Background: The concomitant presence of intestinal parasite infections (IP) and tuberculosis (TB) has relevance. M. tuberculosis immune response is associated with type 1 T helper cell (Th1) while IP is associated with type Th2 cell. However, there are several contradictory reports on cytokine production under coinfection and this could be in association to the mycobacterial antigens used in the studies. Aim: To get insight into the effects of different M. tuberculosis-specific antigens (ESAT-6/CFP-10 and 38 kDa/CFP-10) in generating of appropriate cytokines on peripheral blood mononuclear cells of IPTB co infected patients. Method: ELISA assessed IFN-γ and other 16 cytokines production and plasm IgE. In 18 months, we documented demographic, economic, clinical characteristics and IP frequency in individuals from Brazil. Results: An overall 10/35 (28.5%) were IPTB co infected and 40/76 (52.6%; p = 0.024) asymptomatic intestinal parasite infected community controls (IPCC). Endo-limax nana (40%) and Entamoeba coli (22%), were the most nonpathogenic protozoan identified and Entamoeba histolytica, Giardia intestinalis, Ascaris lumbricoides and Strongyloides stercoralis were the pathogenic species (40%). IgE was higher in IPCC (p = 0.036). Cytokine profiles were significantly biased toward a Th2 type IL-5 (p = 0.001) and IL-13 (p = 0.033), pro-inflammatory GM-CSF (p = 0.019) and borderline lower IL-1β in IPTB, all associated with ESAT-6/CFP-10, while IL-7 was borderline lower, but 38 kDa/CFP-10 associated; as well as IL-8 higher (p < 0.049) vs CC/IPCC. The TB/IPTB IFN-γ levels were similar to both antigens stimuli (p ≥ 0.208). Conclusion: Therefore, coin-cident IPTB coinfection did not exert a significant inhibitory effect in IFN-γ production in response to either of the two antigens, but the partial discrepancy in Th1/Th2 response, is associated with the antigen priming cells.
Mycobacterium tuberculosis (Mtb) is a facultative intracellular pathogen whose host immune response plays a vital role in infection outcome. The immune control of this infection is dependent upon the cellular immune response, mediated predominantly by CD4+ and CD8+ cells, with T helper (Th) 1 cytokine production, as IFN-γ, the major component in stimulation/activation of macrophages in infection control and bacillus elimination [
Helminths and other parasites, as well as bacteria, fungi and some viruses, perpetrate neglected tropical diseases that affect low-income populations [
From June 2011 to December 2012, blood and one-stool samples were collected from control and TB patients. All attended at Clementino Fraga Filho University Hospital, Pedro Ernesto University Hospital, School Center Germano Sinval Farias/FIOCRUZ, and Family Clinic Victor Valla (CFVV) seated in Manguinhos Complex, an urban slum area with high TB incidence rate, all in Rio de Janeiro, Brazil. TB diagnostic followed the World Health Organization (WHO) criteria [
The National Ethical Committee for Human Research n. 548/10 approved the study and all participants signed a written informed consent before samples donation.
The mycobacterial recombinant antigens early secretory antigenic target 6 (ESAT-6) and 38 kDa, also known as phosphate-specific transporter-1 (PstS1), were separately combined with 10 kDa culture filtrate protein (CFP-10). All kindly donated by LIONEX GmbH (Braunschweig, Germany) and used at a final concentration of 5 µg/mL, as well as Concanavalin A mitogen (ConA; Sigma, St. Louis, MO) [
PBMCs were isolated and cultured as previously described [
The IFN-γ was determined with the commercial kit based on immunoenzyme assay (ELISA), DuoSet IFN-γ kit (R&D, USA). Cut off was previously established by a receiver operating characteristic (ROC) curve as 100 pg/ml [
Plasm IgE levels were measured by AccuBindTM ELISA Microwells Immunoglobulin E (IgE) test system (Monobind, Inc., Lake Forest, CA), according to manufacturer’s instructions.
Statistical analyzes were performed on Statistical Package for Social Sciences (SPSS) software, v. 17.0; differences of means with p ≤ 0.05 was considered significant. The nonparametric test was used to analyze significant differences when comparing the groups (Kruskal Wallis) and pairwise comparisons (Mann-Whitney).
