Prevalence and genotypic structure of mixed cultures of Mycobacterium tuberculosis among patients with HIV-associated tuberculosis in St. Petersburg

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Introduction

Infection of a patient with tuberculosis (TB) with several strains of Mycobacterium tuberculosis (mixed infection) is relatively rare, but with the development of molecular genetic research methods, such cases are attracting increasing attention [1, 2]. In certain regions of the world, especially those with high TB incidence, the frequency of mixed TB infection can reach 20–26% [3, 4].

Mixed TB infection in a patient can occur as a result of infection with several different strains (co-infection), changes in the existing strain during treatment [5], as well as due to reinfection against the background of HIV+TB co-infection or immunosuppression of other etiology [6]. Thus, a study of autopsy material from immunocompetent individuals revealed the following variants of simultaneous infection with two strains: double primary infection; reinfection with two strains; reinfection against the background of TB recurrence; reactivation of primary foci against the background of secondary disease due to reinfection [7]. Similar mechanisms of infection with multiple strains of M. tuberculosis have also been demonstrated in HIV-positive individuals [3, 8].

A significant proportion of mixed samples may reflect the active circulation of strains in risk groups. For example, among individuals in penal institutions, where infection with multiple strains of M. tuberculosis is more likely due to high population density and limited opportunities for effective sanitary measures [9].

Infection with multiple strains of M. tuberculosis reduces the effectiveness of treatment and worsens the prognosis for the patient [1, 10, 11]. It has been shown that mixed infections caused by strains with different phenotypic drug resistance can compromise the results of treatment using standard therapy regimens [8, 12]. The presence of a strain with primary resistance to even one anti-tuberculosis drug increases the risk of acquired resistance in another strain and the likelihood of an unfavorable outcome [12–14]. This is important to consider when evaluating the effectiveness of treatment regimens [15] and analyzing the epidemic situation.

Mixed infection poses a particular danger to patients with HIV+TB coinfection, as these patients are more likely than immunocompetent patients to develop more severe forms of TB, often with multiple organ and system involvement, even in the absence of reinfection and mixed infection [16].

In northwestern Russia, a study of the M. tuberculosis population found a significant predominance of Beijing genotype strains [17], especially among HIV+TB patients [18]. Against the background of immunosuppression, lack of antiretroviral therapy, and the presence of concomitant infections, severe forms of TB were associated with both highly virulent and drug-resistant strains of Beijing B0/W148, as well as with representatives of other Beijing subtypes and various genetic families of M. tuberculosis [18, 19].

In this regard, we have put forward the hypothesis that severe forms of TB in HIV+TB patients may be caused by mixed infection with strains of both the Beijing and other genotypes. Consequently, the ratio of M. tuberculosis genotypes in mixed cultures isolated from autopsy material of HIV+TB patients may differ from that in pure cultures.

The aim of the study was to assess the frequency of mixed infections caused by several strains of M. tuberculosis among HIV+TB patients in St. Petersburg, as well as to conduct a comparative analysis of the genotypic diversity of M. tuberculosis strains in mixed cultures and among pure cultures of M. tuberculosis.

Materials and methods

A retrospective study of M. tuberculosis isolates obtained from autopsy material, as well as medical records and pathological reports, was approved by the Local Ethics Committee of the Pasteur Research Institute of Epidemiology and Microbiology (protocol No. 41 dated December 14, 2017).

The study included 173 M. tuberculosis isolates from autopsy material of 70 patients with stage 4 HIV infection and generalized TB who were admitted to the St. Petersburg City Pathological Anatomy Bureau between 2012 and 2018.

Patients were included in the study based on the following criteria: clinical and pathological signs of active generalized TB; presence of HIV infection; age of patients over 18 years; permanent residence in St. Petersburg. Among the patients, there were 56 men and 14 women, with an average age of 36.4 years.

