Prevalence and association of sIgA in saliva and Pseudomonas aeruginosa infection in TB patients: a cross-sectional study

Objectives: Pseudomonas aeruginosa is pathogenic in immunocompromised individuals . It has several complex mechanisms for evading human immunity. The objective of the study was to examine the secretory immunoglobulin A (sIgA) mediated immune response in saliva to detect P. aeruginosa in pulmonary tuberculosis. Methods: The infection with P. aeruginosa was categorized according to the Leeds criteria in the ﬁ nal 86 individuals who were proven to have pulmonary tuberculosis by polymerase chain reaction. Levels of serum immunoglobulin G (IgG) and sIgA which are speci ﬁ c to P. aeruginosa were measured using the method of ELISA. Results: Patients in the “ free of infection (patients who were infected with P. aeruginosa in the lower respiratory tract at the beginning of the study later became negative) ” and “ intermittent colonized (patients who were infected with P. aeruginosa throughout the study) ” groups had substantially higher median baseline sIgA levels in saliva and a much greater proportion of sIgA positive than patients who were never colonized (patients who were found to be P. aeruginosa negative throughout the study) (p=0.038). Median baseline IgG level was 10.7 (1.7 – 145.0), 8.3 (2.5 – 22.9), and 6.7 (3.3 – 17.1) for the patients categorized as “ intermittent colonization ” , “ free of infection ” and “ never colonized ” , respectively. After 3 years of study, sIgA level was found in signi ﬁ cant high level among the patients with infection of P. aeruginosa (p=0.003). Conclusions: Secretory IgA may be readily collected from saliva and is a useful diagnostic technique for determining whether P. aeruginosa infection has occurred.


Introduction
A variety of bacteria present in lungs of patients infected with pulmonary tuberculosis [1].Frequently isolated and potentially pathogenic microorganisms in pulmonary tuberculosis include H. influenza, Pseudomonas aeruginosa, and Streptococcus pneumonia.In pulmonary tuberculosis, P. aeruginosa is associated with reduced lung function and higher mortality [1,2].Some studies have reported a possible association between the presence of these respiratory microorganisms and poorer prognosis in pulmonary tuberculosis [3].P. aeruginosa has several complex mechanisms for evading human immunity, one of which is the formation of a biofilm that protects the bacterial cell from phagocytosis by human immune cells while also protecting the bacterial cell from antibacterial medications.P. aeruginosa can produce rhamnolipids to kill human Poly Morpho Nuclear (PMN) leukocytes, macrophages, and erythrocytes.It has been shown that immunocompromised individuals, like patients of pulmonary tuberculosis are associated with several intrinsic loopholes in immune mechanisms, which may include a defect in mucociliary clearance, and dysregulation of immune and inflammatory mechanisms [4].The published reports [5][6][7] show that the other bacterial infections are more dominant and P. aeruginosa is not the dominant co-infection in pulmonary tuberculosis.However, P. aeruginosa is the most frequently isolated bacteria among pulmonary tuberculosis [5].Therefore, it is required to study co-infection with P. aeruginosa in pulmonary tuberculosis.
The immune mechanism usually found against the infection of P. aeruginosa includes responses by epithelial cells, T-cells, recruited neutrophils, Type-I interferon-mediated innate immunity, etc. [8].Other mechanisms include activation of NF-κB-mediated proinflammatory chemokines and cytokines and activation of inflammasomes signaling.Production and release of IL (Interleukin)-1β and IL-18 primarily from the alveolar macrophages (stimulated by the flagellar antigens of P. aeruginosa) results in clearance of bacterial invasion.But the disadvantage of this production is that both IL-1β and IL-18 play a role in increasing cytotoxicity in the lung and thus increasing mortality [9].In patients with pulmonary tuberculosis, P. aeruginosa causing lung infection is found to grow in places where oxygen is deficient, like mucus plugs from the lower respiratory tract of the patients [10].This is found that P. aeruginosa is prone to be present in oxygen-deficient areas which favors them to have biofilm formation around them under an anaerobic environment [11,12].
To test P. aeruginosa infection, ELISA can be done to measure the specific IgG against the bacterium.Antibodies may be found in the upper airways, tears, and saliva, all of which can be readily retrieved from the patient for diagnosis.Secretory immunoglobulin A (sIgA) is easily detected from saliva in patients with chronic lung infection with P. aeruginosa.It has been well noted that the diagnostic value of sIgA obtained from saliva is significant [13].
The objective of the study was the early detection of P. aeruginosa in pulmonary tuberculosis patients by assessing the secretory IgA-mediated immune response in saliva.

