Tuberculosis (TB) continues to be a serious public health problem in many parts of the world including China, despite multiple efforts have been done to combat it. However, the steady increase in drug resistance including multidrug resistance (MDR), i.e., resistance to at least isoniazid (INH) and rifampin (RIF), and extensive drug resistance (XDR; resistance to at least INH, RIF, a fluoroquinolone, and a second line injectable) has exacerbated the problem.1 Diagnosis and appropriate treatment of multidrug-resistant TB (MDR-TB) remain major challenges.2 Because of airborne transmission of Mycobacterium tuberculosis (M. tuberculosis),3 persons who share a household with drug resistance tuberculosis patients are at high risk for primary drug resistance tuberculosis. Mycobacterial minisatellite interspersed repetitive unit (MIRU) and variable-number tandem repeat (VNTR) have contributed significantly to understanding of epidemiology on transmission dynamicsand investigating outbreaks.4-6 MIRU-VNTR is based on analysis of tandemly repeated sequences of multiple loci that are amplified using primers flanking regions of each locus, followed by size determination of the resulting PCR products, which indicates the numbers of the targeted MIRU-VNTR copies.7,8 Isolated strains of M. tuberculosis were defined as identical if spoligotype and MIRU-VNTR patterns were the same in the household and index case-patients. In this report we use 12-locus MIRU and 7-locus VNTR9 to identify household transmission of MDR and XDR in three families. In addition, we sought to elucidate how to prevent these outbreaks.
Patients and bacterial strains
This study have been reviewed by the Ethics Committee of Tongji University and been performed in accordance with the ethical standards. All persons in this study gave their informed consent prior to their inclusion in the study and details that might disclose the identity of the subjects under the study have been omitted. Six tuberculosis patients from three families admitted in Shanghai Pulmonary Hospital affiliated with Tongji University were subjects of this study. Patient 1 was the mother of patient 2 (daughter of patient 1) from family 1. Patient 3 was the son-in-law of patient 4 (mother-in-law of patient 3) from family 2. Patient 5 was the father of patient 6 (daughter of patient 5) from family 3. All the subjects were human immunodeficiency virus-seronegative and not pregnant. A total of six M. tuberculosis isolates from samples collected from these patients were isolated and genotyped in our microbiology laboratory, which is the reference laboratory for tuberculosis diagnosis in southeastern China. Clinical data were obtained from the subjects’ medical records.
Drug susceptibility test
Drug susceptibility tests were done by the modified proportion method in the MGIT 960 system (Becton Dickinson Microbiology systems, USA).10
Genomic DNA isolation
Strains grown in sauton culture medium were sterilized at 80°C for 30 minutes and then collected by centrifugation for 5 minutes at 12 000 ´g. Bacterial precipitate were washed three times with sterilized saline water by centrifugation for 10 min at 12 000 ´g each time, treated with DNA Lysis Buffer (10 mmol/L NaCl, 1 mg/ml SDS, 0.15 g/ml Chelex-100 glass beads, 1% Tween 20) at 50°C for 1 hour, then at 100°C for 10 minutes. After the mixture was centrifuged for 10 minutes at 5000 ´g, aqueous phase containing genomic DNA was transferred into new tube used for PCR amplification.
Identification of Beijing strains
Identification of Beijing strains followed the Deletion-Targeted Multiplex PCR multiplex PCR (DTM-PCR) on the genomic deletion RD105, which defines the Beijing/W family as a separate lineage within M. tuberculosis.11,12 The primers of DTM-PCR P1 (5′-GGAGTCGTTGAGGGTGTTCATCAGCTCAGTC-3′) and P2 (5’-CGCCAAGGCCGCATAGTCACGGTCG-3′) amplified a 1466 bp product for non-W-Beijing strains, while P1 and P3 (5′-GGTTGCCCACTGGTCGA- TATGGTGGACTT-3′), amplified 761 bp fragment for the W-Beijing Genotype.
The primers from 12 MIRU loci (MIRU2, MIRU4, MIRU10, MIRU16, MIRU20, MIRU23, MIRU24, MIRU26, MIRU27, MIRU31, MIRU39, MIRU40) and 7 VNTR loci (VNTR3820, QUB-11b, QUB-18, MIRU26, QUB-11a, QUB-26, Mtbu21) were designed according to the reported protocols (11) and synthesized and HPLC-purified by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The PCR reaction mixtures (20 μl) consisted of 1´ taq PCR buffer, 3 mmol/L MgCl2, 0.2 mmol/L dNTP, 0.2 mmol/L of each primer, 1 U Taq DNA polymerase, 10 ng of template DNA. The reaction conditions were the following: 94°C for 5 minutes, followed by 35 cycles of 94°C for 1 minute, annealing for 30 seconds at temperature range 55°C–64°C, 72°C for 1 minute, and finally 72°C for 10 minutes. The PCR products were electrophoresed on 6% polyacrylamide gel with silver staining.
