Tuberculosis (TB) is a deadly human disease caused by
Patients with MDR-TB require second-line drugs, which are much more expensive, cause more adverse events, and have lower cure rates [9-12]. Therapeutic responses among MDR-TB patients are not all the same. Independent of socioeconomic factors, malnutrition, and coinfection with other pathogens, genetic factors in MDR-TB patients exert significant influence [13, 14]. One of the individual genetic factors involved in the control of TB infection is a polymorphism in the gene coding for the major histocompatibility complex (MHC), also known as human leukocyte antigen (HLA) [15].
HLA-G is a nonclassical HLA class I antigen that exerts immunomodulatory activity by inhibiting the immune response and inflammatory processes, such as CD8+ T cell and natural killer (NK) cell activities, CD4+ T cell proliferation, antigen-presenting cell (APC) functions, B cell differentiation, and regulatory T cell induction [16-21]. In certain pathological conditions, HLA-G is expressed on APCs (antigen presenting cells including macrophages and dendritic cells [DCs]), thereby suppressing the host immune response necessary to control infection and malignancy [18,22-25]. Given that macrophages are the main targets of
We enrolled 57 Indonesian Javanese patients with MDR-TB patients in this cohort study, TB-infected patients showing resistance to at least isoniazid and rifampisin, at Dr. Moewardi General Hospital, Central Java province, Indonesia, from May 2012 through January 2013. The patients came from various districts in Java island, mostly from Central Java province. There was no consecutive case observed, and none of the subjects were from the same family. Each patient was administered appropriate drugs (taking into account the patient’s drug resistance status) and was monitored from the time of MDR-TB diagnosis until sputum conversion (when a bacterial microscopic test has a negative result) or until January 2014. As control groups, 34 healthy individuals without TB and 69 individuals with a history of TB infection, although not MDR-TB, who were cured during the study period were enrolled to assess a possible association between HLA-G polymorphism status and susceptibility to either TB or MDR-TB. Like the group of MDR-TB patients, the control groups consisted of Javanese people, predominantly from Central Java province.
Blood samples were obtained from all patients to examine their CD4+ T cell counts and percentages, hematologic profiles (hemoglobin, hematocrit, erythrocytes, leukocytes, and thrombocytes), sodium (Na+), potassium (K+), interferon gamma (IFNg), and interleukin-10 (IL-10) levels. Patient sputum samples were evaluated initially at MDR-TB diagnosis and during routine follow-up.
Approval was obtained from the institutional ethical committee review boards of the Faculty of Medicine of Sebelas Maret University and Dr. Moewardi General Hospital, Central Java province, Indonesia (No: E.C. 129/VII/2009). Written informed consent was obtained from all study participants. All procedures were conducted according to the principles of the Declaration of Helsinki.
Genomic DNA from each patient was isolated from peripheral blood using a High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany). For HLA-G 14-bp in/del polymorphism analysis, genomic samples were genotyped by polymerase chain reaction (PCR) using a FastStart HiFi PCR System dNTPack (Roche) with primer pairs that have been previously described [27]. The PCR products were electrophoresed in 3% agarose gels for 30 minutes at 100V and stained with ethidium bromide (EtBr). For the 14-bp insertion (+14bp), a PCR product of 214bp was obtained, while the deletion (–14bp) resulted in a PCR product of 200 bp. All samples were tested at least twice.
The chi-square (χ2) test was used to assess whether the HLA-G 14-bp in/del genotype distributions in the study population were in Hardy-Weinberg equilibrium. The χ2 test, Fisher’s exact test, and the Kolmogorov-Smirnov test were used to analyze categorical variables. Logistic regression was performed to reveal associations between polymorphism status and TB/MDR-TB outcome and susceptibility. All data analyses used a 95% confidence interval (CI), and a two-tailed
The group of MDR-TB patients consisted of 26 (46%) men and 31 (54%) women aged 19-62 years (mean: 41.1 + 10.708). All patients suffered from intrapulmonary TB that was resistant to rifampicin and isoniazid. Other patients were also resistant to ethambutol (46%; 26/57) and streptomycin (54%; 31/57). In one patient, the resistance status to ethambutol and streptomycin was unknown. All patient bacterial cultures indicated the presence of
Evaluation of patient blood profiles was successfully conducted for 93% (53/57) of MDR-TB patients (
Distribution of the HLA-G 14-bp in/del polymorphism in the context of hematologic profiles of MDR-TB patients.
