Both P. gingivalis crude extract and HmuY protein were recognized by specific salivary IgA in individuals with and without leprosy. However, the specific recognition of P. gingivalis crude extract was stronger in the samples of individuals with periodontitis than in those without this oral disease, which makes this validated test an apt tool for the study of periodontitis in individuals with leprosy. In contrast, the HmuY recombinant protein did not show an equal performance.
Although both antigens were recognized, the first, made up of somatic proteins of the bacteria, seems to favor a more specific recognition, possibly due to the salivary IgA, which had undergone affinity maturation, found in individuals who had more previous contact with the bacteria, that is, those clinically diagnosed with periodontitis (Carvalho-Filho et al. 2019). Regarding HmuY, as it is an isolated protein, it is possible that individuals with periodontitis were not sensitized to this protein at the moment of pathogen-host interaction (Śmiga et al. 2015). It should be stressed that it is not a constitutive protein of the bacterium. Rather, it is expressed when there is lack of iron in the environment (Hagewald et al. 2002).
ELISA checkerboard tritation made it possible to determine where the antigen and antibody concentrations meet to achieve an equivalence zone, in the responses of healthy and diseased individuals. This method favors the possibility of eliciting responses with high titers in individuals with greater previous exposure to the antigen, while those with less previous exposure responded with low levels of antibodies.
It is worth noting that the use of ELISA for investigations concerning the biological plausibility of an association between two conditions, such as periodontitis and leprosy reactions in individuals diagnosed with leprosy, is of interest, as it is easily executed, low cost and has good reproducibility (Lyn 2015). Furthermore, saliva collection is simple, avoiding invasive procedures. It is known that saliva marker analyses reflect a local response, result of a bacteria-host interaction in periodontal tissues (Matos et al. 2018; Carvalho-Filho et al. 2019).
On the other hand, saliva can contain contaminants, such as food and cosmetic micro-residues, which interfere in test sensitivity (Zhou et al. 2018), as well as presenting some limitations, as it is a fluid with low stability due to the presence of enzymes (Matos et al. 2018 ). Additionally, antigens taken from bacterial extract can suffer compositional variations depending on the cultivation condition employed, which can reflect indirectly in the ELISA result.
It is important to note that the test validation was carried out in individuals with leprosy, which may have interfered in the performance of the test, especially in relation to sensitivity. Studies have shown that IgG and IgA levels were higher in patients with chronic periodontitis than in healthy individuals. The average levels of serum IgG and salivary IgA increased as the seriousness of the disease increased (Gadekar et al. 2018; Carvalho-Filho et al. 2019).
The individuals in this study sample were undergoing PQT treatment or taking drugs to control leprosy reactions, which may have interfered in the bacterial load, consequently, modulating the humoral response. These drugs can also reduce saliva flow, influence the composition of saliva or provoke systemic and local alterations that impact on the buccal cavity (Femiano et al. 2008; Zhou et al. 2018), and, as such, the findings should be treated with caution.
However, it is worth noting that before this study there was no previous evidence that had analyzed salivary IgA antigen specific to P. gingivalis levels in individuals with leprosy, with the exception of some studies concerning diabetes, acute alcoholic hepatitis and other systemic conditions 20,34. Therefore, a comparison group was used to evaluate the IgA formed by individuals without leprosy diagnosis, as gold standard, with and without periodontitis, so as to ascertain the best conditions for the test employed, free from the response interference provoked by this chronic infectious neurological disease.
Based on the above considerations, indirect ELISA can be used as a tool to detect humoral immune response against P. gingivalis and its virulence factors, contributing, as such, to an avaluation of the association between periodontitis and leprosy.
This standardized ELISA to detect levels of salivary IgA antibody specific to P. gingivalis antigens was capable of discriminating between individuals with periodontitis and without periodontitis and with a leprosy diagnosis.