Vasyl D. Moskaliuk, Tetiana R. Kolotylo, Khrystyna I. Pudiak, Ivanna V. Rudan, Oksana I. Goliar, Iryna V. Balanyk

HSEI Bukovininan state medical university, Chernivtsi, Ukraine

ABSTRACT

Introduction: Epidemics of such dangerous diseases as HIV infection and tuberculosis continue to develop annually. Tuberculosis has become the main cause of mortality in AIDS patients. Both diseases have a negative effect on the state of the immune system, affecting the cells of the lymphatic system.

The aim of the work is to carry out a comparative comprehensive immunological examination of HIV-infected and immunocompetent patients with active tuberculosis.

Materials and methods: It was carried out a comprehensive immunological examination of 231 patients, in particular 155 HIV-infected patients with active TB and 76 cases with TB only. The HIV / TB group was divided into 3 subgroups depending on the time of TB attachment to HIV infection.

It was compared with CD4 + T-lymphocytes, CD8 + T-lymphocytes, CD4 + / CD8 +, levels of interleukin-4 (IF-4), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and results delayed-type hypersensitivity reactions (TGS) with tuberculin for groups with a combined HIV / TB infection and TB-mono-infected patients.

Results: The difference between CD4 + T-lymphocytes, CD4 + / CD8 + with associated HIV / TB infection, and also in the 1st and 3rd HIV / TB subgroups, as compared to those with TB monoinfection, was shown to be significantly more pronounced CD4 + T lymphocytes and a higher index of CD4 + / CD8 + in patients with TB monoinfection.

Conclusions: CD4 + T-lymphocyte ratios, CD4 + / CD8 + ratios are significantly lower with associated HIV / TB infection in comparison with patients with TB monoinfection.

The level of CD4 + T-lymphocytes is significantly lower in infiltrative, fibrous-cavernous and extra pulmonary (peripheral and central lymph nodes) TB, which is combined with HIV infection compared to TB monoinfection.

KEY WORDS: HIV infection, tuberculosis, CD4 + T-lymphocytes, CD8 + T-lymphocytes

Wiad Lek 2019, 72, 10, 1942-1946

Introduction

As you know, the relationship between tuberculosis and HIV infection at the cellular level is very complicated and insufficiently studied. Reducing the number of CD4 + T-lymphocytes in HIV infection significantly increases susceptibility to tuberculosis infection or reactivation of primary latent tuberculosis infection. The severity of clinical manifestations of tuberculosis, including the development of severe extra pulmonary and disseminated forms, correlates with a decrease in the level of CD4 + T-lymphocytes in peripheral blood [1].

A serious problem is the annual increase of the number of patients with tuberculosis in the stage of clinical exposure to HIV (72.5%) and a high proportion of overdue and destructive forms of tuberculosis (31.3%) in newly diagnosed patients with HIV / TB infection.

The aim

The aim of the work is to carry out a comparative comprehensive immunological examination of HIV-infected and immunocompetent patients with active tuberculosis.

Materials and methods

There were 231 people – 184 (79.7%) men and 47 (20.3%) women at the age of 23 to 60 years under observation. The average age was (38.8 ± 1.2) years.

All patients were subjected to a comprehensive laboratory examination and divided into such groups.

I. Non-HIV-infected (immunocompetent) people with active first-time TB – 76 patients (TB group).

II. HIV-infected with active for the first time detected tuberculosis – 155 patients (HIV / TB group). Within the HIV / TB group, 3 subgroups were identified depending on the time of TB attachment to HIV infection:

1st subgroup – HIV-infected, in which TB was detected at different stages of HIV infection (primary illness) – 72 patients;

2nd subgroup – persons in whom for several years before the registered HIV infection and the time of hypothetical infection (according to the epidemiological history) TB was diagnosed, that is, the primary disease was TB – 26 people;

The third subgroup – HIV-infected, in which the disease was recorded at about one time and it was impossible to find out which ailment was the primary one – 57 people.

We compared CD4 + T-lymphocyte, CD8 + T-lymphocytes, CD4 + / CD8 +, IL-4, IFN-γ, TNF-α and the results of the reaction of TBGT with tuberculin for groups with combined HIV / TB and TB patients, monoinfection. The immune status was studied in a group of HIV / TB patients within 3-6 months since the diagnosis of associated infection.

