Clinical and Drug Resistance Characteristics of New Pediatric Tuberculosis Cases in Northern China

Abstract

Aim:

The aim of this study was to evaluate the clinical features and characteristics of drug resistance in newly diagnosed pediatric tuberculosis (TB) patients in northern China.

Methods:

Mycobacterium tuberculosis isolates were collected from September 2010 to October 2016 at the Beijing Children's Hospital. Patients were divided into two groups (resistant to at least one drug and pan-susceptible) according to drug susceptibility testing (DST) results.

Results:

A total of 132 new cases, mainly from northern China (87.9%), were included in the study. The median age was 1.9 years (1 month–15 years). Resistance to at least one drug was detected in Mycobacterium tuberculosis isolates from 33 (25%) cases. Eight cases of multidrug-resistant TB (MDR-TB) (6.1%) were detected. The two groups did not differ in clinical presentations (disease site, fever >2 weeks, and cough >2 weeks) or in chest imaging (lesion location, lymphadenitis [mediastinal], and pleural effusion).

Conclusions:

The rate of Mycobacterium tuberculosis drug resistance in new pediatric TB cases was as high as in the new adult patients surveyed in the national drug resistance survey conducted in 2007. No significant difference was observed in clinical features between patients infected with drug-resistant and drug-susceptible strains. Routine DST is important for prescribing effective antituberculosis treatment regimens.

Introduction

Drug-resistant tuberculosis (TB), especially multidrug-resistant TB (MDR-TB, caused by strains resistant to at least isoniazid [INH] and rifampicin [RIF]), is a major threat to global TB control. Globally, an estimated 4.1% of new TB cases in 2016 and 19% of previously treated TB cases had MDR-TB or rifampicin-resistant TB (MDR/RR-TB).¹ China is one of the top six high TB burden countries and had an estimated 895,000 incident cases and 73,000 MDR/RR-TB cases in 2016.¹ Drug-resistant TB is also a severe threat to children's health. In 2014, Jenkins et al.² published the first global estimate of the annual incidence of pediatric MDR-TB, indicating that 3.2% of the estimated 1 million children who developed TB in 2010 had MDR-TB.

Early detection of TB, especially drug-resistant TB, among new pediatric TB cases is important to the success of TB treatment. Studies on clinical and drug resistance characteristics of new pediatric TB patients are rare in China, partly due to the known difficulty of obtaining Mycobacterium tuberculosis isolates from pediatric patients. Neither the national survey of drug-resistant TB in China in 2007³ nor the 5th national TB epidemiological survey in 2010⁴ included children younger than 15 years. In this study, we report the analysis of clinical and drug-resistant features of new childhood TB cases from Beijing Children's Hospital, the National Children's Health Center in Beijing, China.

Methods

Setting

In 2000, the prevalence of TB in children <14 years in China was 91.8 cases per 100,000.⁵ According to the national survey of drug-resistant TB, the MDR-TB rate in new adult TB cases in China in 2007 was 5.7%, and the MDR-TB rate in new adult TB cases in Beijing in 2004 was 2.3%.⁶ By the end of 2011, 780,000 of China's population of 1,344.83 million were living with HIV/AIDS (an incidence rate of 58/100,000), of which 1.1% were children infected through mother-to-child transmission.⁷

Study sample

This retrospective study was conducted on children aged ≤15 years admitted to Beijing Children's Hospital, China, and diagnosed with TB, between September 2010 and December 2016. The Beijing Children's Hospital is the National Children's Health Center (Beijing) and draws patients from all over the country. All new pediatric TB patients with positive cultures were included.

Demographic, epidemiological, and clinical information, including gender, age, residence area, contact history, BCG vaccination, tuberculin skin testing, disease site, chest imaging, initial diagnosis, delay in diagnosis, length of hospitalization, and the main symptoms, was obtained from medical records.

New cases were patients diagnosed with TB who had never been treated with TB drugs or had been treated for less than 1 month. Previously treated cases were patients who had been treated for TB for 1 month or longer.⁸ Pulmonary TB (PTB) cases were defined as those with exclusively intrathoracic involvement (i.e., confined to the lung parenchyma, pleura, and intrathoracic lymph nodes), while extrapulmonary TB (EPTB) cases were defined as those where disease was found in organs or tissues outside the thorax, and included cases that also had pulmonary involvement.^9,10 BCG vaccination status was determined by either the presence of a scar on the upper left arm or a record of BCG vaccination. Chest imaging included both chest radiography and computed tomography. Parenchymal infiltrates were divided into three types: right side, left side, and both sides.

