INT J TUBERC LUNG DIS 18(3):298–301 © 2014 The Union http://dx.doi.org/10.5588/ijtld.13.0558
SHORT COMMUNICATION
Molecular epidemiology of Mycobacterium tuberculosis strains circulating in the penitentiary system of Kazakhstan A. Ibrayeva,* U. Kozhamkulov,* D. Raiymbek,* A. Alenova,† S. Igilikova,† E. Zholdybayeva,* T. Abildaev,† K. Momynaliev* * National Center for Biotechnology, Astana, † National Tuberculosis Centre, Almaty, Kazakhstan SUMMARY
A total of 60 Mycobacterium tuberculosis isolates collected from patients in prisons in Kazakhstan and 125 from the civilian sector were examined using mycobacterial interspersed repetitive units-variable number of tandem repeat analysis in 2012. The proportion of tuberculosis strains with unique genotypes isolated from the civilian patients was 50.4%, while that in the prison patients was 31.7%. This difference was statistically significant (χ2 4.42, P = 0.035), and may reflect a low
genetic diversity of M. tuberculosis strains isolated from prison patients. The frequencies of mutations in the rpoB531 and katG315 genes of the M. tuberculosis strains isolated from the civilians and in the penitentiary system were not significantly different (rpoB531: 82.4% vs. 88.3%, and katG315: 98.4% vs. 100%, respectively). K E Y W O R D S : Mycobacterium tuberculosis; penitentiary system; MIRU-VNTR genotyping; DNA sequencing
THE PREVALENCE of registered tuberculosis (TB) cases in the penitentiary system is nearly 100 times higher than in the civilian population.1 The incidence of TB in 2012 among the prison population in Kazakhstan was also high, at 1073 per 100 000 population, compared to 81.7/100 000 in the civilian population.2 A closed system is known to present a particularly high risk for many infectious diseases, including TB. The probability of transmission is increased by overcrowding, poor ventilation and nutrition, and delays in medical examinations and treatment.3 Detention facilities also serve as vehicles for the circulation and dissemination of multidrug-resistant TB (MDR-TB, defined as resistance to at least isoniazid and rifampicin) and are associated with a high risk of disease outbreaks.4 Investigation of the structure of Mycobacterium tuberculosis strains circulating in a closed system may not only detect drug resistance, it will also shed light on the pathogenicity and virulence of strains as well as the spread of TB in the general population. It has been shown previously that genetic diversity is significantly lower in closed systems than in open systems; in several studies, a lower genetic variety of M. tuberculosis has been observed in prisoners compared to the civilian population, particularly those who are physically separated from the rest of population.3,4 This low genetic diversity of M. tuberculosis strains shows that TB occurs within small groups of prisoners.4 To confirm this hypothesis, we investigated the genetic variability of M. tuberculosis strains circulating in the prison system of Kazakhstan and examined
their anti-tuberculosis drug resistance profiles compared with the civilian sector.
METHODS In 2012, clinical isolates of M. tuberculosis were collected from male patients aged 25–64 years with pulmonary TB at two prisons run by the State-Established Committee for the Management of the Penitentiary System of the Karaganda Region and the Committee for the Management of the Penitentiary System of the Akmola Region. Among the prison TB patients, 14 (23.3%) were new TB cases and 46 (76.7%) were recurrent cases; infiltrative and fibro-cavernous forms of TB were detected in respectively 43 (71.7%) and 17 (28.3%) patients. The total duration of TB among the patients ranged from 5 to 9 years, which included 2–5 years in prison. To compare the characteristics of the circulating strains of M. tuberculosis in the civilian and penitentiary systems, a total of 125 clinical isolates of M. tuberculosis were collected from the civilian sector. Among the TB patients, 54 (43.2%) M. tuberculosis isolates were selected from newly diagnosed patients and 71 (56.8%) from patients with recurrent TB. Infiltrative and fibro-cavernous forms of TB were detected in respectively 97 (77.6%) and 28 (22.4%) patients. The total TB disease duration ranged from a few months to 10 years among the TB patients in the civilian sector. Clinical isolates of M. tuberculosis were collected during 2012 and deposited at the Reference Laboratory of the National Tuberculosis Centre (Table 1).