As showed in
Of 111 individuals enrolled in the study, 50 (45%) tested positive for IP, concurrent IPTB infection being 28.5% (10/35) and 52.6% (40/76) asymptomatic IPCC. In both IP infected groups, Endolimax nana (20/50, 40%) and Entamoeba coli (11/50, 22%), followed by E. dispar (4/50, 8%) and Iodamoeba butschili (1/50, 2%), were the most protozoan nonpathogenic identified. Among IPTB co infected subjects, 40% had pathogenic species Ascaris lumbricoides, Giardia intestinalis and Strongyloides stercoralis. The IPCC individuals had pathogenic
Number (%) | ||||
---|---|---|---|---|
TB (n = 25) | IPTB (n = 10) | CC (n = 36) | IPCC (n = 40) | |
Age (mean ± SD) | 41.8 ± 12.55 | 40.3 ± 15.83 | 55.1 ± 12.05*** | 44.9 ± 13.82 |
Gender | ||||
Female (n = 56) | 8 (32) | 5 (50) | 23 (63.9) | 20 (50) |
Male (n = 55) | 17 (68) | 5 (50) | 13 (36.1) | 20 (50) |
Smoker: | ||||
Positive | 5 (20) | 5 (50)* | 8 (22.2) | 7 (17.5) |
Negative | 20 (80) | 5 (50) | 28 (77.8) | 33 (82.5) |
AFB smear | ||||
Negative | 14 (66.7) | 5 (62.5) | ||
Positive | 7 (14.3) | 3 (12.5) | ||
No Information | 4/25 | 2/10 | ||
TB Site of infection: | ||||
Pulmonary | 18 (72) | 8 (80) | ||
Extra pulmonary | 7 (28) | 2 (20) | ||
Family monthly Income (One basic salary = US$325.72) | ||||
2 (8) | 3 (30) | 1 (2.8) | 2 (5) | |
one | 8 (32) | 3 (30) | 14 (38.9) | 10 (25) |
two - four | 13 (52) | 3 (30) | 18 (50) | 23 (57.5) |
>four | 2 (8) | 1 (10) | 3 (8.3) | 5 (12.5) |
Education: | ||||
Illiterate | 1 (4) | 1 (10) | 3 (8.4) | 2 (5) |
Incomplete elementary School | 4 (16) | 3 (30) | 21 (58.3) | 15 (37.5) |
Elementary school | 6 (24) | 2 (20) | 4 (11.1) | 5 (12.5) |
Incomplete high school | 4 (16) | 1 (10) | 1 (2.8) | 4 (10) |
High school | 9 (36) | 2 (20) | 7 (19.4) | 10 (25) |
Technical or higher | 1 (4) | 1 (10) | 0 (0) | 4 (10) |
Body Mass Index - BMI (mean ± SD) (n = 95) | 22.4 ± 3.54*** | 22.0 ± 3.47* | 27.6 ± 5.97 | 28.0 ± 5.93 |
Dwelling House: | ||||
Coated masonry | 15 (60)*** | 7 (70)*** | 35 (97.2) | 34 (85) |
Uncoated masonry or wood | 10 (40) | 3 (30) | 1 (2.8) | 6 (15) |
Water for consumption: | ||||
Public treated water | 25 (100) | 8 (80) | 35 (97.2) | 36 (90) |
Mineral or artesian well | 0 (0) | 2 (20) | 1 (2.7) | 4 (10) |
Garbage Public Collection: | ||||
One(time per week) | 3 (12) | 1 (10) | 5 (13.9) | 2 (5) |
Two - three (time per week) | 19 (76) | 6 (60) | 21 (58.3) | 27 (67.5) |
Daily | 2 (8) | 1 (10) | 8 (22.2) | 9 (22.5) |
≥to Fortnightly | 0 (0) | 1 (10) | 1 (2.8) | 0 (0) |
Without information | 1 (4) | 1 (10) | 1 (2.8) | 2 (5) |
*p < 0.05; ***p < 0.001. SD: Standard Deviation. TB: tuberculosis patients; IPTB or IPCC: intestinal parasites TB co infected or asymptomatic intestinal parasitic infected community control.
