Tikki Pang

Typhoid fever—important issues still remain

Abstract The Third Asia Pacific Symposium on Typhoid Fever and Other Salmonellosis was held in Bali, Indonesia on 8–10 December 1997.

Genome size variation among recent human isolates of Salmonella typhi

We performed genome size estimation of 17 recent human isolates of Salmonella typhi from geographically diverse regions using pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with restriction endonucleases XbaI (5-TCTAGA-3), AvrII (5-CCTAGG-3) and SpeI (5-ACTAGT-3), and summation of the sizes of restriction fragments obtained. All 17 isolates had circular chromosomes, and genome sizes differed by as much as 959 kb, ranging from 3,964 to 4,923 kb (mean genome size = 4,528 kb). The data obtained confirm the usefulness of PFGE in studies of bacterial genome size and are in agreement with recent results indicating considerable genetic diversity and genomic plasticity of S. typhi. The variation in genome sizes noted may be relevant to the observed biological properties of this important human pathogen, including its virulence.

Molecular characterisation of Sargassum polycystum and S. siliquosum (Phaeophyta) by polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers

Genomic DNA was extracted from 13 samples of Sargassum polycystum and S. siliquosum collected from various localities around Peninsular Malaysia and Singapore by using four different extraction methods. The yields and the suitability of the DNA to be used as template for the polymerase chain reaction (PCR) was compared. DNA samples were subjected to PCR analysis by using random primers. Only DNA samples that were extracted using the CTAB method were successfully amplified by random amplified polymorphic DNA (RAPD)-PCR. Five of 31 random primers (OPA02, OPA03, OPA04, OPA13 and OPM10) tested amplified sequences of DNA from the DNA samples. Reproducible, amplified products were obtained using these primers and showed some potential to be useful in discriminating individual samples within the genus, in determining relationships between species within a genus and in developing individual fingerprints for individual samples.

Isolation of dengue viruses by intracerebral inoculation of mosquito larvae

The isolation of dengue viruses from clinical specimens has always posed a particularly difficult problem. The use of invertebrate cell cultures such as AP-61 and C6/36 has reduced the time required for definitive diagnosis to within a week. More recently, inoculation of adult mosquitoes has been used but it requires more than a week to reach a confirmed laboratory diagnosis. We describe a method using intracerebral inoculation of immobilized fourth instar of Toxorhynchites splendens larvae for the isolation of dengue viruses from clinical specimens which yields results within a few days following incubation at 32 degrees C.

Genetic Variation among Malaysian Isolates of Salmonella typhi as Detected by Ribosomal RNA Gene Restriction Patterns

Genetic variation among Malaysian isolates of Salmonella typhi was determined by analysis of ribosomal RNA gene restriction patterns. Of the 20 isolates analyzed, eight different pattern combinations were detected. The amount of variation observed was also dependent upon the restriction endonuclease used; PstI produced more different patterns than did SmaI. The results suggested that disease activity was due to a number of different clones circulating simultaneously rather than a single strain. Further implications of the data are discussed.

Comparison of safety and immunogenicity of a Vi polysaccharide typhoid vaccine with a whole-cell killed vaccine in Malaysian Air Force recruits

OBJECTIVEnTo carry out a comparative study of the safety and immunogenicity of Vi polysaccharide vaccine against whole-cell killed (WCK) typhoid vaccine.nnnMETHODSnThe study was carried out on young adult recruits (aged 18-25 years) of the Malaysian Air Force. A total of 125 subjects received the Vi polysaccharide vaccine and 114 received the WCK vaccine.nnnFINDINGSnThe Vi vaccine was significantly less reactogenic than the WCK vaccine with regard to systemic and local reactions. Following administration of the Vi vaccine, seroconversion rates (defined as the percentage of subjects with a 4-fold rise of baseline antibody level) of 75.5% and 67% were observed at 2 weeks and 6 weeks, respectively, after immunization, compared with 25% and 31.3% among recipients of the WCK vaccine. Of the 110 Vi vaccinees with serological data, 21 (19%) had high, seroprotective, pre-immunization levels of anti-Vi antibodies (> or = 1 microgram/ml). The majority of subjects in this group came from a region in Malaysia which is known to have high typhoid endemicity. Interestingly, these antibody levels were boosted considerably following administration of vaccine at a level that was 5-fold higher than in subjects with low pre-immunization levels. In contrast, the seroconversion rates in those receiving the Vi vaccine were higher in subjects with low pre-immunization levels of anti-Vi antibodies (76-84%), compared to those with protective levels of > or = 1 microgram/ml prior to immunization (48-57%).nnnCONCLUSIONSnThe study reaffirms the safety and efficacy of the Vi polysaccharide vaccine and identifies a hitherto unrecognized advantage in its use, i.e. it is a potent immunogen that boosted considerably the protective antibody levels among a significant number of immunologically sensitized individuals living in typhoid-endemic regions.