Cultures of the pathogen were isolated from the affected organs of patients; 1 culture was obtained from 11 patients, 2 cultures from 16 patients, 3 cultures from 42 patients, and 1–4 cultures from 1 patient. M. tuberculosis DNA was isolated as described previously [20]. The isolates were classified as belonging to the Beijing genotype based on the detection of the IS6110 insertion in the dnaA-dnaN locus [21], subtypes were initially determined using IS6110-RFLP typing [20], followed by comparison of the obtained profiles with the nomenclature developed at the Pasteur Research Institute of Epidemiology and Microbiology [22]. Other M. tuberculosis genotypes (non-Beijing) were identified using spoligotyping [23]. The spoligotyping profiles obtained were compared with the international SITVIT2 database (http://www.pasteur-guadeloupe.fr:8081/SITVIT2/). The LAM genotype was additionally identified by PCR-RFLP analysis with MboII endonuclease (Fermentas), detecting the LAM-specific 309G> A substitution in the Rv0129c gene [24]. Real-time PCR was performed to determine membership in the main Beijing subtypes — Central-Asian/Russian (CAR) and B0/W148 [25]. Mutations conferring resistance to isoniazid and rifampicin in the rpoB, katG, and inhA genes were determined using the Amplitube-MDR-RT test (Syntol).

Data were processed using the https://www.medcalc.org resource, calculating the odds ratio (OR). Differences between groups were considered statistically significant at a 95% confidence interval (p < 0.05).

DNA samples were classified as mixed cultures if the results of spoligotyping and IS6110-RFLP typing indicated the presence of two different genotypes (e.g., Beijing and LAM) or different subtypes of the same genotype (e.g., Beijing A0 and Beijing B0). A patient was considered to be infected with multiple strains if, based on the results of the study, an isolate from at least one affected organ could be classified as belonging to two different genotypes. Cases where M. tuberculosis isolates belonging to different genotypes or subtypes were isolated from the tissues of several organs were also classified as mixed infections.

Results

According to medical records, out of 70 HIV+TB patients with multidrug resistance (MDR), 12 (17.1%) were admitted with recurrent TB, 6 (8.6%) with chronic TB, 16 (22.9%) had recurrent TB after interrupted treatment, and 36 (51%) were diagnosed with TB for the first time, including 17 (24.3%) during their last hospitalization. Also, 8 (11.4%) patients had previously been in the penal institution, where they were first diagnosed.

The dispensary registration group was assigned in accordance with Order No. 109 of the Ministry of Health of Russia dated March 21, 2003 (as amended on June 5, 2017) “On the improvement of anti-tuberculosis measures in the Russian Federation”:

• I-A — patients with newly diagnosed disease;
• I-B — patients with recurrent TB;
• I-B — patients who interrupted treatment or were not examined at the end of the course of treatment;
• II — patients with active forms of TB of any localization with a chronic course of the disease.

Of the 173 M. tuberculosis isolates, 61 (35.3%) were isolated from the lungs, 50 (28.9%) — from the lymph nodes (LN), 28 (16.2%) from the spleen, 15 (8.6%) from the kidneys, 10 (5.8%) from the brain and meninges, and other organs.

A comprehensive assessment of the results of genotyping 173 M. tuberculosis DNA samples allowed HIV+TB patients to be divided into two groups. The first group included 15 patients with mixed infection (21.4%), from whom 42 M. tuberculosis isolates were obtained. The second group included 55 patients infected with a single pathogen variant, i.e., without mixed infection, from whom 131 M. tuberculosis isolates were obtained.

Spoliotyping revealed 22 unique profiles of different spoliotypes (SIT), of which 17 were characteristic of pure cultures and 5 had mixed profiles, indicating the presence of several strains of M. tuberculosis (Table 1).

 

Table 1. Characteristics of M. tuberculosis isolates from HIV+TB patients

 

Due to the limited resolution of spoligotyping for the Beijing genotype, all isolates of this genotype (n = 104) were further analyzed using IS6110-RFLP typing and PCR detection of specific markers to determine the main subtypes. Eleven IS6110-RFLP profiles were obtained, among which A0 (42.3%; n = 44) and B0 (19.2%; n = 20) predominated, corresponding to the CAR and B0/W148 subtypes, respectively.

Examples of IS6110-RFLP profiles are shown in Fig. 1. In Fig. 1, b, the IS6110-RFLP profiles contain additional fragments of weak intensity, indicating the presence of a second strain in the bacterial culture obtained.

