Mycobacterium leprae – PCR

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Background
The etiologic agents of Hansen’s disease (leprosy) are acid fast bacilli in the Mycobacterium leprae complex, Mycobacterium leprae and Mycobacterium lepromatosis. These organisms have a predilection for macrophages, endothelial and Schwann cells resulting in dermatological manifestations as well as axonal dysfunction and demyelination. Although less common, the disease may also affect the upper respiratory tract, eyes, bones and testes. As these organisms are slow growing, it may take over a decade to develop signs of infection.

The World Health Organization (WHO) declared Hansen’s disease “eliminated” in 2000; however, the global incidence has remained relatively stable over the last decade. In 2021, there were 140,594 new cases reported in over 120 countries, with the WHO South East Asia region accounting for the majority (66.5%).¹ From 2011-2021, 67 Hansen’s disease cases were reported across Canada.² For information about Leprosy in Ontario, see Leprosy at Diseases and Conditions Index of Public Health Ontario.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 21 nov. 2024.

Testing Indications

A diagnosis of Hansen’s disease is primarily based on clinical findings (e.g. loss of sensation in a hypopigmented or reddish skin patch, thickened or enlarged peripheral nerve); however, obtaining a skin biopsy can provide a definitive diagnosis and aid in disease classification.³ Specimens should be submitted for detection of M. leprae DNA for individuals with a compatible exposure history, clinical presentation, and histopathological findings for Hansen’s disease.

Acceptance/Rejection Criteria

Testing is only performed upon approval by a PHO microbiologist.
All samples meeting the specimen requirements below will be accepted for testing.
Formalin fixed tissues and/or paraffin-embedded samples are not recommended for testing due to DNA fragmentation and will only be considered for testing under exceptional circumstances.

Specimen Collection and Handling

This testing is not performed at Public Health Ontario’s (PHO) laboratory and is sent for M. leprae PCR by PHO to the National Reference Centre for Mycobacteriology (NRCM) in Winnipeg, Manitoba for analysis.

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Mycobacterium leprae – PCR	General Test Requisition	CSF or other fluids	5 mL	Tuberculosis Kit order#: 390042
Mycobacterium leprae – PCR	General Test Requisition	Fresh sterile tissue/biopsies (see note #5)	1-2 grams	Tuberculosis Kit order#: 390042

Submission and Collection Notes

Contact PHO’s laboratory prior to submission for approval by the microbiologist. Specimens submitted without prior approval will automatically be cancelled. Do not send specimens directly to the National Reference Centre for Mycobacteriology.

Complete all fields of the requisition form, including:

Test(s) requested and indication for testing
Patient country of origin
Patient travel history
Compatible exposure history (e.g. infected contacts, armadillo)
Current treatment, if applicable

For clinical specimens, label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. Failure to provide this information may result in rejection or testing delay.

If routine mycobacterial culture work is also required, please indicate testing on the requisition.

Frozen tissue/biopsy samples may be accepted if they have been handled in an aseptic environment.

Timing of Specimen Collection

Skin biopsies should be taken from the most recent and active skin lesions.

Limitations

PCR should be used to support clinical and histopathological diagnosis of Hansen’s disease. A negative M. leprae PCR result may occur due to sampling bias, especially in paucibacilliary specimens. PCR results must be interpreted within clinical context, including travel history and possible exposure, dermatological findings, and the presence of nerve enlargement or sensory impairment.

M. leprae PCR cannot be used for follow up during or after treatment as dead bacilli can persist for several years in tissues.⁴ The detection of M. leprae DNA may also indicate passive carriage of the bacilli or contamination in non-sterile specimens and must be interpreted within the clinical context.⁵

Storage and Transport

Fresh specimens should be stored at 2-8 °C following collection and shipped to PHO’s laboratory as soon as possible. All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

M. leprae molecular detection is performed at the National Reference Centre for Mycobacteriology (NRCM), Winnipeg, Manitoba. Results are available within 7 to 14 days from the submission date of the PHO laboratory to the NRCM.

Test Methods

Real-time PCR is used for the detection of the unique RLEP repeat sequence and the ITS region for M. leprae from specimens.

Interpretation

Real Time PCR Result	Real Time PCR Result 2	Result Interpretation	Comments
Positive for M. leprae	Positive for Mycobacteria	Mycobacterium spp. (pan target) DNA DETECTED, M. leprae DNA DETECTED	Detection of M. leprae DNA must be interpreted within the clinical context. Passive carriage and amplification of non-viable bacilli (DNA may persist for several years after treatment) may also cause positive results.
Positive for M. leprae	Negative for Mycobacteria	Mycobacterium spp. (pan target) DNA NOT DETECTED, M. leprae DNA DETECTED	Detection of M. leprae DNA must be interpreted within the clinical context. Passive carriage and amplification of non-viable bacilli (DNA can persist for several years after treatment) may also cause positive results. A negative Mycobacterium spp. result does not exclude the possibility that other mycobacteria are present in the same sample.
Negative for M. leprae	Positive for Mycobacteria	Mycobacterium spp. (pan target) DNA DETECTED, M. leprae DNA NOT DETECTED	A negative M. leprae PCR result does not exclude the possibility that M. leprae is present in the sample. A positive PCR result for Mycobacterium spp. may suggest infection with another mycobacteria. Interpret PCR results within the clinical context and histopathological findings.
Negative for M. leprae	Negative for Mycobacteria	Mycobacterium spp. (pan target) DNA NOT DETECTED, M. leprae DNA NOT DETECTED	Negative M. leprae and Mycobacterium spp. PCR results do not exclude the possibility that M. leprae or other Mycobacterium spp. are present in the sample. Interpret PCR results within the clinical context and histopathological findings.

Reporting

Results are reported to the ordering physician, authorized health care provider (General O. Reg 45/22, s.18), or submitter as indicated on the requisition. Specimens with molecular detection of M. leprae are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

References

World Health Organization (WHO). Global leprosy (Hansen disease) update, 2021: moving towards interruption of transmission [Internet]. Geneva: WHO; 2022 [cited 2024 Jan 4]. Available from: https://www.who.int/publications/i/item/who-wer9736-429-450
Public Health Agency of Canada. Reported cases from 1924 to 2021 in Canada – notifiable diseases online. Ottawa, ON: Government of Canada; 2023 [cited 2024 Jan 4]. Available from: https://diseases.canada.ca/notifiable/charts?c=pl
Maymone MBC, Laughter M, Venkatesh S, et al. Leprosy: clinical aspects and diagnostic techniques. J Am Acad Dermatol. 2020;83(1):1-14. Available from: https://doi.org/10.1016/j.jaad.2019.12.080
Scollard DM. Chapter 2.4: pathogenesis and pathology of leprosy. In: Scollard DM, Gillis TP, editors. International textbook of leprosy. Greenville, SC: American Leprosy Missions; 2016. Available from: https://doi.org/10.1489/itl.2.4
Fontes AB, Lara FA, Santos AR, Suffys P. Chapter 7.2: pathogen detection. In: Scollard DM, Gillis TP, editors. International textbook of leprosy. Greenville, SC: American Leprosy Missions; 2016. Available from: https://doi.org/10.1489/itl.7.2