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Research Article | Volume 15 Issue 2 (Feb, 2025) | Pages 13 - 19
Investigating Drug Resistance Patterns of Mycobacterium Tuberculosis Isolates in Pediatric Pulmonary Tuberculosis Patients
 ,
 ,
 ,
1
Assistant Professor, National Institute of Medical Sciences & Research, Jaipur, Rajasthan, India
2
Associate Professor, Lokmanya Tilak Municipal Medical College & General Hospital, Sion, Mumbai, India
Under a Creative Commons license
Open Access
Received
Dec. 29, 2024
Revised
Jan. 3, 2025
Accepted
Jan. 20, 2021
Published
Feb. 3, 2025
Abstract

Introduction: Tuberculosis (TB) remains a significant public health concern, particularly in pediatric populations, where diagnosis and treatment are often challenging. The emergence of drug-resistant Mycobacterium tuberculosis strains complicates TB management. This study aimed to investigate the drug resistance patterns of M. tuberculosis isolates in pediatric pulmonary TB patients at a tertiary care hospital. Material & Methods: The present study was a prospective, observational study undertaken in a tertiary care hospital, Jaipur among 150 clinically suspected cases of pulmonary tuberculosis in paediatrics age group. Clinically relevant samples were collected depending on history and clinical findings with all aseptic precautions. Results: Out of the 150 suspected pulmonary TB specimens, 7.33% were positive for acid-fast bacilli. On Lowenstein Jensen medium, mycobacterial growth was observed in 4% specimen. The Gene Xpert positivity was observed in (8.66% patients. Resistance to first-line anti TB drugs was observed in 03 (60%) of the MTB isolates. The overall prevalence of mono resistance was observed in 02 (40%) isolates  showing resistance to Isoniazid and prevalence of multidrug-resistant TB (MDR TB) was observed as 4%. 60% concordance was observed between Gene Xpert and DST for the detection of rifampicin. Discordance in the detection of rifampicin resistance by DST, Gene Xpert, and was observed in 40%. Discordance in the detection of isoniazid resistance by DST and Gene Xpert was observed 40%. Conclusion: Management of drug-resistant tuberculosis represents a potential challenge for clinicians. Antitubercular drugs act as a gold standard in this situation. the use of conventional DST along with Gene Xpert seems promising for the detection of drug resistance in pulmonary TB cases, particularly in the scenario of the rising number of MDR TB cases. Continuous surveillance system to be advocated for evaluation of drug resistance patterns in pediatric age group.

 

Keywords
INTRODUCTION

Tuberculosis (TB) is a communicable disease that is a major cause of ill health and one of the leading causes of death worldwide. Until the coronavirus (COVID-19) pademic, TB was the leading cause of death from a single infectious agent, ranking above HIV/AIDS. 1

 

WHO emphasized the need to prioritise the quality of information on tuberculosis in children, young children more often have less transmissible tuberculosis than do adolescents and adults, on the other, they are at greater risk of disease and severe forms of tuberculosis, particularly children younger than 5 years and without BCG vaccine. 2 TB is caused by the bacillus Mycobacterium tuberculosis, which is spread when people who are sick with TB expel bacteria into the air while coughing and sneezing in the form of droplet nuclei. The disease typically affects the lungs causing pulmonary TB but can affect other sites as well known as extra pulmonary TB. 3

 

TB disease in children under 15 years of age (also called pediatric tuberculosis) is a public health problem. Once infected with TB bacteria, children are more likely to get sick with TB disease and to get sick more quickly than adults. In comparison to children, TB disease in adults is usually due to past TB infection that becomes active years later, when a person’s immune system is weakened for some reason. 4 The source of infection for children is usually an adult in their household who has active TB, is coughing and is infectious.

 

The diagnosis of infection and disease due to Mycobacterium tuberculosis in infants and children presents many clinical challenges. The distinction of infection from disease (tuberculosis) in children is often unclear. There is difficulty in obtaining positive microbiological confirmation of infection in sputum, gastric, or bronchial aspirates. 38 Conventional methods find it difficult to diagnose Pulmonary TB in children primarily because of the long time frame required for testing, and also due to sampling difficulty and the paucibacillary nature of samples. PTB in a patient with active tuberculosis in children is often misdiagnosed as pneumonia. 5

 

India bears 27 percent of the burden of tuberculosis (TB) amongst high burden countries. 2 Limited studies have been available citing the prevalence of drug resistance in Pulmonary TB especially from high burden settings like India. The reasons for this include the difficulty in obtaining diagnostic specimens and the limited number of laboratories in the country having the facility to perform culture and drug susceptibility testing (DST) for Mycobacterium tuberculosis in paediatric population specimens. Hence the high clinical suspicion and Clinical symptoms play very important role in diagnosis and treatment of paediatric TB and physicians suspect drug resistance only after failure or non-response to first-line therapy. 6

 

With this in the background, the present study is conducted which aims at the isolation of Mycobacterium tuberculosis samples from known cases of pulmonary tuberculosis in paediatrics population and determination of their drug resistance pattern.

