Several methods based on phage D29 replication have been reported for rapid drug resistance detection in M. tuberculosis isolates [4,6,13-17]. They differ with respect to the assay format, drug concentration and criteria for classifying isolates as resistant or susceptible. The reported studies indicate that phage replication technology offers the potential of a rapid, sensitive and specific test for detecting resistance to rifampicin. The methods reported initially required three to four days to complete and each isolate was tested in a single tube format, which is not convenient for screening large number of isolates. The phage method we have tested is a simple, low cost assay taking 48 hours to complete. For our study, the test was transferred to the Joint Clinical Research Centre (JCRC) in Kampala, Uganda, to assess its applicability and performance in a developing country setting.
When used with rifampicin concentrations at 4 or 10 μg/ml the phage assay was able to correctly detect all isolates classified as resistant to rifampicin by the BACTEC 460 (n = 35). Four additional isolates, found susceptible by Bactec 460, were identified as resistant by the phage assay. All 4 isolates were found to harbor mutations in the rpoB gene predictive of resistance, in addition they were found to have MIC's of over 50 μg/ml suggesting they were truly resistant. Thus the BACTEC 460 failed to detect 4/39 (10.2%) of rifampicin resistant isolates identified in the study. It could be postulated that the BACTEC culture was a mixed population, consisting of predominantly susceptible organisms but with a low level of (emerging) rifampicin resistant organisms. Retrospective analysis of BACTEC data showed that one isolate with a Leu533Pro mutation appeared to have a low level resistant population, yet the growth rate (change in growth indices) was not indicative of borderline resistance. Results of the other three isolates were clearly susceptible. It is of concern that the BACTEC was unable to satisfactorily detect four resistant isolates. These isolates were found resistant to other first line anti-tuberculosis drugs by BACTEC 460 and such patterns should prompt careful interpretation of the rifampicin result and/or additional testing.
Initial comparison between the phage assay and BACTEC methods suggested an estimated sensitivity and specificity for the phage assay of 100% and 96.5% respectively if the BACTEC was taken as the "gold standard". However, further analysis suggests that the BACTEC may record false negative results and might not, by itself, be an adequate method with which to evaluate new tests for drug resistance. In a previous study Albert et al reported a strain found negative by BACTEC 460 that was positive by a phage test that was subsequently found to harbor a mutation predictive of resistance [14]. BACTEC 460 has previously been considered as one of the gold standard tests for assessing susceptibility to anti-tuberculosis drugs. However, proficiency testing of traditional culture based methods undertaken by the WHO Supranational Laboratory Network indicates variable accuracy when testing susceptibility to rifampicin with an overall sensitivity and specificity of 97.2% and 96.8% respectively [1].
One isolate that was classified as resistant by both BACTEC 460 and the phage assay did not have a mutation in the rpoB core region. It has previously been reported that mutations in other regions of the gene may be responsible for resistance in a small proportion of M. tuberculosis strains [18]. Thus our results suggest the accuracy of the phage assay may be higher then that of molecular methods that are limited to screening the 81 bp genomic region of the rpoB gene.
When the phage assay was used with a drug concentration of 2 μg/ml 19 susceptible isolates were falsely identified as resistant. With this drug concentration the phage assay had a sensitivity of 100% but a specificity of 79.8%. When used at drug concentration of 4 or 10 μg/ml no false positives or false positive results were observed and the phage assay was more accurate than either the BACTEC 460 or the sequence analysis. Previous studies using mycobacteriophage D29 have applied a working concentration of 10 μg/ml [15,16] while a similar study in Spain applied a cut-off of 5 μg/ml [13]. The results presented here suggest a drug concentration of 4 μg/ml is adequate for this assay.
For implementation in resource poor settings such as sub-Saharan Africa new technology should preferably have low running costs and avoid need for major investment. The phage assay does not require sophisticated equipment other than that required for culture of tuberculosis. The estimated reagent and consumable costs, excluding labour and overheads, for testing a batch of 20 isolates for their susceptibility to rifampicin in a single 96 well plate format was 26 USD i.e. 1.3 USD per isolate.
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