Ethics approval and consent to participate
The study by approved by the YongChuan Hospital of Chongqing Medical University ethical and research board.The study reporting adheres to the law of China and the V2008 Declarations of Helsinki.All participants or their legally authorized persons signed an informed consent form regarding diagnosis, pathology, and publication of the patients' personal information in the article form.

Study population
The range of age of the patients varies between 30 and 45 years of age.The present cross-sectional study was done with 86 patients who had pulmonary tuberculosis.Although 102 pulmonary tuberculosis patients were selected initially, 16 patients were excluded or did not show interest as a volunteer.

Diagnosis of pulmonary tuberculosis
The diagnosis of pulmonary tuberculosis was established using chest X-ray, sputum microscopy, culture in both liquid and solid media, and nucleic acid amplification, polymerase chain reaction.Chest computed tomography, and histopathological examination of biopsy samples have also been done in patients with smear-negative pulmonary tuberculosis and drug-resistant tuberculosis patients.A 165 bp was targeted for "nucleic acid amplification" and "polymerase chain reaction.
P. aeruginosa classification P. aeruginosa was classified according to Leeds criteria as follows [14]: (1) Patients who never had P. aeruginosa infection confirmed from the microbiological culture with negative screening serum Ig G result (P1) (2) Patients who had P. aeruginosa infection confirmed in microbiological culture, but the patients are free from P. aeruginosa infection 12 months from the present study (P2) (3) Patients from whom P. aeruginosa could be isolated from less than 50 % of cultures in the last 12 months with serum Ig G negative or P. aeruginosa was isolated with serum Ig G positive (P3).
Pus was the clinical sample used for microbiological culture.These were inoculated on Mac Conkey agar at 37 °C for 48 h.Standard microbiological techniques were used for interpretation [15].

ELISA test
The pad was brushed briefly on the lower gums and then held between the gum and the cheek for 3 min.Levels of serum Ig G and sIgA, which are specific to P. aeruginosa are measured using the method of ELISA (NIF-1: Cusabio Biotech CO. LTD, Wuhan, Hubei Province 430206, P.R.China (Catalogue no.CSB-EL026683HU); analytical measuring range: 80-120 %).Antichlamydial antibody linked with enzyme was added.Substrate color was then measured by an ELISA reader.The intensity of the color was directly proportional to the concentration of antigen present in the specimen.A 96-well ELISA plate was used to measure the levels.A horseradish peroxidase (HRP) conjugated polyclonal rabbit anti-human Ig G and sIgA was added.Ig G was added for measuring antibodies in serum, while sIgA was added to measure antibodies in saliva.The saliva samples were collected for one time and Ig G and sIgA were measured one time in patients.One time collect sputum cultures for P. aeruginosa from patients.