Genotypes (n,%) | ||||
---|---|---|---|---|
D/D(n = 19) | I/D(n = 30) | W(n = 8) | ||
Hemoglobin (g/dL) some data were missing.γ IFN γ = interferon-gamma; IL-10 = interleukin-10 | - | |||
<13.3 for men | 9(47) | 16(53) | 4(50) | |
13.3-16.75 or 11.8-14.8 for women | 9(47) | 11(37) | 3(38) | |
>16.7 for men for women | 0(0) | 1(3) | 0(0) | |
Erythrocytes (×l06/μL) some data were missing.γ IFN γ = interferon-gamma; IL-10 = interleukin-10 | 0.999 | |||
<4.37 for men for women | 5(26) | 5(17) | 1(13) | 0.56 |
4.37-5.66 for men for women | 11(58) | 20(67) | 3(38) | |
>5.66 for men for women | 2(11) | 3(10) | 3(38) | |
Leukocytes (×l06μL) some data were missing.γ IFN γ = interferon-gamma; IL-10 = interleukin-10 | 0.45a | |||
3.9-11.1 for men for women | 12(63) | 22(73) for women | 4(50) | |
>11.1 for men for women | 6(32) | 6(20) | 3(38) | |
Thrombocytes (×105μL) some data were missing.γ IFN γ = interferon-gamma; IL-10 = interleukin-10 | 0.64 | |||
1.5-4.0 | 12(63) | 15(50) | 6(75) | 0.23 |
>4.0 | 6(32) | 13 (43) | 1(13) | |
Hematocrit (%) some data were missing.γ IFN γ = interferon-gamma; IL-10 = interleukin-10 | 0.999 | |||
<33 | 4(21) | 5(17) | 2(25) | 0.999 |
33-45 | 13 (68) | 19(63) | 5(63) | |
>45 | 1(5) | 4(13) | 0(0) | |
Na+ (mEq/L) some data were missing.γ IFN γ = interferon-gamma; IL-10 = interleukin-10 | 0.37 | |||
<135 | 4(21) | 11(37) | 1(13) | 0.66 for men for women |
135-145 | 14(74) | 17(57) | 6(75) | |
K+(mEq/L) some data were missing.γ IFN γ = interferon-gamma; IL-10 = interleukin-10 | 0.88 | |||
<3.7 | 5(26) | 7(23) | 1(13) | 0.92 |
3.7-5.2 | 6(32) | 12(40) | 2(25) | |
>5.2 | 7(37) | 9(30) | 4(50) | |
CD4+ T cell count (cells/μL) | - | |||
<200 | 1(5) | 0(0) | 0(0) | |
200-500 | 3(16) | 7(23) | 4(50) | |
>500 | 15(79) | 23 (77) | 4(50) | |
%CD4+Tcells | 0.96 | |||
14-28 | 6(32) | 10(33) | 3(38) | |
>29 | 13 (68) | 20(67) | 5(63) | |
IFNa(pg/mL) | 0.34 | |||
<10 | 13 for women | 19(63) | 2(25) | 0.05 for men for women |
>10 | 6(32) | 11(37) | 6(75) | |
IL-10(pg/mL) | ||||
<10 | 18(95) | 30(100) | 8(100) | - |
>10 | 1(5) | 0(0) | 0(0) |
Data presented as n (%)
Data for the thorax radiographical examination were obtained for 95% (54/57) of MDR-TB patients. Lung cavity infection was observed in 29/54 (54%)
patients, infiltration in 48/54 (89%), nodules in 1/54 (2%), fibrosis in 15/54 (28%), pleural effusion in 3/54 (6%), atelectasis in 5/54 (9%), and consolidation in 1/54 (2%). None of the patients suffered fibrothorax. During follow-up of MDR-TB patients, sputum conversion was observed mostly after two months of treatment (19/57, 33%). Other patients showed sputum conversion after three months (13/57, 23%), one month (11/57, 19%), four months (7/57, 12%), and five months (1/57, 2%) of treatment. Six (11%) patients died before sputum conversion.
In the total study population (n = 160; 57 MDR-TB patients, 69 controls with a history of TB infection, and 34 healthy controls), the D/D genotype was identified in 71 (44%) patients, I/D genotype in 68 (43%), and I/I genotype in 21 (13%) patients. The frequencies of the deletion (D) and insertion (I) alleles were 0.66 and 0.34, respectively. These frequencies complied with Hardy-Weinberg equilibrium (
The number of MDR-TB patients with the D/D genotype was 19 (33%), I/D genotype was 30 (53%), and I/I genotype was 8 (14%), which resulted in D and I allele frequencies of 0.60 and 0.40, respectively. Among controls with a history of TB infection and healthy controls, the frequency of the D/D genotype was 30/69 (44%) vs 22/34 (65%), I/D genotype was 26/69 (38%) vs 12/34 (35%), and I/I genotype was 13/69 (19%) vs 0/34 (0%), respectively, with D and I allele frequencies of 0.62 vs 0.82 and 0.38 vs 0.18, respectively.