Correspondence to: Albina Ibrayeva, National Center for Biotechnology, 13/1 Valikhanov St, Astana 010000, Republic of Kazakhstan. Tel: (+7) 7172 21 40 20. Fax: (+7) 7172 21 46 33. e-mail: [email protected] Article submitted 29 July 2013. Final version accepted 29 October 2013.
Molecular epidemiology of M. tuberculosis strains
299
Table 1 Susceptibility patterns of 60 M. tuberculosis isolates recovered from patients with tuberculosis in Kazakhstan penitentiary and civil systems in 2012* Penitentiary system isolates
INH
SM
EMB
Total (n = 60) n (%)
R R S S S R R S
R R R R R R S S
R S R S R R R S
45 (75) 3 (5) 3 (5) 3 (5) 3 (5) 2 (3) 1 (2) 0
Drug RMP R R S R R S R S
Civil system isolates
Group N (n = 14) n (%)
Group P (n = 46) n (%)
Total (n = 125) n (%)
Group N1 (n = 54) n (%)
Group P1 (n = 71) n (%)
7 (50) 1 (7.1) 2 (14.3) 1 (7.1) 3 (21.5) 0 0
38 (82.6) 2 (4.3) 1 (2.25) 2 (4.3) 0 2 (4.3) 1 (2.25)
79 (63.2) 10 (8) 2 (1.6) 1 (0.8) 4 (3.2) 4 (3.2) 6 (4.8) 19 (15.2)
22 (40.7) 6 (11.1) 2 (3.7) 1 (1.9) 3 (5.6) 2 (3.7) 4 (7.4) 14 (25.9)
57 (80.4) 4 (5.6) 0 0 1 (1.4) 2 (2.8) 2 (2.8) 5 (7)
* Susceptibility patterns were determined using the radiometric method (BACTEC™ 460TB System, BD, Sparks, MD, USA). RMP = rifampicin; INH = isoniazid; SM = streptomycin; EMB = ethambutol; R = resistant; S = susceptible.
All bioethical norms were adhered to in this study; approval was granted by the local committee on medical ethics. DNA was isolated using standard methods according to the online mycobacterial interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) manual (http://www.miru-vntrplus. org). MIRU-VNTR typing (24 VNTR loci) was performed as previously described.5 DNA sequencing and analysis of the rpoB, katG and fabG-inhA genes was conducted as previously described.6 The polymerase chain reaction products obtained were sequenced with an automated ABI 3730 Genetic Analyzer DNA sequencer (Applied Biosystems, Foster City, CA, USA) using the BigDye terminator kit (Applied Biosystems), per the manufacturer’s instructions. The sequences were compared with their respective wild-type sequences using SeqScape software (Applied Biosystems).6,7
RESULTS AND DISCUSSION Among the 60 M. tuberculosis isolates collected from the patients in the penitentiary system, 27 distinct MIRU-VNTR profiles were detected; 19 (31.7%) unique profiles (detected only in one isolated strain in this study), and 8 profiles comprised of clusters containing 2–14 isolates each (41 isolates, 68.3%). All of the clusters belonged to the W-Beijing family. The largest cluster comprised 14 strains, while the other clusters included 13, 3 and 2 strains each (Figure). In the TB patients from the civilian sector, 125 isolates were analysed, and a total of 77 distinct MIRU-VNTR profiles were detected; 63 (50.4%) were unique, while 14 profiles were comprised of clusters containing 2– 15 isolates each (n = 41, 49.6%). The largest cluster comprised 15 isolates, while the other clusters included 10, 6 and 2 isolates each. According to the DNA sequencing results from the 185 M. tuberculosis strains circulating in the penitentiary and civilian systems in Kazakhstan, there was a high prevalence of mutations in the rpoB gene in codon 531, which were found in 156 strains (84.3%),
leading to the replacement of serine by leucine (TCG→ TTG); in the katG gene, 183 (98.9%) mutations were found in codon 315, leading to the substitution of serine by threonine (AGC→ACC; Table 2). The percentage of strains with unique genotypes among the civilian sector was 50.4%, while that among the prison sector was 31.7%. This difference is statistically significant (χ2 4.42, P = 0.035), and may reflect low genetic diversity among M. tuberculosis strains isolated from prison TB patients, as TB transmission can occur in small groups of prisoners. However, it should be noted that we did not detect statistically significant differences in the frequency of mutations in the rpoB531 and katG315 codons between M. tuberculosis strains isolated from the civilian and penitentiary systems. The majority of nucleotide substitutions were found at codon 531 of the rpoB gene and codon 315 of the katG gene.7 These genes encode enzymes that are of biological significance in the bacteria, and mutations in these genes could impair enzyme function, leading to a fitness