Yun-Fong Ngeow

Molecular Typing of Mycobacterium abscessus Based on Tandem-Repeat Polymorphism

ABSTRACT A variable-number tandem-repeat (VNTR) typing assay for the differentiation of Mycobacterium abscessus strains was developed. This assay showed complete reproducibility, locus stability, and a discriminatory power (Hunter-Gaston discriminatory index [HGDI] of 0.9563) that is superior to that of multilocus sequencing. It is a promising tool for the investigation of Mycobacterium abscessus epidemiology and nosocomial outbreaks.

Heat inactivation of serum potentiates anti-cardiolipin antibody binding in ELISA

Heat treatment of sera at 56 degrees C for 30 min results in positive ELISA reactions for anti-cardiolipin antibody (aCL) in sera that had undetectable or low levels of aCL before heat inactivation. The positive, potentiated reactivity of the heated sera in the aCL ELISA could be inhibited with the cardiolipin antigen and was abolished by prior IgG depletion using staphylococcal protein A. The heat-potentiating effect of aCL binding in ELISA was evident in both normal human sera and clinical sera including sera from patients with systemic lupus erythematosus and syphilis.

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.

Evaluation of PCR-RFLP analysis targeting hsp65 and rpoB genes for the typing of mycobacterial isolates in Malaysia.

In this study, PCR-RFLP analysis (PRA) targeting hsp65 and rpoB gene regions was evaluated for the identification of mycobacterial species isolated from Malaysian patients. Overall, the hsp65 PRA identified 92.2 % of 90 isolates compared to 85.6 % by the rpoB PRA. With 47 rapidly growing species, the hsp65 PRA identified fewer (89.4 %) species than the rpoB PRA (95.7 %), but with 23 slow-growing species the reverse was true (91.3 % identification by the hsp65 PRA but only 52.5 % by the rpoB PRA). There were 16 isolates with discordant PRA results, which were resolved by 16S rRNA and hsp65 gene sequence analysis. The findings in this study suggest that the hsp65 PRA is more useful than the rpoB PRA for the identification of Mycobacterium species, particularly with the slow-growing members of the genus. In addition, this study reports 5 and 12 novel restriction patterns for inclusion in the hsp65 and rpoB PRA algorithms, respectively.

MOMP-based PCR reveals presence of Chlamydia pneumoniae DNA in respiratory and serum samples of patients with acute C. pneumoniae-associated infections

Abstract Diagnosis of Chlamydia pneumoniae infections has always been hindered by difficulties in isolating and culturing the organism, and by poorly standardized serological techniques. Because of this, the polymerase chain reaction (PCR) is now being investigated as a quicker, more sensitive diagnostic tool. In this study, PCR was performed on DNA extracted directly from the serum and tracheal secretions of patients. The PCR reaction amplified a portion of the major outer membrane protein (MOMP) gene of C . pneumoniae . This gave rise to a 487-bp fragment which was detected in all enzyme immunoassay (EIA) Chlamydia positive tracheal secretions and 90.3% (159/176) of the C . pneumoniae microimmunofluorescence (MIF) positive serum samples tested. Also, 24.6% (29/118) of MIF-negative serum samples were positive by PCR. The specificity of this product was confirmed by transferring various PCR products (PCR having been performed with C . pneumoniae DNA, DNA from other Chlamydia spp., other organisms and from patient samples) by Southern blotting and subsequently hybridizing with radioactively labelled PCR product as well as an inter-primer specific oligonucleotide probe. As further confirmation of the identity of the organism, a second PCR using C . pneumoniae -specific rRNA primers was performed on randomly selected samples. The samples that were positive with the MOMP primers were also positive by the rRNA primers and vice versa. The PCR products were also sequenced and matched with published data as further confirmation. The presence of C . pneumoniae in serum extracted DNA strongly suggests the possibility of a systemic spread of the organism. PCR could be an important diagnostic alternative for the sensitive and specific detection of C . pneumoniae infections.

Ethnic distribution of Chlamydophila pneumoniae antibodies in a Malaysian population and possible correlation with coronary heart disease

Chlamydophila pneumoniae, an important respiratory pathogen causing lower respiratory tract infections, has also been implicated in coronary heart disease (CHD). This study reports a cross-sectional, demographic, serological analysis of the prevalence of Chlamydophila pneumoniae antibodies in a multiracial Malaysian population. Generally, Malaysian Indians had the highest degree of seropositivity (58%) followed by the Chinese (54%) and the Malays (32%), results which were statistically significant (CI: 95%; p < 0.01). Interestingly, this trend was also reflected in the study group consisting of patients with acute myocardial infarctions (AMI) and chronic CHD. Again, the Indians were more frequently sero-positive (65%), with more than 50% having IgG titres ≥ 128. Comparatively, the Chinese and Malays showed 51 and 17% seropositivity respectively. These results were also statistically significant (CI: 95%; p < 0.01). Malaysian Indians are more commonly afflicted with CHD. A variety of factors have been suggested to explain this prevalence including diet, social habits, genetics and the possible role of infectious agents. This study notes an interesting association between this pattern of racial prevalence and the possible role of C. pneumoniae infections as a contributory/predisposing factor in the development of cardiovascular disease.

Mycobacterium abscessus to treat or not to treat

A patient with Mycobacterium abscessus lung disease was mistaken to have pulmonary tuberculosis with airway colonization by the non‐tuberculous mycobacterium. Appropriate antibiotics were only given when the patients signs and symptoms worsened while on anti‐tuberculosis therapy. Despite treatment with a combination of antibiotics showing in vitro susceptibility, the pathogen persisted in the respiratory secretions for longer than 6 months and the patient suffered a spontaneous pneumothorax 14 months into treatment. This case illustrates the chronic course of M. abscessus lung infection, the tendency for flare‐ups, the inadequacy of current treatment regimens, and the necessity for prolonged patient follow‐up.

Plasmid profiles of Neisseria gonorrhoeae in Peninsular Malaysia.

The plasmid profiles of 160 strains of Neisseria gonorrhoeae isolated in Peninsular Malaysia, comprising 80 penicillinase‐producing (PPNG) and 80 non‐penicillinase‐producing (non‐PPNG) isolates, were determined. The 80 PPNG isolates were divided into two plasmid groups. All of them harbored two common plasmid species, a 4.4 megadalton (Md) R plasmid previously associated with β‐lactamase production in PPNG strains from the Far East and a 2.6 Md multicopy plasmid of unknown function. In addition to these two plasmids, 60 (75%) PPNG isolates also carried a large 24.5 Md conjugative plasmid.