Leprosy: anti-PGL-I serology for Mycobacterium leprae infection and qPCR RLEP to confirm diagnosis
Serological tests may act as assistants in the surveillance of contacts and/or population at risk14/04/2022
In 2020, 127,396 new cases of leprosy worldwide were reported to the World Health Organization (WHO). Of these, 19,195 (15.1%) occurred in the Americas region and 17,979 were notified in Brazil, which corresponds to 93.6% of the Americas. Also according to WHO data, 62 countries reported new cases in people under 15 years of age. Brazil, India and Indonesia reported more than 10 thousand new cases, which corresponds to 74% of new cases detected in 2020. Brazil ranks second among the countries with the highest number of cases in the world, behind only India. According to data from the 2022 Leprosy epidemiological bulletin of the Ministry of Health, between 2016 and 2020, 155,359 new cases were diagnosed. Of these, 86,225 occurred in men, which corresponds to 55.5% of the total. This predominance was observed in most age groups and years of evaluation, with a higher frequency in individuals between 50 and 59 years, totaling 29,587 new cases. Historically, leprosy presents more occurrences in the North, Northeast and Midwest regions. However, the diagnosis remains a challenge, the tests are limited, especially for early identification.
A study published in late March entitled “NDO-BSA, LID-1, and NDO-LID Antibody Responses for Infection and RLEP by Quantitative PCR as a Confirmatory Test for Early Leprosy Diagnosis” reveals active case findings to validate three potential antigen candidates and one molecular target: anti-PGL-I serology for Mycobacterium leprae infection and qPCR RLEP that can confirm the diagnosis of leprosy. Dr. Cláudio Salgado, professor at the Federal University of Pará (UFPA), dermatologist, leprosy specialist, PhD in Skin Immunology from the University of Tokyo and president of the Brazilian Society of Leprosy (SBH), one of the authors of the article, explains that the three molecules available for use in serology are NDO-BSA, a synthetic molecule that is the main antigenic determinant of phenolic glycolipid I (PGL-I), specific to the wall of M. leprae, discovered in the 1980s, and with many studies showing that it can assist in the diagnosis and follow-up of patients with leprosy; LID-1, a synthetic antigen produced from the fusion of two molecules of M. leprae (ML0405 and ML2331) and NDO-LID, which is the combination of the previous two.
For the study, fieldwork was carried out in Mosqueiro, one of the 36 islands of Belém (PA), where 894 people were examined and 105 new cases (11.7%) were diagnosed, a prevalence considered very high by Dr. Salgado. After the closed diagnosis, a peripheral blood sample was serologically tested for all three molecules in all participants. For the initial cases (early diagnosis) there was no difference in the median compared to the median of healthy contacts in the three antigens studied, therefore, these antigens cannot assist in the early diagnosis, explains the researcher. However, when comparing the medians of early cases, diagnosed in the field, with those well-defined, diagnosed late in the reference, important differences were found, with higher titers in the reference cases. This means that the later the diagnosis, the greater the possibility of having a positive result in serology, with increasingly higher titers, especially for anti-NDO-BSA antibodies, that is, anti-PGL-I IgM, he points out. On the other hand, the medians of anti-NDO-BSA IgM in schoolchildren and people living out of leprosy endemic area were similar, indicating that, in an area with many cases, all are similar to contacts in serology. Although it does not help in the diagnosis, our data confirm that the quantification of anti-NDO-BSA antibodies can serve as an indicator of endemicity in a community, adds Dr. Salgado.
Regarding the qPCR, the researcher details that dermal scraping (formerly known as lymph bacilloscopy) of the earlobes was performed in 79 of the 105 cases diagnosed in the field. Of the 79 cases, 68 (86.07%) were positive for amplification of the RLEP marker, which is specific to M. leprae, which is repeated 29 times in the bacillus genome, therefore, according to him, a good marker because in addition to being specific, it is found in large quantities. “The technique of amplification by qPCR is the same. However, we used 45 cycles instead of the 40 normally used, which enhanced the diagnosis without loss of specificity. It is worth noting that the examination was only performed after clinical confirmation of the case, which is the current diagnostic mode. With this, we obtained an 86.07% positivity between the cases, he points out. Other studies of the team demonstrated this same positivity for clinically defined cases, against 20 to 30% of positivity among contacts of cases of the disease, without clinical leprosy.
