According to World Health Organization (WHO), 210,671 new cases of leprosy were reported globally in 2017.1 In the same year, 26,875 new cases occurred in Brazil,2 which represented 12.8% of the global burden with Brazil being responsible for 92.3% of new cases in Americas.1 Leprosy diagnosis in children indicates an active transmission of the bacteria among the community and represents a public health failure.3,4 Seven to eight percent of the new cases diagnosed in Brazil are represented by children.5 Disease varies by municipality in Brazil with multibacillary (MB) clinical forms ranging from 23.4% to 75% of the pediatric patients; moreover, about 3.7% of them are diagnosed with grade 2 disability.5 In the beginning of 2018, the WHO stated the importance of focusing early case detection among children.6 Household contacts are more prone to be infected and to develop the disease; nonetheless, more recent data reveal the importance of social contact (neighborhood and classmates).7,8 Serology does not differentiate infection and disease, but it is known that the presence of serum antibodies leads to an increased risk for developing disease.9 In a study performed in hyperendemic municipalities of the Brazilian Amazon region, Barreto et al10 demonstrated that 48.8% of children attending school and 41.8% of leprosy household contacts were seropositive for anti-PGL-1 antibody. According to Brasil et al,11 the risk of developing leprosy is 8.6-fold times higher in household contacts presenting with positive serum antibodies and 4.4-fold times in noncontacts, in comparison to those who are seronegative. Most of the literature focuses the IgM isotype against the phenolic glycolipid-1 (PGL-1), antigen specific to Mycobacterium leprae. For these reasons, we have proposed in the present study of a 3-year follow-up of children who were 4–15 years of age living inside or nearby a leprosy index case’s home regardless of the clinical form of leprosy. Besides dermato-neurologic evaluation, serum anti-PGL-1 IgM, IgG, and IgA were measured.
MATERIAL AND METHODS
A prospective cohort study of leprosy contacts 4–15 years of age was carried out the years 2015–2017. Dermato-neurologic evaluation and serologic measurement were performed once a year.
Santana de Ipanema is located in Alagoas state, in the Northeast region of Brazil. It is the main city of the Alagoas hinterland, with a population of 47,486 inhabitants.12 Its economy is mostly based on agriculture and services. The areas where the index cases live have poor access to sewerage systems.
Participants of the Study
Children who were 4–15 years of age living inside an index case’s home [household contacts (HH)] or in the neighborhood [peridomiciliary (PD) contacts] were included in the study. The index cases diagnosed between 2011 and 2015 were classified as either paucibacillary (PB) or MB, regardless of the treatment period, and were selected the National Notifiable Diseases System from both urban and rural areas. Case contacts were invited to join the study. The PD contacts were considered to be those living up to five houses on either side of the index case’s home. Friends and relatives who spent at least two a week in the index case’s home were also included. Those participants who did not complete the follow-up study for any reason were excluded from the analysis. After informed consent, contacts were enrolled in the study and an enrollment questionnaire administered. The participants were invited to go to the health center to be examined by a trained health professional, before blood collection. In the presence of suggestive lesions, thermal, painful and tactile sensitivity were tested, according to the protocol of the Brazilian Health Ministery.13 Nerve thickness was also evaluated.
Five milliliters of venous blood were collected in a clot activator tubes containing a gel separator. After 30 minutes, the samples were centrifuged at 1500g for 10 minutes, and the serum was separated and stored at −20°C.
