NAT-HANS: the first PCR-based diagnostic test for Hansen’s disease using Good Manufacturing practice (GMP)

There are no diagnostic tests for Hansen’s disease that can be considered ‘gold standard’. Indirect tests such as serological analyses to detect antibodies against Mycobacterium leprae have been developed,¹ but these are positive in 20-30% of healthy individuals and the false negative rate (paucibacillary patients who have no antibodies) mean that these tests are unsuitable for clinical diagnostic use.² Cellular techniques, such as measuring increased secretion of the cytokine interferon-gamma in blood cell cultures stimulated with M. leprae proteins are promising, but are still in the development phase.³ Other methods, such as specific molecular signatures of active disease through mRNA detection by real-time PCR (RT-PCR) are advancing rapidly but depend on training and infrastructure.⁴ The main challenges with these novel methods are a lack of controlled multicenter studies, and the need for quality control of reagents with good manufacturing practices to ensure reproducibility of results.

Direct detection of the pathogen is another approach, traditionally by bacilloscopy, which lacks sensitivity but is relatively easy to perform. Over the past 30 years, the National Hansen’s Disease Laboratory in Brazil, through the work of Adalberto Santos, has pioneered the introduction of DNA detection by PCR of Mycobacterium leprae for the diagnosis of the disease.⁵ Later, our group improved this method using RT-PCR. Finally, 15 years after the publication of the first article on the subject,⁶ we were able to produce the diagnostic test according to Good Manufacturing Practice (GMP) processes and approved by the Health Surveillance Agency of Brazil (ANVISA) in a partnership between the Hansen’s Disease Laboratory of the Oswaldo Cruz Institute and the Institute of Molecular Biology of Paraná (IBMP) - both of which are part of the Oswaldo Cruz Foundation (FIOCRUZ).

In research articles published in 2006⁶ and 2011⁷, first-authored by Alejandra Martinez, then a PhD student in the National Hansen’s Disease Laboratory, different regions of the genome (targets) were tested for sensitivity and specificity. RT-PCR methods had the advantages of reduced sample handling and semi-automation, eliminating processing time-consuming steps and increasing throughput.

The National Hansen’s Disease Laboratory is a reference center for the Brazilian Ministry of Health, hosting an outpatient clinic for diagnosis and treatment of patients and their household contacts. Using the new diagnostic methods, a routine for monitoring difficult-to-diagnose cases, such as paucibacillary cases, and for monitoring contacts was established. The test process was refined and internal quality control with standard operating procedures were developed by Suelen Moreira.

Further studies, forming the basis of Raquel Barbieri's doctoral thesis, demonstrated that, in difficult-to-diagnose cases, quantitative PCR (RT-PCR) had increased sensitivity (57%) compared to histopathology (35%), in contrast to other dermatological diseases.⁸ A subsequent contact follow-up study, led by Fernanda Manta, confirmed the validity of the molecular test for diagnosis of the disease when suspicious lesions were identified in these patients' household contacts. However, RT-PCR positivity in healthy household contacts (without lesions) was not a good predictor of disease.⁹

Based on these data, we improved the test with reagents produced according to GMP by our partners at IBMP. Fernanda Manta, together with Alexandre Costa and Thiago Jacomasso from IBMP, led reproducibility, stability, repeatability, specificity and sensitivity studies, in which clinical skin biopsy samples were used. This work resulted in a dossier submitted to ANVISA, which approved the NAT-HANS test for use in skin biopsies from people with suspected Hansen’s disease lesions. Projects evaluating other clinical samples, such as blood and slit skin smears from the lesions, as well as variations in DNA extraction to increase the sensitivity and accuracy of the test are underway.

The NAT-HANS test is intended to be used in the network of central state laboratories with the support of the Ministry of Health. Use of an accurate and sensitive test within Brazil’s national health system (SUS) may prevent late diagnosis of Hansen’s disease. It is hoped that this will reduce disabilities and improve the diagnosis of difficult forms of the disease, enabling interruption of the chain of transmission and culminating in a reduction in the incidence of Hansen’s disease in Brazil.