Stuart M. Wilson

APPLICATION OF A SIMPLE MULTIPLEX PCR TO AID IN ROUTINE WORK OF THE MYCOBACTERIUM REFERENCE LABORATORY

ABSTRACT A PCR specific for spacer regions 33 and 34 of the direct repeat region of the Mycobacterium tuberculosis complex was developed to complement the biochemical differentiation ofM. tuberculosis, Mycobacterium bovis, M. bovis BCG, andMycobacterium africanum subtypes I and II. In addition, this approach was incorporated into a multiplex PCR that included primers specific for IS6110 and the 65-kDa antigen gene in order to differentiate members of the M.tuberculosis complex from atypical mycobacteria.

Use of a Phage-Based Assay for Phenotypic Detection of Mycobacteria Directly from Sputum

ABSTRACT The phage amplified biologically assay is a new method for rapid and low-cost phenotypic determination of the drug sensitivities of Mycobacterium tuberculosis isolates and the detection of viable organisms in patient specimens. Infection of slowly growing mycobacteria with phage (phage D29) was followed by chemical virucide destruction of extracellular phage. Infected mycobacteria were mixed in culture with rapidly growing sensor cells, which the phage can also infect; i.e., lytic amplification of phage occurs. The aims of the present study were to optimize the speed and sensitivity of the assay and reduce its cost for developing countries by using an M. tuberculosis-spiked sputum model with (i) identification of inhibitory components of sputum and optimization of decontamination methods; (ii) simplification of the washing and development steps; (iii) reduction of the use of high-cost components, e.g., oleate-albumin-dextrose-catalase (OADC) supplement; and (iv) optimization of virucide treatment. The following results were obtained. (i) An inhibitory factor in sputum which could be removed by treatment of the sample with sodium dodecyl sulfate or NaOH decontamination was identified. (ii) A microcentrifuge-based approach with thixotropic silica as a bedding and resuspension agent was developed as an alternative to conventional centrifugation medium exchange. The yield was increased 228-fold, with increased speed and reduced cost. (iii) At present, after extracellular inactivation of phage, the ferrous ammonium sulfate (FAS) virucide is sequestered by dilution with an expensive supplement, OADC. Sodium citrate with calcium chloride was found to be a cost-effective after treatment with the FAS protectant and offered greater protection than OADC. Kinetic-lysis experiments indicated that an infection time of 1 to 3 h prior to FAS addition was optimal. (iv) Amplification of the signal (which corresponded to the burst size) was shown by allowing lysis prior to plating in a spiked medium model (up to 20-fold) and a spiked sputum model (up to 10-fold). A liquid culture detection method capable of detecting approximately 60 viable M. tuberculosis organisms in 1 ml of sputum was developed. Taken together, these improvements support the routine application of the assay to sputum specimens.

Evaluation of a Rapid PCR-Based Epidemiological Typing Method for Routine Studies of Mycobacterium tuberculosis

ABSTRACT Restriction fragment length polymorphism (RFLP) based on the insertion sequence IS6110 is used to investigate episodes of suspected transmission of infection of tuberculosis but usually takes a number of weeks from receipt of request to obtain a result. Often investigations would benefit from a more rapid method, possibly one containing an amplification step. The method employed uses a simple DNA extraction followed by a PCR step involving a single primer. Restriction enzyme analysis was performed when the patterns obtained from the PCR products were indistinguishable, especially when only single similar-size bands were obtained. The isolates used were strains of Mycobacterium tuberculosis submitted for epidemiological investigations as part of (i) possible contact-outbreak (22 episodes involving between 2 and 20 patients), (ii) possible incidents of laboratory cross-contamination (21 episodes), and (iii) possible change in drug resistance pattern or a case of reinfection (1 patient). The PCR products giving similar patterns were then subjected to restriction enzyme analysis. In conclusion it has been shown that this method is rapid, with results within 1 to 2 days of the request being received; is reproducible; and gives the same results as does RFLP. The restriction enzyme analysis stage has improved the efficiency of the technique.

Application of nucleic acid-based technologies to the diagnosis and detection of disease.

Introduction Before 1988, hybridization with nucleic acid probes was the only available nucleic acid-based technique for the detection and diagnosis of infectious agents. Since 1988, the widespread ivailability of the polymerase chain reaction (PCR) has offered a rapid and sensitive alternative. Has PCR completely superseded probing technology or is there room for both methods in today’s diagnostics? This article discusses the relative advantages and disadvantages of both methods and argues that there is still a role for both in the detection and diagnosis of disease. In order to allow a discussion in depth of the technologies, a basic knowledge of the principles of nucleic acid hybridization and of PCR will be assumed. Those wishing to refresh their memories of these techniques can find excellent books on the subject (KELLER & MANAK, 1989; KRICKA, 1992; PERSING et al., 1993).

Increased case finding of tuberculosis from sputum and sputum deposits after magnetic bead concentration of mycobacteria

Concentration of mycobacteria from sputum by centrifugation prior to acid-fast microscopy increases case finding compared to direct microscopy of the sputum (direct smear). However, centrifugation has to be performed outside the safety cabinet and many laboratories do not have access to a centrifuge. Magnetic bead extraction of the mycobacteria is an alternative method that can be performed in a cabinet with just a magnet. Magnetic TB-Bead (Microsens Medtech Ltd) extraction of mycobacteria from sputum prior to microscopy was compared to direct smear on 78 sputum samples. Microscopy of the TB-Bead extracts identified all of 26 of the direct smear positive samples either with the same microscopy score or, in 19/27 of samples, with an increased microscopy score which aided microscopy detection. In addition, microscopy of the TB-Bead extracts identified 10 additional positive samples compared to direct smear; which represents a statistically significant increase in case finding of 38% (p = 0.002) compared to direct smear. In a separate study, TB-Beads enabled further 4 positive samples to be detected from 30 centrifuged pellets that were originally smear negative; two of these were subsequently found to be positive when the original deposits were reinvestigated by smear microscopy. By concentrating mycobacteria from sputum and sputum deposits, TB-Beads have been demonstrated to increase the number of positive sputum samples which could increase case-finding. The TB-Bead method is simple and rapid and compatible with use within a safety cabinet.