- Volume 46, Issue 10, 1997

Mycobacterium tuberculosis strains resistant to two or more of the first line antituberculosis drugs (MDR) are a serious threat to successful tuberculosis control programmes. For this retrospective study, 85 follow-up drug resistant isolates from 23 patients residing in a community with a high incidence of tuberculosis were collected and the level of in-vitro resistance to antibiotics determined quantitatively. PCR-SSCP and sequencing techniques were used to screen for gene mutations associated with resistance in 31 follow-up samples from a smaller group of eight patients. DNA fingerprint analysis was done on sequential isolates to confirm identity. Although treatment had a profound effect on changes in drug resistance patterns, the MIC for a particular agent remained constant in follow-up isolates. DNA fingerprinting and mutational analysis (14 different loci) showed that the genome of MDR strains of M. tuberculosis is relatively stable during the course of therapy. The rpoB gene was the most frequently mutated structural gene involved in drug resistance and a novel C to T mutation upstream of open reading frame (ORF)1 of the inhA operon was detected. No evidence was found of the presence of sfrain W (New York) in this group of MDR strains. The results stress the importance of confirming individuality of strains for the accurate calculation of frequencies of particular mutations associated with drug resistance, particularly in a high incidence area. Approximately one-half (47.8%) of the patients had isolates resistant to concentrations just above the critical concentration for isoniazid (MICs of 0.2–5 mg/L). Therefore, these patients and their contacts who develop primary drug-resistant tuberculosis may respond to higher dosages of treatment which could have a considerable impact on the cost and the ease of management of resistant tuberculosis.

A modified chocolate blood agar medium incorporating cefsulodin, a semi-synthetic cephalosporin, was developed and compared with non-selective chocolate blood agar and selective haemin-bacitracin blood agar for the routine isolation of Haemophilus influenzae from the respiratory secretions of patients with cystic fibrosis. The results showed that cefsulodin chocolate blood agar improved the recovery rate of H. influenzae in this group of patients. The medium was stable on storage for 10 days at 4°C.

Fusobacterium necrophorum strains from human infection (21) were compared with strains from animals (17 biotype A, 2 biotype AB, 4 biotype B, 1 biotype unknown), and the type strain NCTC 10575 in conventional tests reaction patterns (CTRPs), SDS-PAGE and pyrolysis mass spectrometry (PMS). Classifications from the three approaches showed one major consensus group comprising all human strains, and another comprising animal biotype A strains. Animal biotype B strains and one animal strain, designated with some doubt to biotype A, were outliers of the consensus ‘human strain’ group. Again, animal biotype AB strains were outliers of the consensus ‘animal biotype A group’, as was the type strain, which was clearly atypical in conventional tests and PMS. Colonial and microscopic characters showed good discrimination between the major consensus groups. However, only haemagglutination and the API-ZYM leucine arylamidase of the biochemical tests discriminated well between these groups. The ‘animal biotype A group’ clearly corresponds to F. necrophorum subsp. necrophorum, but synonymy of F. necrophorum subsp. funduliforme with the group of human strains was less certain. The latter subspecies was described solely on the basis of animal strains, all of biotype B, but each of four animal biotype B strains in this study was an outlier of the ‘human strain group’ in one or more of the characterisation approaches. Strains of F. necrophorum causing human infection were clearly distinct from the biotype A strains commonly found in animal infection. This has implications for the validity of animal models of human necrobacillosis. In view of these differences, it would be useful to have a validated designation for strains causing human infection. However, it would be premature to assume that the definition of F. necrophorum subsp. funduliforme encompasses the human strains in the absence of confirmatory DNA-homology and 16S rRNA-sequencing studies.

Two monoclonal antibodies (MAbs), designated as H9 (IgG2a) and H20 (IgM), directed against heat-shock protein 60 (HSP60) of Helicobacter pylori strain TK1029 were established. Affinity-purified antigens cross-reacted in immunoblots with MAb H9 and MAb H20 respectively. These antigens also reacted with the 3C8 MAb previously established in this laboratory, which recognised Yersinia enterocolitica HSP60. By amino-acid sequence analysis, the N-terminal amino-acid sequence of the protein recognised by both H9 and H20 MAbs was confirmed as the amino-acid sequence of H. pylori HSP60 reported previously. Both MAbs reacted with nine strains of H. pylori in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. In addition, MAb H9 reacted with extracts of other bacteria including H. mustelae, Pseudomonas aeruginosa, Vibrio cholerae, Serratia marcescens, Proteus mirabilis, Escherichia coli and Shigella sonnei. In contrast, MAb H20 reacted only with strains H. pylori. These results suggest that both the species-specific epitope recognised by MAb H20 and the common epitope recognised by MAb H9 exist on HSP60 of the bacterial cell. Both MAbs also reacted with the 60-kDa protein in the lysate of human gastric carcinoma (MKN45) cells. It was shown by immunohistochemical staining that gastric epithelial cells of four out of six biopsy specimens examined stained positively with MAb H20. These results suggest that there is a common epitope in H. pylori HSP60 and human gastric epithelial cells.

This study was performed to differentiate serologically between patients with hydatid disease which is active, and which has been successfully cured. A total of 18 cases was included. Pre-treatment serum samples were collected before surgery or chemotherapy. Post-treatment serum samples were collected at various intervals (3 days, 7 days, 1 month, 6 months, 1 year and 2 years) after surgery or chemotherapy. These sera were tested for the presence of circulating hydatid antigen (CAg) by bacterial co-agglutination (Co-A) and counter-current immunoelectrophoresis (CIEP) tests, and for circulating hydatid antibodies (CAb) by indirect haemagglutination assay (IHA). Ten and eight sera, respectively, were positive out of 11 pre-operative and pre-chemotherapeutic sera tested for CAg by the Co-A and CIEP tests. Post-operative sera collected from these cases did not show any CAg by the CIEP test. However, CAg was detected by Co-A in three and four serum samples collected on the third and seventh day, respectively, after surgical removal of the cyst. However, the CAg levels in these post-operative sera showed a gradual decline by the seventh day and were completely absent in the serum specimens collected 1 month after surgery and 6 months after chemotherapy. All the post-operative serum samples except.two, collected 2 years after surgical removal of the cyst, in seven cases of old hydatid disease, were negative for CAg by both the CIEP and Co-A tests. Unlike the CAg profile, no marked differences were noted between the CAb profile of the pre-and post-treatment sera, as shown by the IHA test. Even 1 year after surgery or chemotherapy, two sera showed a marginal decrease in their CAb titre. CAb at varying titres was still detectable in all seven serum samples from old cases of hydatid disease, even 2 years after surgical removal of the cyst. This study shows the value of serial pre-and post-operative or chemotherapy estimation of CAg by Co-A and CIEP as an index of cure or of continuing hydatid infection.