Various scenarios of patients
The change in the level of sIgA in saliva was correlated with the status of P. aeruginosa in the lower respiratory tract based on the levels and their increase as the severity of the disease increased.The status of P. aeruginosa was considered in terms of microbiological and serological results of P. aeruginosa.The changes in the status of sIgA were noted periodically.The analysis of sIgA and the status of P. aeruginosa in the lower respiratory tract were made in various scenarios.
In the first scenario, for study the patients effectively, they were divided into 3 groups (a) Never colonized, (b) Free of infection, (c) intermittent colonized: firstly, patients found to be P. aeruginosa negative throughout the study or they never encountered P. aeruginosa infection (categorized as "Never colonized").Secondly, patients who were infected with P. aeruginosa in the lower respiratory tract at the beginning of the study later became negative (categorized as "Free of infection").Thirdly, patients infected with P. aeruginosa throughout the study (categorized as "Intermittent Colonized").
In the second scenario, the patients who became P. aeruginosa negative later during the study were analyzed to observe any change in the level of sIgA.For this purpose, the patients were divided into 2 groups as follows: firstly, the patients who had ≥1 positive result of P. aeruginosa (in microbiological culture) or serum IgG, and secondly, the patients who never had any positive result of culture or presence of specific serum IgG.
In the third scenario, the patients were classified based on their history of exposure to P. aeruginosa in the lower respiratory tract.Even with a single occurrence of P. aeruginosa colonization, the history of the patient was positive in this scenario.The patients who never showed any sign of exposure in terms of positive culture or serum IgG level were considered to have no history of exposure to P. aeruginosa.

Statistical analysis
The linearity of continuous parameters was evaluated using the method of Kolmogorov and Smirnov.According to the scenarios, sIgA serum levels and the status of P. aeruginosa were compared.The nonparametric Mann-Whitney U test (for two independent samples) and one-way Kruskal-Wallis' test (for more than two independent samples) were employed for comparison.Using the Wilcoxon test (for two different periods) and the Friedman test (for more than two different periods), the assessment of sIgA level variation was well analyzed.Differences in the percentages are assessed by employing the Chi-square test.Odd-ratio and 95 % confidence intervals were analyzed by using SPPS 20.0 software and a p-value less than 0.05 was statistically significant for all the tests.

Study population
Out of 86 patients, 46 (53 %) were female patients, while the number of males was 40 (47 %).The median age of the patients was 38.1±1.6 years.Sputum was obtained from 25 (29 %) patients, while respiratory swabs were obtained from the rest of them (61 (71 %) patients).
The patients who were "free of infection" showed the following outcomes: out of 26 such patients (as shown in Table 1), 8 patients became intermittent colonization (sIgA positive) while 4 patients turned into chronic cases (sIgA negative).The rest of the 14 patients (out of 26 patients) remained free of infection.Five of the 14 individuals were positive for sIgA, whereas the others tested negative (Figure 1).
Next, 23 patients were classified as "Never Colonized".Out of 23 patients, 11 patients were colonized by P. aeruginosa during the study.This group of patients who got colonized during the study had different outcomes finally.Out of the 6 patients who got free from the colonization, 4 showed sIgA negative while 2 showed sIgA positive.Again, out of 11 patients who were categorized as "colonized during the study", the remaining 5 patients were categorized as "Intermittent colonization".Of these 5 patients, 3 were sIgA positive while 2 were sIgA negative.
Again, out of 23 patients who belonged to the "Never Colonized" group, the remaining 12 patients remained as it is.But out of 12 patients, 9 showed sIgA negative while 3 were sIgA positive (Figure 2).
Again, 37 patients were categorized as "Intermittent Colonized", out of which, 14 patients remained colonized at the end of the study.Out of 8 patients who became chronic cases, 4 showed sIgA negative, while another 4 showed sIgA positive.Out of 14 patients who remained as colonized, 5 patients were sIgA positive, while 9 patients were found to be sIgA negative.Out of 15 patients who became free of colonization later, 9 patients were found to be sIgA positive while 6 patients were found to be sIgA negative (Figure 3).

First scenario
Median baseline IgG level was 10.7 U/mL (1.7-145.0),8.3 (2.5-22.9),6.7 (3.3-17.1)for the patients categorized as "Intermittent colonization", "Free of infection" and "Never colonized", respectively.After 3 years of study, sIgA level was found to significantly decrease levels among the patients with infection of P. aeruginosa.The level of sIgA increased prominently among patients who were positive for P. aeruginosa infection and those who had the infection but later became negative.