Clinical presentation of patients
The patients’ characteristics are listed in the Table 1. Patient 1 had smear- and culture-positive pulmonary TB diagnosed in January 2001 but she completed treatment with streptomycin, isoniazid, rifampicin, pyrazinamid for only 1 month due to poor compliance. She relapsed in the next year and received a standard four-drug regimen for 1 month again. In May 2007 she developed signs and symptoms consistent with pulmonary TB and had abnormal chest radiographs with multiple cavitaries. Her isolates were found to be resistant to streptomycin, isoniazid, rifampicin, ethambutol and she was diagnosed MDR-TB. She received anti-MDR-TB regimen but sputum smear were consistently positive. The isolates were found to be also resistant to amykacin, capreomycin and ofloxacin in October 2008 and she was diagnosed XDR-TB. Patient 2 is the daughter of patient 1. Pulmonary MDR-TB was diagnosed based on bacteriology in March 2008. She received anti-MDR TB regimen but sputum smear were consistently positive. In January 2009 we performed drug susceptibility test (DST) of her isolates to second-line anti-TB medications and foundthe isolates developed additional resistance to amykacin, capreomycin and ofloxacin. Patient 3 had sputum smear positive pulmonary TB in February 1996. He had received irregular treatment due to poor compliance for 14 years. MDR-TB was diagnosed in July 2008 and XDR-TB was diagnosed in May 2010. Patient 4 is the mother-in-law of patient 3. She was diagnos
ed as pulmonary TB in August 2007 based on bacteriology and received regular treatment, but sputum smear were consistently positive. XDR-TB was diagnosed in August 2009. Patient 5 was diagnosed as pulmonary tuberculosis and tuberculous pleurisy, received treatment with ethambutol, isoniazid, rifampicin, pyrazinamid in May 2009. The isolates were found to be resistant to streptomycin, isoniazid, rifampicin, ethambutol, pyrazinamid in December 2009. Patient 6 is the daughter of patient 5. She was also diagnosed as pulmonary tuberculosis and tuberculous pleurisy in April 2010, M. tuberculosis
isolates cultured from pleural fluid were susceptible to all first- and second-line anti-TB medications. Chest X-rays of the six patients are shown in Figure 1.
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Table 1. Clinical characteristics of the six patients
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Figure 1. Chest X-rays of the six patients. A: Patient 1. B: Patient 2. C: Patient 3. D: Patient 4. E: Patient 5. F: Patient 6.
All of the six strains were defined as Beijing genotype by the deletion-targeted multiplex PCR (DTM-PCR) identification on the genomic deletion RD105.12,13 Strains from patient 1 and patient 2 had the same MIRU pattern (232225172531) and the same MIRU pattern (3677235). Strains from patient 3 and patient 4 had the same MIRU pattern (2212261553323) and the same MIRU pattern (3685134). MIRU pattern from patient 5 (223326173533) and patient 6 (223325173533) differed at only one locus (MIRU-23 locus) and VNTR pattern differed at two loci (3667233, 3677234).
Drug resistance profile
The drug resistance profiles of isolates from the six patients are summarized in Table 1. Patient 1 and patient 3 were XDR-TB because of their poor compliance. Patient 2 and patient 4 contracted the drug-resistant isolates of patient 1 and patient 3, and become primary XDR-TB. Patient 5 mutated to MDR-TB after the strains had transmitted to patient 6. Patient 6 were pansusceptible TB.