Clinical isolates were cultured from body fluid samples, mainly including bronchoalveolar lavage fluid, gastric aspirates, and cerebrospinal fluid. Isolates were collected in the hospital's bacteriology laboratory. Isolates included were positively identified as Mycobacterium tuberculosis. Nontuberculous mycobacteria (NTM) and other members of the Mycobacterium tuberculosis complex were identified based on the para-nitro benzoic acid/thiophene-2-carboxylic acid hydrazide (PNB/TCH) test and multilocus PCR.¹¹

Drug susceptibility testing

Strains were cultured on Lowenstein–Jensen medium for 4 weeks at 37°C. Drug susceptibility testing (DST) against the 4 first-line anti-TB drugs (INH, RIF, ethambutol [EMB], and streptomycin [STR]) and 1 second-line drug (ofloxacin [OFL]) was performed using the proportion method as recommended by the WHO.¹² Concentrations of these drugs in the media were as follows: INH 0.2 μg/mL, RIF 40 μg/mL, EMB 2 μg/mL, STR 4 μg/mL, and OFL 2 μg/mL. Strains were considered resistant to a specific drug when the growth rate was more than 1% compared with the control. Pyrazinamide (PZA) resistance was assessed by sequencing the pncA gene.¹³

Gene sequencing

In this study, gene regions associated with resistance to INH, RIF, STR, PZA, EMB, and OFL were tested, including katG, the mabA-inhA promoter, the rpoB RIF-resistance-determining region (rpoB RRDR), rpsL, embB, pncA, and gyrA. Genomic DNA was extracted from freshly cultured Mycobacterium tuberculosis cells by the cetyltrimethyl-ammonium bromide-lysozyme (CTAB) method.¹⁴ All primers used for amplification of target nucleotide positions and DNA sequencing are listed in the Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/mdr).

Statistical analyses

Enrolled patients were divided into two groups, a resistant to at least one drug (DR) group and a pan-susceptible (DS) group, according to DST results for the six drugs tested. Clinical characteristics were compared using the chi-square or rank sum tests. Statistical analyses were performed using SPSS statistics 20.0. p Values of less than 0.05 were considered to be significant.

Ethics approval

This study was approved by the Ethics Committee of Beijing Children's Hospital (no. 2015-45).

Results

A total of 996 pediatric TB patients were hospitalized in Beijing Children's Hospital between September 2010 and December 2016. Of these, 203 cases were culture positive, but 71 cases were excluded for the following reasons: 35 isolates failed to reculture, 33 isolates were identified as NTM or other species of the Mycobacterium tuberculosis complex, and 3 were previously treated cases (Fig. 1). The remaining 132 cases were included in the final analysis. Bacterial culture identified the presence of two isolates from 16 of these 132 cases, but only one isolate was included in the final analysis, as DST and genotyping results were the same, indicating they were duplicate isolates.

FIG. 1.

Patients with tuberculosis during the study period from September 2010 to December 2016. NTM, nontuberculous mycobacteria.

Of the isolates included, 43.2% (57/132) were isolated from bronchoalveolar lavage fluid, 31.8% (42/132) from gastric aspirates, and 8.3% (11/132), 5.3% (7/132), 4.5% (6/132), 4.5% (6/132), and 2.4% (3/132) were from cerebrospinal fluid, sputum, pleural effusion, blood, and other origins (pyogenic fluids, urine, or synovial fluid), respectively.

Patient characteristics

Most enrolled patients (87.9%, 116/132) were from northern China, defined according to administrative divisions in China. Demographic data and data on clinical manifestations of disease obtained on hospital admission are summarized in Table 1. The median age was 1.9 years (range from 1 month to 15 years), and 51.5% (68/132) of cases were younger than 2 years. A total of 38.2% (26/68) of patients <2 years had a history of contact with TB patients. Of the 132 patients enrolled in the study, 80 were diagnosed with PTB and 52 with EPTB, tuberculous meningitis being the most common diagnosis among EPTB cases (48.1%, 25/52). Chest imaging indicated that 53.0% (70/132) of cases had bilateral lesions, 52.3% (69/132) had lymphadenitis (mediastinal) in the mediastinum or hilus pulmonis, and 17.4% (23/132) had pleural effusion. A total of 40.2% (53/132) of the cases were not suspected of TB before admission to the hospital, and half of these 53 cases were previously diagnosed as having pneumonia before hospitalization. The most common symptoms observed were fever and a cough that had lasted longer than 2 weeks (63/132 and 61/132, respectively).