 

Fig. 1. Examples of IS6110-RFLP profiles of M. tuberculosis isolates from a single patient.

a — profiles of pure isolates Beijing A0 (lane 1) and Beijing B0 (lane 2) obtained from different organs (lung and meninges) of patient No. 5. M — molecular weight marker (strain Mt14323);

b — profiles of mixed M. tuberculosis isolates obtained from different organs (lung and kidney) of patient No. 13. Lane 1 — main profile B0 with the presence of weak signal characteristic of profile A0 (arrow a). Lane 2 — main profile A0 with the presence of weak signals characteristic of profile B0 in the upper part of the profile (arrows b, c).

 

The number of mixed cultures and pure cultures of M. tuberculosis isolated from various organs is shown in Fig. 2. The difference between the frequency of isolation of mixed cultures from different organs was statistically insignificant (in all cases p > 0.05).

 

Fig. 2. Ratio of mixed cultures and pure cultures of M. tuberculosis isolated from different organs (data presented in absolute values).

 

Among patients in group 1, 12.1% of mixed isolates (n = 21) and 12.1% of pure isolates (n = 21) were identified, for a total of 24.3% of isolates (n = 42) from 15 (21.4%) patients. In 4 (5.7%) patients, mixed cultures were found in each organ examined, and in 7 (10%) patients, a mixed isolate was detected in at least one organ. In 4 (5.7%) patients, pure cultures belonging to different genotypes or subtypes were isolated from different organs, which also allowed them to be classified as patients with mixed infection. Detailed information about this group is presented in Table 2.

 

Table 2. Characteristics of patients with mixed infection, genotypes, and spectrum of resistance of M. tuberculosis strains

Patient No.	Dispensary registration group	Penal institution	CD4, cells/ μL	Number of affected organs	Organ from which cultures were isolated	M. tuberculosis Genotype, Beijing subtype	SIT	Mutations in M. tuberculosis genes
1	I-Б	Yes	20	6	Meninges	LAM	252	rpoB D516V, inhA C(-15)T
					Spleen	LAM + Beijing other*	252+1	rpoB S531L, D516V, katG S315T, inhA C(-15)T
					Lung	LAM + Beijing other	252+1
2	I-B	Yes	272	5	LN	Ural + B0/W148 Beijing	35+1	rpoB S531, H526R, katG S315T, inhA C(-15)T
					Spleen	Ural + B0/W148 Beijing	35+1
					Lung	Ural + B0/W148 Beijing	35+1
3	I-A	No	43	7	Left lung	CAR Beijing	265	rpoB S531L, katG S315T, inhA C(-15)T
					Right lung	CAR Beijing	265
					Spleen	CAR Beijing	1
4	I-B	Yes	–	6	Lung	Ural + B0/W148 Beijing	35+1	rpoB S531L, H526R, katG S315T, inhA C(-15)T
					Para-aortic LN	Ural + CAR Beijing	35+1
					Mesenteric LN	Ural + CAR Beijing	35+1
5	I-Б	Yes	78	4	Lung	CAR Beijing	1	rpoB S531L, katG S315T
					Meninges	B0/W148 Beijing	1
6	I-B	No	788	7	LN	LAM	266	rpoB H526D, katG S315T, inhA C(-15)T
					Spleen	LAM + B0/W148 Beijing	266+1	rpoB S531L, H526R, katG S315T, inhA C(-15)T
					Lung	LAM + B0/W148 Beijing	266+1
7	I-A	No	–	6	Spleen	Haarlem + B0/W148 Beijing	50+1	rpoB S531L, katG S315T
					Lung	Haarlem + CAR Beijing	50+1
8	I-Б	No	8	7	Spleen	B0/W148 Beijing	1	rpoB S531L, katG S315T
					Meninges	LAM + B0/W148 Beijing	252+1
					Lung	LAM + B0/W148 Beijing	252+1
9	I-A	No	153	10	Lung	LAM + CAR Beijing	252+1	rpoB S531L, D516V, katG S315T
					Brain	LAM	252	rpoB D516V, katG S315T, inhA C(-15)T
					Spleen	LAM	252
10	II	Yes	416	2	Lung	LAM + CAR Beijing	266+1	rpoB S531L, katG S315T
					LN	LAM	266	rpoB H526R, inhA C(-15)T
11	I-B	No	180	8	Intrathoracic LN	CAR Beijing	1	rpoB S531L, katG S315T
					Mesenteric LN	CAR Beijing	1
					Lung	Х1	2327	rpoB L516P, katG S315T
12	I-A	No	–	6	LN	LAM + B0/W148 Beijing	267+1	rpoB S531L, L516P, katG S315T, inhA C(-15)T
					Spleen	LAM	267	rpoB L516P, katG S315T, inhA C(-15)T
					Lung	B0/W148 Beijing	1	rpoB S531L, katG S315T
13	I-A	No	76	6	Kidney	B0/W148 Beijing	1	rpoB S531L, katG S315T, inhA C(-15)T
					Lung	CAR Beijing	1	rpoB S531L, katG S315T
					LN	CAR Beijing	1
14	I-B	Yes	226	3	LN	LAM + B0/W148 Beijing	252+1	rpoB S531L, L516P, katG S315T, inhA C(-15)T
					Kidney	LAM + B0/W148 Beijing	252+1
					Lung	LAM + B0/W148 Beijing	252+1
15	I-Б	Yes	18	6	Spleen	LAM	252	rpoB D516V, katG S315T, inhA C(-15)T
					LN	LAM	252
					Lung	LAM + Beijing other	252+1	rpoB S531L, D516V, katG S315T, inhA C(-15)T

