Transmission of Drug-Resistant Tuberculosis between ...Central rii cellece i e ccess JSM Bioavailability and Bioequivalence Cite this article: Kozińska M, Napiórkowska A, Brzezińska

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Cite this article: Kozińska M, Napiórkowska A, Brzezińska S, Zabost A, Borkowska D, et al. (2017) Transmission of Drug-Resistant Tuberculosis between House-hold Contacts. JSM Bioequiv Bioavailab 1(1): 1004.

*Corresponding authorMonika Kozińska, Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Plocka 26, 01-138 Warsaw, Poland; Tel: 48-22-4312182; Fax: 48-22-43182; Email:

Submitted: 04 April 2017

Accepted: 09 May 2017

Published: 11 May 2017

Copyright© 2017 Kozińska et al.

OPEN ACCESS

Keywords• Tuberculosis; Drug-resistant; Household; Transmission;

Genotyping

Research Article

Transmission of Drug-Resistant Tuberculosis between Household ContactsMonika Kozińska*, Agnieszka Napiórkowska, Sylwia Brzezińska, Anna Zabost, Dagmara Borkowska, Zofia Zwolska, and Ewa Augustynowicz-KopećDepartment of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Poland

Abstract

Objective: The most important measures in the control of the spread of drug-resistant tuberculosis (DR-TB) are the early detection of smear-positive patients, implementation of appropriate treatment, and tracing the chain of transmission of infection. Research on TB transmission in the environment of patients indicates that the risk of infection between close contacts, especially family members, is very high. The purpose of this study was to investigate the transmission of DR-TB within 6 family-households identified during the period of 2006-2016 in Poland.

Methods: Two PCR-based genotyping methods were used: spoligotyping, and mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) typing.

Results: According to spoligotyping and MIRU-VNTR results, in all households, patients had identical Mycobacterium tuberculosis isolates, implying intra-familial transmission. Isolates from 2 families with Beijing-TB represented the pre-XDR and XDR phenotypes.

Conclusions: This study demonstrates the household setting as an important pathway of drug-resistant Mycobacterium tuberculosis transmission, and thereby reinforces the need for routine extensive screening of the housemates of TB patients.

ABBREVIATIONSTB: Tuberculosis; IGRA: Interferon Gamma Release Assay;

MIRU-VNTR: Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats; DR: Drug Resistant; DS: Drug Sensitive; NTRL: National Tuberculosis Reference Laboratory; MTB: Mycobacterium Tuberculosis; AFB: Acid-Fast Bacilli; L-J: Lowenstein-Jensen; INH: Isoniazid; RMP: Rifampicin; SM: Streptomycin; EMB: Ethambutol; OFL: Ofloxacin; KAN: Kanamycin; AMK: Amikacin; CAP: Capreomycin; CTAB: Cetyltrimethyl-Ammonium Bromide; Pre-XDR: Pre-Extensively Drug-Resistant; XDR: Extensively Drug-Resistant; MDR: Multidrug-Resistant; H: Haarlem; LAM: Latin American Mediterranean; ST: Shared Type; NR: Not Registered

INTRODUCTIONTuberculosis (TB) is an infectious disease most commonly

transmitted by airborne transfer of bacteria in droplet nuclei. The most important measures in the control of the spread of the disease are the early detection of smear-positive patients, implementation of appropriate treatment, and tracing the chain of transmission of infection. Many years of research on TB transmission indicate that the risk of infection among close contacts in prisons, hospitals, schools, homeless shelters, migrants’ shelters, and other space-restricted and crowded environments, is very high [1-5]. Epidemiological investigation

should therefore be extended to all persons who have contact with the TB patient, and special attention should be paid to the people living in the close proximity of patients with drug-resistant tuberculosis. Normally, the closest contacts of a positively diagnosed TB patient, including housemates, are the first to be investigated, and depending on the number of positive contacts traced in subsequent circles around the patient, this contact tracing is extended. Generally, conventional contact tracing by interview and skin and/or interferon gamma release assay (IGRA) testing of possibly exposed individuals is considered an effective strategy for identifying recent TB transmission.

However, although proximity to a smear-positive patient is a known risk factor for infection and disease, early detection of subsequent infection and disease close to TB patients remains poor in many countries. In Poland, detection has improvement recently (2012−2015), but still only 3% of cases are detected [6]. The purpose of this study was to investigate the transmission of drug-resistant TB within family-households in Poland, by using two PCR-based genotyping methods – spoligotyping, and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing [7,8].

