Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 25, Num. 4, 2007, pp. 358-363

Indian Journal of Medical Microbiology, Vol. 25, No. 4, October-December, 2007, pp. 358-363

Original Article

Comparison of radiorespirometric budemeyer assay with ATP assay and mouse foot pad test in detecting viable Mycobacterium leprae from clinical samples

Agrawal VP, Shetty VP

The Foundation for Medical Research, Worli, Mumbai-400 018

Correspondence Address: The Foundation for Medical Research, Worli, Mumbai-400 018, India. Email: fmr@vsnl.net.

Date of Submission: 22-Dec-2006
Date of Acceptance: 18-May-2007

Code Number: mb07099

Abstract

Keywords: adenosine triphosphate assay, M. leprae, mouse foot pad test, radiorespirometric Buddemeyer assay, viability

There are no easy tools to determine the viability of Mycobacterium leprae . Mouse foot pad (MFP) test developed by Shephard ^[1],[2] is still considered a gold standard for the detection of viable M. leprae , but is laborious, takes 6 months to yield results and involves usage of animals. The morphological index (MI) is widely used as an index of M. leprae viability in field conditions but besides its poor sensitivity, the method is highly subjective. ^[3]

Among the various in vitro methods available, adenosine tri-phosphate (ATP) bioluminescence assay has been shown to be more sensitive than MFP test in the hands of some investigators. ^[4],[5],[6] Jagannathan and Mahadevan ^[7] showed a good correlation between ATP and Fc receptor assay. Katoch et al. , ^[8] found ATP, an easy and a better tool for viability determination than morphological index (MI) and fluorescence di-acetate (FDA-EB) staining. Gupta et al. , ^[6] found a good correlation with gene amplification assay.

The Buddemeyer assay based on evolution of ¹⁴ CO ₂ from 1- ¹⁴ C palmitic acid by viable cells has been used to determine the viability and efficacy of antileprosy drugs using M. leprae derived from nude mice and armadillos. ^{[9],[10],[11],[12]} We standardized this assay system in our laboratory and applied it to determine the viability of M. leprae in clinical samples. The results indicated good applicability particularly in low bacteriological index (BI), i.e., tuberculoid cases. ^[13] To further validate the assay system, three tests, i.e., MFP test, Buddemeyer and ATP assay were compared. The results thus obtained in 60 clinical samples are presented and discussed in this paper.

Materials and Methods

Lesional skin biopsies obtained from 60 cases, including 20 untreated borderline lepromatous to lepromatous leprosy (BL-LL), 13 treated BL-LL, 12 untreated borderline tuberculoid to mid-borderline (BT-BB) and 15 treated BT-BB were studied using all the three methods. The treated cases under study had received 12 months of WHO multibacillary multidrug therapy (MB-MDT) and the biopsies were collected after 4-6 months of completion of treatment. The classifications of cases were done using Ridley-Jopling scale. ^[14] In each case, approximately 0.15 g of the biopsy tissue was homogenized using a constant weight/volume ratio (i.e., 1 mL/0.1 g wt of tissue). Bacterial count/mL of tissue, BI and MI were determined using the standard protocol. ^[15] The homogenate thus obtained was divided in to three parts and used with in 48 h, for Buddemeyer assay, ATP assay and MFP test, respectively.

Radiorespirometric Buddemeyer assay

Palmitic acid oxidation assay described by Buddemeyer et al. , ^[16] was standardized in our laboratory with slight modifications. In brief, glass fibre filter papers were soaked in liquid scintillation fluid, dried and overlaid with 2 N methylated NaOH. They were then rolled and inserted into scintillation vials. For each sample, in a glass roller tube, 3.5 mL of Dubos [with 10% fetal calf serum (FCS) and without glycerol] containing 0.1 μCi/mL of 1- ¹⁴ C palmitic acid [Board of Radiation and Isotope Technology (BRIT), specific activity - 20.06 mCi/mmol] was taken and 0.3 mL of homogenate sample (neat), regardless of bacterial count, was added. After thorough mixing, 1 mL each was dispensed into three plastic vials (triplicates) of 4 mL capacity and each vial was then gently placed (without cap) within the scintillation vial. The readings of ¹⁴ CO ₂ evolution were recorded at an interval of 24-48 h for 3 weeks using a liquid scintillation counter (Wallac 1409 DSA). The vials were maintained at 33 °C and were checked for sterility at the end, using nutrient broth, Sabouraud broth and Loweinstein Jensen slants. If found contaminated, the test results were not considered for analysis.

