Leprosy diagnosis is mainly based on clinical evaluation [1]. This approach represents a low-cost strategy; however, it is subjective and dependent upon the experience of the examiner, a difficult task for untrained physicians [2].

Early recognition is critical for the prompt initiation of multidrug therapy (MDT) to prevent irreversible adverse outcomes [1]. Although the number of new cases registered annually by the leprosy surveillance program of the World Health Organization (WHO) have been dramatically reduced since the introduction of multidrug therapy, MDT [3], endemic leprosy pockets linger in countries such as Brazil, India and Indonesia, and current case detection strategies require clinical expertise for diagnosis. Many national leprosy-specific control programs have been integrated into the general health system to facilitate patient care. Nevertheless, these control programs have not reduced the expected number of new cases, as they have been implemented in high endemic countries, where the actual number of cases is 6-8-fold higher than currently reported [4]. The present recommendation is to extend surveillance following the “index” case and to examine close contacts [5], particularly those who are blood relatives of multibacillary (MB) patients [6]. Not only are the logistics of such an approach extremely difficult, such activities are complicated by the need to recognize an index case to trigger them.

Bacilloscopy, histopathological examination, serology and polymerase chain reaction are used to recognize leprosy [7, 8]. Phenolic glycolipid-I (PGL-I) is a specific antigen from Mycobacterium leprae (M. leprae) that induces the formation of IgM class antibodies [9]. Anti-PGL-I antibodies are found in high concentrations in the sera of MB patients [9] and are correlated with the bacterial index [10, 11]. This specific humoral response has been investigated as an auxiliary tool for diagnosis [12, 13], although its utility in active surveillance remains uncertain [14]. Antibody responses to other antigens, such as Leprosy IDRI Diagnostic (LID)-1 protein, a fusion of two well-recognized M. leprae proteins (ML0405/ML2331) [15], have been studied to improve the accuracy of serological tests [16, 17]. Therefore, the combination of anti-LID-1 IgG and anti-PGL-I IgM detection is a strategy to improve test sensitivity in the entire clinical spectrum of disease [18]. Similarly, NDO-LID, a commercial immunochromatographic lateral flow test, has been developed to detect anti-PGL-I and anti-LID1 antibodies for initial clinical screening [19, 20] and is applied easily in the field with minimal training.

In the current study, we conducted clinical examinations during a brief active campaign in midwestern Brazil to determine the extent of undiagnosed leprosy. In parallel, we tested all individuals using and comparing three leprosy serological methods: anti-PGL-I and anti-LID-1 by ELISA and the NDO-LID commercial rapid diagnostic test (RDT).

A total of 434 individuals were evaluated, and 44 (10.1%) were diagnosed with leprosy. According to the WHO operational classification, 88.6% were classified as MB. The Borderline forms were the most represented, with 36 cases (81.8%; Fig 1; Table 1) distributed among BT (n = 5), BB (n = 24) and BL (n = 7) patients. Blood collection was performed on 381 individuals, while 53 individuals declined, including a confirmed leprosy patient. Table 2 shows the individual demographics.

Among the leprosy patients, 34 resided within FD (77.3%), whereas the remaining patients lived in either neighboring Goiás (n = 9) or Mato Grosso State (n = 1) (Fig 1A). Cases in the FD were distributed throughout at least 16 different administrative regions, with the most cases from any one administrative region being only 4 (Fig 1B). Furthermore, although more than half were originally from other states and had migrated into the Federal District (Fig 1C), the majority of patients had been in residency for extended periods of time. The 44 patients had a mean time of residency in their administrative regions of 13.5 years, and 32 of them had lived more than 5 years in the same place.

Twenty-two (50%) of the leprosy patients did not report any known leprosy patient contact. Among the individuals without any clinical signs of disease (n = 390), 99 (25.4%) had a leprosy contact, whereas 291 (74.6%) declared no known contact.

Regarding the blood samples collected from healthy individuals (n = 338), 93 (27.5%) were contacts of persons with leprosy, whereas 245 (72.5%) did not have any known contact. The results of the anti-PGL-I and anti-LID-1 ELISA and the NDO-LID are characterized for each clinical form in Table 1. In leprosy diagnosis, considering all disease forms, each of the tests demonstrated a specificity of approximately 70% for anti-PGL-1 and anti-LID ELISA and of approximately 40% for NDO-LID. Sensitivity was less than 65% in all tested techniques. (Table 3). Considering only healthy subjects (n = 338), with the aim of identifying leprosy contacts (Table 3), the sensitivy values of anti-PGL-I and anti-LID-1 ELISA were 30.11% and 23.66% respectively, while NDO-LID sensitivity was 51.61%. Specificity values were lower than 75% in all the three tests.