 

Aims and Objectives:

  • To isolate and identify Mycobacterium tuberculosis from pulmonary specimens in paediatric age group by conventional methods.
  • To determine the drug resistance pattern of Mycobacterium tuberculosis isolates to first-line Anti-tubercular Treatment.
MATERIALS AND METHODS

Study Design:

The present study was a prospective, observational study undertaken to determine the drug susceptibility pattern of Mycobacterium tuberculosis to first-line Anti-tubercular treatment in a tertiary care hospital, Jaipur.

 

Study Place:

The present study was conducted at the Department of Microbiology, at a tertiary care hospital, Jaipur.

 

Study Period:

The present study period was conducted from September 2020 to August 2021.

 

Study Population:

The study population consisted of clinically suspected cases of pulmonary tuberculosis in paediatric age group.

 

Selection Criteria:

Inclusion criteria

  • Patients of paediatric age group (0-12 yrs) registered at the outpatient department (OPD)
  • Paediatric patients admitted towards examined by the attending clinician for diagnosis of pulmonary tuberculosis.

 

Exclusion criteria:

  • Patients positive for extrapulmonary tuberculosis.
  • Not willing for giving consent for study

 

Methodology:

Sample Collection: 7

Clinically relevant PTB samples were collected depending on history and clinical findings with all aseptic precautions in a sterile container and properly labeled. Samples included were Sputum and GL (gastric lavage)

  1. GL (gastric lavage)- Withdraw the gastric contents (ideally at least 5–10 ml). Transfer gastric fluid from the syringe into a sterile container (sputum collection cup). Add an equal volume of sodium bicarbonate solution to the specimen (in order to neutralize the acidic gastric contents and so prevent destruction of tubercle bacilli).
  2. Sputum: 2 early morning samples of sputum are collected. 1 on the spot and second sample on the next day early morning. Or two samples on the spot one hour apart can be taken.

 

Materials required for decontamination procedure: 8

  • Disposable 50 ml plastic tubes (Falcon tubes)
  • Sterile NaOH-NALC-sodium citrate solution(Annexure-II) [4% NaOH solution was mixed with sodium citrate solution (2.9%) to make a working solution (NaOH concentration in this solution is 2%)].
  • Phosphate buffer pH 6.8 (0.067M), laboratory prepared and sterilized
  • Centrifuge with a minimum 3000-3500x g force and safety shield
  • (refrigerated centrifuge preferred)
  • Vortex mixer, shaker
  • Timer
  • Pipettes with tips

 

Modified Petroff’s Method: 8

The decontamination procedure was carried out as follows:

  • Samples were transferred into screw-capped test tubes.
  • An equal volume of freshly prepared digestant of 2% NaOH and NALC powder was added to the samples.
  • The solution was vortexed for 30 seconds and was allowed to stand at room temperature for 15 minutes.
  • Phosphate buffer (pH 6.8) was overlaid to form a 1 cm column and mixed properly.
  • The tubes were centrifuged at 3000 rpm for 15 minutes.
  • The supernatant was discarded in a container containing 1% sodium hypochlorite solution.
  • The sediment was further divided into aliquots and the LJ medium was inoculated likewise.

 

AFB Smear microscopy:

3 aliquots of the sediment were made: 1st loopful of sediment was inoculated on a Lowenstein Jenson (LJ) medium slope, 2nd was taken to make a smear and 3rd was processed for Gene Xpert.