Normal skin biopsy samples obtained from 15 donor non-leprosy cases were processed in a similar manner. The cut-off counts per minute (CPM) value for Buddemeyer assay was determined using these samples.

ATP assay

Adenosine triphosphate estimation was carried out using Dhople′s method. ^[5] Briefly, 0.3 mL of homogenate was centrifuged, bacillary pellet obtained was decontaminated with 2% NaOH, followed by treatment with 0.4% w/v trypsin, 0.1% w/v triton x-100 and 0.34% ATPase. After each step, the pellet was washed with 0.05 m phosphate buffer. ATP was extracted by adding Tris EDTA-HCl buffer and boiling it in water bath for 10 min. The amount of ATP was estimated using ATP monitoring kit (Biothema). The statistical significance was determined using unpaired t test by comparing test readings with the blank readings.

Mouse foot pad inoculation and harvesting of M. leprae

The hind foot pads of 8-10 Swiss white (S/W) mice per sample were inoculated with 0.03 mL of appropriately diluted tissue homogenates containing ≤1 x 10 ⁴ M . leprae cells. All the mice were fed with standard mouse diet and kept in a 12 hourly light-dark cycle in an air-conditioned room. Foot pads were harvested at 6, 7 and/or 8 and 12 months post-inoculation using Shepherd′s method. ^[1] Briefly, foot pads were disinfected, cut and collected in sterile cups containing sterile saline (0.5 mL/foot pad). This was then electrically homogenized and the bacterial counts were taken by Ziehl Neelsen Carbol Fuchsin staining on a spot slide using the standard method. A minimum of 200 microscopic fields/sample was covered for counting acid fast bacilli (AFB). The lower limit of detectability by this method is 1 x 10 ⁴ M. leprae per foot pad. A minimum of two counts per foot pad per interval was obtained at 6, 7 and 8 months. The remaining mice were harvested at the twelfth month. Any foot pad count exceeding 1 x 10 ⁵ was considered as significant increase.

Statistical methods

The statistical significance of the difference between the results of all the three methods was determined by using Fisher-exact test. The statistical tests were performed using the programme InStat 3.06 (GraphPad, Inc.). P ≤ 0.05 was considered statistically significant.

Ethical approval

The study had a clearance from the institutional ethics committee. All the biopsies were done using local anaesthesia, with informed consent from the patients and controls before recruitment. Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) cleared the use of animals in the study. The Foundation for Medical Research is registered with CPCSEA and has a valid registration number 424/01/a/CPCSEA).

Results

In case of Buddemeyer assay, the average CPM value for the 15 non-leprous skin biopsy samples was 300.17 ± 175.09 (results not shown). Applying 2SE, any value above 390.1 CPM at the peak interval was considered as an indication of positive metabolic activity and hence presence of viable M. leprae .

It was generally noted that, an initial lag phase was followed by a log phase with peak CPM output, followed by a plateau. The peak CPM output was observed between 11 and 14 days. The results of three representative cases are depicted in the Figure.

The results of the three tests in four groups of cases are given in [Table - 1],[Table - 2],[Table - 3],[Table - 4], while the comparison between the three methods with respect to positivity is given in [Table - 5].

Comparison of results between Buddemeyer assay and ATP assay

Buddemeyer assay showed better sensitivity than ATP assay in all the four groups [Table - 1],[Table - 2],[Table - 3],[Table - 4],[Table - 5], but the differences were not statistically significant. One-to-one concordance between the two tests was seen in 33/60 (55%) cases. Of the remaining 27 cases, 21 (35%) were positive in Buddemeyer assay but negative in ATP assay, while 6/60 (10%) were positive in ATP assay but negative in Buddemeyer assay. Number of cases positive in Buddemeyer assay was more in the discordance group, indicating better sensitivity of this detection method.

The differences in the results of Buddemeyer assay and MFP test were not significant in untreated groups [Table - 1],[Table - 3], indicating good agreement between the two. In treated groups [Table - 2],[Table - 4], Buddemeyer assay showed better sensitivity and the differences were statistically significant ( P < 0.05). Overall one-to-one concordance between the results of two tests was observed in 38/60 (63%) cases. Remaining 21 cases (35%) showed discordance, of which 20 (33%) were positive in Buddemeyer assay but negative in the MFP test. One case (1.6%) was positive in MFP test that was negative in Buddemeyer assay [Table - 1].

In treated BL-LL cases, the ATP assay showed a significant difference when compared with MFP test results ( P < 0.05), whereas in the other three groups the results were comparable [Table - 5]. One-to-one concordance between two tests was observed in 41 (68%) cases. Remaining 19 (32%) cases showed discordance, of which 12 (20%) were positive in ATP assay but negative in MFP test and 7 (11.6%) were negative in ATP assay but positive in MFP test.