cost. Previous studies have shown that this biological cost can vary according to the particular mutation (i.e., the amino acid and position involved) and the genetic background of the strain. The degree to which the mutation affects the fitness of the organism varies depending on the specific drug resistance mutation, the environment and the genetic background of the strain.8 Genetic clustering has been applied extensively in molecular epidemiological studies as a measure of ongoing TB transmission.8 In recent years, numerous molecular epidemiological studies have been conducted to clarify the epidemiology of TB among different population groups.4,6 Previously published data show that the genetic diversity of TB strains in open systems ranges from 50% to 80%, whereas in closed systems (e.g., hospitals and prisons) it ranges from 20% to 40%.4,9 Our results are consistent with these findings regarding the genetic diversity of TB among civilians in Kazakhstan (50.4%) vs. the penitentiary system (31.7%). Comparing the strains circulating in the open vs. closed systems, we detected
Figure Dendogram of the phylogenetic similarity of Mycobacterium tuberculosis strains isolated in the Kazakhstan penitentiary and those in civilian populations according to MIRU cluster analysis. The order of the 24 MIRU loci is as follows, from left to right: 154, 580, 960, 1644, 2059, 2531, 2687, 2996, 3007, 3192, 4348, 802, 2156, 2461, 577, 424, 1955, 2347, 2401, 3171, 3690, 2136b, 4052 and 4156. NC1 = non-clustered isolates; K (2–15 is the number of isolates) = MIRU clusters; MIRU = mycobacterial interspersed repetitive units.
Molecular epidemiology of M. tuberculosis strains
301
Table 2 Mutations in genes involved in rifampicin and isoniazid resistance of M. tuberculosis isolates in the Kazakhstan penitentiary and civil system in 2012 Isolates with mutations
Gene Rifampicin-resistant isolates rpoB
Penitentiary system (n = 60) n (%)
Civil system (n = 125) n (%)
Total (n = 185) n (%)
Affected codon
Nucleotide changes
Amino acid change
531 526
TCG→TTG CAC→TAC CAC→GAC CAC→CTC CTG→CCG
Ser→Leu His→Arg His→Asp His→Leu Leu→Pro
53 (88.3) 2 (3.3) 2 (3.3) 1 (1.8) — 2 (3.3)
103 (82.4) 9 (7.2) 2 (1.6) 3 (2.4) 1 (0.8) 7 (5.6)
156 (84.3) 11 (6.0) 4 (2.1) 4 (2.1) 1 (0.5) 9 (5.0)
AGC→ACC
Ser→Thr
60 (100) — —
123 (98.4) — 2 (1.6)
183 (98.9) 0 2 (1.1)
533 None Isoniazid-resistant isolates katG 315 fabG-inhA None
the same spectrum of mutations causing multidrug resistance. In contrast, the percentage of genetic heterogeneity in prisons was significantly lower, indicating circulation of the most virulent strains in this closed system. The main reason for the formation of these clusters is exogenous infection resulting from recent transmission and clonal expansion.9,10 An additional factor is the long persistence of the bacteria in the closed system, during which time natural selection occurs, favouring the most virulent strains and leading to the formation of a homogeneous environment. It can be concluded that individuals showing the same spectrum of mutations harbouring clustered isolates are expected to belong to a transmission chain involving the most virulent TB strains in a closed system, whereas strains found in individuals harbouring unique isolates are expected to circulate in an open system. In M. tuberculosis, different strains have been shown to differ in their immunogenicity and virulence in animal models.11 There is also increasing evidence that strain diversity can influence the outcome of infection and disease in humans. Genomic analyses of strain collections from global sources have revealed that M. tuberculosis shows a phylogeographic population structure in which different strain lineages are associated with particular geographic regions. A recent theoretical study found that simulated populations with immunologically distinct strain groups displayed a higher risk of drug resistance than populations without strain diversity, even when the quality of TB control was the same.12 Patients with infectious TB who are imprisoned and those who are released from prison before the completion of treatment may play an important role in the epidemiology of TB disease. Close coordination between TB programmes targeting prison and civilian communities is thus important.
Conflict of interest: none declared.