Statistical processing of the obtained results of research was carried out with the help of the software complex Windows, Word and Excel; STATISTICA 6.0 using the method of variation statistics with validity determination using Student’s criterion, and with the number of observations less than 20, the non-parametric Wilcoxon method for independent aggregates was used, or the dispersion analysis of Kraskele-Wallis. U-Mann-Whitney Criteria were used to compare two independent groups. The differences were considered statistically significant at P <0.05. Spearmen rank method was used for correlation analysis [2].

Results and discussion

The average number of CD4 + T lymphocytes was significantly lower in the HIV / TB group (389.1 ± 24.5) kl / mm3 than in patients with TB alone (542.2 ± 27.4) kl / mm3 (P <0.001). But the level of CD8 + T-lymphocytes in the groups with combined and monoinfection did not differ – (464.8 ± 25.6) kl/mm3 and (426.9 ± 24.9) kl/mm3, respectively.

Comparing the indicators of CD4 + T-lymphocytes in patients with primary TB with a combined infection in three subgroups, depending on the time of TB attachment, it can be noted that the patterns typical for the whole HIV / TB group are traced to all three subgroups as compared to monoinfection (P <0 , 05-0.01, Table 1).

The spontaneous production of IL-4 was higher – (7.1 ± 1.0 pg / ml / 106) in the HIV / TB group compared to TB monoinfection – (3.2 ± 0.6) pg / ml / 106 (P <0.05) with the caseogenesis pneumonia, while the spontaneous production of IFN-γ also exceeded the corresponding indicator in patients with TB alone – (90.7 ± 32.6) pg / ml / 106 vs. (7.3 ± 1.1) kp / ml / 106 (P < 0.001). HHV was kept at a moderate level – (92450 ± 22890) kp / ml.

When comparing the indicators of CD8 + T-lymphocytes in patients with primary TB with a combined infection in three subgroups, depending on the time of TB attachment, it can be noted that the smallest number of cells appeared in the 1st subgroup – (378.5 ± 18.2) kl / mm3, and the largest in the 2nd – (526,7 ± 32,6) kl / mm3 subgroup of HIV-infected people. However, in general, the average number of CD8 + T-lymphocytes in patients with mono-TB and associated infections did not differ.

The CD4 + / CD8 + index in patients with associated infections, as well as in each of the three subgroups, was statistically significantly lower than with TB monoinfection.

The indicators of CD4 + T-lymphocytes for different forms of TB were analyzed. The differences in CD4 + T-lymphocyte levels, CD8 + T-lymphocytes were not detected at focal TB in patients with HIV-infection, in its subgroups, and in patients with TB monoinfection. In infiltrative TB, CD4 + T-lymphocyte indexes are significantly lower in patients with HIV / TB – (441.6 ± 36.7) kl / mm3 compared to TB monoinfection – (619.8 ± 67.6, P <0, 01). The same pattern can be traced to all subgroups of HIV + TB infected (P <0.05-0.01, Table 2).

The significant differences were observed with respect to CD8 + T-lymphocytes in caseogenesis. Thus, this indicator was significantly lower in the patients with TB monoinfection than in the HIV-infected group – (279.6 ± 40.3) kl / mm3 vs. (404.9 ± 43.6) kl / mm3 (P <0.01). The same pattern was inherent in the 3rd subgroup of HIV-infected patients as compared to patients with TB monoinfection.

A significantly lower number of CD4 + T lymphocytes was detected in a group of people with fibro-cavernous tuberculosis (FCT) with a combination of HIV infection and TB – (320, 9 ± 43, 9) kl / mm3 compared with a group of patients with TB alone – (473.2 ± 56.0) kl / mm3 (P <0.05). But the level of CD8 + T-lymphocytes was significantly higher with concomitant HIV infection – (615.3 ± 52.6) kl / mm3 compared with patients only with TB – (236.4 ± 70.8, P <0.001). The same regularities were true for the 3rd subgroup of HIV-infected people.