MATERIALS AND METHODSPatients

The study included 14 patients with pulmonary drug-resistant

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(DR) tuberculosis, who were members of 6 separate family-households (Table 1). Selected family members represented 9% of all TB cases detected and bacteriologically confirmed (n=158) among 62 households during 2006-2016 in Poland. Family-households (n=56) not included in this analysis represented drug sensitive (DS) TB cases and were the subject of other studies. Microbiological data were obtained from the laboratory records, and patient information (medical history, demographic data, including age, gender, ethnicity, socio-economic status, and marital status) was gathered from clinical documents. All patients were Polish, except two who were Chechens. In the study group, 7 (50%) patients were males and 7 (50%) were females. The patients were aged between 12 and 59 years. Eleven (79%) patients had no history of TB treatment, whereas 3 (21%) patients had received anti-TB therapy previously.

The term ‘family’ refers to a group of two or more people related by blood ties or marriage. The term ‘household’ was applied to a group of two or more people residing in the same dwelling. An ‘index case’ was defined as the first TB case diagnosed in a household, according to the date of collection of the first culture-positive sample. All subsequently identified cases in the same household were defined as secondary cases.

Bacterial isolates

All Mycobacterium tuberculosis (MTB) isolates used in this study were collected by the National Tuberculosis Reference Laboratory (NTRL) in Warsaw, Poland. Primary processing of specimens, which included sputum (n = 13) and bronchoalveolar lavage (BAL, n = 1), isolation and cultivation, species-level identification, and drug susceptibility testing (DST) were carried out in local TB laboratories using classical mycobacteriological procedures. The isolates were subcultured and sent to the NTRL, where confirmatory identification and drug susceptibility testing were performed. The strains were identified as MTB on the basis of a niacin test and a DNA strip assay (GenoTypeMTBC (Hain Lifescience, Germany)). The acid-fast bacilli (AFB) smear status of the sputum or BAL specimen was assessed by Ziehl-Neelsen staining and microscopy. Drug susceptibility testing was performed by use of the 1% proportion method on Lowenstein-Jensen (L-J) medium described elsewhere [9]. The following concentrations were used: isoniazid (INH) 0.2 mg/mL; rifampicin (RMP) 40.0 mg/mL; streptomycin (SM) 4.0 mg/mL; ethambutol (EMB) 2.0 mg/mL; ofloxacin (OFL) 2.5 mg/mL; kanamycin (KAN) 30.0 mg/mL; amikacin (AMK) 40.0 mg/mL; and capreomycin (CAP) 40 mg/mL.

Genotyping methods

Genomic DNA was isolated from L-J slants using the cetyltrimethyl-ammonium bromide (CTAB) method, as described elsewhere [10]. Spoligotyping was performed with a commercially available kit (Isogen Bioscience BV, Maarssen, The Neetherlands) according to manufaturer’s instructions and as described by Kamerbeek et al., [7]. MIRU-VNTR typing was done by the amplification of the 15 independent MIRU-VNTR loci, i.e.: 580 (MIRU04 or ETRD), 960 (MIRU10), 1644 (MIRU16), 2996 (MIRU26), 3192 (MIRU31 or ETRE), 802 (MIRU40), 424 (VNTR42), 577 (VNTR43 or ETRC), 2165 (ETRA), 2401 (VNTR47), 3690 (VNTR52), 4156 (VNTR53 or QUB-4156c), 2163b (QUB-11b), 1955, and 4052 (QUB-26), as described elsewhere [11,12].

RESULTS AND DISCUSSION It was found that, according to spoligotyping and MIRU-VNTR

results, all patients within the same household had identical Mycobacterium tuberculosis isolates, thus implying the intra-familial transmission.

Drug susceptibility testing revealed that MTB isolates from a total of 14 patients studied were resistant at least to one antimicrobial agent tested (Table 1). In two families (A and E) with two patients each, infected family members were infected with INH-mono-resistant phenotype MTB. All isolates from family B exhibited resistance to SM. In family D, isolates from wife and husband were resistant to INH and SM. In the remaining 2 families (C and E), family members were infected with pre-XDR (pre-extensively drug-resistant) bacteria, defined as MTB resistance to at least INH and RMP, which is obligatory for multidrug resistance in addition to either a fluoroquinolone or one of the injectable aminoglycosides (AMK, KAN, or CAP), and XDR (extensively drug-resistant) phenotype, defined as resistance to INH, RMP, one of the fluoroquinolone and one of the injectable aminoglycosides [13].