References 1 World Health Organization. Global tuberculosis report, 2012. WHO/HTM/TB/2012.6. Geneva, Switzerland: WHO, 2012. 2 Ministry of Health, Republic of Kazakhstan. [Statistic TB review]. Almaty, Kazakhstan: National TB Center, 2012. [Russian] 3 Pfyffer G, Strassle A, van Gorkum T, et al. Multidrug-resistant tuberculosis in prison inmates, Azerbaijan. Emerg Infect Dis 2001; 7: 855–861. 4 Chernyaeva E, Dobrynin P, Pestova N, Matveeva N, Zhemkov V, Kozlov A. Molecular genetic analysis of Mycobacterium tuberculosis strains spread in different patient groups in St. Petersburg, Russia. Eur J Clin Microbiol Infect Dis 2012; 31: 1753– 1757. 5 Supply P, Allix C, Lesjean S, et al. Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variablenumber tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 2006; 44: 4498–4510. 6 Sekiguchi J, Miyoshi-Akiyama T, Augustynowicz-Kopec´ E, et al. Detection of multidrug resistance in Mycobacterium tuberculosis. J Clin Microbiol 2007; 45: 179–192. 7 Kozhamkulov U, Akhmetova A, Rakhimova S, et al. Molecular characterization of rifampicin and isoniazid-resistant Mycobacterium tuberculosis strains isolated in Kazakhstan. Jpn J Infect Dis 2011; 64: 253–255. 8 Gagneux S, Burgos M, De Riemer K, et al. Impact of bacterial genetics on the transmission of isoniazid-resistant Mycobacterium tuberculosis. PLOS Pathog 2006; 2: 603–610. 9 Franzetti F, Codecasa L, Matteelli A, et al. Genotyping analyses of tuberculosis transmission among immigrant residents in Italy. Clin Microbiol Infect 2010; 16: 1149–1154. 10 Cooksey R C, Abbadi S H, Woodley C L, et al. Characterization of Mycobacterium tuberculosis complex isolates from the cerebrospinal fluid of meningitis patients at six fever hospitals in Egypt. J Clin Microbiol 2002; 40: 1651–1655. 11 Gagneux S, Small P M. Global phylogeography of Mycobacterium tuberculosis and implications for tuberculosis product development. Lancet Infect Dis 2007; 7: 328–337. 12 Borrell S, Gagneux S. Infectiousness, reproductive fitness and evolution of drug-resistant Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2009; 13: 1456–1466.
Molecular epidemiology of M. tuberculosis strains
i
RÉSUMÉ
Une série de 60 souches de Mycobacterium tuberculosis recueillis auprès de patients détenus dans des prisons du Kazakhstan et 125 souches de patients non détenus a été examinée avec l’analyse par la méthode des unités répétitives dispersées sur le génome mycobactérien– nombre variable de répétitions en tandem en 2012. Nous avons constaté que le pourcentage de souches de TB avec un génotype unique isolé chez des patients civils était de 50,4% contre 31,7% chez les détenus. La différence
était statistiquement significative (χ2 4,42 ; P = 0,035) et pourrait refléter une faible diversité génétique des souches de M. tuberculosis isolées chez des détenus. La fréquence des mutations des gènes rpoB531 et katG315 de M. tuberculosis isolées chez des civils et les détenus n’était pas significativement différente (rpoB531 : 82,4% contre 88,3% et katG315 : 98,4% contre 100%, respectivement).
RESUMEN
Se practicó la genotipificación con marcadores para locus múltiples de las secuencias repetitivas en tándem (MIRU-VNTR) de 60 aislados clínicos de Mycobacterium tuberculosis recogidos en pacientes de las prisiones de Kazakstán y 125 aislados de pacientes del sector civil en el 2012. Se observó que el porcentaje de cepas de M. tuberculosis con genotipos únicos aislados de los pacientes del sector civil era 50,4% y en los pacientes en prisión era 31,7%. Esta diferencia alcanzó significación
estadística (χ2 4,42; P = 0,035) y puede corresponder a una baja diversidad genética de las cepas de M. tuberculosis aisladas en los pacientes de las prisiones. La diferencia de frecuencia de las mutaciones en los genes rpoB531 y katG315 de las cepas de M. tuberculosis aisladas en personas en el sistema penitenciario y en la población civil no fue estadísticamente significativa (rpoB531: 82,4% contra 88,3% y katG315: 98,4% contra 100%, respectivamente).
