclonal antibodies for detection and quantitation of rotaviruses in a tetravalent rotavirus vaccine.

A fluorescent focus identification assay (FFIDA) was developed for use in experimental studies and for quantitation of the components in a tetravalent live oral rotavirus vaccine. The assay utilizes four serotype-specific neutralizing monoclonal antibodies (MAb) to detect and quantify individual rotaviruses by immunofluorescence staining of fixed virus-infected monkey kidney cells. In mixed virus infections, all four MAb, W1 (serotype 1), 1C10 (serotype 2), R1 (serotype 3), and S4 (serotype 4), specifically stain the relevant homologous serotype without exhibiting any cross-reactivity against the other serotypes. Furthermore, the test is sensitive enough to differentiate at least twofold (0.3 log) differences in virus titer. The results of testing four individual experimental vaccine lots three or more consecutive times showed that all four lots contained similar proportions of the four vaccine strains as detected by the classical plaque neutralization identification test. The rapidity and efficiency of the FFIDA are desirable attributes that make it suitable for use in studies requiring identification and quantitation of one or more of the four major rotavirus serotypes.

Animals↗
PubMed2006

Two complex, adenovirus-based vaccines that together induce immune responses to all four dengue virus serotypes.

Dengue virus infections can cause hemorrhagic fever, shock, encephalitis, and even death. Worldwide, approximately 2.5 billion people live in dengue-infested regions with about 100 million new cases each year, although many of these infections are believed to be silent. There are four antigenically distinct serotypes of dengue virus; thus, immunity from one serotype will not cross-protect from infection with the other three. The difficulties that hamper vaccine development include requirements of the natural conformation of the envelope glycoprotein to induce neutralizing immune responses and the necessity of presenting antigens of all four serotypes. Currently, the only way to meet these requirements is to use a mixture of four serotypes of live attenuated dengue viruses, but safety remains a major problem. In this study, we have developed the basis for a tetravalent dengue vaccine using a novel complex adenovirus platform that is capable of expressing multiple antigens de novo. This dengue vaccine is constructed as a pair of vectors that each expresses the premembrane and envelope genes of two different dengue virus serotypes. Upon vaccination, the vaccine expressed high levels of the dengue virus antigens in cells to mimic a natural infection and induced both humoral and cellular immune responses against multiple serotypes of dengue virus in an animal model. Further analyses show the humoral responses were indeed neutralizing against all four serotypes. Our studies demonstrate the concept of mimicking infections to induce immune responses by synthesizing dengue virus membrane antigens de novo and the feasibility of developing an effective tetravalent dengue vaccine by vector-mediated expression of glycoproteins of the four serotypes.

Adenoviridae↗
PubMed2006

Use of miniaturized protein arrays for Escherichia coli O serotyping.

Serological typing of Escherichia coli O antigens is a well-established method used for differentiation and identification of O serotypes commonly associated with disease. In this feasibility study, we have developed a novel somatic antibody-based miniaturized microarray chip, using 17 antisera, which can be used to detect bound whole-cell E. coli antigen with its corresponding immobilized antibody, to assess the feasibility of this approach. The chip was tested using the related 17 control strains, and the O types found by the microarray chip showed 100% correlation with the O types found by conventional typing. A blind trial was performed in which 100 E. coli isolates that had been O serotyped previously by the conventional assay were tested by the array approach. Overall, the O serotypes of 88% of isolates were correctly identified by the microarray method. For several isolates, ambiguity of O-type designation by microarray arose due to increased sensitivity of this method, allowing signal intensities of cross-reactions to be quantified. Investigation of discrepancies between conventional and microarray O serotyping indicated that some isolates upon storage had become untypeable and, therefore, gave poor signal intensity when tested by the microarray or retested by conventional means. For all 20 serotype O26 and O157 isolates, the apparent discrepancy in O serotyping was analyzed further by a third independent test, which confirmed the microarray results. Therefore, the use of miniaturized protein arrays increases the speed and efficiency of O serotyping in a cost-effective manner, and these preliminary findings suggest the microarray approach may have a higher accuracy than those of traditional O-serotyping methods.

Antigens, Bacterial↗
PubMed1982

Serotypes of enterotoxigenic Escherichia coli in Thailand and the Philippines.