There was a difference in the proportions of the HLA-G 14-bp genotype between controls with a history of TB infection and healthy controls (
The frequency of the HLA-G genotype was not different in terms of hematocrit, erythrocytes, leukocytes, thrombocytes, Na+, K+, or IFNγ levels (
Distribution of the HLA-G 14-bp in/del polymorphism in the context of MDR-TB patient drug resistance status and the results of the thorax radiographical imaging.
Genotypes (n,%) | ||||
---|---|---|---|---|
D/D(n = 19) | I/D(n = 30) | W(n = 8) | ||
Drug Resistances some data were missing. | ||||
Ethambutol | 0.18 | |||
Sensitive | 12(63) | 14(47) | 4(50) | respectively |
Resistant | 6(32) | 16(53) | 4(50) | |
Streptomicin | 0.26 | |||
Sensitive | 10(53) | 12(40) | 3(38) | respectively |
Resistant | 8(42) | 18(60) | 5(63) | |
Rontgen of thorax some data were missing. | ||||
Cavity | 0.70 | |||
Negative | 9(47) | 13 (43) | 3(38) | respectively |
Positive | 9(47) | 16(53) | 4(50) | |
Infiltration | - | |||
Negative | 2(11) | 3(10) | 1(13) | |
Positive | 16(84) | 26(87) | 6(75) | |
Nodule | - | |||
Negative | 18(95) | 29(97) | 6(75) | |
Positive | 0(0) | 0(0) | 1(13) | |
Fibrosis | 0.02 | |||
Negative | 14(74) | 23 (77) | 2(25) | |
Positive | 4(21) | 6(20) | 5(63) | |
Consolidation | - | |||
Negative | 17(90) | 29(97) | 7(88) | |
Positive | 1(5) | 0(0) | 0(0) | |
Pleural effusion | - | |||
Negative | 17(90) | 28(93) | 6(75) | |
Positive | 1(5) | 1(3) | 1(13) |
Data presented as n (%)
The numbers of cured and uncured patients did not differ based on HLA-G genotype status (
In some pathological conditions, such as in cytomegalovirus and human immunodeficiency virus infections, lung carcinoma, psoriasis, multiple sclerosis, breast cancer, and nontumorous pulmonary disease, HLA-G is expressed in macrophages [28,29]. Because macrophages are the main immune cell type involved in the pathogenesis of M
The HLA-G 14-bp in/del polymorphism has been suggested to be associated with HLA-G mRNA stability and HLA-G expression [30-33]; however, data regarding the impact of this polymorphism on MDR-TB are lacking. In this study, the HLA-G 14-bp polymorphism did not influence drug resistance status, time to sputum conversion, treatment results, or IKNγ and IL-10 levels of Indonesian Javanese patients with MDR-TB. Notably, an association was identified between pulmonary fibrosis and the HLA-G 14-bp deletion allele; MDR-TB patients carrying the deletion allele were less likely to develop pulmonary fibrosis, suggesting a possible role for the HLA-G polymorphism in MDR-TB pathogenesis that will need to be characterized in further studies. The results of this study also showed that patients with pulmonary fibrosis were less likely to be cured; thus, clinicians must be more concerned by this manifestation.
This study identified a difference in the proportion of the HLA-G genotype distribution between healthy people and people with a history of TB infection (
Notably, D/D genotype carriers were less susceptible to TB infection (OR 0.4, 95% CI 0.179-0.981,
This study provides information about the frequencies of the HLA-G 14-bp polymorphism among Indonesian Javanese; most Indonesian participants in the study carried the D/D genotype (44%), followed by the I/D (43%), and I/I genotypes (13%). The frequencies of the deletion and insertion alleles were 0.66 and 0.34, respectively. These early data contribute to our knowledge of the status of the HLA-G polymorphism in the Indonesian Javanese population, which is currently limited.
This study has several limitations, such as the limited number of participants and missing data from a number of participants. The results of this study require confirmation in studies using larger sample sizes and different populations.
The results of this study indicated that D/D genotype carriers are less susceptible to TB. Furthermore, the absence of the HLA-G 14-bp deletion allele may be a biomarker for the development of lung fibrosis in patients with MDR-TB.