Abstract
Objectives
Tuberculosis (TB) is an airborne infectious disease transmitted through droplets from coughing or sneezing. The risk of Mycobacterium tuberculosis complex infection is high among close contacts sharing living or working spaces with patients with TB. The Korea Disease Control and Prevention Agency conducts close contact investigations targeting household contacts to prevent TB spread through early detection and treatment. As part of these investigations, TB strain genotyping is performed to analyze the molecular epidemiological links.
Methods
From 2016 to 2024, the epidemiological information and molecular epidemiological characteristics of 20 suspected cases of household transmission involving 43 individuals were analyzed following their identification through investigations. Spoligotyping and 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat were used for TB genotyping.
Results
Each case of suspected intrafamilial TB transmission involved 2–3 patients, including parent-child (15 cases), sibling (4 cases), and cohabitant (1 case) transmissions. At the time of index notification, 12 cases (60%) involved cohabitation, while 7 cases (35%) did not. The time difference between the index and additional case notifications ranged from 2 to 2,241 (average 291) days. The TB genotyping results confirmed molecular epidemiological links between each suspected case. Among the 20 cases, 5 cases (involving 10 individuals) presented drug-resistant TB, with 4 cases of multidrug-resistant TB and 1 of extensively drug-resistant TB. Drug resistance profiles were consistent within each case.
Conclusions
Based on the TB genotyping and contact investigations, cases of TB transmission within the household appear to have epidemiological links. To support the national TB control efforts, we plan to continue molecular epidemiological analyses of household transmission and outbreak cases.
Keywords: Tuberculosis, Household, Transmission, Molecular epidemiology
Key messages
① What is known previously?
Family members sharing living environments with patients with tuberculosis (TB) are known to be at a higher risk of TB infection.
② What new information is presented?
Genotyping of the Mycobacterium tuberculosis complex (MTBC) was performed for 20 suspected cases of household transmission between 2016 and 2024, and molecular epidemiological links between MTBC strains isolated from household members were confirmed.
③ What are implications?
For the prevention and management of TB transmission within households, MTBC genotyping and contact investigations are necessary to analyze the TB transmission routes.
Introduction
Tuberculosis (TB) is designated and managed as a second-class legal infectious disease in Republic of Korea (ROK) caused by the Mycobacterium tuberculosis (MTB) complex. As of 2022, ROK ranked second in TB incidence and fourth in mortality among the Organisation for Economic Co-operation and Development member countries [1,2]. From 39,557 cases in 2011 (78.9 per 100,000 population) to 15,640 cases in 2023 (30.6 per 100,000 population), there has been a steady decline in new cases. However, continued efforts are still needed to prevent and control TB [2].
The MTB complex is primarily transmitted through airborne droplets without a vector, and because symptoms often develop slowly, the difficulties in early recognition of TB can lead to a delayed diagnosis, making it easier to spread. The transmission of TB is known to be high, especially in congregate settings, such as homes, schools, and hospitals, where people may have frequent contact with patients with TB and share daily routines [3-5]. TB outbreaks in congregate settings can lead to large-scale epidemics, making it a significant public health concern.
In 2023, the Korea Disease Control and Prevention Agency (KDCA) conducted an epidemiologic investigation of 11,596 TB cases, in which TB testing was performed on 21,502 household contacts and 86,302 contacts in congregate settings. As a result, 200 additional patients were identified, of which 99 were family contacts (460.4 per 100,000 population), which is approximately 15 times higher than the 2023 new case rate (30.6 per 100,000 population) [6]. In addition, genetic analysis of the MTB complex isolated from the index and additional cases in families in Poland and the United Kingdom has shown high concordance rates of 90% and 75%, respectively, indicating high levels of familial transmission [7,8]. The risk and importance of managing TB outbreaks have been emphasized more in households than in other populations because households may include immunocompromised members, such as infants, the elderly, and chronically ill individuals, who live closely together for a long time in an enclosed space [7-9].