SHORT COMMUNICATION
Molecular epidemiology of Mycobacterium tuberculosis strains circulating in the penitentiary system of Kazakhstan A. Ibrayeva,* U. Kozhamkulov,* D. Raiymbek,* A. Alenova,† S. Igilikova,† E. Zholdybayeva,* T. Abildaev,† K. Momynaliev* * National Center for Biotechnology, Astana, † National Tuberculosis Centre, Almaty, Kazakhstan SUMMARY
A total of 60 Mycobacterium tuberculosis isolates collected from patients in prisons in Kazakhstan and 125 from the civilian sector were examined using mycobacterial interspersed repetitive units-variable number of tandem repeat analysis in 2012. The proportion of tuberculosis strains with unique genotypes isolated from the civilian patients was 50.4%, while that in the prison patients was 31.7%. This difference was statistically significant (χ2 4.42, P = 0.035), and may reflect a low
genetic diversity of M. tuberculosis strains isolated from prison patients. The frequencies of mutations in the rpoB531 and katG315 genes of the M. tuberculosis strains isolated from the civilians and in the penitentiary system were not significantly different (rpoB531: 82.4% vs. 88.3%, and katG315: 98.4% vs. 100%, respectively). K E Y W O R D S : Mycobacterium tuberculosis; penitentiary system; MIRU-VNTR genotyping; DNA sequencing
THE PREVALENCE of registered tuberculosis (TB) cases in the penitentiary system is nearly 100 times higher than in the civilian population.1 The incidence of TB in 2012 among the prison population in Kazakhstan was also high, at 1073 per 100 000 population, compared to 81.7/100 000 in the civilian population.2 A closed system is known to present a particularly high risk for many infectious diseases, including TB. The probability of transmission is increased by overcrowding, poor ventilation and nutrition, and delays in medical examinations and treatment.3 Detention facilities also serve as vehicles for the circulation and dissemination of multidrug-resistant TB (MDR-TB, defined as resistance to at least isoniazid and rifampicin) and are associated with a high risk of disease outbreaks.4 Investigation of the structure of Mycobacterium tuberculosis strains circulating in a closed system may not only detect drug resistance, it will also shed light on the pathogenicity and virulence of strains as well as the spread of TB in the general population. It has been shown previously that genetic diversity is significantly lower in closed systems than in open systems; in several studies, a lower genetic variety of M. tuberculosis has been observed in prisoners compared to the civilian population, particularly those who are physically separated from the rest of population.3,4 This low genetic diversity of M. tuberculosis strains shows that TB occurs within small groups of prisoners.4 To confirm this hypothesis, we investigated the genetic variability of M. tuberculosis strains circulating in the prison system of Kazakhstan and examined
their anti-tuberculosis drug resistance profiles compared with the civilian sector.
METHODS In 2012, clinical isolates of M. tuberculosis were collected from male patients aged 25–64 years with pulmonary TB at two prisons run by the State-Established Committee for the Management of the Penitentiary System of the Karaganda Region and the Committee for the Management of the Penitentiary System of the Akmola Region. Among the prison TB patients, 14 (23.3%) were new TB cases and 46 (76.7%) were recurrent cases; infiltrative and fibro-cavernous forms of TB were detected in respectively 43 (71.7%) and 17 (28.3%) patients. The total duration of TB among the patients ranged from 5 to 9 years, which included 2–5 years in prison. To compare the characteristics of the circulating strains of M. tuberculosis in the civilian and penitentiary systems, a total of 125 clinical isolates of M. tuberculosis were collected from the civilian sector. Among the TB patients, 54 (43.2%) M. tuberculosis isolates were selected from newly diagnosed patients and 71 (56.8%) from patients with recurrent TB. Infiltrative and fibro-cavernous forms of TB were detected in respectively 97 (77.6%) and 28 (22.4%) patients. The total TB disease duration ranged from a few months to 10 years among the TB patients in the civilian sector. Clinical isolates of M. tuberculosis were collected during 2012 and deposited at the Reference Laboratory of the National Tuberculosis Centre (Table 1).
Correspondence to: Albina Ibrayeva, National Center for Biotechnology, 13/1 Valikhanov St, Astana 010000, Republic of Kazakhstan. Tel: (+7) 7172 21 40 20. Fax: (+7) 7172 21 46 33. e-mail: [email protected] Article submitted 29 July 2013. Final version accepted 29 October 2013.