The serotypes of 386 enterotoxigenic Escherichia coli (ETEC) isolated from 82 individuals with and without diarrhea in Thailand and the Philippines were determined. The 136 strains producing both heat-labile toxin (LT) and heat-stable toxin (ST) belonged to 12 different O serogroups; however, 83% (113/136) were of one of four serogroups (O6, O8, O25, and O78), and 76% of (104/136) belonged to one of seven O:K:H serotypes. Only 14% (28/196) of LT-only-producing ETEC belonged to serogroups most common among LT and ST strains, and these 196 strains belonged to 35 different O:K:H serotypes. Three O serogroups (O20, O27, and O78) accounted for 94% (52/54) of strains producing only ST. Although only 4% (2/54) of ST-only ETEC belonged to the seven serotypes most commonly found among strains which produced LT and ST, 85% of ETEC belonged to three other serotypes, O20:K?:H21, O27:K?:H7, and O78:H-. A total of 46% (37/80) of ETEC of serotypes O6:H16, O8:H9, O25:H42, and O78:H12 were resistant to two or more antibiotics in comparison to 68% (208/306) of ETEC of other serotypes (P less than 0.001). In Thailand and the Philippines, E. coli which produced LT and ST or ST alone, but not those which produced LT alone, were restricted in their O:K:H serotypes.

Diarrhea↗
PubMed1984

Characterization of monoclonal antibodies to Streptococcus mutans antigenic determinants I/II, I, II, and III and their serotype specificities.

Monoclonal antibodies (McAb) were developed to four protein components of Streptococcus mutans serotype c, some of which are significant in the protection against dental caries. The six McAb used in this investigation support the identities of streptococcal antigens (SA) I/II, I, II, and III. The specificities of these antigenic determinants were established both by direct binding and inhibition with the pure SA with a solid-phase radioassay. Whereas conventional antisera to S. mutans serotype c cross-react with serotypes c, e, and f (and g), McAb to serotype c-derived SA I/II react predominantly with serotype c and show some low-titer reactivity with serotype f. The slight cross-reactivity between S. mutants cells of serotypes c and f could be further differentiated by absorption of any of the three McAb to SA I/II with cells of serotype c. Parallel studies of McAb with cells of S. mutans and their ammonium sulfate-precipitated culture supernatants suggest that some SA determinants are retained predominantly on the cell surface, but others are readily shed into the culture medium, so that they are detected both on the cell surface and culture medium. Unlike polyclonal antibodies, McAb are capable of discriminating single antigenic determinants and can be applied to the study of shedding of antigens from microorganisms into the environment, such as the gut or gingival sulcus.

Animals↗
PubMed1986

Serotype-related HEp-2 cell interaction of Yersinia enterocolitica.

The ability of human clinical isolates of Yersinia enterocolitica to interact with HEp-2 cells was found to vary considerably between the serotypes O:3, O:8, and O:9. Although all three serotypes adhered initially to the cell surface, regardless of incubation temperature of the bacterial inoculum or presence of the 40- to 48-megadalton virulence plasmid, the ability to localize intracellularly was not uniformly expressed. By using a combined light optical method based on differential interference contrast and UV incident light microscopy, we found that in serotype O:3, resistance to internalization was dependent upon prior growth at 37 degrees C and carriage of the virulence plasmid; in serotype O:9, this property was plasmid dependent but not temperature dependent; in serotype O:8, it was constitutive. The ability of serotype O:3 to resist internalization was correlated with the expression of plasmid-associated fibrillae on the bacterial surface. No relationship between fibrillation and HEp-2 cell interaction was apparent for serotype O:8 or O:9. Serotypes O:8 and O:9, unlike the O:3 strains studied, associated with HEp-2 cells in greater number after cultivation at 22 degrees C than after cultivation at 37 degrees C. We failed to establish a correlation between the expression of surface fibrillae and the ability to evoke guinea pig conjunctivitis.

Animals↗
PubMed1987

Genetic relationships and clonal population structure of serotype 2 strains of Neisseria meningitidis.

Two hundred and thirty-four strains of Neisseria meningitidis, including 94 serotype 2a, 111 serotype 2b, and 19 serotype 2c isolates, together with 10 isolates that were serotyped as 2 with polyvalent antiserum but did not react with monoclonal antibodies, were characterized by the electrophoretic mobilities of 15 metabolic enzymes. Of these enzymes, 14 were polymorphic, and 56 distinctive combinations of alleles at the enzyme loci (electrophoretic types) were identified, among which the mean genetic diversity per locus was 0.413, or about 75% of that recorded for the species N. meningitidis as a whole. Mean genetic diversity among electrophoretic types of the same serotype (2a, 2b, or 2c) was, however, on average, less than half the total species diversity, and no multilocus genotypes were shared between isolates of the different serotypes, which belong to distinctive clonal lineages. Recent temporal changes in the frequencies of recovery of pathogenic strains of serotypes 2a and 2b in South Africa and North America resulted from clone replacement in these populations rather than evolutionary modification of the serotype protein of the initially dominant clones.

