Study of KIR expression and HLA ligands in cd56+ lymphocytes of drug resistant tuberculosis patients

Analysis of receptor-ligand interactions in the context of diseases necessitates to understand how HLA-KIR genotypes function in diseases. Although CD56+ lymphocytes are derived from multiple lineages, they share a functional association with immunosurviellance and antimicrobial responses. The present study aimed to determine whether KIR phenotype in CD56 lymphocytes and corresponding HLA-class 1 ligands are associated with multidrug resistance tuberculosis [MDR-TB]. We compared the frequencies of HLA-C and HLA-BW4 genes, the expression of KIRs 2DL1/2DS1, 2DL2/2DL3, 3DL1, and 2DS4 and the combinations of HLA/KIR in 32 Nifamycin and Isoniazid-resistant TB with those in 68 drug non resistant [NR] sputum smear positive pulmonary TB patients. PCR-SSP and flow cytometry were performed for HLA and KIRs typing, respectively. We showed no significant differences between inhibitory or activating KIRs as well as HLA ligands in MDR TB patients compared with NR-TB. The combinations of inhibitory KIR-HLA ligands in MDR-TB were much more prevalent, but not statistically significant than in NR patients [p=0.07]. The frequency of MDR patients with all HLA-C and HLABW4 ligands was higher than NR-TB [p<0.009]. Conversely, the percentage of MDR patients having only one kind of HLA gene was significantly lower than NR-TB [p<0.01]. We conclude that the expression of inhibitory KIRs with corresponding HLA ligands genes, and/or co-existence of three HLA class 1 ligands for inhibitory KIRs may be associated with drug resistance in pulmonary tuberculosis

Elevation of CD56[bright]CD16[-] lymphocytes in MDR pulmonary tuberculosis

Protective immune responses induced in the majority of people infected with Mycobacterium tuberculosis enable them to control TB infection. The aim of this study was to investigate CD56 and CD16 positive peripheral blood mononuclear cells [PBMCs] and leukocyte subsets from multi-drug resistant pulmonary tuberculosis [MDR-TB], and compare them with nonresistant [NR] TB patients and healthy controls. 13 MDR-tuberculosis patients, 20 NR-TB individuals and 40 healthy subjects were included. Peripheral blood mononuclear cells were double stained with fluorochrome conjugated antibodies against CD56 and CD16 cell surface markers. The phenotype of positive cells was then analyzed by flow cytometry and the percentages of CD56[+] CD16[+], CD56[-] CD16[+], CD56[dim]CD16[ +/- ], and CD56[bright]CD16[ +/- ] subsets were calculated. There was a significant decline in the percentage of CD56[]+CD16[+] lymphocytes in both MDR and NR-TB patients compared with healthy controls. We also observed lower proportions of CD56[dim]/[bright]CD16[+] in addition to higher percentages of CD56dim/brightCD16-subsets in all TB patients [p

[Correlation study of phospholipase C genes and pathogenicity of Mycobacterium tuberculosis strains of the Beijing family and non- Beijing family]

The Beijing strain of Mycobacterium tuberculosis is responsible for more than 25% of cases of Tuberculosis in the world, it has a high transmission potential and there is a significant correlation between Beijing strain and multidrug resistance. In this study we compared the presence of Phospholipase C genes in Beijing and Nonbeijing strains of Mycobacterium tuberculosis. CTAB method was used to extract DNA from positive culture specimens from patients with pulmonary tuberculosis. Spoligotyping was used to identify Beijing from non-Beijing strains. Presence of Phospholipase C genes was ascertained by using PCR. Of 200 specimens, 19 were Beijing strain, [9.5%] and 181 non Beijing strains, [90.5%]. In Beijing strains, 16 specimens, [84.2%]were positive for plcA,17 [89.5%] were positive for plcB and 17 [89.5%] were positive for plcC genes and in non Beijing strains 17 specimens, [9.4%], were positive for plcA, 18[9.9%], for plcB and 18[9.9%] for plcC. Majority of Beijing strains have phospholipase C genes that may be responsible for the virulence of this strain

Comparison of Nramp1 gene polymorphism among TB health care workers and recently infected cases; Assessment of host susceptibility

A link between polymorphisms in the natural resistance -associated macrophage protein gene 1 [Nramp] and susceptibility to tuberculosis [TB] has been demonstrated worldwide. This study aimed to investigate the Nramp1 gene variants among workers exposed to TB bacilli [1-2 hours per day for 1 to 20 years] who did not develop the diseases with those who developed the disease through recent transmission. The polymorphism of Nramp1 at INT4, D543 and 3'UTR was examined in 71 newly smear-positive TB cases and 39 healthcare workers exposed to TB. Polymerase chain reaction [PCR] and restriction fragment length polymorphism [RFLP] were used to genotype Nramp1 polymorphism. Patients' clinical and demographical data were collected. The heterozygote patterns of INT4 [G/C], D543 [G/A] and 3'UTR [+/del] occurred more frequently in control subjects than in patients [P =0.012], respectively [odds: 1.9 CI95%] [1.13-3.12]. Although, the homozygous patterns of INT4 [C/C; 8.5%], D543 [A/A; 1.4%] and 3'UTR [del/del; 1.4%] were only seen in patients [sensitivity 11% and specificity 100%]. The other risk factors like gender, age, resistance and PPD were not associated with Nramp1 gene polymorphism. Individuals with homozygous type mutation have an increased risk of developing tuberculosis. Therefore, we suggest detection of Nramp1 variants in high-risk groups i.e., health workers and close contact cases