 

Ziehl-Neelsen Staining Procedure: 9

  1. A new unscratched slide was selected and the slide was labeled with the Laboratory Serial Number with a glass marking pencil.
  2. A smear was made from the specimen which was spread evenly, 2 cms x 3 cms in size. Smear preparation was done near a flame, as six inches around the flame is considered as a sterile zone which coagulates the aerosol raised during smear preparation.
  3. The slide was allowed to air dry for 15–30 minutes.
  4. The slide was fixed by passing it over a flame 3–5 times for 3–4 seconds each time.
  5. 1% filtered carbol fuchsin was poured to cover the entire slide.
  6. The slide was gently heated with carbol fuchsin on it, until vapors rose, but not boiled.
  7. Carbol fuchsin was left on the slide for 5 minutes.
  8. The slide was gently rinsed with tap water until all free carbol fuchsin stain was washed away. The smear on the slide now looked red.
  9. 25% of sulphuric acid was poured onto the slide and allowed to stand for 2–4 minutes.
  10. The slide was gently rinsed with tap water and tilted to drain off the water.
  11. A properly decolorized slide appeared light pink. If the slide was still red, sulphuric acid was reapplied for 1–3 minutes and then rinsed gently with tap water.
  12. The back of the slide was wiped with a swab dipped in sulphuric acid.
  13. 1% methylene blue was poured onto the slide and left for 30 seconds. Then the slide was rinsed gently with tap water and allowed to dry.
  14. The slide was examined under the binocular microscope using an x40 lens to select a suitable area and then examined under the x100 lens using a drop of immersion oil.
  15. The results were recorded.

 

Interpretation:

The smears were reported as presence/absence of AFB on direct smear examination.

 

GeneXpert:

The Xpert® MTB/RIF Assay, performed on the GeneXpert® Instrument Systems, is a qualitative, nested real-time polymerase chain reaction (PCR) in-vitro diagnostic test for the detection of Mycobacterium tuberculosis complex DNA and Rifampicin resistance in samples.

 

Principle: 10

The Xpert MTB/RIF Assay simultaneously detects MTB-complex and RIF resistance. The primers in this test amplify an MTB-complex specific sequence of the rpoB gene containing the 81 base pair core region, which is probed with five molecular beacons (Probes A – E) for mutations within the rifampin resistance determining the region (RRDR). Each molecular beacon is labeled with a different fluorophore. The valid maximum cycle threshold (Ct) of 39.0 for Probes A, B and C and 36.0 for Probes D and E are set for MTB/RIF data analysis.

 

Sample processing:

The samples like GL and sputum samples were subjected to decontamination procedure and were processed according to manufacturer instructions (Cepheid Gene Xpert®System, CA, USA).

  1. Briefly, 1.5 ml of the sample was transferred to a new 15 ml centrifuge tube. The centrifuge tubes were labeled with an individual patient identification number.
  2. Sample reagent buffer at 2:1 (v/v) ratio was added to a 15 ml centrifuge tube containing the sample and hand mixing was performed by inverting the tube for 20 times at different intervals.
  3. The specimen was incubated at room temperature for an additional 5 minutes.
  4. Using a fresh transfer pipette, 2 ml of the processed sample was transferred to the Xpert MTB/RIF cartridge.
  5. The cartridge  was  loaded  into  the  Gene  Xpert  instrument  following  the manufacturer instructions
RESULTS

A total 150 clinically relevant PTB samples of clincically suspected pediatrics cases of pulmonary tuberculosis were observed. The maximum number of cases were belonged to age group of 2-6 years (60.0%), followed by in 7-12 years (26.6%). The mean age among the distribution of cases was 5.5 years.

 

Table 1: Distribution of patients according to Age

Age in years

No. of Patients

Percentage %

 

 

 

0-1 yrs

20

13.33

 

 

 

2-6 yrs

90

60.0

 

 

 

7-12 yrs

40

26.66

 

 

 

Total

150

100

 

Figure 1: Distribution of patients according to Age

 

Above table shows sex distribution among patients. Out of 150 , 76 (51%) where females and 74 (49%) where males which shows slightly higher incidence in females ie1.1:1  in paediatric age group.

 

Table 2: Distribution of patients according to Gender

 

Gender

 

Frequency

 

Percentage

 

Male

 

74

 

49

 

Female

 

76

 

51

 

Total

 

150

 

100

 

Figure 2: Distribution of patients according to Gender

 

On observing findings of laboratory test parameters of 150 study samples  ie; ZN stain, Culture results and Gene Xpert results has showing that 11 (7.33%) are ZN staining positive. 06 were culture positive (04%) and 13 were Gene Xpert positive (8.6%).