Molecular epidemiology of M. tuberculosis strains
299
Table 1 Susceptibility patterns of 60 M. tuberculosis isolates recovered from patients with tuberculosis in Kazakhstan penitentiary and civil systems in 2012* Penitentiary system isolates
INH
SM
EMB
Total (n = 60) n (%)
R R S S S R R S
R R R R R R S S
R S R S R R R S
45 (75) 3 (5) 3 (5) 3 (5) 3 (5) 2 (3) 1 (2) 0
Drug RMP R R S R R S R S
Civil system isolates
Group N (n = 14) n (%)
Group P (n = 46) n (%)
Total (n = 125) n (%)
Group N1 (n = 54) n (%)
Group P1 (n = 71) n (%)
7 (50) 1 (7.1) 2 (14.3) 1 (7.1) 3 (21.5) 0 0
38 (82.6) 2 (4.3) 1 (2.25) 2 (4.3) 0 2 (4.3) 1 (2.25)
79 (63.2) 10 (8) 2 (1.6) 1 (0.8) 4 (3.2) 4 (3.2) 6 (4.8) 19 (15.2)
22 (40.7) 6 (11.1) 2 (3.7) 1 (1.9) 3 (5.6) 2 (3.7) 4 (7.4) 14 (25.9)
57 (80.4) 4 (5.6) 0 0 1 (1.4) 2 (2.8) 2 (2.8) 5 (7)
* Susceptibility patterns were determined using the radiometric method (BACTEC™ 460TB System, BD, Sparks, MD, USA). RMP = rifampicin; INH = isoniazid; SM = streptomycin; EMB = ethambutol; R = resistant; S = susceptible.
All bioethical norms were adhered to in this study; approval was granted by the local committee on medical ethics. DNA was isolated using standard methods according to the online mycobacterial interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) manual (http://www.miru-vntrplus. org). MIRU-VNTR typing (24 VNTR loci) was performed as previously described.5 DNA sequencing and analysis of the rpoB, katG and fabG-inhA genes was conducted as previously described.6 The polymerase chain reaction products obtained were sequenced with an automated ABI 3730 Genetic Analyzer DNA sequencer (Applied Biosystems, Foster City, CA, USA) using the BigDye terminator kit (Applied Biosystems), per the manufacturer’s instructions. The sequences were compared with their respective wild-type sequences using SeqScape software (Applied Biosystems).6,7
RESULTS AND DISCUSSION Among the 60 M. tuberculosis isolates collected from the patients in the penitentiary system, 27 distinct MIRU-VNTR profiles were detected; 19 (31.7%) unique profiles (detected only in one isolated strain in this study), and 8 profiles comprised of clusters containing 2–14 isolates each (41 isolates, 68.3%). All of the clusters belonged to the W-Beijing family. The largest cluster comprised 14 strains, while the other clusters included 13, 3 and 2 strains each (Figure). In the TB patients from the civilian sector, 125 isolates were analysed, and a total of 77 distinct MIRU-VNTR profiles were detected; 63 (50.4%) were unique, while 14 profiles were comprised of clusters containing 2– 15 isolates each (n = 41, 49.6%). The largest cluster comprised 15 isolates, while the other clusters included 10, 6 and 2 isolates each. According to the DNA sequencing results from the 185 M. tuberculosis strains circulating in the penitentiary and civilian systems in Kazakhstan, there was a high prevalence of mutations in the rpoB gene in codon 531, which were found in 156 strains (84.3%),
leading to the replacement of serine by leucine (TCG→ TTG); in the katG gene, 183 (98.9%) mutations were found in codon 315, leading to the substitution of serine by threonine (AGC→ACC; Table 2). The percentage of strains with unique genotypes among the civilian sector was 50.4%, while that among the prison sector was 31.7%. This difference is statistically significant (χ2 4.42, P = 0.035), and may reflect low genetic diversity among M. tuberculosis strains isolated from prison TB patients, as TB transmission can occur in small groups of prisoners. However, it should be noted that we did not detect statistically significant differences in the frequency of mutations in the rpoB531 and katG315 codons between M. tuberculosis strains isolated from the civilian and penitentiary systems. The majority of nucleotide substitutions were found at codon 531 of the rpoB gene and codon 315 of the katG gene.7 These genes encode enzymes that are of biological significance in the bacteria, and mutations in these genes could impair enzyme function, leading to a fitness cost. Previous studies have shown that this biological cost can vary according to the particular mutation (i.e., the amino acid and position