Genetic Variation↗
PubMed1993

Characterization of neuraminidases produced by various serotypes of Pasteurella haemolytica.

Neuraminidases produced by 16 strains of Pasteurella haemolytica (serotypes 1 to 16) were characterized by molecular weight, antigenic identity, and substrate specificity. After growth in a chemically defined medium, stage I (lyophilized) culture supernatants were assayed for activity with N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Neuraminidase produced by P. haemolytica serotype A1 (Ph A1) was purified by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. Purified Ph A1 neuraminidase was used to immunize rabbits, and the resultant antiserum reduced the activity of Ph A1 neuraminidase by 46%. This antiserum also reduced the activity of neuraminidase produced by the other serotypes by between 15 and 66%. Molecular weight estimates of the neuraminidases produced by the various serotypes were obtained by gel filtration chromatography on Sephadex G-200. Fifteen of the 16 serotypes examined produced a neuraminidase with a molecular weight of approximately 150,000 to 200,000. One serotype (serotype 11) produced no material with neuraminidase activity. In addition, all 15 high-molecular-weight neuraminidases showed similar substrate specificities. That is, they were all most active against N-acetylneuramin lactose and least active against bovine submaxillary mucin. On the basis of these results, it appears that the high-molecular-weight neuraminidases produced by the different P. haemolytica serotypes are quite similar.

Mannheimia haemolytica↗
PubMed2001

Identification, characterization, and variation in expression of two serologically distinct O-antigen epitopes in lipopolysaccharides of Campylobacter fetus serotype A strains.

Monoclonal antibodies (MAbs) to the lipopolysaccharide (LPS) O-antigens of Campylobacter fetus serotype A and B strains were produced. Eight MAbs specific for serotype A LPS were characterized on immunoblots of C. fetus serotype A LPS. Two immunoblot patterns were observed and were used to divide the eight MAbs into two groups. MAbs M1177 and M1194 were selected as representative of the two groups and were used in an enzyme-linked immunosorbent assay (ELISA) to examine the LPS O-antigen epitopes of 37 serotype A C. fetus subsp. fetus and C. fetus subsp. venerealis strains. Thirty-three strains (89%) reacted with both M1177 and M1194, 2 strains reacted only with M1177, and 2 strains reacted only with M1194. To further characterize the O-antigen epitopes, purified serotype A LPS was treated using various temperature and pH conditions and the effect of the treatments on the reactivity of the LPS with MAbs M1177 and M1194 was evaluated by ELISA. While no difference among several treatments was observed, heating serotype A LPS under alkaline conditions decreased the reaction with M1177 to background levels and increased the reaction with M1194. MAbs M1177 and M1194 were also used with ELISA to investigate in vivo and in vitro expression of the two O-antigen epitopes. There was substantial variation in expression of the two epitopes among weekly isolates of two C. fetus serotype A strains recovered from experimentally infected heifers. There was minimal variation in expression of the two epitopes in successive subcultures of three C. fetus serotype A strains.

Animals↗
PubMed2001

Isogenic serotypes of Borrelia turicatae show different localization in the brain and skin of mice.

Mice with severe combined immunodeficiency (scid mice) and infected with the relapsing fever agent Borrelia turicatae develop manifestations that resemble those of disseminated Lyme disease. We have characterized two isogenic serotypes, A and B, which differ in their variable small proteins (Vsps) and disease manifestations. Serotype A but not serotype B was cultured from the brain during early infection, and serotype B caused more severe arthritis, myocarditis, and vestibular dysfunction than serotype A. Here we compared the localization and number of spirochetes and the severity of inflammation in scid mice, using immunostained and hematoxylin-and-eosin-stained coronal sections of decalcified heads. Spirochetes in the brain localized predominantly to the leptomeninges, and those in peripheral tissues localized mainly to the extracellular matrix. There were significantly more serotype A than B spirochetes in the leptomeninges and more serotype B than A spirochetes in the skin. The first tissue where spirochetes were observed outside the vasculature was the dura mater. Inflammation was more severe in the skin than in the brain. VspA, VspB, and the periplasmic flagellin protein were expressed in all tissues examined. These findings indicate that isogenic but antigenically distinct Borrelia serotypes can have marked differences in their localization in tissues.