 

Table 3: Distribution of patients according to laboratory test findings

 

Laboratory Test parameters

 

 

Frequency

 

Positive

 

Negative

 

Percentage(%)

 

ZN staining

 

150

 

11

 

139

 

7.33

 

Culture results

 

150

 

06

 

144

 

04

 

Gene Xpert

 

150

 

13

 

137

 

8.66

 

Figure 3: Distribution of patients according to laboratory test findings

 

Table 4: Gene Xpert results compared with culture results

 

Table 5: Comparative results of DST with Gene Xpert and rifampicin resistance

 

Above table shows comparison between Gene Xpert results and DST. Total 13 samples where positive by Gene Xpert and 6 samples where positive by Gene Xpert and DST. Out of 6 samples positive on culture 3 samples are RR and 3 samples are RS. whereas the 7 samples positive by  only by Gene Xpert are RS. Seven which were shown to be RS only by Gene Xpert; amongst these 2 where found resistant to INH on DST Out of 3 RR according to DST 1 is RR which was detected by Gene Xpert. And 2 where missed by Gene Xpert and detected by DST.

DISCUSSION

Isolation of mycobacteria from clinical specimens is considered to be the best method for the diagnosis of pulmonary tuberculosis. Culture isolation followed by reliable and accurate DST is essential to determine an effective treatment regimen and to avoid further development of drug resistance. The present prospective observational study was undertaken to determine the drug susceptibility pattern of isolates of Mycobacterium tuberculosis from pulmonary TB cases in paediatric patients to first-line Anti-tubercular treatment in a tertiary care hospital.

In the present study, the age distribution among patients showed that the maximum number of cases was in the age group of 2-6 years (60%), followed by in 7-12 years (26.6%). The mean age among the distribution of cases was 5.5 years. A study conducted by Ira shah et al. in 2010 shows included children from 0–15 years of age suffering from TB,  A total of 242 children were included in the study. Mean age of presentation was 5 ± 3.8 years, which is comparable with present study. In the study conducted by Ira shah et al in 2016 the gender wise distribution among 242 patients showed that female : male ratio is 1.1:1 that is slightly higher in females, which is comparable to present study. In the study conducted by Allah Rakhia et al in 2020 in Pakistan and a study by Sandhya v et al. in 2017 the results of studies comparable with present study. 11, 12

 

In present study total 13 out of 150 samples are Gene Xpert positive and 6 samples are found culture positive out of which 4 are medium bacillary load, 1 low bacillary load and 1 very low bacillary load in gene expert result and 7 which have come negative on culture results but found positive on gene expert with very low bacillary load. 

 

A retrospective study in  Children were defined as having DR-TB on the basis of Gene Xpert done by Ira shah et al  The prevalence of DR-TB was 110 of 1145 cases (9.6%), which showed an increase, compared with 5.6% pre-2010 and 7% in 2010-2013 . Eight cases (7.2%) were monoresistant, 7 (6.3%) poly resistant, MDR-TB seen in 28 patients (25.45%), 32 (29.09%) had pre-XDR-TB, 9 (8.18%) had XDR-TB and 12 (10.9%) were rifampicin resistant. Ethionamide resistance increased from 26.1% pre-2013 to 60.8% post-2013. In present study, total 13 samples where positive by Gene Xpert and 6 samples where positive by Gene Xpert and DST. Out of 6 samples positive on culture 3 samples are RR and 3 samples are RS, whereas the 7 samples positive   only by Gene Xpert are RS. 7 which were shown to be RS only by Gene Xpert amongst these 2 where found resistant to INH on DST. Out of 3 RR according to DST 1 is RR which was detected by Gene Xpert. and 2 where missed by Gene Xpert  and detected by DST.

 

This may be due to reasons like Resistance to rifampicin is mediated by mutations clustered in a small region of the rpoB gene.  There are other mechanisms of resistance, possibly efflux pumps, may exist. Which are not detected on Gene Xpert hence it is missed on Gene Xpert assay but it is detected by conventional method of DST.

 

Although DST by conventional culture method has a disadvantage that it takes several weeks (28 days for culture and 42 days for DST) but its importance cannot be ignored when Gene Xpert shows false-positive results due to patients on treatment, relapse or treatment after lost to follow-up cases while screening patients on routine basis as this misguides the clinician about the patient’s response to treatment.

 

Recommendations:

  1. Implement universal DST for all pediatric TB patients.
  2. Intensify contact tracing and screening.
  3. Promote public awareness and education.
  4. Foster collaboration between healthcare providers and policymakers.

