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Influence of intestinal parasites on cytokine profile of patients with pulmonary tuberculosis, including cases complicated...
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Davis Nikolay Aleksandrovich, Research Institute of Epidemiology, microbiology and infectious diseases, researcher Toychiev Abdurakhim Khodjiakbarovich, Research Institute of Epidemiology, microbiology and infectious diseases, researcher Djuraeva Zulfiya Baratovna, Tashkent Medical Academy, assistant Parpieva Nargiza Nusratovna, Republican specialized scientific research medical center of phthisiology andpulmonology, director Osipova Svetlana Olegovna, Research Institute of Epidemiology, microbiology and infectious diseases, head of the department E-mail: davisnikolay@gmail.com
Influence of intestinal parasites on cytokine profile of patients with pulmonary tuberculosis, including cases complicated with aspergillosis
Abstract: influence of intestinal parasites on immune imbalance in pulmonary tuberculosis (PT), including cases complicated by pulmonary aspergillosis (PA) was studied. 300 and 111 patients with active PT were examined for intestinal parasites. Group of comparison: 200 residents of Tashkent and Tashkent region. The group of111 patients was examined for PA (clinical, X-ray, mycological and serological data). Serum IFN-y and IL-4 were assessed by ELISA. Ascariasis was found in patients with PT and PT with invasive PA respectively 5 and 12 times as frequent as in population. Decrease of IFN-y and increase of IL-4 level (P < 0.05) was observed in these groups in comparison with healthy individuals and patients with PT free of intestinal parasites and PA. Conclusion: Ascariasis as a concomitant disease enhances immune imbalance, typical for PT and PT + invasive PA.
Keywords: Ascariasis, pulmonary tuberculosis, intestinal parasites, aspergillosis, cytokines.
Control of Mycobacterium tuberculosis replication is mainly nity in helminthiasis is mediated by Th2-response [6, 459-466]. mediated by production of Th1-cytokines IFN-y and TNF-a and Protective mechanisms in pulmonary tuberculosis (PT) and as-cytotoxicity of CD8+-lymphocytes, directed against infected mac- pergillosis are similar and are based on elevated IFN-y produc-rophages. Control efficiency is specified by the balance of Th1-and tion [5, 403-413]. Our previous study showed that pulmonary Th2-response (IL4, IL-10, IL-13) [7, 694-701]. Protective immu- aspergillosis (PA) complicates course of the disease in 40 % of the
Section 5. Medical science
patients with PT and the majority of Aspergillus spp. strains were resistant to widely applied antimycotics. Maximal susceptibility was detected to voriconazole [2, 113-118]. It was of interest to study influence of intestinal parasites on immune imbalance specific for active PT, including cases complicated by PA under conditions of the region endemic on intestinal parasitic diseases.
The purpose of the communication is to determine level of serum cytokines IFN- y and IL-4 in patients with PT including cases with concomitant intestinal parasitic diseases and complicated with PA.
Materials and methods. Two groups of patients with active PT, hospitalized at Republican specialized scientific research medical center of phthisiology and pulmonology of the Ministry of Public Health of the Republic of Uzbekistan (respectively included 300 and 111 patients) were examined, the last group was additionally examined for PA. Patients with infiltrative PT dominated in both groups. Diagnosis of PT was based on clinical, X-ray and bacteriological data. HIV-infected and individuals with viral hepatitis were excluded. All the patients were at the age of 18-64 and received standard antituberculosis therapy: isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin.
Group of comparison included 200 inhabitants of Tashkent city and Tashkent region. Family where patients with PT, HIV-infected individuals and patients with chronic viral hepatitis were excluded. Control groups for IgG to Aspergillus spp. and cytokines detection consisted of 30 and 20 healthy persons respectively. Sexual and age structure of groups of comparison were analogous to the patients with PT and PA.
Intestinal parasites were diagnosed by the triple coproscopy, stool samples were collected with 2-3 days interval.
The cause for examination for PA was sudden aggravation of patients condition against the background of antituberculosis therapy or torpidly current tuberculosis.
Diagnosis of PA was based on clinical, radiological (chest computerized tomography (CT) scan), and laboratory findings: isolation ofAspergillus spp. of sputum, bronchoalveolar lavage and blood samples, detection of IgG antibodies to Aspergillus spp. by ELISA (kits from LLC Vector-Best, Novosibirsk, Russian Federation).