The average number of CD8 + T-lymphocytes was significantly higher in patients with associated TB-disseminated infection compared to the TB monoinfection group (323.6 ± 24.8) kl / mm3 against (222.7 ± 26.8) kl/mm3 (P <0.01).

The big attention is drawn to the fact that in the structure of extra pulmonary TB in HIV / TB patients there were no such forms as TB of the urogenital system, bone and ligament. The level of CD4 + T-lymphocytes was significantly below the above indicator only with TB – (287.8 ± 28.3) kl / mm3 against (539.6 ± 77.4) kl/mm3 (P <0.001) at TB of peripheral and central lymph nodes against the background of HIV infection.

The frequent development of complicated forms of the tuberculosis process, in particular dissemination and generalization in HIV-infected patients at later stages, is fixed by many authors [3-6]. Moreover, the conditions in the HIV-infected organism (progressive decrease of CD4 + T-lymphocytes level and their functional failure) are created, when even a moderate immune deficiency, even the infiltrative tuberculosis process often leads to unfavorable results.

The results of the study of the level of cytokines – serum concentration and spontaneous production of IL-4, IFN-γ, TNF-α in HIV / TB and TB patients only were analyzed. Simultaneously with the study of the level of cytokines, the burden of HIV in patients with different forms of TB was also determined.

The differences were observed in various forms of TB. Thus, with focal TB, serum IL-4 concentration was higher with combined HIV / TB infection than with TB monoinfection – (2.8 ± 1.2) kp / ml against (1.5 ± 0.2) kp / ml (P <0.05), and the serum IFN-γ concentration was lower (24.2 ± 8.9) kp / ml versus (50.6 ± 9.4) kp / ml (P <0.05). Viral load (VH) of HIV was the lowest – (33115 ± 9896 kp / ml).

The serum IL-4 concentration significantly exceeded the indicator for TB monoinfection – (3.8 ± 0.7) kp / ml versus (1.5 ± 0.3) kp / ml (P <0, 05) in infiltrative TB in a group with a combined infection. At the same time, the viral load was quite high – (201834 ± 53984) kp / ml (high VN is considered at a level> 100 thousand kp/ml).

The serum concentration and spontaneous production of TNF-α in TB and HIV / TB groups were investigated. The significant differences were found between the levels of tumor necrosis factor-α in HIV-infected patients and patients with TB alone. Thus, at serous, infiltrative, fibro-cavernous and generalized TB in association with HIV infection, the serum concentration of TNF-α was statistically significantly higher than that for TB monoinfection (P <0.05-0.001). In the case of caseous pneumonia, the spontaneous production of TNF-α in the HIV / TB group was (181.0 ± 62.2) pc / ml / 106, significantly exceeding the study level in patients with TB alone – (11.3 ± 1.5) pc / ml / 106 (P <0.001, Table 3).

In general, the serum concentration and spontaneous production of TNF-α was higher in the group of patients with a combined infection (29.5 ± 6.4) pc / ml and (82.6 ± 32.8) pc / ml / 106 compared with TB – monoinfection – (6,5 ± 1,4) pc / ml and (16,1 ± 4,7) pc / ml / 106 (in both cases P <0,001).

Thus, with the progression of HIV infection (a decrease in the number of CD4 + T-lymphocytes and an increase in viral load of HIV), an increase in serum IFN-γ and TNF-α is observed, which is likely to indicate a decrease in the number of anti-inflammatory T-regulatory cells, or about reducing their suppressor activity [7].

Conclusions

1. CD4 + T-lymphocyte ratios, CD4 + / CD8 + ratios are significantly lower with associated HIV / TB infection in comparison with patients with TB monoinfection.

2. The level of CD4 + T-lymphocytes is significantly lower than that of TB monoinfection, in infiltrative, fibrous-cavernous and extra pulmonary (peripheral and central lymph nodes) TB, which is combined with HIV infection.

3. An increase in serum IFN-γ and TNF-α is observed with the progression of HIV infection (a reduction in the number of CD4 + T-lymphocytes and an increase in the burden of HIV), which is likely to indicate a decrease in the number of anti-inflammatory T-regulatory cells or a decrease their suppressor activity.