Since TB takes a long time to develop symptoms after the infection, it is difficult to analyze the route of infection among the patients, recognize outbreaks early, and differentiate between relapses and reinfections using conventional epidemiologic surveys. For this reason, molecular epidemiological analysis methods are being used worldwide to identify the infection route of MTB. The KDCA currently operates the Korean National TB Surveillance System to identify the phylogenetic links through the genetic characterization of domestic outbreaks and establish a database to provide scientific evidence for the prevention and control of MTB transmission.
This study aims to identify the molecular associations and characteristics of suspected familial transmission cases identified through TB epidemiologic surveillance from 2016 to 2024 by genotyping to serve as a basis for national TB control.
Methods
1. Study Participants and DNA Extraction
Of the 521 cases identified as clusters from 2016 to 2024, 20 cases (43 individuals) with suspected familial transmission were selected, and patient information, drug susceptibility test results, and culture strains were additionally collected via epidemiologic surveillance.
The collected strains were inactivated in a biosafety level 3 laboratory, and DNA was extracted using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research) in a biosafety level 2 laboratory and used in the study.
2. Spoligotyping and 24-loci MIRU-VNTR Genotyping
Spoligotyping was performed according to the manufacturer’s instructions using a spoligotyping kit (Ocimum Biosolutions), and the results were analyzed for MTB phylogeny using the SITVIT2 (http://pasteur-guadeloupe.fr.8081/SITVIT2) and TB insight (https://tbinsight.cs.rpi.edu) databases.
In the 24-loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) genotyping process, the amplified polymerase chain reaction (PCR) products were fragment analyzed on an ABI 3500xL DNA Analyzer (Applied Biosystems), and the copy number of each locus was determined using GeneMapper 4.0 (Applied Biosystems) based on GeneScan 1200 (Applied Biosystems) [9].
3. Analysis of Molecular Typing
The spoligotyping and MIRU-VNTR genotyping patterns were quantified in Excel spreadsheets (Microsoft) and analyzed using the BioNumerics 8.0 (Applied Maths) software. To calculate the similarity between patterns, the unweighted pair group method with arithmetic mean (UPGMA) algorithm was applied to create a dendrogram, and the TB genotypes were categorized by sequentially assigning four-digit numbers. A molecular link was determined to exist if two or more patients with TB had contact and the isolates were genotypically identical or within two alleles of each other at one locus by MIRU-VNTR genotyping analysis [10,11].
Results
1. Distribution of Tuberculosis Transmission Cases within Families
From 2016 to 2024, 20 suspected cases of family-to-family transmission were identified through TB epidemiologic surveillance, 43 patients were identified. The nationalities of these patients were 38 in ROK, 3 in Uganda, 2 in Vietnam, with 26 (60.5%) being men and 17 (39.5%) being women. The patients ranged in age from 0 to 61 years (mean, 31.7 years), with three patients under 5 years of age, one patient in the 5–12 age group, nine patients in the 13–19 age group, and 13 (30.2%) minor patients. The patients included 15 parents (34.9%), 17 children (39.5%), eight siblings (18.6%), one relative (2.3%), and two cohabitants (4.7%).
Each of the 20 suspected cases consisted of two to three patients with TB. The largest number of suspected TB transmission cases were parent-to-child with 15 cases (75%), followed by siblings with 4 cases, and cohabitants with 1 case. The living arrangements at the time of the reporting of the index patient were cohabitation in 12 cases (60%) and non-cohabitation in 7 cases (35%). The remaining case involved a family of three, in which two were cohabiting and one was living separately (Table 1).
Table 1. Epidemiologic and molecular characteristics of 43 patients, members of 20 families in household transmission in Republic of Korea.
