Summary: Three medieval bone samples with osteological evidence of tuberculosis infection were analysed for the presence of DNA sequences from Mycobacterium tuberculosis using a series of PCRs. In each case amplification of IS6110 and part of the β-subunit of RNA polymerase identified infection with a bacterium belonging to the M. tuberculosis complex. Amplification of the mtp40 genome fragment and the presence of a guanine residue at position 285 in the oxyR pseudogene, demonstrated the infecting strain to be similar to present day M. tuberculosis isolates rather than to Mycobacterium bovis. Spoligotyping, based on amplification of the direct repeat (DR) region of the mycobacterial genome, provided further evidence of similarity to M. tuberculosis and indicated a close relationship between isolates associated with two separate medieval burials. The study demonstrates the feasibility of amplifying multiple M. tuberculosis loci in ancient human remains and suggests important applications in the study of the palaeoepidemiology and virulence of tuberculosis in past populations.

A number of 1,2-benzothiazines have been synthesized in a three-step process. Nine chalcones 1–9 bearing methyl, fluoro, chloro and bromo substituents were chlorosulfonated with chlorosulfonic acid to generate the chalcone sulfonyl chlorides 10–18. These were converted to the dibromo compounds 19–27 through reaction with bromine in glacial acetic acid. Compounds 19–27 were reacted with ammonia, methylamine, ethylamine, aniline and benzylamine to generate a library of 45 1,2-benzothiazines 28–72. Compounds 28–72 were evaluated for their antimicrobial activity using broth microdilution techniques against two Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and two Gram-negative bacteria (Proteus vulgaris and Salmonella typhimurium). The results demonstrated that none of the compounds showed any activity against Gram-negative bacteria P. vulgaris and S. typhimurium; however, compounds 31, 33, 38, 43, 45, 50, 53, 55, 58, 60, 63 and 68 showed activity against Gram-positive bacteria Bacillus subtilis and Staphylococcous aureus. The range of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was 25–600 µg/mL, though some of the MIC and MBC concentrations were high, indicating weak activity. Structure activity relationship studies revealed that the compounds with a hydrogen atom or an ethyl group on the nitrogen of the thiazine ring exerted antibacterial activity against Gram-positive bacteria. The results also showed that the compounds where the benzene ring of the benzoyl moiety contained a methyl group or a chlorine or bromine atom in the para position showed higher antimicrobial activity. Similar influences were identified where either a bromine or chlorine atom was in the meta position.

Transmission of multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB) presents a serious problem for infection control in hospitals, particularly in the context of co-infection with the human immunodeficiency virus (HIV). We report on the use of molecular genetic tools to allow rapid assessment of samples from patients potentially infected with MDR-TB. Sputum and bronchoalveolar lavage samples were obtained from two HIV-positive patients with suspected tuberculosis, who had previous contact with a known MDR-TB index case. Polymerase chain reaction (PCR) was used directly on clinical samples to amplify genetic loci associated with rifampicin resistance (rpoB), and strain-specific polymorphisms (the direct repeat (DR) region). Drug resistance was determined using a commercially available kit for detection of point mutations in the rpoB gene (Inno-Lipa RifTB; Innogenetics, Belgium), and confirmed by nucleotide sequencing. Strain variation was determined using the spoligotyping method, based on the presence or absence of variable linker sequences within the DR region. In one patient, infection with a MDR strain identical to that of a known index case was demonstrated. A second patient, although positive for M. tuberculosis, was found to be infected with a rifampicin-sensitive strain. Results were obtained within 48 h, allowing appropriate treatment to be initiated and infection control measures to be implemented. PCR-based tests for strain-typing and for identification of rifampicin resistance provide important tools for identifying patients with MDR-TB and for rapid monitoring of potential nosocomial spread of MDR-TB. Prompt confirmation or exclusion of possible transmission allows early clinical intervention to prevent future outbreaks of multidrug-resistant M. tuberculosis.

BACKGROUND Over the last 10 years there has been a fourfold increase in cases of tuberculosis in Harare, Zimbabwe. The use of molecular epidemiology to understand tuberculosis transmission in this epidemic has been hampered by the availability of suitable culture facilities. A study was therefore undertaken to explore the potential of spoligotyping, a polymerase chain reaction based technique that does not require tuberculosis culture. METHODS Adults attending a chest clinic with clinical or radiological pulmonary tuberculosis and one smear positive sputum were enrolled over one month. Demographic, socioeconomic, and clinical data were gathered using a standardised questionnaire. Molecular fingerprinting of genomic DNA recovered from sputum was performed by spoligotyping. RESULTS Sixty one subjects (median age 28 years (range 18–73); 61% men) were recruited and 57 provided adequate sputum samples. Recent rural-urban migration or immigration was not common; 40% of subjects lived in crowded living conditions. DNA suitable for spoligotyping was recovered from 28 patients and 20 different genotypes of Mycobacterium tuberculosis were identified. Fifteen patients were infected with an M tuberculosis strain shared by one or more individuals. Patients infected with a shared spoligotype were not closely linked geographically within Harare, but were more likely to live in overcrowded conditions (69% versus 23%; odds ratio 6.85 (95% CI 1.2 to 47), p = 0.026). Analysis of the patients’ original rural family homes revealed two geographically related spoligotype clusters. CONCLUSIONS Spoligotyping may yield valuable molecular typing information in populations where tuberculosis culture is not available. This novel technique requires further development and evaluation in larger epidemiological studies.