Species | Number of infected participants (%) | ||
---|---|---|---|
IPTB | IPCC | TOTAL | |
Any parasite | 10/35 (28.5) | 40/76 (52.6) | 50/111 (45) |
Nonpathogenic species | 5/10 (50) | 30/76 (39.4) | 35/86 (70) |
Entamoeba coli | 2/10 (20) | 9/40 (22.5) | 11/50 (22) |
Endolimax nana | 3/10 (30) | 17/40 (42.5) | 20/50 (40) |
Entamoeba díspar | 0 | 4/40 (10) | 4/50 (8) |
Iodamoeba butschili | 1/10 (10) | 1/50 (2) | |
Pathogenic species | 4/10 (40) | 20/76 (26.3) | 24/50 (48) |
Ascaris lumbricoides | 2/10 (20) | 1/40 (2.5) | 3/50 (6) |
Giardia intestinalis | 1/10 (10) | 5/40 (12.5) | 6/50 (12) |
Strongyloides stercoralis | 1/10 (10) | 3/40 (7.5) | 4/50 (8) |
Entamoeba histolytica | 0 | 7/40 (17.5) | 7/50 (14) |
Taenia sp. | 0 | 2/40 (5) | 2/50 (4) |
Schistossoma mansoni | 0 | 1/40 (2.5) | 1/50 (2) |
Enterobius vermicularis | 0 | 1/40 (2.5) | 1/50 (2) |
Dual parasitism | 0 | 10/40 (25) | - |
IPTB or IPCC: intestinal parasites TB co infected patients and asymptomatic intestinal parasitic infected community controls.
species Entamoeba histolytica, Giardia intestinalis, Strongyloides stercoralis, Taenia sp. and three participants, respectively infected by Schistossoma mansoni, Ascaris lumbricoides and by Enterobius vermicularis. Dual parasitism accounted for 25% (
All 111 participants had serum IgE tested and was significantly higher in IPCC vs CC (80.18 ± 144.8 vs 36.33 ± 75.03, p = 0.036), and slightly increased in IPTB com-pared to TB (93 ± 132.2 vs 57.36 ± 78.17, p = 0.673). The IgE systemic higher level (>300 UI/ml) was associated with either nonpathogenic (E. coli and E. nana) or pathogenic (G. intestinalis, E. histolytica) protozoa infection.
Of 108 participants tested for IFN-γ production 36 were CC, 38 IPCC, 25 TB patients and 9 IPTB (
The Th type 2 cytokines IL-5 (p = 0.001), IL-13 (p ≤ 0.029) and also the pro-inflammatory chemokine GM-CSF (p ≤ 0.026) exhibited significantly higher mean levels in IPTB compared to that TB and controls, stimulated with ESAT-6/ CFP-10. However, no significant difference to both antigens in the average level of IL-4 (p > 0.097). Moreover, IL-5 production was significantly high in response to 38 kDa/CFP-10 compared to ESAT-6/CFP-10 only in TB group (p = 0.041) (
The IPTB gave rise to a strong reduction of IL-1β compared to all others groups (p ≤ 0.029), although borderline to TB (p = 0.068). Moreover, TB (p = 0.006) and IPCC (p = 0.029) showed low levels compared to CC/IPCC, all associated with ESAT-6/CFP-10. In addition, 38 kDa/CFP-10 elicited higher IL-1β expression in all groups compared to ESAT-6/CFP-10 but significant only to TB group (p = 0.029) (