| No. | Case | Difference of notification date | Sex | Age (yr) | Relationship | History of TB | Living arrangement | Nationality | TBG | |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | A | A1 | 0 | M | 14 | Son | - | Cohabitation | Korea | TBG0338 |
| 2 | A2 | 9 | M | 51 | Father | - | Cohabitation | Korea | TBG0338 | |
| 3 | A3 | 38 | F | 19 | Daughter | - | Cohabitation | Korea | TBG0338 | |
| 4 | B | B1 | 0 | M | 51 | Father | - | Cohabitation | Korea | TBG5882 |
| 5 | B2 | 133 | M | 22 | Son | - | Cohabitation | Korea | TBG5882 | |
| 6 | C | C1 | 0 | F | 54 | Mother | - | Non-cohabitation | Korea | TBG0084 |
| 7 | C2 | 116 | M | 28 | Son | - | Non-cohabitation | Korea | TBG0084 | |
| 8 | D | D1 | 0 | M | 20 | Brother | - | Non-cohabitation | Vietnam | TBG1054 |
| 9 | D2 | 84 | M | 20 | Brother | - | Non-cohabitation | Vietnam | TBG1054 | |
| 10 | E | E1 | 0 | F | 27 | Live together | - | Cohabitation | Korea | TBG2397 |
| 11 | E2 | 2,241 | F | 30 | Live together | - | Cohabitation | Korea | TBG2397 | |
| 12 | F | F1 | 0 | M | 57 | Brother | O | Cohabitation | Korea | TBG5964 |
| 13 | F2 | 88 | F | 55 | Sister | O | Cohabitation | Korea | TBG5965 | |
| 14 | G | G1 | 0 | M | 29 | Son | - | Cohabitation | Korea | TBG0014 |
| 15 | G2 | 188 | F | 61 | Mother | - | Cohabitation | Korea | TBG0014 | |
| 16 | H | H1 | 0 | F | 20 | Daughter | - | Cohabitation | Korea | TBG5916 |
| 17 | H2 | 7 | M | 52 | Father | - | Cohabitation | Korea | TBG5916 | |
| 18 | I | I1 | 0 | F | 18 | Daughter | - | Cohabitation | Korea | TBG0035 |
| 19 | I2 | 6 | F | 50 | Mother | O | Cohabitation | Korea | TBG0035 | |
| 20 | J | J1 | 0 | M | 48 | Father | O | Cohabitation | Korea | TBG0644 |
| 21 | J2 | 14 | M | 14 | Son | - | Cohabitation | Korea | TBG0644 | |
| 22 | K | K1 | 0 | M | 28 | Father | - | Non-cohabitation | Korea | TBG0548 |
| 23 | K2 | 186 | M | 57 | Son | - | Non-cohabitation | Korea | TBG0546 | |
| 24 | L | L1 | 0 | M | 57 | Father | O | Non-cohabitation | Korea | TBG5676 |
| 25 | L2 | 15 | M | 25 | Son | - | Non-cohabitation | Korea | TBG5676 | |
| 26 | M | M1 | 0 | F | 18 | Daughter | - | Non-cohabitation | Korea | TBG5675 |
| 27 | M2 | 34 | M | 51 | Father | - | Non-cohabitation | Korea | TBG5675 | |
| 28 | N | N1 | 0 | F | 17 | Daughter | - | Cohabitation | Korea | TBG2121 |
| 29 | N2 | 8 | M | 49 | Father | - | Cohabitation | Korea | TBG2121 | |
| 30 | O | O1 | 0 | M | 23 | Brother | - | Cohabitation | Korea | TBG0938 |
| 31 | O2 | 29 | M | 21 | Brother | - | Cohabitation | Korea | TBG0938 | |
| 32 | P | P1 | 0 | F | 24 | Daughter | - | Non-cohabitation | Korea | TBG0569 |
| 33 | P2 | 484 | F | 55 | Mother | O | Non-cohabitation | Korea | TBG0569 | |
| 34 | Q | Q1 | 0 | M | 7 | Brother | - | Non-cohabitation | Korea | TBG4550 |
| 35 | Q2 | 7 | M | 4 | Brother | - | Non-cohabitation | Korea | TBG4550 | |
| 36 | R | R1 | 0 | F | 35 | Mother | - | Cohabitation | Uganda | TBG4706 |
| 37 | R2 | 2 | F | 0 | Daughter | - | Cohabitation | Uganda | TBG4706 | |
| 38 | R3 | 2 | M | 0 | Son | - | Cohabitation | Uganda | TBG4706 | |
| 39 | S | S1 | 0 | M | 52 | Father | - | Cohabitation | Korea | TBG1050 |
| 40 | S2 | 1,444 | M | 16 | Son | - | Cohabitation | Korea | TBG1050 | |
| 41 | T | T1 | 0 | F | 18 | Cousin | - | Non-cohabitation | Korea | TBG1462 |
| 42 | T2 | 778 | F | 14 | Daughter | - | Cohabitation | Korea | TBG1462 | |
| 43 | T3 | 778 | M | 53 | Father | - | Cohabitation | Korea | TBG1462 | |
TB=tuberculosis; TBG=tuberculosis genotype; M=male; female.