4. The signs of the progression of combined HIV / TB infection should be considered as a rapid decrease in the number of CD4 + T-lymphocytes, the ratio of CD4 + / CD8 +, increased HIV load, increased serum levels of TNF-α and IFN-γ.

5. It was found that CD4 + T lymphocyte levels were significantly lower in those who died soon in the case of primary or simultaneous diagnosis of HIV infection, compared to patients who had tuberculosis with primary background illness.

References

1. Sarrazin H, Wilkinson KA, Andersson J et al. Association between tuberculin skin test reactivity, CD4 cell subset memory and circulating FoxP3-expressing cells in HIV-infected individuals: J. Infect. Dis. 2009;199 (5):702-710.

2. Lapach SN, Chubenko AV, Babich PN. Statistical methods in medical-biological research using Excell: Kyiv: MORION, 2000, p.320.

3. Alexandrina TA. The features of the TB epidemic in Ukraine: Tuberculosis, pulmonary diseases, HIV-infection. 2012;2:7-12.

4. Redzwan M, Rashid Ali S, Ralph AP et al. Individualized second line anti-tuberculosis therapy for an extensively resistant pulmonary tuberculosis (XDR PTB) in East Malaysia: Med. J. Malaysia. 2015;70 (3):200-204.

5. Sulis G, Centis R, Sotgiu G et al. Recent developments in the diagnosis and management of tuberculosis: NPJ Prim. Care Respir. Med. 2016;26:1-8.

6. Petrenko VI, Protsiuk RG. The problem of tuberculosis in: Tuberculosis, pulmonary diseases, HIV-infection. 2015;2 (21):16-29.

7. Chase AJ, Yang HC, Zhang H et al. Preservation of FoxP3 + regulatory T cells in the peripheral blood of human immunodeficiency virus type 1-infected elite suppressors con-elating with low CD4 + T-cell activation: J Virol. 2015;82:8307-8315.

The work is performed within the limits of the HSEI Bukovininan state medical university scientific work “Molecular-genetic and clinical-pathogenetic features of the combined the pathology of internal organs, the role of infectious and metabolic factors in its development”.

Authors’ contributions:

According to the order of the Authorship.

ORCID numbers:

Vasyl. D. Moskaliuk – 0000-0002-4104-8153

Tetiana R. Kolotylo – 0000-0002-0821-7904

Khrystyna I. Pudiak – 0000-0002-0157-1433

Ivanna V. Rudan – 0000-0002-4985-5363

Oksana I. Goliar – 0000-0001-9165-4840

Iryna V. Balanyk – 0000-0002-3258-9791

Conflict of interest:

The Authors declare no conflict of interest.

CORRESPONDING AUTHOR

Tetiana Kolotylo

HSEI Bukovininan state medical university, Chernivtsi, Ukraine

tel: +380664669273

e-mail: taniakolotylo15@gmail.com

Received: 19.03.2019

Accepted: 20.09.2019

Table 1. Indicators CD4+, CD8+, index CD4+/CD8+ in the groups of patients under investigation

Indicator

HIVB

TB monoinfection,

n=76

1st subgroup, n=72

2nd subgroup, n=26

3rd subgroup, n=57

all, n=155

1

2

3

4

5

abs. number

M

m%

abs. number

M

m%

abs. number

M

m%

abs. Number

M

m%

abs. number

M

m%

CD4+

339,6±

23,32, 5

19,

0,7

434,2±

25,81, 5

33,

0,9

393,6±

24,35

20,

0,8

389,1±

24,55

24,6±

0,8

542,2±

27,41-4

38,

1,0

СD8+

378,5±

18,22, 3

22,6±

0,9

526,7±

32,61, 5

28,

1,0

489,3±

26,11

25,

0,9

464,8±

25,6

25,

0,9

426,9±

24,92

24,7±

0,7

CD4+/

CD8+

0,90±0,065

0,82±0,085

0,80±0,075

0,84±0,075

1,27±0,091-4

Note (here and below). The digit in power denotes a subgroup with which the difference is significant (P <0,05-0,001).