There was no significant difference to both antigens average level of Th1 (IL-2, TNF-α), Th17 (IL-17A), pro-inflammatory IL-6 and regulatory IL-10 cytokines, as well as of MIP1β and MCP-1 chemokine, among tested groups. However, 38 kDa/CFP-10 showed higher immune regulatory cytokine (IL-10, p = 0.001) expression compared to ESAT-6/CFP-10 in TB and IPCC (
The mean level of IL-8 was significantly increased associated to 38 kDa/CFP-10 stimuli in IPTB compared to both controls groups (p < 0.049). However, in response to ESAT-6/CFP-10 antigen, which compared with 38kDa/CFP-10, there was a significant expression in both CC groups (p ≤ 0.035). On the other hand, G-CSF chemokine lower mean level related to ESAT-6/CFP-10 stimuli in IP infected groups, but significance only for IPCC compared to both IP uninfected
Mean ± SD | p values | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
TB (N = 13 and 25▲) | IPTB (n = 9 and 9▲) | CC (n = 5 and 36▲) | IPCC (n = 9 and 38▲) | IPTB × TB | IPTB × IPCC | IPTB × CC | TB × IPCC | TB × CC | IPCC × CC | ||
38kDa/CFP-10 | |||||||||||
IL-1beta | 778 ± 715.5* | 1211 ± 2572 | 1415 ± 1208 | 692.6 ± 494.4 | 0.191 | 0.254 | 0.189 | 0.973 | 0.298 | 0.297 | |
IL-2 | 34 ± 36.4 | 35 ± 43.9 | 31.7 ± 23.5 | 34.6 ± 32.5 | 0.703 | 0.716 | 0.673 | 0.810 | 0.792 | 0.944 | |
IL-4 | 46.1 ± 40.6 | 56.3 ± 73.7 | 28.8 ± 21.5 | 26.2 ± 33.4 | 0.963 | 0.175 | 0.508 | 0.882 | 0.388 | 0.429 | |
IL-5 | 40.5 ± 29.1* | 49.2 ± 38.7 | 30.6 ± 28.6 | 19.7 ± 22.8 | 0.610 | 0.124 | 0.350 | 0.084 | 0.477 | 0.646 | |
IL-6 | 1456 ± 1060 | 2024 ± 2008 | 1544 ± 1127 | 1670 ± 1062 | 0.331 | 0.812 | 0.677 | 0.366 | 0.944 | 0.566 | |
IL-7 | 297 ± 135.3*** | 201.3 ± 200 | 102.3 ± 154.3 | 90.3 ± 135.5 | 0.067 | 0.421 | 0.772 | 0.004 | 0.037 | 0.537 | |
IL-8 | 2098 ± 2221 | 3311 ± 2366 | 523.6 ± 416 | 1022 ± 1602 | 0.244 | 0.049 | 0.038 | 0.156 | 0.089 | 0.497 | |
IL-10 | 500.5 ± 311.3** | 381.2 ± 386.6 | 392.8 ± 143.4 | 372.6 ± 181** | 0.402 | 0.775 | 0.629 | 0.348 | 0.580 | 0.629 | |
IL-12p70 | 728.4 ± 678.9 | 402.4 ± 392.4 | 128.2 ± 286.5 | 174.8 ± 354.3 | 0.188 | 0.294 | 0.286 | 0.01 | 0.021 | 0.835 | |
IL-13 | 83.8 ± 43 | 142 ± 150.2 | 47.2 ± 12.3 | 62.6 ± 51.7 | 0.480 | 0.195 | 0.089 | 0.360 | 0.185 | 0.572 | |
IL-17A | 681.6 ± 507.7 | 670.3 ± 571.7 | 277.1 ± 282.6 | 344.3 ± 296.6 | 0.753 | 0.2 | 0.174 | 0.065 | 0.06 | 0.740 | |
IFN-gamma▲ | 440 ± 392.3 | 262.3 ± 270 | 15.7 ± 39.7 | 3.9 ± 11.3 | 0.208 | >0.001 | >0.001 | >0.001 | >0.001 | 0.992 | |
TNF-alpha | 221.6 ± 120.3 | 229 ± 127.7 | 155 ± 85 | 249 ± 362 | 0.986 | 0.281 | 0.251 | 0.396 | 0.376 | 0.766 | |
G-CSF | 367.3 ± 223.5* | 411.6 ± 760.2 | 357.6 ± 200.8 | 217.3 ± 119.9** | 0.129 | 0.651 | 0.311 | 0.069 | 0.995 | 0.436 | |
GM-CSF | 206.6 ± 713.6 | 265.7 ± 673.6 | 3.69 ± 0.93 | 141.2 ± 301.7 | 0.193 | 0.105 | 0.328 | 0.812 | 0.999 | 0.670 | |