In this study we used MIRU-VNTR genotyping to identify household tuberculosis transmission in 3 families. The isolates from family 1 and family 2 were indistinguishable on MIRU-VNTR typing. Although the genotype of isolates from Patient 5 did not match that of patient 6, they differed only at one or two loci. Therefore we assumed this difference was caused by evolution of the MIRU-VNTR pattern or a processing error or they may share remote common clonal ancestors Therefore we think that patient 2 and patient 4 and patient 6 acquired TB within household. There were household transmissions in the three families. This result suggests that persons who share a household with TB patients are at high risk for infection. The results differed from observations in high-incidence settings but are similar to those in low-incidence settings. In Morocco a retrospective molecular epidemiological study of Mycobacterium tuberculosis found heterogeneity in members from a same patient family.14 In South Africa the proportion of transmission in the community that took place in the household was 19%, and therefore, tuberculosis transmission occurs mainly outside the household.15 In Vietnam eighty-three percent of persons with recent household TB were infected by different strains of Mycobacterium tuberculosis than were their household members.16
Patient 2 had TB diagnosed 7 years later than patient 1 and patient 4 had TB diagnosed 11 years later than patient 3. So these outbreaks could have been prevented with adequate treatments of patient 1 and patient 3. Their irregular treatments resulted in their progression to XDR-TB and consequently primary XDR-TB for patient 2 and patient 4. Resistance spectrum of patient 2 and patient 4 are the same as patient1 and patient 3. It can be seen that resistance of M. tuberculosis to these drugs is relatively stable. They did not regain susceptibility after undergoing the change of living environment and replacement of host. So controlling the spread of drug-resistant tuberculosis not only reduce the incidence of tuberculosis but also reduce drug resistance rate, especially primary drug resistance rate.
Several molecular epidemiology studies found genetic factors are implicated in susceptibility to Mycobacterium tuberculosis infection. Genetic susceptibility not only varies endemic prevalence levels, but also drastically alters the effects of treatment for tuberculosis patients.17 FokI polymorphism is possibly related to host susceptibility to TB among Han population.18 Macrophage receptors are involved in host resistance to M. tuberculosis.19 In this study patient 1 and patient 2 were diagnosed pulmonary tuberculosis, patient 5 and patient 6 were diagnosed pulmonary tuberculosis and tuberculous pleurisy. It is probable that genetic susceptibility may affect TB pathogenesis. The authors suggested patient 5 and patient 6 may have genetic susceptibility of pulmonary tuberculosis and tuberculous pleurisy. Patient 1 and patient 2 may have genetic susceptibility of pulmonary tuberculosis. But we did not measure the tuberculosis susceptibility gene of them and the sample size was so small that these results must be considered preliminary. Since the current data were gathered as part of a longitudinal cohort study, we are addressing the following questions.
M. tuberculosis itself may be another factor of deciding TB transmitted in a family. It has been suggested that genetically distinct M. tuberculosis strains may invoke different immune responses, although how these differences influence the immune responses is still poorly understood. Rakotosamimanana et al20 compared the IFN-gamma responses with the spoligotype of the infecting clinical strains showed that “modern” M. tuberculosis strains, like Beijing and Central Asian (CAS) strains, tended to induce lower IFN-gamma responses than “ancient” strains, like East African-Indian (EAI) strains, in index patients and their household contacts. Abadía et al21 found that genetically distinct M. tuberculosis strains invoke different immune responses. Patients with SIT 17 were likely to have had specimen positive for Acid Fast Bacilli on microscopy and patients with SIT 53 were older and more commonly smear negative.21 Xu et al22 found that the multi-drug resistant M. tuberculosis strains have been more frequently clustered. There are a growing number of observations indicating that some strains may have the ability to disseminate more easily than others. According to literature, 223325173533 (the isolates of patient 6) showed significantly higher frequencies of MDR and increased transmissibility in China.23 Furthermore, a cell culture study in Uganda separated isolates on the basis of their frequency of transmission to household contacts, and those which transmitted efficiently showed more rapid growth in human acute monocytic leukemia cell line than nontransmitted isolates.24 However, so far there are few data on the effects of M. tuberculosis isolates with different genotypes on transmissibility. We further sequenced Rv0050 gene of these isolates trying to find association between genotypes and transmissibility. The Rv0050 locus encodes the bifunctional penicillin-binding protein ponA1 and is essential to mycobacterial survival.25 But we found nearly all of the W-Beijing strains harbored the allele with the highest repeat numbers. This locus was determined to not represent a good molecular marker for epidemiological investigations of the W-Beijing genotype.26 In addition, there is evidence that T cell responses may contribute directly to human-to-human transmission of Mycobacterium tuberculosis complex.27 In particular, cavitary tuberculosis, which generates secondary cases more efficiently than other disease forms.28 Therefore we are going to study the association between Rv0288, Rv0589, Rv0934, Rv1694 and T-SPOT test result on large scale.
This study has determined that a clear chain of tuberculosis transmission within family exists. Tuberculosis susceptibility should be considered when there is more than one tuberculosis patients in a family. Household tuberculosis transmission could be prevented with adequate treatment of source patients.
Acknowledgements: We thank YUE Jun and JING Ling-jie (Shanghai Dulmonary Hospital Microbiology Laboratory) for their assistance.
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