Table 1.

Demographic and Clinical Characteristics of New Pediatric Tuberculosis Cases from 2010 to 2016: Comparison Between Drug-Susceptible and Drug-Resistant Cases

	Total (N = 132), n (%)	Pan-susceptible (N = 99), n (%)	Resistant to at least one drug (N = 33), n (%)	p
Sex
Male	77 (58.3)	57 (57.6)	20 (60.6)	0.84
Female	55 (41.7)	42 (42.4)	13 (39.4)
Age (years)
Median (range)	1.9 (1 month–15 years)	1.9 (1 month–15 years)	1.6 (2 months–14 years)	0.760
<2	68 (51.5)	50 (50.5)	18 (54.5)	0.841
2–4	12 (9.1)	8 (8.1)	4 (12.1)	0.494
5–9	25 (18.9)	21 (21.2)	4 (12.1)	0.312
10–15	27 (20.5)	20 (20.2)	7 (21.2)	1.000
Residence
Rural	71 (53.8)	49 (49.5)	22 (66.7)	0.108
Urban	61 (46.2)	50 (50.5)	11 (33.3)
Contact with TB source case
Yes	42 (31.8)	30 (30.3)	12 (36.4)	0.517
No	88 (66.7)	68 (68.7)	20 (60.6)
Possible	3 (2.3)	1 (1.0)	2 (6.1)
BCG vaccination
Yes	101 (76.5)	76 (76.8)	25 (75.8)	1.000
No	25 (18.9)	18 (18.2)	7 (21.2)
Uncertain	6 (4.5)	5 (5.1)	1 (3.0)
TST
Positive	87 (65.9)	71 (71.7)	16 (48.5)	0.187
Negative	15 (11.4)	10 (10.1)	5 (15.2)
No result	30 (22.7)	18 (18.2)	12 (36.4)
Disease site
PTB	80 (60.6)	65 (65.7)	15 (45.5)	0.063
EPTB	52 (39.4)	34 (34.3)	18 (54.5)
Chest imaging^a
Right	40 (30.3)	30 (30.3)	10 (30.3)	1.000
Left	20 (15.2)	17 (17.2)	3 (9.1)	0.401
Both	70 (53.0)	51 (51.5)	19 (57.6)	0.688
Lymphadenitis (mediastinal)	69 (52.3)	50 (50.5)	19 (57.6)	0.549
Pleural effusion	23 (17.4)	19 (19.2)	4 (12.1)	0.436
Initial diagnosis
Suspected TB	79 (59.8)	59 (59.6)	20 (60.6)	1.000
Pneumonia	27 (20.5)	21 (21.2)	6 (18.2)	0.807
Other	27 (20.5)	19 (19.2)	8 (24.2)	0.619
Outside hospital diagnosis delay (days)
Main symptoms	30 (3–1095)	30 (3–1095)	31 (9–180)	0.414
Median (range)
Fever >2 weeks	63 (47.7)	48 (48.5)	15 (45.5)	0.842
Cough >2 weeks	61 (46.2)	48 (48.5)	13 (39.4)	0.423
Shortness of breath or wheezing	21 (15.9)	17 (17.2)	4 (12.1)	0.592

Chest imaging includes both chest radiography and computed tomography.

EPTB, extrapulmonary TB; PTB, pulmonary TB; TB, tuberculosis; TST, tuberculin skin testing.

There was no significant difference in demographic and epidemiologic data between the DR and DS groups. We did not find a significant difference between the two groups in terms of chest imaging (lesion site or presence of lymphadenitis [mediastinal] and pleural effusion) or clinical symptoms (fever >2 weeks, cough >2 weeks, and shortness of breath or wheezing).

Phenotypic and genotypic drug resistance

The drug resistance profiles of the studied Mycobacterium tuberculosis isolates are shown in Table 2. Resistance to at least one drug was identified in 33 isolates (33/132, 25%). Fourteen isolates (10.6%) were resistant to INH and 8 (6.1%) to RIF. All the RR-TB isolates were resistant to INH, that is, they were true cases of MDR (6.1%, 8/132). Of the MDR cases, one was resistant only to RIF and INH, the remaining seven being resistant to other drugs.

Table 2.