MCP-1 (CCL2) | 467.7 ± 241.1 | 673 ± 579.7 | 333 ± 274.6 | 546 ± 266.4 | 0.707 | 0.842 | 0.410 | 0.481 | 0.360 | 0.181 | |
MIP-1beta (CCL4) | 1092 ± 1269* | 1348 ± 1876 | 479.4 ± 244.6 | 693.7 ± 401.2 | 0.299 | 0.729 | 0.677 | 0.555 | 0.112 | 0.410 | |
ESAT-6/CFP-10 | |||||||||||
IL-1beta | 282.2 ± 272.4 | 138.3 ± 154.5 | 930.8 ± 427.2 | 398.6 ± 323.4 | 0.068 | 0.023 | 0.004 | 0.402 | 0.006 | 0.029 | |
IL-2 | 27.7 ± 23.9 | 49.2 ± 72.4 | 25.3 ± 21.5 | 20.4 ± 21.8 | 0.831 | 0.456 | 0.875 | 0.561 | 0.972 | 0.572 | |
IL-4 | 28.9 ± 29 | 43.4 ± 31 | 25.7 ± 15.2 | 18.5 ± 18.4 | 0.248 | 0.097 | 0.361 | 0.495 | 0.779 | 0.408 | |
IL-5 | 19.5 ± 18.1 | 77 ± 71.5 | 33.9 ± 14.3 | 33.2 ± 25.4 | >0.001 | 0.085 | 0.125 | 0.197 | 0.127 | 0.812 | |
IL-6 | 1377 ± 821.1 | 1390 ± 1105 | 1341 ± 336.7 | 1343 ± 986.4 | 0.882 | 0.975 | 0.722 | 0.882 | 0.552 | 0.603 | |
IL-7 | 128.2 ± 176 | 199.4 ± 216.3 | 189.3 ± 164.4 | 146 ± 146 | 0.323 | 0.674 | 0.772 | 0.332 | 0.293 | 0.880 | |
IL-8 | 3445 ± 2313 | 4100 ± 2172 | 2697 ± 1914* | 3058 ± 2220* | 0.604 | 0.509 | 0.262 | 0.834 | 0.501 | 0.897 | |
IL-10 | 148.2 ± 108.4 | 184.8 ± 132.4 | 246.7 ± 162.8 | 122.4 ± 122.5 | 0.479 | 0.194 | 0.580 | 0.238 | 0.298 | 0.089 | |
IL-12p70 | 509.8 ± 595.3 | 407.2 ± 422.3 | 128.2 ± 286.4 | 148.1 ± 238 | 0.745 | 0.181 | 0.237 | 0.109 | 0.157 | 0.999 | |
IL-13 | 70.4 ± 101 | 170.6 ± 157.2 | 70.7 ± 28.3 | 51.4 ± 36.4 | 0.033 | 0.029 | 0.409 | 0.699 | 0.231 | 0.201 | |
IL-17A | 392.7 ± 364.3 | 644 ± 764.2 | 372.8 ± 193 | 237.5 ± 309.1 | 0.700 | 0.313 | 0.984 | 0.375 | 0.999 | 0.262 | |
IFN-gamma▲ | 448.3 ± 481.4 | 420.4 ± 634.8 | 14.6 ± 42.2 | 7.9 ± 24.3 | 0.474 | >0.001 | >0.001 | >0.001 | >0.001 | 0.277 | |
TNF-alpha | 137.6 ± 80 | 191 ± 129 | 120.5 ± 49 | 119.3 ± 55.3 | 0.253 | 0.160 | 0.286 | 0.937 | 0.882 | 0.999 | |
G-CSF | 218.2 ± 185.2 | 173.9 ± 177.6 | 348.2 ± 198 | 88.3 ± 54.5 | 0.522 | 0.423 | 0.119 | 0.045 | 0.074 | 0.006 | |
GM-CSF | 173.2 ± 467.6 | 218.5 ± 572 | 3.69 ± 0.93 | 11.8 ± 25.5 | 0.019 | 0.026 | 0.055 | 0.999 | 0.999 | 0.999 | |
MCP-1 (CCL2) | 497.5 ± 293.2 | 821.8 ± 812.8 | 230.2 ± 201.3 | 392.2 ± 179.6 | 0.390 | 0.160 | 0.141 | 0.348 | 0.071 | 0.080 | |
MIP-1beta (CCL4) | 505.3 ± 346.1 | 459.2 ± 361.8 | 503.4 ± 444.8 | 442.3 ± 384.4 | 0.706 | 0.777 | 0.824 | 0.402 | 0.832 | 0.972 |
Cytokine mean results in units of pg/ml together with standard deviation (SD). N▲ = Number of cases tested INF-gamma release assay. Significant difference of cytokine/chemokines production between groups and between antigen tested was calculated by Mann Whitney (***, p ≤ 0.0001; **, p ≤ 0.01; *, p ≤ 0.05). TB: tuberculosis patients; IPTB or IPCC: TB-intestinal parasites co infected or asymptomatic IP infected community control.