involved) and the genetic background of the strain. The degree to which the mutation affects the fitness of the organism varies depending on the specific drug resistance mutation, the environment and the genetic background of the strain.8 Genetic clustering has been applied extensively in molecular epidemiological studies as a measure of ongoing TB transmission.8 In recent years, numerous molecular epidemiological studies have been conducted to clarify the epidemiology of TB among different population groups.4,6 Previously published data show that the genetic diversity of TB strains in open systems ranges from 50% to 80%, whereas in closed systems (e.g., hospitals and prisons) it ranges from 20% to 40%.4,9 Our results are consistent with these findings regarding the genetic diversity of TB among civilians in Kazakhstan (50.4%) vs. the penitentiary system (31.7%). Comparing the strains circulating in the open vs. closed systems, we detected
Figure Dendogram of the phylogenetic similarity of Mycobacterium tuberculosis strains isolated in the Kazakhstan penitentiary and those in civilian populations according to MIRU cluster analysis. The order of the 24 MIRU loci is as follows, from left to right: 154, 580, 960, 1644, 2059, 2531, 2687, 2996, 3007, 3192, 4348, 802, 2156, 2461, 577, 424, 1955, 2347, 2401, 3171, 3690, 2136b, 4052 and 4156. NC1 = non-clustered isolates; K (2–15 is the number of isolates) = MIRU clusters; MIRU = mycobacterial interspersed repetitive units.
Molecular epidemiology of M. tuberculosis strains
301
Table 2 Mutations in genes involved in rifampicin and isoniazid resistance of M. tuberculosis isolates in the Kazakhstan penitentiary and civil system in 2012 Isolates with mutations
Gene Rifampicin-resistant isolates rpoB
Penitentiary system (n = 60) n (%)
Civil system (n = 125) n (%)
Total (n = 185) n (%)
Affected codon
Nucleotide changes
Amino acid change
531 526
TCG→TTG CAC→TAC CAC→GAC CAC→CTC CTG→CCG
Ser→Leu His→Arg His→Asp His→Leu Leu→Pro
53 (88.3) 2 (3.3) 2 (3.3) 1 (1.8) — 2 (3.3)
103 (82.4) 9 (7.2) 2 (1.6) 3 (2.4) 1 (0.8) 7 (5.6)
156 (84.3) 11 (6.0) 4 (2.1) 4 (2.1) 1 (0.5) 9 (5.0)
AGC→ACC
Ser→Thr
60 (100) — —
123 (98.4) — 2 (1.6)
183 (98.9) 0 2 (1.1)
533 None Isoniazid-resistant isolates katG 315 fabG-inhA None
the same spectrum of mutations causing multidrug resistance. In contrast, the percentage of genetic heterogeneity in prisons was significantly lower, indicating circulation of the most virulent strains in this closed system. The main reason for the formation of these clusters is exogenous infection resulting from recent transmission and clonal expansion.9,10 An additional factor is the long persistence of the bacteria in the closed system, during which time natural selection occurs, favouring the most virulent strains and leading to the formation of a homogeneous environment. It can be concluded that individuals showing the same spectrum of mutations harbouring clustered isolates are expected to belong to a transmission chain involving the most virulent TB strains in a closed system, whereas strains found in individuals harbouring unique isolates are expected to circulate in an open system. In M. tuberculosis, different strains have been shown to differ in their immunogenicity and virulence in animal models.11 There is also increasing evidence that strain diversity can influence the outcome of infection and disease in humans. Genomic analyses of strain collections from global sources have revealed that M. tuberculosis shows a phylogeographic population structure in which different strain lineages are associated with particular geographic regions. A recent theoretical study found that simulated populations with immunologically distinct strain groups displayed a higher risk of drug resistance than populations without strain diversity, even when the quality of TB control was the same.12 Patients with infectious TB who are imprisoned and those who are released from prison before the completion of treatment may play an important role in the epidemiology of TB disease. Close coordination between TB programmes targeting prison and civilian communities is thus important.
Conflict of interest: none declared.