Trends of Drug-Resistant Tuberculosis in Beijing Children's Hospital

	n (%)
Resistance to at least one drug	33 (25)
Any RIF	8 (6.1)
Any INH	14 (10.6)
Any STR	19 (14.4)
Any EMB	3 (2.3)
Any OFL	5 (3.8)
Any PZA (pncA)	8 (6.1)
Monoresistance	21 (15.9)
INH	2 (1.5)
PZA	5 (3.8)
STR	11 (8.3)
OFL	3 (2.3)
All MDR	8 (6.1)
MDR	1 (0.8)
MDR+EMB	1 (0.8)
MDR+PZA	1 (0.8)
MDR+STR	2 (1.5)
MDR+EMB+PZA	1 (0.8)
MDR+STR+EMB	1 (0.8)
MDR+STR+OFL	1 (0.8)
Other polyresistance	4 (3.0)
INH+STR	2 (1.5)
INH+STR+OFL	1 (0.8)
INH+STR+PZA	1 (0.8)

EMB, ethambutol; INH, isoniazid; MDR, multidrug-resistant tuberculosis; OFL, ofloxacin; PZA, pyrazinamide; RIF, rifampicin; STR, streptomycin.

Mutation profiles in drug-resistant isolates

Concordance of the phenotypic DST results with genotypic drug resistance detection for RIF, INH, EMB, and OFX was 99.2% (131/132), 96.2% (127/132), 97.0% (126/132), and 97.7% (129/132), respectively (Table 3). Specific mutation types observed in the drug resistance-related genes tested are shown in Table 4. The most common mutated codons were katG315 (INH resistance) and rpoB531 (RIF resistance). Two mutations, affecting codons 43 and codon 88 of rpsl, were observed in STR-resistant isolates. Mutations in embB codons 306 and 406 were found in EMB-resistant isolates. Mutations in pncA were highly diverse and scattered across the gene, being found in codons 41, 57, 63, 114, 148, and 171.

Table 3.

Drug Resistance Profiles of Mycobacterium tuberculosis Isolates from New Pediatric Tuberculosis Cases

	Total (N = 132)			Concordance
Drug	No. of drug-resistant by DST (%)	% (No. of concordant isolates/N)	Gene	No. of drug-resistant by genotypic way (%)	Kappa value
RIF	8 (6.1)	rpoB	7 (5.3)	99.2 (131/132)	0.929
INH	14 (10.6)	katG and/or mabA-inhA promoter	12 (9.1)	96.2 (127/132)	0.794
STR	19 (14.4)	rpsl	20 (15.2)	90.2 (119/132)	0.609
EMB	3 (2.3)	embB	7 (5.3)	97.0 (126/132)	0.308
OFL	5 (3.8)	gyrA	6 (4.5)	97.7 (129/132)	0.716
PZA	—	pncA	8 (6.1)	—	—

DST, drug susceptibility testing.

Table 4.

Mutations in Drug Resistance-Related Genes

Drug (No. of drug-resistant isolates)	Locus	Mutated position	Mutation	N
INH (14)	katG	codon 315	AGC→ACC	10
		codon 335	ATC→ACC	1
	mabA-inhA promoter^a	−15	−15 C → T	1
RIF (8)	rpoB RRDR	codon 531	TCG→TTG	4
		codon 516	GAC→GTC	2
		codon 511	CTG→CCG	1
		codon 482^b	CAG→CGG	1
STR (19)	rpsL	codon 43	AAG→AGG	7
		codon 88	AAG→AGG	6
EMB (3)	embB	codon 306	ATG→GTG	1
		codon 406	GGC→GAC	1
PZA (8)	pncA	codon 57	CAC→GAC	2
		codon 41	TAC→TAT	2
		codon 63	GAC→GGC	1
		codon 114	ACG→GCG	1
		codon 148	CGC→AGC	1
		codon 171	GCG→GAG	1
OFL (5)	gyrA	codon 94	GAC→GGC	3
		codon 91	TCG→CCG	1

One INH-resistant isolate has mutations in both −15 in mabA-inhA promoter and codon 315 in gene katG.

One RIF-resistant isolate has mutations in both codon 482 and codon 531 in gene rpoB.

RRDR, RIF-resistance-determining region.

Discussion

Childhood TB is a neglected problem threatening the health of children. Globally, increasing efforts are made to improve the diagnosis of TB in children. In this study, we report the clinical and drug resistance characteristics of new pediatric TB cases from Beijing Children's Hospital.

Young children are at high risk of developing active TB. In this study, the most prevalent age subgroup was <2 years, possibly because patients were enrolled from a children's hospital where many patients with fever and/or a cough were young children. On the contrary, young children easily develop active TB because of the immaturity of their immune system.