Table 2. The average quantity CD4+ і CD8+ in the study groups of patients (kl/mm3)

Form of TB

HIV/TB

TBmonoinfection,

n=76

1st subgroup,

n=72

2nd subgroup,

n=26

3rd subgroup,

n=57

all,

n=155

1

2

3

4

5

CD4+

CD8+

CD4+

CD8+

CD4+

CD8+

CD4+

CD8+

CD4+

CD8+

Fulminant

447,6±

54,9

471,3±

34,8

556,4±

77,6

659,9±

89,2

439,8±

69,1

478,5±

67,3

481,3±

67,2

536,6±

63,8

648,3±

65,4

489,6±

67,6

N

13

4

7

24

8

Infiltrative

419,5±

52,05

512,5±

25,5

444,0±

29,55

567,0±

43,0

461,4±

28,75

573,3±

21,6

441,6±

36,75

550,9±

30,0

619,8±

67,61-4

492,9±

79,5

N

20

10

19

49

14

Caseous pneumonia

433,5±

36,5

340,0±

49,5

370,3±

32,7

344,8±

36,4

492,9±

28,3

529,8±

44,75

432,2±

32,5

404,9±

43,65

411,3±

58,5

279,6±

40,33, 4

N

6

3

9

18

6

FCT

155

340

560,0±

92,03

584,5±

70,53

247,8±

39,62, 5

921,3±

87,32, 5

320,9±

43,95

615,3±

52,65

473,2±

56,03, 4

426,4±

70,83, 4

N

1

2

3

6

12

Disseminated

208,3±

36,5

269,0±

38,32

311,5±

39,0

410,0±

26,01, 5

243,6±

43,9

291,7±

48,3

254,5±

39,8

323,6±

24,85

337,4±

41,2

222,7±

26,84

N

15

4

9

28

14

Generalized

145,6±

32,6

252,3±

25,3

423

403

120,0±

19,0

245,0±

20,0

229,5±

17,2

300,1±

15,1

337,0±

80,0

208,5±

66,5

N

6

1

4

11

4

Extra-pulmonary

252,8±

33,1

339,2±

35,3

218,7±

0,0

252,0±

0,0

207,9±

42,0

356,7±

36,9

226,5±

25,0

316,0±

24,1

360,3±

77,2

248,2±

31,5

N

11

2

6

19

18

Pleurisy

378,5±

22,5

511,0±

35,03

254

263

252,0±

84,5

279,5±

76,01

294,8±

32,1

351,2±

37,0

541,2±

154,2

403,6±

57,8

N

4

1

2

7

8

Meningitis

156,3±

33,3

261,6±

34,5

134,0

255,0

96,8±

11,1

172,2±

96,5

N

3

0

1

4

0

Peripheral and central lymph nodes

223,6±

43,4

245,0±

36,43

402,0

493,0

237,8±

41,6

535,7±

34,61

287,8±

28,35

424,6±

23,7

539,6±

77,44

341,1±

36,8

N

4

1

3

8

10

Table 3. The level of TNF-α in the patients with HIV / TB and TB

The form of TB

HIV/TB

TB-momoinfection

TNFα

TNFα

serum,

pc/ml

spontaneous,

pc/ml/106

serum,

pc/ml

spontaneous,

pc/ml/106

1-1

1-2

2-1

2-2

Fulminant

22,6±4,32-1

209,2±76,3

5,3±1,01-1

60,9±13,2

N

24

8

Infiltrative

40,5±7,72-1

43,5±20,2

6,5±1,31-1

7,1±3,4

N

49

14

Caseous pneumonia

20,2±4,4

181,0±62,22-2

9,9±2,7

11,3±1,51-2

N

18

6

FCT

46,4±8,12-1

47,9±22,7

4,3±0,81-1

4,5±2,2

N

6

12

Disseminated

20,8±6,9

25,1±10,7

6,6±1,4

7,8±1,7

N

28

14

Generalized

32,7±6,22-1

40,6±19,2

4,0,81-1

11,3±7,0

N

11

4

Extra-pulmonary

23,3±7,3

31,1±18,5

9,1±1,9

9,8±3,6

N

19

18

Together

29,5±6,42-1

82,6±32,82-2

6,5±١,٤1-1

16,1±٤,٧1-2

N

155

76