Sputum smears for AFB were positive in 7 patients in 5 families (71% of all families under study). Four of them represented ‘index case’ status. In 2 (29%) families, all patients were smear positive, and in the remaining 1 (14%) family 2 patients were negative for AFB microscopy (Table 1).

As demonstrated in other studies, in a household with a person with active tuberculosis, 40-50% of the remaining members of the family can be infected, and approximately 5% of them will develop the active form of the disease [14-16]. Families with a history of drug resistant TB and young children are particularly at risk. Such families should be monitored closely and, according to experts, surveillance of family members and others in close proximity should last 2 years in the case of MDR-TB, and 4 years in the event of XDR-TB [14]. Adults are generally responsible for infection of children due to the fact that children always live in a household with other people, whereas adults may live alone. In our study there was a family-household where the mother (index case) was the source of the TB infection in the 12-year-old son. It is possible that both infections could have happened outside the household, and the child developed symptoms faster. However, as child transmitters were more likely to be smear and culture positive (and thereby have the potential to transmit) compared to non-transmitters, we should not assume that adults within the household are always the index case. Exposure to infectious TB may have occurred within the community, with the age of the child influencing both their risk of exposure and the likelihood of transmitting to others.

In Poland, research has focused on the transmission of drug-resistant TB as an accidental phenomenon, rather than the result of close, frequent, long lasting contact. It has been proven that close populations also transmit TB, and supply on average 30-40% of study subjects [17,18]. Although incidence rates for multidrug-resistant-TB (MDR-TB) in Poland remain stable, it is disturbing from an epidemiological point of view to identify MDR-TB forms among newly diagnosed patients, as is observed in our analysis. This may indicate, among other things, an insufficient control of transmission of MDR-TB in Poland.

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Table 1: Selected demographic and epidemiologic characteristics of 14 TB patients, members of 6 families, investigated in the studyFam-

ilyCity of

residenceEthnic-

ityPa-

tientFamily

member Sex Age History of TB Specimen

AFB Date of isolation Drug resistance

smear

A Warszawa Polish

1A mother* F 36 No plwocina + 05.08.2006 INH

2A son M 12 Nobronchoal-

veolar - 23.08.2006 INHlavage

B Koszalin Polish

1B daughter* F 23 No sputum - 01.03.2008 SM2B father M 58 Yes sputum - 01.09.2008 SM3B son M 29 No sputum - 01.09.2008 SM4B son M 27 No sputum +++ 20.10.2008 SM

C Łódź Polish2C mother* F 52 Yes sputum + 06.08.2008 pre-XDR1C daughter F 24 Yes sputum - 22.08.2008 (INH+RMP+SM+EMB+OFL)

D Warszawa Chech-nya

1D wife* F 28 No sputum - 05.02.2009 INH+SM2D husband M 39 No sputum - 21.02.2010 INH+SM

E Radom Polish1E sister* F 26 No sputum + 01.09.2016 INH2E sister F 26 No sputum + 26.09.2016 INH

F Warszawa Polish

1F father in law* M 36 No sputum + 01.03.2015

XDR(INH+RMP+SM+EMB+OFL+

AMK+KAN+CAP)

2F son in law M 59 No sputum + 04.02.2017XDR

(INH+RMP+SM+EMB+OFL+AMK+KAN+CAP)

Abbreviations: TB: Tuberculosis; AFB: Acid-Fast Bacilli; INH: Isoniazid; RMP: Rifampicin; SM: Streptomycin; EMB: Ethambutol; OFL: Ofloxacin; KAN: Kanamycin; AMK: Amikacin; CAP: Capreomycin; Pre-XDR: Pre-extensively Drug-Resistant; XDR: Extensively Drug-Resistant

Table 2: MIRU-VNTR profiles and spoligotypes identified among Mycobacterium tuberculosis isolates evaluated in this study. Comparison with the SITVIT database and clade assignment