Measurement of serum anti-PGL-1 antibodies was performed as described by Macedo et al.14 Ninety-six well flat-bottom microplates (Costar 3590, Cambridge, MA) were coated with 5 mg/L of native PGL-1 in absolute ethyl alcohol (50 L per well), kindly donated by Bei Resources/ATCC (Manassas, VA). The microplates were incubated for 2 hours at 37°C. After, they were incubated with 1% fetal bovine serum (FBS; LGC Bio, Cotia, SP, Brazil) in phosphate buffered saline (PBS, pH 7.4) for 2 hours at 37°C in a humid chamber. After four washes with PBS-0.05% FBS, serum samples previously diluted (1:200 for IgG and IgM and 1:50 for IgA) in PBS-0.5% FBS were added to the plates (50 L per well, in duplicate). The plates were then incubated for 2 hours at 37°C. After washing, peroxidase-labeled anti-IgG (A0170; Sigma, Saint Louis, MO) anti-IgM (SAB3701404; Sigma, Saint Louis, MO) or anti-IgA (A0295; Sigma, Saint Louis, MO), previously diluted to 1:2500, 1:9000, and 1:1000, respectively, was added to the plates and incubated for 1.5 hours at 37°C. After washing, the plates were incubated for 30 minutes with the substrate solution (100 L per well) which contained 0.4 mg orthophenylenediamine/mL of 0.01 M citrate-phosphate buffer, pH 5.0 and 0.01% H2O2 final concentration. The reaction was interrupted by adding 25 L 2.5 N sulfuric acid. The analysis was performed at 492 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (ASYS Expert Plus; Biochrom, Cambridge, United Kingdom). An aliquot of pooled normal human serum was used as cutoff sample and tested in all assays. A negative and a positive control were included in the assays.
The results were expressed as optical density (OD) mean of the test sample (minus blank) divided by the OD mean of the normal human serum pool (minus blank). Values ≥1.2 were considered to be positive for IgG and IgM and ≥1.1 for IgA, according to Macedo et al.14 The cutoff sample was obtained from 50 healthy blood seronegative donors to HIV, Chagas, hepatitis B and C, HTLV, syphilis and that did not present with leprosy at the time of sample collection.
The data were analyzed using nonparametric tests as the data did not follow a Gaussian distribution (Kolgomorov–Smirnov test). The Spearman test was used to correlate the antibody isotype titers in paired groups. The Mann–Whitney U test was used to compare the antibody isotype titers between household and PD leprosy contacts. The Friedman test was used to compare the isotype antibody titers among three paired groups. The Fisher test was used to analyze the association between the frequency of positive serum antibody isotypes with the presence of lesions, and also with leprosy diagnosis. The relative risk (RR) with a confidence interval of 95% was calculated to evaluate the risk of presenting positive serum antibody isotype and the presence of lesions, and also with leprosy. All statistical analyses were performed using GraphPad Prism version 6.0. The level of statistical significance was 5% (P < 0.05).
The project was approved by the National Committee for Research Ethics (process 532,850; February 18, 2014). After explanation of the project, the guardians of the participants were asked to sign a consent form. The participants who were 8–15-year-olds also signed an assent form.
In 2015, 155 young people who were 4–15-year-olds living in Santana do Ipanema municipality were enrolled in the project. In 2016, many of them did not return. In the follow-up study, a total of 68 participants completed annual follow-up dermato-neurologic evaluation and serologic testing. In 2017, those participants who presented lesions during the follow-up study were examined by a dermatologist. Twenty-five contacts of PB cases (PB contacts) and 43 contacts of MB cases (MB contacts) were evaluated. Thirty-two participants were HH contacts and 36 were PD contacts.
Among the PB contacts, no significant variation in respect to the IgM isotype titers was found in the follow-up study (Fig. 1A). In respect to IgG (Fig. 1B) and IgA antibodies (Fig. 1C), a significant decrease in the antibody titers was observed comparing 2015 and 2016 (P < 0.05 and P < 0.01, respectively). Among the MB contacts, the antibody titers also decreased comparing 2015 and 2016, for both IgM (P < 0.01; Fig. 1A) and IgG isotypes (P < 0.001; Fig. 1B). The lowest antibody titers were found in 2017 in comparison to 2015 for both IgG (P < 0.05; Fig. 1B) and IgA (P < 0.001; Fig. 1C), but also in comparison to 2016 in relation to IgA (P < 0.05; Fig. 1C).
There was a positive correlation between anti-PGL-1 IgG and IgM levels among MB contacts in 2015 (Spearman correlation; r = 0.56 and P = 0.001) (Fig. 2A), and between anti-PGL-1 IgM and IgA levels among PB contacts in 2016 (r = 0.45 and P = 0.02) (Fig. 2B), and between IgG and IgA anti-PGL-1 among MB contacts in 2017 (r = 0.37 and P = 0.02) (Fig. 2C).