groups (p ≤ 0.045). Higher expression of this chemokine was significant in TB and IPCC in response to 38 kDa/CFP-10 compared to ESAT-6/CFP-10 stimuli (
The regulatory cytokines IL-7 and IL12p70 were significantly high in TB subjects compared to both controls groups (p ≤ 0.037), but IPTB elicited slightly IL-7 lower level (p = 0.067), all related to 38 kDa/CFP-10. Moreover, 38 kDa/CFP-10 elicited a significantly higher level of IL-7 than to ESAT-6/CFP-10 in TB group (p = 0.008) (
In the present study, 45% of the studied individuals tested positive for IP, and among TB patients 28.5% were coinfected, a similar frequency to that in other studies [
The impact of IP infections in the suppression of the Th1 immune response, essential for protection against TB, deregulating the immune system toward the Th2 branch remains contradictory. In order to test the hypothesis that part of this variable response may be associated to specific antigens of M. tuberculosis, we investigated the effects of 38kDa/CFP-10 and ESAT-6/CFP-10 on Th1/Th2 patterns in intestinal parasite infected TB patients. Several studies have demonstrated a significant decrease in the production of IFN-γ in IPTB [
The mean levels of all cytokines were not significantly different in IPTB vs TB, mainly for 38 kDa/CFP-10. However, we noted significant levels of the cytokines IL-1β, IL-5, IL-7, IL-10 and G-CSF for this antigen comparing to ESAT-6/ CFP-10 in TB patients. Thus possibly suggesting differential modulation in TB host.
The Th2 immunity arm activates several downstream effector mechanisms that promote parasite eradication or at least minimize host tissue damage. The Th2 cytokines, such as IL-4, IL-5, IL-6, and IL-13, leading to the production of B cell, immunoglobulin IgE and IgG subtypes, eosinophilia and mast cells inhibiting parasite invasion or dissemination [
Previous studies have shown the TB gravidity associated with disease progression and with a low immune response in a broad range of T cell to pathogens stimulation. Beside TNF-α, it was detected lower IL-1β and IL-7 levels in patients with poor TB clinical evolution after dead M. tuberculosis cell stimulation [
The IL-8, or CXC chemokine, an important neutrophil-activating factor, provides the chemotaxis of inflammatory cells to the site of infection [
Neutrophils are the most numerous types of lymphocytes, and the factors involved in their differentiation are G-CSF, IL-6, GM-CSF, and IL-13, which are critical in the activation of the antibacterial neutrophils response [
This study has, as major limitations, the reduced number of IPTB, the absence of prevalent pathogenic IP species and no surveillance of the evolution of the immune response after IP-specific therapy. However, our study suggests that in addition to parasite infections, mycobacterial antigens immune responses inducer may be an extra reason for these controversies.
This study underlines that: i) Coincident TB-intestinal parasites did not exert a significant inhibitory effect on IgE levels, as well as in IFN-γ production in response to either of two antigens, ii) however, cytokine profiles were partially significantly biased toward a Th2 type (IL-5 and IL-13) and a pro-inflammatory GM-CSF, and borderline lower to IL-1β, but associated with ESAT-6/CFP-10; while IL-7 was borderline lower, and IL-8 was significantly high related to both CC groups, with 38 kDa/CFP-10. Last, further studies before and after anthelminthic treatment are needed.
We are grateful to Mitchell Raymond Lisbon for editing the manuscript and LIONEX GmbH (Braunschweig, Germany) for antigens donated. This research was supported by the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Oswaldo Cruz Institute (Fiocruz). The authors would like to thank to all those who participated in the study.
The authors declare that there is no conflict of interests. The authors alone are responsible for the writing and content of the paper.
Silva, R.J., Mello, F.C.Q., Leung, J.A., Moraes Neto, A.H.A., Fonseca, L.S., Siqueira, H.R., Health Care Victor Vala and CSEGSF Team and Saad, M.H.F. (2018) Insights on Blood Cytokines Production under Different In Vitro Mycobacterial Antigens in Tuberculosis Intestinal Parasites Co Infected Patients. Advances in Microbiology, 8, 161-174. https://doi.org/10.4236/aim.2018.83011