Animals↗
PubMed2003

Actinobacillus actinomycetemcomitans serotype d-specific antigen contains the O antigen of lipopolysaccharide.

Actinobacillus actinomycetemcomitans is a gram-negative, facultatively anaerobic bacterium which is associated especially with aggressive forms of periodontitis. Contradictory results on the localization of the A. actinomycetemcomitans serotype-specific antigen have been reported. The aim of the present study was to characterize the A. actinomycetemcomitans serotype d-specific antigen. The antigen was isolated by affinity chromatography. The affinity column was prepared from immunoglobulin G isolated from rabbit antiserum raised against A. actinomycetemcomitans serotype d. The isolated antigen was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and silver staining, all of which revealed a ladder-like structure typical for the O antigen of lipopolysaccharide (LPS). In a displacement enzyme-linked immunosorbent assay (ELISA), the isolated antigen displaced in a concentration-dependent manner the binding of the polyclonal rabbit antiserum raised against A. actinomycetemcomitans serotype d to the competing whole-cell serotype d antigen. The isolated antigen contained LPS, and an equal concentration of LPS isolated from A. actinomycetemcomitans serotype d gave a similar displacement curve in the ELISA. In order to test the immunogenic properties of the isolated antigen, it was used to immunize a rabbit. The antiserum raised against the isolated antigen displayed specificity in Western blotting and ELISA similar to that of antibody raised against LPS isolated from A. actinomycetemcomitans serotype d. In conclusion, our results show that the A. actinomycetemcomitans serotype d-specific antigen contains the O-antigenic structure of LPS.

Actinobacillus Infections↗
PubMed2004

Ability of pneumococcal serotypes and clones to cause acute otitis media: implications for the prevention of otitis media by conjugate vaccines.

The relative abilities of pneumococcal serotypes and strains (clones) to cause acute otitis media (AOM) were investigated by comparing the serotypes and genotypes of pneumococci recovered from cases of AOM (n = 149) in children <2 years of age with those from nasopharyngeal carriage (n = 288) in age-matched controls from the same region. The odds ratio (OR) for association of pooled vaccine serotypes with AOM was found to be slightly elevated over unity, although this was not significantly different from that of pooled nonvaccine or vaccine-related serotypes. Comparing individual serotypes, 19F and 23F had 2- to 2.5-fold higher ORs, although these were not markedly different from the ORs of nonvaccine serotypes. None of the major clones had an OR that was significantly greater than the average, and the differences in ORs among serotypes and clones were much less than those for invasive disease, suggesting little variation in their ability to cause AOM. We conclude that serotype replacement may reduce the long-term efficacy of these vaccines against AOM.

Acute Disease↗
PubMed2007

Cost-effective real-time reverse transcriptase PCR (RT-PCR) to screen for Dengue virus followed by rapid single-tube multiplex RT-PCR for serotyping of the virus.

Virus detection methodology provides detection of dengue virus in the early phase of the disease. PCR, targeting cDNA derived from viral RNA, has been used as a laboratory-based molecular tool for the detection of Dengue virus. We report the development and use of three real-time one-step reverse transcriptase PCR (RT-PCR) assays to detect dengue cases and serotype the virus involved. The first RT-PCR assay uses SYBR green I as the reporting dye for the purpose of cost-effective screening for dengue virus. The detection limit of the SYBR green I assay was 10 PFU/ml (0.01 equivalent PFU per assay) for all four dengue virus serotypes. The second RT-PCR assay is a duplex fluorogenic probe-based real-time RT-PCR for serotyping clinical samples for dengue viruses. The detection threshold of the probe-based RT-PCR format was 0.1 PFU for serotypes Dengue-1 and Dengue-2, 1 PFU for serotype Dengue-3, and 0.01 PFU for serotype Dengue-4. The third is a fourplex assay that detects any of the four serotypes in a single closed tube with comparable sensitivity. Validation of the assays with local clinical samples collected from 2004 to 2006 revealed that there was an 88% positive correlation between virus isolation and RT-PCR with regard to dengue virus detection and a 100% correlation with seroconversion in subsequent samples. The serotyping results derived from duplex and fourplex assays agree fully with each other and with that derived from immunofluorescence assays.

Aedes↗