Phage as a diagnostic – the use of phage in TB diagnosis

Abstract

Rapid TB diagnosis and drug susceptibility testing is limited by the slow growth of the causative organism, Mycobacterium tuberculosis (MTB). Effective and rapid methods for the detection and drug susceptibility testing of TB, that are suitable for implementation in low income countries, which bear the highest TB burden, are urgently needed for effective identi®cation, treatment and control of the disease. Mycobacteriophages have the potential to become useful tools in the diagnosis of TB, as they are speci®c for mycobacteria and only replicate in, and hence detect, viable cells. Phage-based techniques involve simple manual manipulations that typically require little capital expense and yield results rapidly. Two techniques have been developed recently which use mycobacteriophage for the antibiotic susceptibility testing and direct diagnosis of TB from sputum. Luciferase Reporter Phages (LRP) are recombinant phages that contain the gene for the ®re-¯y luciferase enzyme. Upon infection of mycobacteria by these phages, this enzyme is produced within a cell. In the presence of this enzyme, light is produced by viable cells that also contain the substrates for the enzyme: ATP and luciferin (added exogenously). The`Bronx Box' has been developed as a low cost means of detecting light signal from mycobacteria infected with LRP using photographic ®lm. A MTB strain's susceptibility to an antibiotic can be assessed by comparing light production, as an indication of the presence of viable cells, in samples pre-incubated in the presence and absence of the test antibiotic. Susceptibility testing of some of the front line drugs for TB chemotherapy has given excellent results (detection of all resistant isolates), using LRP and the Bronx Box. Phage ampli®cation uses the production of progeny phage, instead of a reporter gene product, to re¯ect the presence of target bacteria. The technique has been successfully applied to drug susceptibility testing and for diagnosis to the TB from sputum specimens. Viable MTB cells within a specimen are uniquely infected by a bacteriophage reagent. A potent selective virucide destroys all the phage particles that remain outside the cells while remaining inactive against the MTB bacilli (and the phages that have infected them). After the virucide has been neutralised, and phages that are released from infected cells are indicative of TB cells being present in the original specimen. These phages can be easily detected by propagation on lawns of non-pathogenic rapidly growing mycobacteria. A commercially available test, termed FASTPlaqueTB-RIF, determines the rifampicin susceptibility of isolates by comparing the viability of cells pre-treated with and without rifampicin. Results give excellent agreement (97±98% overall) with conventional`gold standard' methods, but in 2 days rather than 3 weeks. Preliminary work has shown that it should be possible to reduce the assay time to 24 h. A variation to this method, FASTPlaqueTB, detects TB directly from sputum specimens and is capable of rap...

Evaluation of the usefulness of phage amplification technology in the diagnosis of patients with paucibacillary tuberculosis

“…The test showed poorer sensitivity in our study than in earlier studies [3][4][5][6][7][8][9] primarily because in those studies, a majority of the patients included had been sputum-smear positive. It had been demonstrated by Alcaide et al, [3] that the test is more likely to give positive results for specimens in which high number of acid-fast bacilli were observed in the smear and from which recovery of the bacilli was highest (>30 colonies isolated/slant).…”

“…Several studies have been carried out to assess the diagnostic utility of the test in various countries. [3][4][5][6][7][8][9] But, no study has yet focussed exclusively on the group of sputum-negative paucibacillary patients, who constitute a sizeable fraction of tuberculosis patients attending a referral hospital of a developing country. Establishment of a deÞ nitive diagnosis is often problematic in this group of patients.…”

Usefulness of a New Mycobacteriophage-Based Technique for Rapid Diagnosis of Pulmonary Tuberculosis

“…Only a few studies have assessed the diagnostic utility of the phage technology by using clinical samples (1,3,18,19). Although the results of those studies have shown a high sensitivity and specificity of the test, some important issues should be a Negative, no acid-fast bacilli observed; ϩ, 1 to 9 bacilli/10 fields; ϩϩ, 1 to 9 bacilli/field; ϩϩϩ, Ͼ9 bacilli/field.…”

Bacteriophages of Ralstonia solanacearum: Their Diversity and Utilization as Biocontrol Agents in Agriculture

“…(ii) The phage amplification assay detects the increase in phage particles in target bacterial cells after infection (Mole & Maskell, 2001). (iii) The phage-tagging method is another phage-based approach for bacterial detection.…”