We used as differential diagnostic sign a rapid improvement of patient's condition on the 3rd - 5th day of empiric therapy: decrease of temperature, relief of dyspnea, significant diminution of weakness in the cases without mycological confirmation (absence of sputum and bronchoalveolar lavage refusal) and nonspecific results of CT (progression of infiltrative process) against the background of clinical impairment and positive serology.
Blood samples for mycological study were taken at fever period once a day (5-7 ml.) for 3 days. For prevention of contamination we used flasks of the system HiSafe, Hi-Media, India, with diphase system Hi-Combi for fungi with CC addition). In 2, 5 and 10 days blood samples from flasks were inoculated on Petri dishes with Sab-ouraud agar which were incubated at 37 °C up to 3 days, Sabouraud agar with glucose (pH 5.0), (Hi-Media, India) was used for inoculation of sputum and bronchoalveolar lavage samples.
IFN-y and IL-4 in serum samples were assessed using commercially available enzyme-linked immunosorbent assay (ELISA) kits from LLC Vector-Best, Novosibirsk, Russian Federation.
Statistics. Comparison between indices under investigation were made using Student's t-test. The significance was determined at P < 0.05.
Results. Results of intestinal parasites diagnosis in patients with PT are represented in table 1. Ascariasis was diagnosed in
10.0 ± 1.7 % of patients with PT but in 2.0 ± 0.9 % of population (P < 0.001). This index was even higher in patients with infiltrative PT: in 23 (l2.3 ± 2.4 %) of 187 patients.
Table 1. - Prevalence of intestinal parasites in patients with PT (n/M ± m)
Parasites Patients with PT (n = 300) Population of Tashkent city and Tashkent region (n = 200)
Ascaris lumbricoides 30/10.0 ± 1.7* 4/2.0 ± 0.9
Enterobius vermicularis 14/4.7 ± 1.2 9/4.5 ± 1.4
Giardia lamblia 14/4.7 ± 1.2* 32/16.0 ± 2.6
Blastocystis hominis 161/53.7 ± 2.8* 36/18.0 ± 2.7
Note: * — significant difference between patients with PT and population (P < 0.05).
Prevalence of Enterobius vermicularis in patients with PT and population was the same in spite of more efficient mode of transmission in enterobiasis, belonging to contact parasitic diseases.
Morbidity with giardiasis in patients with PT was 4 fold lower, than in population. This phenomenon appears to be stipulated by the influence of antituberculosis therapy. Aminoglycosides are known to possess a limited antigiardial activity (paromomycin) [9, 8-10], probably other antituberculosis preparations exert analogous effect.
Prevalence of Blastocystis hominis was 3 fold higher than in population. It seems to can be connected with an expressed immune imbalance and decrease of activity of local immunity to control the B. hominis number. It is confirmed by analogous tendency in HIV-infected individuals, a high intensity of B. hominis infection (> 5-6 parasites in a field of view, oc x 10, ob x 40) being detected only in patients with PT and HIV-infected individuals, not in healthy persons and patients with various pathology of gastrointestinal tract (gastritis, enterocolitis, cholecystitis) [3, 8-11].
Table 2. - Level of serum IFN-y and IL-4 in patients with PT, invasive PA and intestinal parasites (pg/ml)
Cohort under study IFN-y IL-4
Healthy individuals (n = 20) 125.7 ± 6.7 2.6 ± 0.7
Patients with PT without intestinal parasites and aspergillosis (n = 15) 70.0 ± 6.1* 8.0 ± 2.1*
Patients with PT and ascariasis (n = 17) 54.6 ± 2.8* ** 18.2 ± 3.0* **
Patients with PT and enterobiasis (n = 14) 69.3 ± 3.8* 9.3 ± 1.8*
Patients with PT and invasive PA (n = 12) 41.6 ± 3.1* ** 27.6 ± 4.7* **
Patients with PT, ascariasis and invasive PA (n = 4) 22.8 ± 4.7* ** 31.7 ± 4.9* **
Patients with PT and giardiasis (n = 12) 58.7 ± 3.8* 15.4 ± 2.4*
Patients with PT and blastocytosis (n = 12) 62.8 ± 3.8* 22.4 ± 5.7* **
Note: *—significant difference with healthy individuals (P < 0.05); ** — significant difference in comparison with patients with PT without intestinal parasites and aspergillosis (P < 0.05).