Higher levels of serum antibodies were found among HH contacts than among the PD contacts for all isotypes (Fig. 3). This difference was statistically significant for anti-PGL-1 IgM and IgA (P = 0.03), and also for anti-PGL-1 IgG (P = 0.04) in 2016, and for anti-PGL-1 IgM (P = 0.01) in 2017.
The Table 1 shows the number of participants who presented with suspected leprosy skin lesions (n = 23) and who did not (n = 45) during the follow-up study and the percentages of seropositivity or negativity for each isotype. The IgM isotype was positive in 26.1% to 43.5% of contacts with lesions over the years of the study period. Nonetheless, a high percentage of seropositive IgM in the absence of lesions (40.0%–46.7%) was observed. A low frequency of positive IgA was observed in the contacts with the presence of lesions (8.6%). A high frequency of seronegative IgA was found among those contacts that did not present any lesion (95.6%–100.0%). A strong association was found between IgG isotype seropositivity and the presence of lesions (Fisher´s test, P = 0.035). The RR for contacts presenting with lesions and positive anti-PGL-1 IgG was 3.25 (IC95: 2.26–4.68).
In 2017, the contacts with suspicious lesions were evaluated by a dermatologist. Eight cases of leprosy (11.8%) were diagnosed among the 68 participants. One of them was diagnosed with lepromatous leprosy, one with borderline leprosy, six with the indeterminate leprosy. Four were HH contacts and four were PD contacts with two being MB contacts and six PB contacts. Five of the eight contacts diagnosed with leprosy (62.5%) had positive anti-PGL-1 antibodies.
One of the contacts (Table 3) was seropositive for anti-PGL-1 IgA in 2015. In 2016, he began to present positive IgG and IgA, but no lesions. In 2017, all isotypes were positive. In that year, he presented a slight hypochromic lesion with alopecia behind the neck. He was diagnosed with the indeterminate form of the disease.
The Table 2 shows the accuracy of the anti-PGL-1 tests in the newly diagnosed leprosy cases. Anti-PGL-1 IgG was considered to the best serologic marker because of its strong association with confirmed leprosy (Fisher´s test, P < 0.05). The RR of developing leprosy and presenting with positive serum anti-PGL-1 IgG was 8.5 times (95% confidence interval: 4.0–18.0). The diagnostic sensitivity and specificity for the test were 25.0% (3.2%–65.0%) and 100.0% (92.1%–100.0%), respectively. The positive and negative predictive values were 100.0% (15.8%–100.0%) and 88.2% (76.1%–95.6%), respectively. There was no significant association between anti-PGL-1 IgM isotype and disease (Fisher’s test: P = 0.46), nor between anti-PGL-1 IgA and disease (Fisher’s test: P = 0.15).
Leprosy in children and in young people indicates active transmission in the community.15 As we demonstrated recently in Rio Largo, another municipality of the Alagoas state, in 2016, 36% of leprosy contacts had a positive anti-PGL-1 IgM in saliva. This likely indicates presence of M. leprae in community and consequently active transmission.16 Additionally, in 2015 the detection rate in that municipality was about 20 cases per 100,000 inhabitants. For this reason, it will be almost impossible to achieve the goal of breaking transmission during the next few years as intended by the WHO strategy.17
In reality in this setting, active case finding is rarely carried out with people who have suspicious lesions having to self report to a health center to be diagnosed. Unfortunately, untrained professionals often miss the diagnosis there. It is important to remember that the type of research we performed requires a close partnership with the municipality. In our study, eight new cases of leprosy were diagnosed among the 68 participants, that is, 11.8%. Although the participants were referred to a doctor every time suspected lesions were found, none of them got his/her diagnosis until the dermatologist invited by the project went to examine them in the last year of the study (2017). This reveals the challenge faced by these communities in controlling the disease. Hopefully research such as ours will stimulate increased effort in training and diagnosis of leprosy by municipal health authorities. Some problems of this nature have already been reported by other researchers.18
Besides the fact that leprosy has a long incubation period, it is rather difficult to diagnose early stages of the disease.19 Some factors that may favor the development of the disease include crowded homes with little exposure to sunlight or natural ventilation, which guarantees a favorable environment for subsistence of mycobacteria in the environment.
