Identification of Mycobacteria using per and reverse hybridisation

Abstracts

and specific results very quickly. diagnostic

techniques

1 Journal

of Microbiological

At present, however, -inolecular

of infectious

diseases have a limi’ted impact

on the daily routine of general practitioners and specialists, particularly because 1) major laboratory problems of these techniques are still unresolved (lack of standardization and reproducibility, contamination); 2) the sensitivity of most molecular detection procedures is not improved compared to culture techniques: 3) culture techniques are still needed to assess susceptibility: 4) interpretation of results obtained by molecular techniques may be difficult (viable versus dead organisms; versus disease); 5) high costs. Nevertheless,

improved

molecular

diagnostic

infection

used in the state-of-the-art

evaluation

and/or

monitoring of the following diseases: A) Evaluation of 1) diarrhea (enteropathogenic E. co/i, shigella); 2) STD (chlamydia, gonorrhea); 3) memngitis/encephalitis. particularly in immunocompromised oplasma

patients (detection in cerebrospinal

of HSV, VZV, CMV, JC virus, To.rfluid); 4) CMV disease in organ

transplant and HIV infected patients; 5) Tuberculosis: 6) Retinitis (HSV, VZV, Toxoplasma in intraocular fluid). B) Monitoring of I ) HIV infection: 2) Hepatitis C; 3) CMV disease in immunocompromiaed patients.

by PCR-ribotyping. PCR-ribotype

of Irtfection

and Immunity,

University

of Glasgow,

G12

SQQ, Scotland “Scottish

Agru.dtural

Panicuik

EHZOQE,

College

Veterinary

Services,

Bush Estate,

Scotland

somnus, Histophilus ovis and Actinobacillus are Gram-negative bacteria that cause similar clinical conditions in cattle and sheep. They share similar cultural and biochemical properties which make them difficult to differentiate although historically they have been regarded as host-specific. A combination of REP-PCR, ERIC-PCR, and PCR-ribotyping was used to Identify and differentiate these bacterial species and to investigate strain variation. Twenty nine isolates of H. somnus, nineteen isolates of H. ovis and twenty four isolates of A. seminis from cattle and sheep were typed by these PCR methods with supernate derived from boiled cells as template DNA. REP-PCR generated 11 types for H. .somnu.s isolates, nine for H. ovis isolates and five for A. .seminis isolates. ERIC-PCR produced 16 types for H. somnus isolates, seven for H. ovis isolates and nine for A. seminis. The PCRribotyping produced eight types for H. somnus, five for H. ovis and the pattern for A. seminis was identical for all isolates tested except one. The banding patterns of REP- and ERIC-PCR were complex but provided greater discrimination than PCR-ribotyping. The discrimination between isolates was increased by a combination of the three methods. In general the three species were clearly distinct from each other by each of the typing methods. However, Haemophi1u.s

2.X-2%

14s

three sheep strains

(H. ovis) had the same

as one of the H. somnus types and an A. srminis

isolate of bovine origin was identical to the sheep isolates. Amongst these related species, the suggested host-specific relationship may not be absolute. 33 Identification sation T.J. Brown Palace

of Mycobacteria

of

Microbiology,

Road, London,

lmmunocompromised

SEI

using per and reverse hybridi-

St.

Thorno.\

Hospital,

Lamheth

7EH. Englaml

patients

are often Infected by mycobac-

teria other than M. tuberculosis (MOTT). The treatment these patients recezve is dependant on the infectmg species of nrycobacteria. The slow growth rate of mycobacteria mean that identification may take several weeks. The detection of species specific nucleotide sequences provides ,I rapid technique for mycobacterial identification. Genus specific primers were used to amplify by per a DIG labelled fragment of dnaj from mycobactrriul lysates. Labelled fragments were used to probe IO immobilised oligonucleotides specific to M. tuberculosis, M. arium. M. intrac,ellulare. M. kansasii,

32 Identification and differentiation of isolates of Huemophilus somnus, Histophilus ovis and Actinobacillus seminis by PCR methods S. Appuhamy . J.G. Coote”, J.C. Lowh and R. Parton” “Division

30 (1997)

Department

tools open fas-

cinating perspectives such as detection and identification of pathogens that can not be cultured. In addition, molecular techniques are already

Methods

M. xenopii,

M. marinum,

M. .sc~rofitlacu,mr M. gordonar,

and M. haemophilum. The hybridisation between probe and oligonucleotide was determined colormetrically. 60 consecutive mycobacrerial clinical isolates were identified using this rapid scheme and conventional bactermlogy. The identity determined by both methods was in agreement in 55 (92%) isolates. The per failed to amplify a fragment in the remaining 6 isolates. Two of these cultures were M. chelowr a species known to not be amplified by these pnmers. 3 isolates were not acid fast on the slopes used for per. One isolate of M. ,fortuitum was not amplified. >9.5% of clinical isolates of these IO mycobacterial species can be identified using this rapid method. M. fortuitum

.srminis

34 PCR detection of M. tuberculosis in clinically mens T.J. Brown and G.L. French Department Palace

of

Miuobiology,

Road, London.

St.

selected speci-

Thoma Y’ Hospital.

Lambeth

SE1 7EH, En,yland

In uncomplicated pulmonary tuberculosts (TB) diagnoals is achieved using x-ray and smear, then confirmed by culture, after up to 8 weeks when growth of M. tubercrrhxis (Mtb) is observed. The amplification and detection of Mtb specific nucleic acid sequences is a useful tool in the diagnosis of TB. This paper describes the results seen over 12 months using a commercially available per kit (Amplicor) for the detection of Mtb in diagnostically difficult specimens. 55 specimens were examined using Amplicor. PCR had been requested by the clinicians because clinical diagnosis was difticult. 38% of these were respiratory and 62% were non-respiratory specimens. The per was carried out according to the manufacturera instructions. Where non-respiratory specimens were blood stained