PA was diagnosed in 44 (39.6 ± 4.6 %) from 111 patients with PT, 16 of them suffered from invasive PA. Aspergillus fumigatus were isolated from blood samples of 2 patients and from sputum and bronchoalveolar lavage samples of 10 patients. Invasive PA was diagnosed in 4 patients on the base of significant impairment of condition and progression infiltrative process against back-
Influence of intestinal parasites on cytokine profile of patients with pulmonary tuberculosis, including cases complicated.
ground of antituberculosis therapy (increase of temperature up to 38.5-39.9 °C, appearance/intensification of dyspnea and appearance of blood streaked sputum, progressing of infiltrative process (CT), high level of IgG to Aspergillus spp. and rapid positive effect ofvoriconazole therapy. Other cases were presented by aspergillo-ma. We consider positive serology as a test orienting to the disease, because In total positive serology was observed in 4 (20 %) from healthy individuals of control group. Positive results were obtained in 1 (5 %) from 20 nonsmokers and 3 (15 %) from 20 smokers, all three smokers have A. fumigatus colonization of upper part of respiratory tract. Diagnostic level of IgG to A. fumigatus was determined in 31 (70.4 %) patients with PA.
Results of serum cytokines detection are represented in table 2.
Level of serum IFN- y in patients with PT without intestinal parasites and PA was significantly lower, than in healthy individuals (P < 0.05). This index in patients with PT and ascariasis was even lower, and is significantly different from control values as well as values, obtained in patients with PT and without parasites and PA (P < 0.05). Enterobiasis didn't induce significant changes in IFN- y concentration in patients with PT. Invasive PA, diagnosed in 12 patients with PT, was developed against a background of low level of serum IFN-y: 41.6 ± 3.1pg/ml; ascariasis diagnosed in 4 patients with PT and invasive PA induced even more expressed decrease of IFN-y: 22.8 ± 4.7pg/ml.
Level of serum IL-4 was significantly higher in all groups under study in comparison with control value. The highest IL-4 level was detected in patients with PT + invasive PA + ascariasis and PT + invasive PA and PT + ascariasis, respectively 31.7 ± 4.9 pg/ml; 27.6 ± 4.7 pg/ml h 18.2 ± 3.0 pg/ml. In patients with PT without parasites and PT with enterobiasis IL-4 level was similar and significantly higher than in control group.
Significantly higher prevalence of A. lumbricoides in patients with PT can be considered as a factor provoking manifestation or exacerbation of tuberculosis process due to augmentation of IFN-y and IL-4 imbalance. Larvae migration stage through respiratory tree is of importance due to direct affection of lung tissue and probably activation and spreading of mycobacterial infection. Data ofJ. Potian et al. [8, 1863-1874] indicate to such possibility.
They showed in experiment with mice, infected with Nippostron-gylus brasiliensis (model equivalent ascariasis in human) and M. tuberculosis that Th2 response can enhance the intracellular persistence of M. tuberculosis, in part by mediating the alternative activation of macrophages via the IL-4Ra signaling pathway unlike classic one, mediating by IFN-y. May be ascariasis triggers other mechanisms. Diniz L. M. et al. (2010) [4, 142-150] found A. lumbricoides in 12 % of patients with tuberculoid lepra and in 2 % of individuals who were in contact with patients but were not sick. These numbers are completely in agreement with our data. Although tuberculosis and lepra characterized by different target organs the infection gate (respiratory tract) are similar at these infections. Diniz et al. (2010) received analogous dynamics of IFN-y and IL-4 in patients with lepra and concomitant ascariasis and without it [4, 142-150]. Thus we can conclude with good grounds that ascariasis influences susceptibility to Mycobacterium and course of mycobacterial infections. Our data are consistent with results of Abate et al. (2012), showed that one third of bacteriological positive PT patients in Ethiopia are infected by helminths and this level is significant higher than in population [1].
Ascariasis seems to influence development of invasive PA in patients with PT. Absence of significant shifts in the level of IFN-y and IL-4 in patients with PT and concomitant enterobiasis and similar prevalence ofE. vermicularis in patients with PT and population point to lack of enterobiasis effect on susceptibility to M. tuberculosis and course of PT. Influence of concomitant giardiasis and blastocys-tosis (patients only with high intensity of infection were considered) on the level of serum IFN-y was essentially weaker: in significant decrease in comparison with control value, differences with patients with PT without parasites and PA were negligible (P > 0.05). Some other regularities were elucidated in IL-4 detection: if its values in patients with PT + giardiasis and PT without parasites and PA were similar, blastocystosis resulted in a significant increase of IL-4 level (table 2). Thus influence of protozoan infections on cytokine profile of patients with PT is insignificant.
Conclusion
Ascariasis as a concomitant disease influences development of PA due to worsening immune imbalance typical for PT.
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