New tests for diagnosis of leprosy in the Unified Health System (SUS)
The Unified Health System (SUS) will have new types of testing for leprosy this year. The National Commission for the Incorporation of Technologies in the Unified Health System (CONITEC) approved the incorporation of a rapid test that detects anti-PGL-I IgM antibodies, and also a PCR test that detects RLEP. Dr. Salgado acknowledges the advance for the leprosy community, both for patients and for health professionals, especially those who are at the tip of the system, in the Family Health Strategies and in the Basic Health Units, since there was no exam available, but draws attention to the fact that the approved algorithms will need to undergo modifications. Unfortunately, they do not consider the use of tools in contacts, only in suspicious cases, with diagnostic uncertainty. For example, the approved PCR test is to be used only in biopsies, not in scrapes, as we did. Scrapes are already incorporated into our system, as they are used for traditional bacilloscopy to stain acid-fast bacilli (AFB). In addition, early cases often do not even have lesions where a biopsy could be performed. Moreover, biopsies need local anesthesia and, minimally, a procedure room, while the dermal scrape is collected in the same place where blood is collected.” he justifies.
The new tools are in the implementation phase, but are not yet in the system. Dr. Salgado emphasizes that SBH has important restrictions to the Clinical Protocol and Therapeutic Guidelines (PCDT) of leprosy , not yet definitively approved, and that, together with the Brazilian Society of Infectious Diseases (SBI), the Brazilian Society of Clinical Medicine (SBCM) and the Brazilian Academy of Neurology (ABN), made an important technical analysis. The researcher expects the protocol to be urgently revised. The final price fixed is not yet known, but, according to Dr. Salgado, the value should be between R$ 50,00 to R$ 90,00 per test, considered expensive by him. Recalling that the implementation of the rapid test is in primary care and qPCR only in reference centers.
Epidemiological data justify the use of the tool in Brazil
The current scenario of leprosy is the worst possible. For 20 years the numbers have fallen in the world for lack of diagnostic expertise. Since 2000, when the WHO declared leprosy “eliminated as a public health problem,” the numbers have only dropped from 800 thousand new cases per year to less than 200 thousand in 2019, the last year before the pandemic. During the pandemic, what was bad got worse. In Brazil alone, the number of newly diagnosed cases decreased by only 40%. The WHO reports a reduction of 27.7% in the prevalence recorded and 37.1% in the number of new cases compared to 2019. These reductions are likely attributable to the consequences of Covid-19. Due to the pandemic, many countries have stopped or postponed their leprosy control activities, including the active search for cases. A reduction in the active search for cases naturally results in fewer cases being detected. Papers from different groups estimate around 4 million people with leprosy in the world today, without diagnosis.
For Dr. Salgado, the data justify the use not only in Brazil, but worldwide. I advocate a complete restructuring of the leprosy program in the world, and the first step would be to carry out surveillance in hot spots and around the old leprosy colonies, using clinic, serology and qPCR. Only then can we know if it was really eliminated as a public health problem or if we have numbers similar to those we had there in the 1990s, which is our hypothesis, he says. Leprosy is a long-term chronic disease with a huge loss of diagnostic expertise. More treatment means less disability. More diagnosis corresponds to fewer sequelae. For years there have been demonstrations about the need to incorporate serological and molecular tests in the public health network of countries for diagnostic support.
Finally, the researcher is categorical in stating that serology should be quantified, that is, using ELISA. He understands the need for a rapid test for on-site diagnosis, but reports that during the last 10 years he was in the field with his team, in the interior of Pará and the Amazon, collecting material and bringing it to the laboratory to quantify both serology and qPCR, did not present problems. We have to be careful that a rapid test does not rule out cases that in the future will appear with physical disabilities already installed due to a negative result, which may even remain negative even in cases with installed physical disability, he recalls. According to him, it is not clear what the cut off of the offered test is, which is very important. If the cut off is too low, we run the risk of having too many positives. If it is too high, of losing many cases that could be positive with a lower cut off. In our ELISA, we have positive ranges that indicate a higher or lower risk of being ill. In the qPCR, for the first time, the possibility of using a greater number of cycles (45) to detect early cases is published, which will not happen with the current test as approved, he concludes.