Effect of cryo preservation on Mycobacterium leprae growth in the footpads of non-immunosuppressed mice.

OBJECTIVE: To investigate using the mouse footpad system, whether the use of cryopreservants help in retaining the viability of Mycobacterium leprae samples stored at three different temperatures of 4 degrees, -20 degrees and -70 degrees C for 30 days. DESIGN: Biopsies from eight untreated lepromatous leprosy cases were homogenised and inoculated into footpads of normal Swiss White mice within 24 hours (control) and remaining homogenates in each case was divided and stored at 4 degrees C, -20 degrees C and -70 degrees C respectively for 1 month, using either 10% skimmed milk (SM) or Roswell Park Memorial Institute media + 10% glycerol (RPMI) (test). Homogenates adjusted to contain 1 x 10(4) M. leprae/footpad was inoculated into 10 mice per set. Harvestings were done at 6th, 7th, 8th and 12th months. Footpad counts showing > 1 x 10(5) M. leprae at 6th month or later were considered as positive yield. RESULTS: Control All the cases showed > 100 fold growth and 100% take. Viability at 4 degrees C: Only one case (SM) showed a 100 fold increase and 23% take. Viability at -20 degrees C: Two cases showed fold growth that was 40-60 fold less with takes of 63% (SM) and 71% (RPMI) respectively. Viability at -70 degrees C: Positivity was 45% but the fold increase was less as compared to control and takes were between 80-20%, except one RPMI where take was 100%. CONCLUSION: The viability assessed using the mouse footpad was best and consistent in the inoculas that were injected within 24 hours of harvest from the host tissue (control group). None of the storage temperatures used matched with the controls with respect to bacterial yield or % takes. Among the three storage temperatures, -70 degrees C appeared to be better with 45% of the samples showing growth. There was no significant difference noted between the two preservatives used.

Isolation of mycobacterium leprae from untreated borderline tuberculoid, mid-borderline and indeterminate cases using the mouse foot pad technique--a study of 209 cases.

Using the mouse foot pad (MFP) system, isolation of Mycobacterium leprae was attempted in 209 skin biopsies obtained from 114 borderline tuberculoid (BT), 62 mid borderline (BB) and 33 indeterminate (1) untreated cases. Unequivocal growth in the foot pads of mice was seen in 100 (47.8%) cases. Of these 100 cases that showed growth in the mouse foot pad system, in 20 cases acid fast bacilli (AFB) were detected in small numbers (1 + ) in either smear or homogenate. The remaining 80 (42%) cases were negative for AFB in both smear and homogenate. The occurrence of viable bacilli and percentage take at 12 months was highest in BB (76 and 86%) followed by BT (38 and 75%) and I (30% and 52%) cases. In most of the BT (65%) and I (60%) cases, the first peak was seen only at 12 months. These results confirm that viable bacilli can be isolated and expanded from a good proportion of negative BT-BB cases using immunocompetent Swiss White mice.

Clinical, histopathological and bacteriological study of 52 referral MB cases relapsing after MDT.

Fifty-two BB-LL relapse cases referred to our centre during 1997-2003 were investigated in detail. Twenty-four cases had been treated with extended MB-MDT [until smear negativity (NON-FDT)]. The remaining 28 cases (54%) had received one of the fixed duration regimens (FDT), of whom 11 had 24 months and 6 had 12 months of WHO MB-MDT. Eleven cases had received rifampicin/ofloxacin (RO) treatment. Follow-up slit skin smear reports were available for 41 cases, all but three cases had been smear negative at some point after release from treatment. None of the cases showed any clinical or bacteriological evidence of upgrading, i.e. LL to BT where as downgrading BB to BL occurred in five cases. The duration between cessation of treatment and reappearance of lesions (DCTR) varied from 2 to 15 years. The mean DCTR was longest (9.4 years) for the NON-FDT and 24 months MB-MDT cases. The mean DCTR was significantly lower in the 12 months MB-MDT and RO treated cases (6.8 and 6.2 years, respectively). Four of RO treated cases and four cases with multiple episodes of reaction had DCTR less than 5 years. Inadequate treatment/poor killing of Mycobacterium leprae results in early onset relapse, whereas 'persisting' or 'drug resistant mutants' contribute to late onset relapse.

Viability and drug susceptibility testing of M. leprae using mouse footpad in 37 relapse cases of leprosy.

Mycobacteria leprae isolates obtained from 37 referral relapse cases of leprosy (37 skin and 10 nerve biopsy samples) received during the years 1994-2001, were tested for viability and drug sensitivity in the mouse footpad. A significant M. leprae yield in the footpads of control mice was obtained, with 32/47 (68%) isolates (from 26 cases) thus confirming viability. Of the 28 isolates successfully drug tested, 6 (21%) were resistant to one or more drugs. All except one, were multidrug treated cases (5/24 = 21%). One of the isolates was resistant to all three drugs, i.e., dapsone (di-aminodiphenyl sulphone, DDS), rifampin (RFP), and clofazimine (CLF). Two were resistant to two drugs, i.e., DDS and RFP, and each of the others were mono resistant to DDS, RFP, or CLF. Notably, one of the isolates that showed combined resistance to DDS and RFP was derived from a borderline tuberculoid case. Also, in one case skin and nerve showed that discordance viz: M. leprae derived from skin were resistant to RFP, while those derived from nerve tested sensitive to all three drugs, indicating tissue related difference.