Sensitivity and specificity of both the in vitro methods were determined using MFP test as gold standard in the untreated cases only. The calculated sensitivity and specificity with Buddemeyer assay was 75 and 87.5% and with ATP assay it was 85.7 and 61%, respectively.

Discussion

Overall, both the in vitro assays showed a better sensitivity as compared to MFP test in detecting viable M. leprae in clinical samples. Among the two in vitro methods, Buddemeyer assay emerged as more sensitive in all four groups of cases [Table - 1],[Table - 2],[Table - 3],[Table - 4],[Table - 5]. In the untreated group of cases, the differences in the results were not statistically significant when compared to MFP test and ATP assay indicating a good agreement between the three tests. Moreover, in untreated BT-BB group of cases, 9 out of 12 were negative for AFB, but Buddemeyer assay scored positive in 7 (58%) cases as against two cases (16%) positive by both ATP assay and MFP test. Considering the limitation of the microscopic method where in the lower limit of sensitivity is 1 x 10 ⁴ cells/mL, both the in vitro assay systems have detected viable cells in a good proportion of BT-BB cases.

Notably, in the Buddemeyer assay, a good proportion of cases scored positive in both the treated groups ( P < 0.05) while in the ATP assay significant positivity was recorded only in treated BL-LL group ( P < 0.05). On the other hand, none of the treated BT-BB and BL-LL cases scored positive in the MFP test. This finding in turn could be interpreted in two ways. Firstly, there are a good proportion of treated cases that still harbour viable M. leprae , if proven true will have a great implication on treatment efficacy. Secondly, it can be argued that in the in vitro tests, some of the results obtained could be false positives. It should be noted that in the untreated group of cases, the Buddemeyer assay has shown a specificity of 87.5%, which is within the acceptable limits. Never the less a longitudinal study and comparison with another proved sensitive method, probably molecular method based on RNA may help in further validating the assay system.

Presence of viable M. leprae after MB-MDT treatment has been cited in the literature. ^{[17],[18],[19],[20]} Poor sensitivity of MFP test as a viability detection method in treated cases is also well recognized. It is known that the multiplication of few viable organisms among mostly dead cells is hampered due to immune response mounted by the normal mice ^[14] and is the reason why immunocompromised mice are preferred in the detection of persisters. ^[21] Needless to say that the latter is more difficult to maintain besides the cost.

It was observed that there was no linear relationship between the CPM output and BI and MI that is the microscopic hallmark of infectivity and viability of bacteria. Similar lack of correlation between BI/MI and other in vitro viability detection methods like FDA-EB staining, thymidine uptake and ATP assay have been reported. ^[4],[8] Moreover in this study, in few particularly high BI cases, the cpm output was very inconsistent with BI [Table - 1]. Two likely explanations could be inhibition of metabolic activity by host tissue or suppression of metabolic activity due to competitive inhibition in the medium. Although all the earlier studies used purified M. leprae , in this study no purification steps were applied to remove host tissue to prevent the loss of bacterial cells. However, a constant weight to volume ratio during homogenization, i.e., 0.1 g/mL was maintained throughout and a fixed volume of homogenate, i.e., 0.3 mL, was used for the Buddemeyer assay regardless of the bacterial count. A further investigation along this line may be in order that may help in improving the sensitivity and specificity of the assay system.

The usefulness of Buddemeyer assay system in determining the efficacy of various antileprosy agents has been established. ^[11],[12] Radiorespirometric method has also been used for detecting M. tuberculosis . ^[22],[23] In a parallel study on many clinical samples, we record the sensitivity and specificity of Buddemeyer assay as 75% and 74.2% respectively, using MFP test as gold standard. ^[13] The results of that study also indicated its better utility in treated cases, the fact that has been reiterated in this study.

To conclude, among the two in vitro tests, Buddemeyer assay emerged as a better and a more sensitive method. Our findings suggest that it will be a good tool in day-to-day patient care in detecting persisters, relapses and determining the efficacy of chemotherapy but we hasten to add that further verification to rule out false positivity would be needed.

Acknowledgements

We gratefully thank Dr Sunil Ghate, Medical Officer, The Foundation for Medical Research (FMR), for performing the biopsies, Mrs Anju Wakade, Research assistant and Fatema Khambati, Msc Research student, FMR for technical help. This study was supported by a grant (5/8/3(6)/2001-ECD-1) from the Indian council of Medical Research (ICMR).

References

Copyright 2007 - Indian Journal of Medical Microbiology

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