The difference in the date of occurrence between the index patient and the additional patients was 2 to 2,241 days, with an average of 291 days. The average difference in the reported date of occurrence for the four cases where the difference was more than 1 year between the index and additional patients was 1,236 days.
2. Genotyping Results
Lineages and clades were classified using SITVIT2 and TB insight databases to analyze the spoligotyping results. Results showed that the MTB complex identified in the 20 cases (43 patients) belonged to 4 lineages and 5 clades. The lineages and clades of MTB isolated from each suspected case of transmission were all identical. Of the 20 total cases, 18 cases (38 patients) were in the Beijing clade of the East-Asian lineage, one case (3 patients) was in an unknown clade of the Euro-American lineage, and one case (2 patients) was in the Central Asian strain (CAS) clade of the East-African-Indian (EAI) lineage. Of the 38 natives, 36 were in the Beijing clade of the East-Asian lineage, and the remaining two were in the CAS clade of the EAI lineage. Spoligotyping of the MTB complex isolated from three Ugandan nationals among the foreign nationals showed an unknown clade of the Euro-American lineage. Two Vietnamese nationals were categorized in the Beijing clade of the East-Asian lineage.
MTB in all patients was characterized by 22 different MIRU-VNTR patterns. The same pattern was seen in each of the 18 cases (39 patients), with 2 repeat differences in Mtub04 of 24-loci in case F (2 patients) and 1 repeat difference in Qub11b in case K (2 patients).
Based on the spoligotyping and MIRU-VNTR results, the genotypes were analyzed, and all patients were divided into 22 genotypes. In 18 cases, the genotypes of the patients with TB isolated from each case were confirmed to be identical. Cases F (TBG5964, TBG5965) and K (TBG0548, TBG0546), which showed differences in the MIRU-VNTR patterns, were determined to be molecularly related as they showed one or two allelic differences at a single locus in the MIRU-VNTR results (Figure 1).
Figure 1. Comparative dendrogram combining MIRU-VNTR and spoligotype results obtained from 20 family-households in Republic of Korea.
MIRU-VNTR=Mycobacterial interspersed repetitive unit-variable number tandem repeat; SPO=spoligotying; TBG=tuberculosis genotype.
3. Drug Resistance Profiling
Five of the 20 cases (10 patients) were found to be resistant to one or more anti-TB drugs. Four cases (8 patients) were multidrug-resistant to isoniazid and rifampin. Case F (2 patients) was classified extensively resistant TB, because it was resistance to isoniazid and rifampin, along with quinolones (levofloxacin, moxifloxacin) and group A drugs (linezolid). Both patients had a history of TB infection. The index patient in case L and the additional patient in case P had a history of TB; however, the TB drug resistance results of the index and additional patients in all five cases, including these two cases, were consistent (Table 2).
Table 2. Data on cases of household transmission of drug-resistant.
| Case | TBG | Date of notification | History of TB | Drug-resistant profile | |
|---|---|---|---|---|---|
| F | F1 | TBG5964 | 20240510 | O | Isoniazid, Rifampin, Levofloxacin, Moxifloxacin, Ofloxacin, Linezolid |
| F2 | TBG5965 | 20240805 | O | Isoniazid, Rifampin, Levofloxacin, Moxifloxacin, Ofloxacin, Linezolid | |
| G | G1 | TBG0014 | 20230303 | X | Isoniazid, Rifampin |
| G2 | TBG0014 | 20230906 | X | Isoniazid, Rifampin | |
| L | L1 | TBG5676 | 20211122 | O | Isoniazid, Rifampin |
| L2 | TBG5676 | 20211206 | X | Isoniazid, Rifampin | |
| P | P1 | TBG0569 | 20200116 | X | Isoniazid, Rifampin |
| P2 | TBG0569 | 20210513 | O | Isoniazid, Rifampin | |
| S | S1 | TBG1050 | 20160608 | X | Isoniazid, Rifampin |
| S2 | TBG1050 | 20200521 | X | Isoniazid, Rifampin | |
TBG=tuberculosis genotype; TB=tuberculosis.