Note. *Strains that did not belong to either Beijing B0/W148 or Beijing CAR according to PCR results were classified as Beijing other.

 

Of the 15 patients in group 1, 7 (46.7%) fell ill in penal institutions, and 3 were treated irregularly. In 5 patients, TB was detected for the first time, including 2 during their last hospitalization, but based on indirect signs, such as severe condition upon admission and extensive tuberculosis lesions, it can be assumed that at the time of referral, these patients had been ill with TB for a long time.

Of the 55 patients in group 2, 8 (14.5%) were admitted with recurrent TB, 5 (9.1%) with chronic TB, 11 (20%) with recurrent TB after interrupted treatment, and 31 (56.3%) were diagnosed for the first time, including 15 (27%) during their last hospitalization. Only 1 patient from this group had previously served a sentence in a penal institution (1.8% vs. 46.7%; OR = 47.3 [5.1164; 436.3546]; p = 0.0007).

No significant differences were found in the frequency of relapses and discontinuation of therapy (p > 0.05). Patients with severe immunodeficiency (CD4 less than 200 cells/μL) predominated in both groups, but it was not possible to obtain a reliable comparison result due to the lack of information on some patients. In particular, the lack of information on patients No. 4, 7, and 12 in group 1, presented in Table 2, is due to low adherence to treatment (they did not receive antiretroviral therapy, their immune status was not determined), their serious condition during their last hospitalization, sudden death, and the inability to perform a postmortem analysis.

Two different strains were identified in 14 patients, and only one patient (No. 4) had three strains of M. tuberculosis (Table 2). In all 15 cases of mixed infection, at least one of the strains in the mixed culture belonged to the Beijing genotype. Each strain was counted separately, and thus 42 cultures isolated from patients in group 1 contained 63 strains. The proportion of Beijing strains was 55% (n = 33), among which the subtypes Beijing B0/W148 (n = 17) and CAR (n = 16) predominated. Strains of the LAM (33.3%; n = 21), Ural (10%; n = 6), Haarlem (3.3%; n = 2), and X1 (1.7%; n = 1) genotypes were also detected.

Among the M. tuberculosis strains isolated from patients in group 2, the Beijing genotype predominated (70.2%; n = 92): B0/W148 (n = 29), CAR (n = 44), other subtypes (n = 19), and an ancient sublineage (2.3%; n = 3). Strains of the LAM (6.9%; n = 9), Ural, T, Haarlem (6.1% each; n = 8), and X1 (2.3%; n = 3) genetic families were also identified.