We observed a substantial agreement between the NDO-LID and antibody-detection ELISA (anti-PGL-I plus anti-LID-1), with a κ index value of 0.79 (CI 95% 0.36–1.22) for the leprosy patients. A fair agreement (κ index between 0.19 and 0.20) was shown between the NDO-LID and ELISA assays, with concomitant results between individuals with known contact and those without. NDO-LID and the anti-PGL-I ELISA showed a moderate agreement (0.37 κ index value (CI 95% 0.08–0.67)) and a fair agreement when the NDO-LID was compared with the anti-LID-1 ELISA (0.35 CI95% (0.06–0.63)) among the leprosy patients.

We would expect the NDO-LID titers to be represented by the reactivity to each individual component, i.e., approximating the combination of the anti-PGL-I and the anti-LID-1 titers by ELISA. The ELISAs were stratified by indices (Tables 4 and 5), and the results were compared between the results obtained by the Smart Reader and by visual reading. For ELISA indices <1.0, there was much lower concordance with the response given by Smart Reader; however, for ELISA indices >1.0, there was better agreement with the Smart Reader results. Further, there was moderate agreement (0.55 κ index (CI 95% 0.45–0.66)) between the results read with Smart Reader and the results by visual interpretation (Tables 4 and 5).

According to the leprosy prevalence rate, FD has been considered a non-endemic area since 2005 [23]; however, the present recruitment procedure resulted in a different scenario with a very high percentage of new cases of leprosy, similar to what one would expect from a hyperendemic area [12, 24]. This fact can be explained by an expected high sensitivity in the screening strategies incorporating our passive random surveillance strategy. Importantly, the leprosy cases detected were not recent emigrants from areas recognized as being hyper-endemic for leprosy and thus, both our clinical data and serological evaluations indicate that leprosy remains an issue, and that M. leprae transmission continues, in FD.

Leprosy diagnosis is still essentially defined by clinical examination. However, in many cases, the changes can be subtle and are often missed even by specialists. Bacilloscopy, histopathology and serology tests can be used to assist the clinical diagnosis and are also useful in spectral and treatment categorization [25, 26]. Nevertheless, the accuracy of these tests is still debated, and many groups are searching for simple ways to make them more reliable, especially in the identification of leprosy cases among household contacts [15].

NDO-LID was shown to be positive in 62.8% of the clinically diagnosed leprosy patients, although this rapid test presented a lower specificity than anti-PGL-I and anti-LID-1 ELISA, as described in Table 3. Despite being able to identify the dominant responses to both glycolipid (IgM anti-PGL-I) and protein (IgG anti-LID-1), NDO-LID has the same limitation as the previously used RDT, showing a difficulty in screening patients in early stages of disease and/or PB forms. Regardless, the use of these tests in conjunction with, or as a trigger for, clinical examination may promote earlier detection and treatment of leprosy cases.

Our anti-PGL-I ELISA findings were similar to previously described results [13, 27, 28]. The anti-PGL-I ELISA was able to identify all LL cases (n = 3), 54.3% of borderline forms (n = 19), especially BL patients (71.4%) and 66.7% of I form. Individuals with the TT form (n = 2) were not identified. The anti-LID-1 ELISA results showed a lower percentage of positivity in borderline forms (n = 15/35, 42.9%) and indeterminate forms (n = 1/3, 33.3%). Also in borderline patients, this test performed better in BL forms, detecting a similar percentage of patients (71.4%) in comparison to anti-PGL-I ELISA. However, considering the limited number of samples tested, our data showed 100% positivity in both LL and TT forms (n = 3 and 2, respectively). In general, the three tests performed better in the identification of MB patients, especially in the BL and in the LL forms. Many studies showed that borderline and LL patients produce high titers of IgM against PGL-I, whereas Tuberculoid patients have low levels of specific antibody [9]. Serological tests are expected to show better responses in MB; however, such improvements remain challenging.