 

Future Directions:

  1. Investigate novel diagnostic and therapeutic approaches.
  2. Develop pediatric-specific TB treatment guidelines.
  3. Conduct longitudinal studies to monitor drug resistance trends.
CONCLUSION

Management of drug-resistant tuberculosis represents a potential challenge for clinicians. Antitubercular drugs act as a gold standard in this situation. This study yields a piece of important information regarding the comparative results of the conventional DST and Gene Xpert towards first-line antitubercular drugs. The tests were performed in a quality assured reference laboratory thus making the results valid and reliable. Findings of this study demonstrated that Gene Xpert can detect a large number of rpoB mutations, but not all mutations that cause rifampicin resistance. So it acclaims that the conventional method remains the gold standard for culture and DST in cases of tuberculosis. Molecular tests like Gene Xpert assay serve as a tool for early initiation of appropriate therapy which can be followed by phenotypic DST confirmation for MDR TB patients.

 

Hence, the use of conventional DST along with Gene Xpert seems promising for the detection of drug resistance in pulmonary TB cases, particularly in the scenario of the rising number of MDR TB cases. Continuous surveillance system to be advocated for evaluation of drug resistance patterns in pediatric age group.

 

Conflict of interest: None

Funding: None

 

Acknowledgments:

We acknowledge the contributions of hospital staff and laboratory personnel involved in this study.

REFERENCES
  1. Global Tuberculosis Report 2021. World Health Organization, 2021, https://www.who.int/publications-detail-redirect/9789240037021.
  2. Global Tuberculosis Report 2019. World Health Organization, 2019, https://www.who.int/publications-detail-redirect/9789241565714.
  3. Fact Sheets on Tuberculosis. Updates 2019. World Health Organization, 2019, http://www.who.int/features/factfiles/tuberculosis/en/.
  4. Diagnostic Standards and Classification of Tuberculosis in Adults and Children. American Journal of Respiratory and Critical Care Medicine, vol. 161, 2000, pp. 1376-1395.
  5. Khemiri, M., et al. "Tuberculosis in Childhood: Clinical Features and Problems in Diagnosis. Report of 30 Cases." Tunis Med, vol. 87, 2009, pp. 61-67.
  6. Nicol, M. P., et al. "Accuracy of the Xpert MTB/RIF Test for the Diagnosis of Pulmonary Tuberculosis in Children Admitted to Hospital in Cape Town, South Africa: A Descriptive Study." The Lancet Infectious Diseases, vol. 11, no. 11, 2011, pp. 819-824.
  7. Schreckenberger, P. C. Koneman’s Color Atlas Introduction to Microbiology: Guidelines for the Collection, Transport, Processing, Analysis and Reporting of Cultures from Specific Specimen. 6th ed., Lippincott Williams & Wilkins, 2006, pp. 67-110.
  8. RNTCP Manual of Standard Operating Procedure. Culture of M. Tuberculosis and Drug Susceptibility Testing on Solid Medium. Central TB Division, Ministry of Health and Family Welfare, 2017, Version No. 01.01.
  9. Revised National Tuberculosis Control Programme Technical and Operational Guidelines for Tuberculosis Control. Central TB Division, DGHS, 2015, https://tbcindia.gov.in/WriteReadData/l892s/7293794058standard%20operating%20procedures%20for%20C&DST%20labs.pdf.
  10. Kumar, V. G., Urs, T. A., and Ranganath, R. R. "MPT 64 Antigen Detection for Rapid Confirmation of M. Tuberculosis Isolates." BMC Research Notes, vol. 4, 2011, p. 79.
  11. Rakhia Allah, et al. "A Comparative Study of Conventional Methods and GeneXpert Mycobacterium Tuberculosis Rifampicin Assay for Diagnosis of Childhood Pulmonary Tuberculosis." Journal of Tuberculosis, PHRC, Specialized Research Centre on Child Health, National Institute of Child Health, Karachi, Pakistan, https://meddocsonline.org/journal-of-tuberculosis/a-comparative-study-of-conventional-methods-and-genexpert-mycobacterium-tuberculosis-rifampicin-assay-for-diagnosis-of-childhood-pulmonary-tuberculosis.pdf.
  12. Nicol, M. P., et al. "Accuracy of the Xpert MTB/RIF Test for the Diagnosis of Pulmonary Tuberculosis in Children Admitted to Hospital in Cape Town, South Africa: A Descriptive Study." The Lancet Infectious Diseases, vol. 11, no. 11, 2011, pp. 819-824.
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