A simple dipstick assay for the detection of antibodies to phenolic glycolipid-I of Mycobacterium leprae.

Among the many reported applications of the detection of antibodies to phenolic glycolipid-I (PGL-I) of Mycobacterium leprae, in particular, the use of seroprevalence as an indicator of the magnitude of the leprosy problem may turn out to be very useful in leprosy control programs. An operational function of serology within the leprosy control services requires a simple test system. We have developed a simple dipstick assay for the detection of antibodies to PGL-I and compared its performance with that of an ELISA. A high degree of agreement (97.2%) was observed between the ELISA and the dipstick assay when tested on 435 sera; the agreement beyond chance (Kappa value) was 0.92. No significant difference was found between the dipstick assay and the ELISA when seropositivity rates obtained in groups of leprosy patients, household contacts, and controls were compared. The interpretation of the dipstick results as positive or negative was unequivocal, as illustrated by the high agreement between different persons reading the test (Kappa values > 0.88). Storage of the only reagents required, the dipsticks and the stabilized detection reagent, up to three weeks under tropical conditions of high temperatures, high humidity, and exposure to light, did not influence the results of the assay. The dipstick assay described here is an easy-to-perform method for the detection of IgM antibodies to PGL-I of M. leprae; it does not require any special equipment and the highly stable reagents make the test robust and suitable for use in tropical countries. An internal control validates the performance of the assay. This dipstick assay may be the method of choice for epidemiologic mapping of leprosy.

Preservation of Mycobacterium leprae in vitro for four years by lyophilization.

Although the viability of Mycobacterium leprae suspended in distilled water with or without 10% fetal calf serum was reduced approximately 10(-2) to 10(-4) from that of the starting material during the process of lyophilization, bacilli capable of multiplication in nude mouse foot pads were found in the lyophilized samples stored for 4 years at 4 degrees C. The multiplication rate of the lyophilized bacilli which were suspended in 10% serum-water was much higher than that of the bacilli suspended in water only. On the other hand, no reduction of the viability of M. leprae suspended in 10% skim milk-water was demonstrated during the process of lyophilization as well as storage for 2 years at 4 degrees C. From the results obtained here, it could be suggested that M. leprae might be preserved in vitro by means of lyophilized M. leprae was extremely stable during cryopreservation when the bacilli were suspended in 10% skim milk-water. Therefore, the composition of the solution for suspending the bacilli is definitely critical for the maintenance of M. leprae viability by means of lyophilization.

Polymerase chain reaction amplifying DNA coding for species-specific rRNA of Mycobacterium leprae.

The sensitivity of the polymerase chain reaction (PCR) on the DNA coding for the species-specific fragment of 16S rRNA of Mycobacterium leprae studied on mouse foot pad harvests and human skin biopsies varies widely between 1 and 3 x 10(4) organisms. This is probably the result of variations in the proportions of organisms with sufficiently intact DNA suitable for PCR. Preserving human skin biopsies for 3 weeks at an ambient temperature even after boiling for 6 minutes gives rise to a 10-fold decrease in sensitivity. Fixation of tissues in formol 10% or Lowy fixative or preserving in Dubos OAA broth is very harmful to the PCR, mainly due to the enhancement of an inhibitory effect on the PCR reaction. For preservation, the best choice at the moment seems to be alcohol 70%. Sample preparation of five cycles of freeze-boiling is simple and generally more efficient than proteinase K treatment and DNA extraction.

New data on the ultrastructure of the membrane of Mycobacterium leprae.

In previous reports on the ultrastructure of Mycobacterium leprae, we described the occurrence of symmetric membranes in normal-looking bacilli from fresh or frozen samples primarily fixed with aldehydes. In those reports we admitted that such a symmetric profile, which is not found in the other normal mycobacteria, would not represent the structure of the normal membrane of the leprosy bacillus. We, therefore, re-analyzed the ultrastructure of the membrane of M. leprae. In the present work the micromorphology of the M. leprae membrane was studied by transmission electronmicroscopy after the fixation of fresh samples by OsO4 plus calcium followed by glutaraldehyde plus formaldehyde and calcium followed by uranyl acetate. The study of samples from two patients with lepromatous (LL) leprosy, three armadillos with natural leprosy, and one nude mouse with experimental leprosy showed that normal-looking bacilli present in lead-stained sections had asymmetric membranes with a thickness of 6.49 +/- 0.36 nm. These membranes showed periodic acid-Schiff (PAS)-positive components exclusively located in the outer half of the bilayer. We demonstrated that the symmetric profile of the M. leprae membrane described in our previous reports corresponds, as admitted in those reports, to an abnormal membrane structure. Such an abnormality was now found to result from the use of primary fixation with aldehydes or of samples stored frozen before fixation. These results indicate that, although ultrastructurally similar to that of the other mycobacteria, the membrane of M. leprae has a peculiar sensitivity to fixation by aldehydes. Such a characteristic, which was not found in M. lepraemurium, M. aurum, M. avium, and M. tuberculosis H37Ra, must reflect a unique membrane molecular structure, which is presently unknown.

Effect of lyophilization on viability of Mycobacterium leprae multiplied in nude mouse.

In an effort to preserve Mycobacterium leprae in vitro, the effect of freezing and drying, i.e., lyophilization, on viability of M. leprae was studied. The viability of the bacilli was quantitatively measured with foot-pad inoculation method using nude mouse. The results obtained demonstrate that the viability of M. leprae was reduced approximately 10(-2) to 10(-3) from that of the starting material, during the process of lyophilization; no viable bacilli were detected in the lyophilized sample containing less than 1.8 X 10(3) bacilli. On the other hand, the bacilli capable of multiplication in nude mouse foot-pads were found in the lyophilized sample with more than 10(5) bacilli. From the results obtained here, it could be suggested that there might be a possibility to preserve M. leprae in vitro by means of lyophilization.

The effect of freezing and storage in liquid nitrogen on the viability and growth of Mycobacterium leprae.

The effects of rapid and slow rates of freezing in liquid nitrogen, storage in liquid nitrogen for 12 months, and the rate of subsequent thawing on the viability and growth of M. leprae in the mouse footpad were studied. Some loss of viability of M. leprae was detected, and this was found to be associated with the freezing process, rather than with storage or thawing. Slow freezing was less deleterious than quick freezing, with a loss of viability of 90% compared with 98%. The growth pattern of M. leprae was unaffected except for a delay in the appearance of growth caused by the loss of viability, though there was some evidence of an increased lag phase of one strain, possibly due to the repair of sub-lethally damaged organisms.