Strain Family ST CladeSpoligotype description

MIRU-VNTR typing patternBinary Octal

1AA

36 H3 ■■■■■■■■■■■■□■■■■■■■■■■■■■■■■■□■□□□□■■■■■■■ 777737777720771 343635333434337

2A 36 H3 ■■■■■■■■■■■■□■■■■■■■■■■■■■■■■■□■□□□□■■■■■■■ 777737777720771 343635333434337

1B

B

803 LAM9 ■■■■■■■■■■■■■□□□□□□□□□□□■■■■■■■■□□□□■■■■■■■ 777740007760771 343526422123035

2B 803 LAM9 ■■■■■■■■■■■■■□□□□□□□□□□□■■■■■■■■□□□□■■■■■■■ 777740007760771 343526422123035

3B 803 LAM9 ■■■■■■■■■■■■■□□□□□□□□□□□■■■■■■■■□□□□■■■■■■■ 777740007760771 343526422123035

4B 803 LAM9 ■■■■■■■■■■■■■□□□□□□□□□□□■■■■■■■■□□□□■■■■■■■ 777740007760771 343526422123035

1CC

NR ■■■■■■■■■■■■■■■■■■□■■■□□■■■■■■■■□□□□■■□■■■■ 777777347760671 343444333443345

2C NR ■■■■■■■■■■■■■■■■■■□■■■□□■■■■■■■■□□□□■■□■■■■ 777777347760671 343444333443345

1DD

1 Beijing □□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□■■■■■■■■■ 000000000003771 343753544441458

2D 1 Beijing □□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□■■■■■■■■■ 000000000003771 343753544441458

1EE

53 T1 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■■■■■■■ 777777777760771 331634242232355

2E 53 T1 ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■■■■■■■ 777777777760771 331634242232355

1FF

265 Beijing □□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□■■□■■■■■■ 000000000003371 342743543341359

2F 265 Beijing □□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□■■□■■■■■■ 000000000003371 342743543341359

Abbreviations: NR: Not Registered; ST: Shared Type; MIRU-VNTR: Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats

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The molecular strain typing performed in this study also shed some light on the epidemiology of TB in a wider, whole-country context. Comparison of the spoligotyping results with a worldwide database SITVIT (http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE/) provided insights into the phylogenetic diversity of the Mycobacterium tuberculosis strains circulating in Poland. It is of note, that at the phylogenetic level all patient isolates fell into four major clades, i.e. the Haarlem (H) clade (2 patients from family A), T clade (2 from family E), the Latin American Mediterranean (LAM) clade (4 members of family B), and the Beijing clade (4 patients from families D and E). In the remaining family (C), isolates exhibited a spoligotype not registered (NR) in the SITVITWEB database (Table 2). All the clades identified in this study had been reported in Poland previously [19]. Among these, T and H are the most prevalent European genotypes [20,21]. Members of two family-households in the study were infected with MTBC strains of the Beijing genotype, which has been associated with an increased acquisition of drug resistance, enhanced virulence, and high transmissibility [22]. This finding, together with previous reports on the occurrence of the Beijing strains in Poland implies their ongoing transmission within the country. Based on the results from past studies and this one (one family where Beijing strains was noted originated from Chechnya), it seems that the main source of Beijing family strains in Poland is immigration over Poland’s eastern border. It is of note that isolates from 2 families with Beijing-TB represented a pre-XDR and XDR phenotype. This suggests that, in accordance with previously described observations, in Poland the Beijing genotype can be correlated with a drug-resistant phenotype [21]. Although spoligotyping revealed some characteristics of the population structure of Mycobacterium tuberculosis in Poland, a relatively small sample size and the confinement of the study to only one type of setting precluded an accurate estimation of the share of the genotypes identified in cases of recent TB transmission in Poland.

CONCLUSIONThis study demonstrates the household setting as an

important site of drug-resistant Mycobacterium tuberculosis transmission, and thereby reinforces the need for routine, extensive screening of close contacts of TB patients. The most important assumption in this study was that finding at least two patients within the same household whose MTBC isolates had identical genotypes was indicative of the intra-familial transmission. However, neither the source case nor the direction of the transmission within a given family-household could be determined with certainty. Although analysis of clinical and demographic data would make it seem very likely that the location of the source case was within the family-household, the possibility that family members hosting the same Mycobacterium tuberculosis strain could have been infected by a source outside the household cannot excluded.

FUNDINGThis article has been prepared under the research project

granted by the J. hr. Potocki Foundation and partial funding comes from the statutory activity of National Tuberculosis and Lung Diseases Research Institute, Task No 1.5.

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