To compare the genotype ratios in groups 1 and 2, each strain in the mixed culture was considered separately; in the case of a match in SIT and IS6110-RFLP type 2 and more strains isolated from one patient (including in a mixed culture), only one was taken into account in the calculation. The results of the comparison are shown in Fig. 3.

 

Fig. 3. Comparison of the prevalence of different M. tuberculosis genotypes in patients in groups 1 and 2 (data are given in absolute values).

 

In group 1, Beijing B0/W148 subtype strains were detected in 9 (60%) patients, while in group 2, they were detected in 13 (23.6%; p = 0.0103). Furthermore, the proportion of LAM genotype strains was higher in group 1 than in group 2: 53% (n = 8) versus 5.5% (n = 3), p = 0.0002.

When studying M. tuberculosis isolates from the first group for mutations associated with resistance to isoniazid and rifampicin, it was found that in 16 (38%) mixed cultures, double substitutions were detected in the rpoB gene, including S531L (most common in Beijing) and D516V (typical for LAM SIT252). The frequencies of mutations in the rpoB gene were: S531L — 78.6% (n = 33), H526R — 23.8% (n = 10), D516V — 21.4% (n = 9), L516P — 9.5% (n = 4). The katG S315T mutation was found in 39 (92.9%) strains, and inhA C(-15)T — in 27 (64.3%). Mutations in the katG and inhA genes were found simultaneously in 24 (57.1%) isolates, of which 15 (35.7%) were mixed cultures.

Discussion

The prevalence of mixed TB infection caused by several strains (mixed cultures) of M. tuberculosis among patients with HIV-associated generalized TB in St. Petersburg was 21.4%, which corresponds to previously obtained data for countries with a high TB burden [3, 4]. At the same time, no statistically significant differences were found between the frequency of detection of mixed cultures in different affected organs (p > 0.05).

In patients infected with several M. tuberculosis isolates, it is not always possible to diagnose mixed infection, since even in the presence of several M. tuberculosis isolates in the body, both a mixture of isolates and one of them can be isolated from sputum. More accurate data can be obtained by examining surgical material [26], but in generalized TB, cultures of different compositions may also be obtained from different organs. The examination of autopsy material from several organs allows for a more accurate assessment of the prevalence of mixed infection and its composition, which is important for understanding the pathogenesis of the disease and optimizing epidemiological surveillance strategies.

All patients with mixed infection were found to have M. tuberculosis strains of the Beijing genotype, predominantly subtypes B0/W148 (isolated from 9 patients; 60%) and CAR (isolated from 8 patients, 53.3%); among patients without mixed infection, Beijing strains were isolated from 36 (65.5%) patients, and in this group, subtypes B0/W148 (23.6%; n = 13) and CAR (29.1%; n = 16) also predominated. No significant differences in the proportion of CAR were found, while the proportion of subtype B0/W148 was slightly higher in patients with mixed cultures (p = 0.0103). It is known that B0/W148 strains are highly virulent [27] and can cause severe forms of TB both in mixed cultures and without the involvement of other genotypes. The proportions of Beijing strains in the two groups also differed insignificantly, but the total proportion of Beijing among HIV+TB patients significantly exceeded that among strains circulating in northwestern Russia (72.9% vs. 57.5% [17]; OR = 1.9803 [1.1357; 3.4531]; p = 0.0160). Significantly more strains of the LAM genotype (p = 0.0002) were detected in mixed cultures compared to pure cultures isolated from deceased patients. Further and more extensive studies are needed, including studies of the pathogenesis of the disease caused by pure and mixed cultures of M. tuberculosis.

Data on the composition of mixed cultures may also be important for selecting a treatment regimen, since it is known that M. tuberculosis strains belonging to different genotypes have different resistance to anti-tuberculosis drugs. Thus, among strains of the Beijing genotype, the proportion of MDR strains reached 77.8% [28], and among LAM strains, 45.5% [29], with multidrug resistance most frequently observed in LAM SIT252 [30]. The proportion of MDR strains in Ural varied from 30% [28] to 42.2% [29]. Strains of the Haarlem genotype were less resistant to anti-tuberculosis drugs and were less common in northwestern Russia (up to 5%) [28]. However, they also deserve attention because they are found in HIV+TB patients, including in mixed cultures. The combination of strains with different drug resistance profiles can distort the results of drug susceptibility testing [15, 31].