Vaz Cardoso et al. [20] found an anti-PGL-I sensitivity from 69.8% to 92.3% in borderline patients in contrast to our results (54.3%). These differences may be explained by recruiting differences. Although we expected a higher sensitivity in a screening campaign, we used a cross-sectional/cohort strategy in which the diagnosis of controls was previously unknown. This approach generated a more pragmatic environment, reducing sensitivity values.

In addition to the importance of achieving an early diagnosis, a systematic and qualified approach to monitoring household contacts is considered essential for breaking the transmission of M. leprae [5, 29]. Individuals in close contact with untreated MB patients have a 10% lifetime risk of eventually succumbing to leprosy [2, 9, 29–31], particularly when they are consanguineous relatives [29]. Although a positive titre to anti-PGL-I is an infection biomarker, it is not an absolute indicator of disease. Evidence indicates that substantial increases in titers to anti-PGL-I and anti-LID-1 are important indicators of progression, even in the absence of skin lesions or nerve damage [11, 15, 32]. In healthy subjects, a high anti-PGL-I and anti-LID-1 titre suggests that there is a hidden bacillary load driving the response to this antigen. Household contacts demonstrating substantial increases in titre against LID-1 and/or PGL-I should be closely monitored [29, 31].

Worobec et al. [30] reported subclinical M. leprae infection in endemic regions with anti-PGL-I antibody detected in 1.7 to 35% of all individuals. Thus, the high percentage of positivity found in our group may reflect a situation of occult prevalence with many foci of infection in the municipality because many patients may be unknown contacts of leprosy patients. Therefore, considering this rate of exposure, it was not surprising that the rates of positivity in the contact group for all three tests (52% for NDO-LID, 30% for anti-PGL-I and 24% for anti-LID-1) were fairly similar to those among individuals with no history of contact with a leprosy patient, indicative of the generally high rate of exposure in this region.

Among healthy individuals, the concordance of NDO-LID with both the Smart Reader and the visual analysis with the anti-PGL-I ELISA results increased as indices rose (until 95%). Among healthy individuals, the strongest positive responses with NDO-LID showed the best concordance with individuals with the highest anti-PGL-I ELISA index, reaching a maximum where the calculated index was >2.0 (κ = 95%). In general, individuals with very strong positive responses by NDO-LID or with the highest indices in the ELISA, particularly against LID-1, should receive more careful scrutiny during their clinical examination, and regular follow-up examinations would appear prudent.

The tests evaluated in this study demonstrated low specificity and sensitivity values, which would limit their use as stand-alone tests for leprosy diagnosis. However, the tests do appear useful for screening populations as a way to assist clinicians in patient categorization, contacts’ follow-up of patients with high titers, and to assess the risk of treatment failure. Further examination of the use of serological tests for the early diagnosis of leprosy is warranted to attempts break the cycle of M. leprae transmission. This is particularly meaningful given our clinical evidence that M. leprae infection and leprosy remain as a public health concern in FD, a region officially reported having controlled leprosy.

The unexpected high percentage of leprosy cases and the high number of individuals with positive serological responses to M. leprae antigens found in this population, previously considered ‘nonendemic,’ revealed a greater than expected extent of subclinical and frank disease, as well as a continuous contact with M. leprae antigens. These data may point to faults in the health care delivery system and a lack of training by physicians working in the basic health units. Ultimately, more aggressive targeted surveillance strategies, particularly focusing on cities that may experience a large migration of people, should be considered part of a national coordinated leprosy control program.

The positivity of approximately 60% of the anti-PGL-I ELISA and NDO-LID demonstrated that each test is a potential tool to identify suspected patients. The unexpectedly high rates of positivity with NDO-LID among household contacts (50%) and non-contacts (60%) indicates the difficulties in using a single test for diagnosing leprosy.

Despite the limited sensitivity found in all of the diagnosed cases, the anti-PGL-I ELISA and NDO-LID were in complete agreement in identifying all LL and more than 50% of the borderline forms, demonstrating a possible positive impact and the relevance of this type of surveillance campaign to ultimately achieving a reduction in transmission of this disease.

Further studies are necessary to elucidate the efficacy of these serological tests in other diagnostic reference standards; nevertheless, the training of local health professionals to recognize signs of disease and epidemiological surveillance remains the primary strategy for leprosy control.