References 1 World Health Organization. Global tuberculosis report, 2012. WHO/HTM/TB/2012.6. Geneva, Switzerland: WHO, 2012. 2 Ministry of Health, Republic of Kazakhstan. [Statistic TB review]. Almaty, Kazakhstan: National TB Center, 2012. [Russian] 3 Pfyffer G, Strassle A, van Gorkum T, et al. Multidrug-resistant tuberculosis in prison inmates, Azerbaijan. Emerg Infect Dis 2001; 7: 855–861. 4 Chernyaeva E, Dobrynin P, Pestova N, Matveeva N, Zhemkov V, Kozlov A. Molecular genetic analysis of Mycobacterium tuberculosis strains spread in different patient groups in St. Petersburg, Russia. Eur J Clin Microbiol Infect Dis 2012; 31: 1753– 1757. 5 Supply P, Allix C, Lesjean S, et al. Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variablenumber tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 2006; 44: 4498–4510. 6 Sekiguchi J, Miyoshi-Akiyama T, Augustynowicz-Kopec´ E, et al. Detection of multidrug resistance in Mycobacterium tuberculosis. J Clin Microbiol 2007; 45: 179–192. 7 Kozhamkulov U, Akhmetova A, Rakhimova S, et al. Molecular characterization of rifampicin and isoniazid-resistant Mycobacterium tuberculosis strains isolated in Kazakhstan. Jpn J Infect Dis 2011; 64: 253–255. 8 Gagneux S, Burgos M, De Riemer K, et al. Impact of bacterial genetics on the transmission of isoniazid-resistant Mycobacterium tuberculosis. PLOS Pathog 2006; 2: 603–610. 9 Franzetti F, Codecasa L, Matteelli A, et al. Genotyping analyses of tuberculosis transmission among immigrant residents in Italy. Clin Microbiol Infect 2010; 16: 1149–1154. 10 Cooksey R C, Abbadi S H, Woodley C L, et al. Characterization of Mycobacterium tuberculosis complex isolates from the cerebrospinal fluid of meningitis patients at six fever hospitals in Egypt. J Clin Microbiol 2002; 40: 1651–1655. 11 Gagneux S, Small P M. Global phylogeography of Mycobacterium tuberculosis and implications for tuberculosis product development. Lancet Infect Dis 2007; 7: 328–337. 12 Borrell S, Gagneux S. Infectiousness, reproductive fitness and evolution of drug-resistant Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2009; 13: 1456–1466.
Molecular epidemiology of M. tuberculosis strains
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RÉSUMÉ
Une série de 60 souches de Mycobacterium tuberculosis recueillis auprès de patients détenus dans des prisons du Kazakhstan et 125 souches de patients non détenus a été examinée avec l’analyse par la méthode des unités répétitives dispersées sur le génome mycobactérien– nombre variable de répétitions en tandem en 2012. Nous avons constaté que le pourcentage de souches de TB avec un génotype unique isolé chez des patients civils était de 50,4% contre 31,7% chez les détenus. La différence
était statistiquement significative (χ2 4,42 ; P = 0,035) et pourrait refléter une faible diversité génétique des souches de M. tuberculosis isolées chez des détenus. La fréquence des mutations des gènes rpoB531 et katG315 de M. tuberculosis isolées chez des civils et les détenus n’était pas significativement différente (rpoB531 : 82,4% contre 88,3% et katG315 : 98,4% contre 100%, respectivement).
RESUMEN
Se practicó la genotipificación con marcadores para locus múltiples de las secuencias repetitivas en tándem (MIRU-VNTR) de 60 aislados clínicos de Mycobacterium tuberculosis recogidos en pacientes de las prisiones de Kazakstán y 125 aislados de pacientes del sector civil en el 2012. Se observó que el porcentaje de cepas de M. tuberculosis con genotipos únicos aislados de los pacientes del sector civil era 50,4% y en los pacientes en prisión era 31,7%. Esta diferencia alcanzó significación
estadística (χ2 4,42; P = 0,035) y puede corresponder a una baja diversidad genética de las cepas de M. tuberculosis aisladas en los pacientes de las prisiones. La diferencia de frecuencia de las mutaciones en los genes rpoB531 y katG315 de las cepas de M. tuberculosis aisladas en personas en el sistema penitenciario y en la población civil no fue estadísticamente significativa (rpoB531: 82,4% contra 88,3% y katG315: 98,4% contra 100%, respectivamente).