Armadillos are natural hosts of M. leprae and may cause zoonotic leprosy.20 Nonetheless, most of the infected people do not have contact with those animals, and although there are still some doubts in the literature in respect to the mode of transmission, it is widely accepted that individuals who reside in the household and those who have close contact to MB and to PB patients are more prone to develop the disease.10,19 Mohanty et al21 have found genetic material of M. leprae in soil and water around the houses of leprosy patients which provides some evidence of the bacterial persistence in environment.
In the present study, half of the newly diagnosed participants were HH contacts, and the others were PD contacts. Interestingly, two were MB contacts; the others six participants were PB contacts. This evidence reinforces that leprosy contacts are more prone to develop the disease, independently of the clinical form of the index case and independently of living inside the same house. It is enough to have a close contact to the case index. Another point that we have observed in our study is that when children were diagnosed early they presented with the indeterminate form. The fact that two contacts were diagnosed with the MB form likely indicates delayed diagnosis. It is important to note that atopic and malnourished children are at increased risk.22,23 Interestingly, PGL-1 can itself down-regulate the production of interleukin-1 and tumor necrosis factor by macrophages.24
Considering the importance of early diagnosis in children, the question arises is if there is any biomarker that could predict the onset of leprosy in infected people.9
Anti-PGL-1 IgM isotype has been used in searching for infection,7 but not necessarily for the disease, because once positive, IgM can be found in both infection and disease, although it has been demonstrated that there is a good correlation between the IgM antibody and bacillary load.25 Moreover, its positivity suggests an increased risk for developing leprosy.26
Brito e Cabral et al27 demonstrated a good correlation between anti-PGL-1 IgG in cases and their contacts, and suggested its use in follow-up studies. In that study, the authors found that among those leprosy contacts who presented with negative IgG, 58% showed positivity to IgM. On the other hand, 74% of positive IgG samples were also positive for IgM.
Chujor et al28 suggested IgA as a biomarker for early detection of the disease. Macedo et al14 found a strong correlation between serum anti-PGL-1 IgM and IgA in MB patients, and a moderate correlation was found in PB patients. The isotype showed a better performance than IgG. For this reason, the IgA isotype was also recommended to be used in follow-up studies, besides the search for IgG and IgM antibodies.
In the present study, anti-PGL-1 IgM was present in 40%–46% among the participants without lesions and in 26%–44% among those with lesions. The percentage of positive IgA was very low in the presence of lesions. Interestingly, a strong association was found between seropositive IgG and the presence of lesions (RR of 3.25 times).
In respect to the performance of the serologic tests in the newly diagnosed leprosy cases, anti-PGL-1 IgG was considered to the best biomarker, with a RR was 8.5 times for the presence of positive serum anti-PGL-1 IgG and leprosy. No association was found between the IgM isotype and disease, nor between the IgA isotype and disease.
Our data supports the use of anti-PGL-1 IgG isotype in monitoring pediatric leprosy contacts, in addition to IgA and IgM. Although its positivity is low, when it is present, careful examination of the child is warranted as one may be facing a new case of leprosy. In cases without antibody, it is important to have the case evaluated by someone with expertise in diagnosing the disease.29 Thus, while the diagnosis of cases is complex, we must use current serologic tools and clinical expertise to reduce the current large disease burden in Brazil.
We gratefully acknowledge the kindly assistance provided by the health professionals, particularly, Mrs. Ana Lúcia Carneiro Leal, Mrs. Maria do Socorro da Costa Freire, Mrs. Fabilenia Mendes, and Mrs. Gilvânia Franca Vilela.
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Keywords:Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.
leprosy contacts; anti-PGL-1; serum IgA; serum IgG; serum IgM