Discussion
To identify the molecular association and characteristics of intrafamilial TB transmission cases in ROK, MTB isolated from 20 cases (43 patients) suspected of intrafamilial transmission from 2016 to 2024 were genotyped using spoligotyping and MIRU-VNTR.
Spoligotyping revealed that the isolated MTB bacteria belonged to the same strain and clade in each of the 20 cases (43 patients). The the Beijing clade of the East-Asian lineage, identified in 18 cases (38 patients), is the most common clade (80.5%) in ROK [12].
Genotyping confirmed that the MTB genotypes of each case were identical and that the same strain was transmitted within the families. In all 20 cases, the genotypes of each case were identical, both cohabiting and non-cohabiting families. In two cases (cases I and P), a past history of TB was identified in the additional patients. This suggests that the index patient may have been reinfected after the past infection was completely cured, or the index patient may have been infected during the past infection and then relapsed. Continued genotyping will be needed to analyze these cases.
As a result of analyzing the difference in patient reporting dates between the index patient and additional patients for each case, 17 cases were within 2 years and 3 cases were more than 2 years: 2,241 days for case E, 1,444 days for case S, and 778 days for case T. Typically, molecular epidemiological methods are applicable within 2 years from the date of onset [13]. However, a report that used MTB genotyping to determine relapse and reinfection [14] showed that in some relapsed patients, MTB with the same genotype was isolated even when the time between the first and second onset was more than 6 years. In this study, the three cases with more than 2 years between the first and second patients were determined to be transmission cases caused by MTB with the same genotype.
Internationally, countries have reported different rates of MTB genotype concordance in familial transmission cases. In South Africa, the concordance rate was only 18.7% [15], compared to high concordance rates of 90% and 75% in Poland and the United Kingdom, respectively [7,8]. These findings have been attributed to differences in the incidence of TB in each country, in which there are environments where people could easily come into contact with patients with TB [15].
The genotypes identified across all cases were different for each case. The genotype of case D (2 patients of Vietnamese nationality) was TBG1054, and a total of six cases were identified in the domestic genotype database, five of which were strains isolated from patients of Vietnamese nationality. In addition, TBG4706 in case R (comprising Ugandan nationals), was only identified in three of the cases, suggesting that TBG1054 and TBG4706 were not infected in ROK, but developed symptoms in ROK after being infected in Uganda.
In case R, the mother and her 2-month-old twin children were diagnosed almost simultaneously; congenital TB was reported to have been transmitted from the mother, who was an asymptomatic TB carrier, to her children [16].
A combined analysis of molecular epidemiological results and TB contact investigation results confirmed that cases of TB transmission within families had epidemiological links. This suggests that the prevention and management of TB within the home is vital, and the spread of TB can be effectively prevented with prompt investigation and diagnosis of contacts when a patient is infected with TB within the family. The KDCA will continue to provide data for national TB management through molecular epidemiological analyses to identify the infection routes in suspected cases of group outbreaks as well as cases of suspected intrafamilial transmission.
Acknowledgments
None.
Declarations
Ethics Statement: None.
Funding Source: None.
Conflict of Interest: The authors have no conflicts of interest to declare.
Author Contributions: Conceptualization: YMK, SWP, DHK, JIY. Data curation: YMK, JSL, MJL, SWP, JAS, SJC, JEK. Formal analysis: YMK, JSL, MJL, SWP. Investigation: YMK, JSL, MJL, SWP, JAS, SJC, JEK. Methodology: YMK, JSL, MJL, SWP, DHK, JIY. Supervision: DHK, JIY. Visualization: YMK, SWP. Writing – original draft: YMK, SWP. Writing – review & editing: DHK, JIY, JAS, SJC, JEK.
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