Mycobacterium thermoresistibile infection in a child

Mycobacterium thermoresistibile was first reported in 1981 as a human pathogen. Several studies have reported pulmonary infection and cutaneous lesions due to this type of mycobacterium. A five-year-old boy with cough, fever, and abdominal pain was referred to Masih Daneshvari Hospital. He had been treated with diagnosis of histiocytosis x. Gastric lavage was performed and examined by polymerase chain reaction [PCR] and Mycobacterium thermoresistibile was found. It seems that this case is the first report of an atypical tuberculosis caused by Mycobacterium thermoresistibile in a child

Expression of alpha-crystalline protein in the in- vitro model of hypoxically induced nonreplicating persistence of mycobacterium tuberculosis strains

Latent tuberculosis infection [LTBI] is caused by Mycobacterium tuberculosis [MTB] in a state of non-replicating persistence [NRP]. Recent evidence suggests that some very specific adaptations to oxygen depletion occur so that MTB undergoes hypoxic NRP state. In this study the modified slowly stirred, limited Head Space Ratio [0.5HSR] method was used to investigate the physiological response of MTB to different oxygen tension levels. For setting up the various NRP stages, some susceptible and drug resistant clinically isolated strains of MTB were cultivated in Dubos Tween-Albumin medium via hypoxically, slow stirring 0.5 HSR methods. Additionally, the effects of isoniazid, rifampin, pyrazinamide, ciprofloxacin and metronidazole against MTB were examined during NRP-1 and NRP-2 stages. The alpha-crystalline protein was detected during NRP-1 stage of the MTB cultures via performance of the suitable procedures for pellet preparation, washing and cell disruption and SDS-PAGE[Sodium dodecyl sulfate-polyacrylamide gel electrophoresis] technique. NRP-1 and NRP-2 stages of MTB were assessed. The first three of the four drugs mentioned above affected the MTB at actively replicating period and the rifampin effect was continued slightly during NRP-1 stage. Metronidazole affected the MTB at anaerobic NRP-2 stage. Alpha-crystalline protein was detected in NRP -1 stage but was not detected at aerated cultures. Induction of the alpha-crystalline protein during hypoxic shift-down of MTB metabolism, and its function as a chaperone, suggests a critical role for this protein in the ability of MTB to persist without replicating in the hostile regions of the host's tissues. Therefore, for an effective TB control program, it is critical to understand the mechanisms of factors induction associated with the hypoxic condition of tubercle bacilli and some strategies for the identification of new drug targets must be developed and the persistence states in human lesions should be prevented as well

Primary and acquired drug resistance in childhood tuberculosis

Background: Increased rates of multidrug-resistant tuberculosis [MDR-TB] have been reported from developing countries. We evaluated the incidence of drug resistance in children in order to determine the magnitude of the problem, in our region

Objective: To determine the resistance pattern of Mycobacterium tuberculosis to four anti-tuberculosis drugs in childhood pulmonary tuberculosis at National Research Institute of Tuberculosis and Lung Disease [NRITLD] which is a referral centre in Tehran. Treatment of the patients was based on the DOTS strategy according to the WHO protocols since 1989

Materials and Methods: Retrospective analysis of all cases of pulmonary tuberculosis with positive M. tuberculosis culture who had referred to paediatrics ward from January 1999 to August 2004. M. tuberculosis sensitivity testing was performed by the Lowenstein-Jensen medium for isoniazid [INH], rifampicin [RMP], streptomycin [SM], and ethambutol [EMB]

Results: Among 350 children [0-15years] with confirmed tuberculosis, 7 children had resistance to at least one of the four anti-TB drugs. Out of the 7 patients, 6 were Afghan refugees and one patient was Iranian. Among those 85.7% had resistance to RMP, 71.4% to INH, 57.1% to SM, and 28.6% to EMB .In addition, 28.5% of patients had resistance to all four drugs [RMP, INH, SM, EMB], 14.2% to INH, RMP, SM, 28.5% to INH, RMP and 14.2% had resistance to each of SM and RMP. In this study 2% of children with TB had resistance out of which primary resistance was detected in 57.1%. Secondary resistance was found in 42.9% of cases who had previous history of anti-TB therapy

Conclusion: According to 2% prevalence of drug resistance in children and high resistance to RMP in our study, more aggressive interventions should be considered. Further management and supervision in DOTS implementation is highly recommended to prevent transmission of resistant tuberculosis