In our study, MDR was detected in all M. tuberculosis cultures studied, and it was noted that mutations varied depending on the composition of the mixed culture. For example, the S531L mutation in the rpoB gene was found in most isolates containing Beijing strains, but in mixed isolates, other mutations were detected simultaneously with it, such as the D516V substitution typical of LAM SIT252 [30]. The presence of multiple mutations may increase the resistance of M. tuberculosis to anti-tuberculosis drugs, reducing the effectiveness of treatment. There are also studies showing that when determining the drug sensitivity of M. tuberculosis, discrepancies between phenotypic and genotypic resistance to certain drugs are more common in mixed cultures with heteroresistance than in pure cultures (29.1% vs. 17.2%; p > 0.05) [15].

In this study, individuals who had previously served time in prison were significantly more common in the mixed infection group than in the group of patients who did not have mixed cultures (p = 0.0007). This is consistent with previous studies, according to which staying in a penal institution is a risk factor for the development of mixed TB infection [9, 32], which is especially dangerous for patients with immunodeficiency. Furthermore, the widespread prevalence of the Beijing genotype among M. tuberculosis strains circulating in penal institutions [9], including in mixed infections, poses a threat.

Low adherence to treatment — discontinuation of treatment, delayed seeking of medical care — was not a risk factor for the development of mixed infection, however, according to some data, it contributes to the recurrence or exacerbation of TB infection [2], complicates the collection of epidemiological anamnesis, and in the case of mixed infection, can lead to the wrong choice of treatment regimen.

Conclusion

Among patients with HIV-associated generalized TB in St. Petersburg, mixed infection caused by several strains of M. tuberculosis was detected in 21.4% of cases. At the same time, strains of the Beijing genotype of various subtypes (60% — B0/W148) were found in all mixed cultures, and about half of them were found in combination with strains of the LAM genotype.

A significant risk factor for the development of mixed infection was a history of staying in a penal institution (p = 0.0007). Low adherence to treatment among HIV+TB patients makes it difficult to collect the epidemiological anamnesis and diagnose this condition in a timely manner.

About the authors

Alena A. Gerasimova

St. Petersburg Pasteur Institute

Author for correspondence.
Email: kantarelle@mail.ru
ORCID iD: 0000-0001-5246-8658

junior researcher, Laboratory of molecular epidemiology and evolutionary genetics

Russian Federation, St. Petersburg

Anna A. Vyazovaya

St. Petersburg Pasteur Institute

Email: annavyazovaya@gmail.com
ORCID iD: 0000-0001-9140-8957

Dr. Sci. (Biol.), senior researcher, Laboratory of molecular epidemiology and evolutionary genetics

Russian Federation, St. Petersburg

Marina Yu. Mayskaya

City Pathological Anatomical Bureau

Email: pab@zdrav.spb.ru
ORCID iD: 0009-0008-4642-6441

Cand. Sci. (Med.), chief physician

Russian Federation, St. Petersburg

Natalya S. Solovieva

Saint Petersburg State Research Institute of Phthisiopulmonology

Email: baclab@niif.ru
ORCID iD: 0000-0003-1509-0734

Cand. Sci. (Med.), Head, Laboratory of bacteriology

Russian Federation, St. Petersburg

Alexander M. Panteleev

Saint Petersburg State Research Institute of Phthisiopulmonology; City Tuberculosis Dispensary

Email: alpanteleev@gmail.com
ORCID iD: 0000-0001-8940-9758

Dr. Sci. (Med.), leading researcher, Saint Petersburg State Research Institute of Phthisiopulmonology; chief physician, City Tuberculosis Dispensary

Russian Federation, St. Petersburg; St. Petersburg

Igor V. Mokrousov

St. Petersburg Pasteur Institute

Email: imokrousov@mail.ru
ORCID iD: 0000-0001-5924-0576

Dr. Sci. (Biol.), Head, Laboratory of molecular epidemiology and evolutionary genetics

Russian Federation, St. Petersburg

References

Supplementary files