Application of polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers in the molecular identification of selected Sargassum species (Phaeophyta, Fucales)

The random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was used for the molecular characterisation and identification of Sargassum spp. A total of 17 samples of Sargassum (Sargassaceae, Fucales) was obtained from various localities around Peninsular Malaysia and Singapore. On the basis of morphological characteristics, the samples were tentatively grouped into five species: Sargassum baccularia, S. glaucescens, S. oligocystum, S. polycystum and S. siliquosum. By RAPD-PCR, five of 31 random primers tested generated reproducible amplification products, and polymorphic loci were detected by four of them (OPA02, OPA03, OPA04, OPA13). The RAPD-PCR profiles did not correlate with the morphological grouping into five species and extensive variation was detected between different isolates of the same species. A 450 base pair fragment generated using OPA13 was detected in 12 of 17 samples of Sargassum. This fragment was also present in profiles from Turbinaria (Sargassaceae). This study suggests...

Genetic dynamics of Salmonella typhi—diversity in clonality

I acknowledge the work on genetic diversity of S. typhi, which was very ably performed in my laboratory by Dr Kwai-Lin Thong as part of her PhD dissertation (University of Malaya, 1997). I also thank Ken Sanderson, Martin Altwegg and Edmundo Calva for their valuable comments and suggestions during the preparation of this article.

Outbreak of Salmonella enteritidis gastroenteritis: Investigation by pulsed-field gel electrophoresis

OBJECTIVEnPulsed-field gel electrophoresis (PFGE) was used to investigate an outbreak of gastroenteritis caused by Salmonella enteritidis. The outbreak occurred among university undergraduates who consumed contaminated food.nnnMETHODnMolecular typing was done by analyzing DNA band patterns of isolates of S. enteritidis after digestion of chromosomal DNA with infrequently-cutting restriction endonucleases XbaI, AvrII, and SpeI and separation of DNA fragments using PFGE.nnnRESULTSnTwenty-nine outbreak isolates of S. enteritidis had identical or highly similar PFGE patterns, whereas different PFGE patterns were observed among three epidemiologically unrelated isolates obtained during the same period.nnnCONCLUSIONnThe data obtained confirm the value of PFGE in epidemiologic investigations of outbreaks caused by S. enteritidis.

An immunogenic epitope of Chlamydia pneumoniae from a random phage display peptide library is reactive with both monoclonal antibody and patients sera

Random 15-mer peptides displayed on filamentous phages were screened in binding studies using a Chlamydia pneumoniae-specific monoclonal antibody (RR-402) and affinity-purified, polyclonal sera from patients seropositive for C. pneumoniae infections by the microimmunofluorescence (MIF) test. One 15-mer epitope, epitope Cpnl5A (LASLCNPKPSDAPVT) was identified in both the monoclonal and polyclonal screenings, and showed higher ELISA reactivity with C. pneumoniae MIF-positive sera compared to patients with other chlamydial infections, non-chlamydial respiratory infections and normal healthy sera (MIF-negative). Interestingly, epitope Cpnl5A also showed significant (52%) amino acid sequence homology to the 56 kDa type-specific antigen of Rickettsia tsutsugamushi, a protein implicated in the virulence of this organism.