Second scenario
The patients who became P. aeruginosa negative later during the study were analyzed to observe any change in the level of sIgA.In the second scenario, the median sIgA was 26.3 U/mL, which had P. aeruginosa recurrence, while the patients who had no recurrence, showed lesser median sIgA, that is, 23.3 U/mL.The level of median sIgA increased with time, increasing up to 98.3 U/mL in the second year for those who had a recurrence, followed by 74.1 U/mL in the third year.The patients who had no recurrence also showed increased median sIgA levels up to the second year; in the third year, the median sIgA level came much lower (Table 2).

Third scenario
The patients were classified based on their history of exposure to P. aeruginosa in the lower respiratory tract.The secretory IgA positivity in the third year was 48.1 % among the exposed group.Interestingly, the percentage of secretory IgA positivity came down to 21.9 U/mL among the non-exposed group as compared to 29.6 % in the second year.This shows that in the non-exposed patients who did not get P. aeruginosa infection despite the presence of sIgA in the second year and made up to the third year, the risk of getting P. aeruginosa infection lowered among them (Table 3).

Discussion
This study presents an association between the detection of P. aeruginosa in chronic lung infection and sIgA in the saliva.It is a non-invasive method and so it has high patient compliance.Secondly, it has been found that P. aeruginosaspecific sIgA response in the saliva is significant even when there is no significant P. aeruginosa culture in samples obtained from the lower respiratory tract [16].The results of an association between the detection of P. aeruginosa in pulmonary tuberculosis of adult patients and sIgA in the    saliva of the current study are consistent with those of a prospective study of pediatric cystic fibrosis patients [17].
Using saliva to determine P. aeruginosa infection is advantageous in many ways.
In most cases, this organism is found both in the lower and upper respiratory tract.It was also found that P. aeruginosa can be present in both the upper and lower respiratory tract.It is hypothesized that P. aeruginosa first colonizes the upper respiratory tract and then proceeds to the lungs [18].It can be used as an early detection method of P. aeruginosa infection in the respiratory tract.Therefore, sIgA obtained from saliva specific to P. aeruginosa can ease the management of P. aeruginosa by showing an increased risk of P. aeruginosa infection [19,20].Early management of pseudomonal treatment is very important.It was found that early strong antimicrobials can reduce pseudomonal colonization among patients [21].Using sIgA detection, it is possible to detect P. aeruginosa in the early stage.
The usage of three scenarios implied the significance of the determination of sIgA level in these patients to detect P. aeruginosa early.Other studies showed that there is a chance that 42 % of patients may contain the same pathogens in the lower respiratory tract who are yet to undergo transplantation and who has already done transplantation [22].
It cannot be said with full evidence that P. aeruginosa can be attributed to the presence of sIgA obtained from saliva, but some occurrences suggest high genotype similarity between samples of the upper and lower respiratory tract [23].In a way, it confirms that paranasal sinuses may act as reservoirs for bacteria [24,25].
There are limitations of this study to consider.The number of patients was unsatisfactory and it would be better if more patients were there.More patients might have given a more statistically significant result.Also, the salivary sIgA response was not compared with the overall microbiological assessment of the sinuses.Another limitation is that this present study does not provide the long-term outcomes of P. aeruginosa infection and lower respiratory tract infection.

Conclusions
The research was carried out over three years, and there is sufficient evidence to conclude that the detection of secretory immunoglobulin A in saliva may be attributed to increased risk or P. aeruginosa infection.Secretory immunoglobulin A is relatively easy to obtain from the saliva and is an effective diagnostic technique for determining whether a person has been infected with P. aeruginosa.
As a result of this finding, it is possible to commence early intervention.

Figure 1 :Figure 2 :
Figure 1: Patients who were "free of infection" at the start of the study and their outcome after the follow-up.

4Figure 3 :
Figure 3: Patients who were "intermittently colonized" at the start of the study and their outcome during the study and after the follow-up.

Table  :
Status of patients during the time of enrollment.

Table  :
Status of patients during the second scenario.

Table  :
Exposure of Pseudomonas aeruginosa during follow-up study and corresponding changes of sIgA positivity percent and its level in saliva.