In the study population examined here, PTB was the most common diagnosis, and only 39.4% of cases had EPTB. This is in contrast to our earlier study, in which more than a half of the pediatric TB cases (54%) were EPTB.¹⁰ Differences in the structure of the two study populations may account for this difference; in the previous study, all pediatric inpatients diagnosed with TB in the internal medicine department were included,¹⁰ while in the present study only culture-positive cases were included. Moreover, 84.8% of the strains examined here originated from the lungs, further contributing to the observed difference. Although lymph node TB has been reported as the most common type of childhood EPTB, TB meningitis (48.1%) was the most common type found in the present and our previous study population. This may be explained by the fact that this hospital is the National Children's Center in China, and children hospitalized here are thus more likely to have severe diseases. In contrast, patients with lymph node TB may be treated as outpatients or admitted to other hospitals. This study found no difference in the proportion of EPTB cases in the DR- and DS-TB groups, in agreement with research from Thailand¹⁵ but different from research conducted in South Africa.¹⁶ The small sample size in these studies may be the main reason for differences and further studies on larger sample populations are warranted.

Consistent with previous studies, the drug-resistant and drug-susceptible groups did not differ significantly in clinical presentations (fever or cough for more than 2 weeks) and chest imaging (lesion site or the presence of lymphadenitis [mediastinal] and pleural effusion).^15,16 It confirmed once again that it is difficult to diagnose drug-resistant TB based on clinical features.

We found that the rate of MDR-TB in this study was 6.1%, as high as that for new adult TB cases enrolled in the national drug resistance surveillance in 2007 (5.7%).³ The rate of drug resistance in new pediatric cases may also reflect recent transmission of drug-resistant strains.¹⁷ Recently, Jiao et al.⁸ characterized drug resistance profiles of 100 Mycobacterium tuberculosis isolates from pediatric cases collected from different regions of China. They found an alarmingly high prevalence of MDR-TB (11.7%, 9/77) in new pediatric cases. Another hospital-based study from the Chongqing megapolis in central China found that 4.1% of new pediatric cases had MDR-TB.¹⁸ Regional differences may be the primary reason for differences in the results obtained in these studies. The MDR-TB rate in pediatric cases in South Africa, Mexico, Korea, and the United States is reported to be 8.8%, 11.1%, 4.2%, and 1.6%, respectively.^19–22 As these studies included both new and previously treated cases, and the MDR rate in new TB cases is usually lower than that in previously treated cases, the MDR rate found in this Chinese study is indeed very high. This situation emphasizes the importance of routine DST in TB control.

Our phenotypic and genotypic DST results showed good concordance, especially with respect to MDR/RR-TB cases. While DST for PZA resistance is important in TB treatment, it is not routinely performed in clinical work as PZA DST is both expensive to perform but with low reproducibility. The pncA gene is commonly used for genotypic testing of PZA resistance as 72–99% of PZA-resistant isolates are reported to harbor mutations in pncA.²³ In this study, 6.1% cases were genotypically PZA resistant. This rate is much lower than that detected in an earlier population-based study²⁴ of adult patients (14.9%), likely due to the high proportion of previously treated cases in that study (21.1%). A population-based study of adult TB cases in five countries (Azerbaijan, Bangladesh, Belarus, Pakistan, and South Africa) reported PZA resistance rates of 3.0–42.1%.¹³ It is important that clinical doctors should consider possible development of PZA resistance if a patient has an unsatisfactory response to the recommended treatment regimen (INH+RIF+PZA).

This study had several limitations. First, it is a hospital-based study. As a referral hospital, severe TB (such as TB meningitis) cases tend to be overestimated. Second, the isolation time of the strains was quite dispersed. Thus, the results cannot reflect the real TB resistance status of the general pediatric TB population in the same area and time periods.

In conclusion, the proportion of pediatric patients infected with drug-resistant Mycobacterium tuberculosis strains in this study was as high as adult patients in the national baseline survey. We did not observe any significant differences in clinical features or imaging between patients infected with drug-resistant and drug-susceptible strains. Our findings further highlight the importance of performing routine DST to aid selection of appropriate therapeutic agents. Genotypic testing may provide an alternative method for the rapid identification of drug-resistant TB, thus assisting effective therapy.

Footnotes

Acknowledgments

This work was supported by the Beijing Talent Fund (No. 2016000021223ZK38); the Beijing Health System High-level Health Technical Personnel Foundation (no. 2015-3-081); and The Capital Health Development of Scientific Research Projects (no. 2016-1-2092).

Disclosure Statement

No competing financial interests exist.

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