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.
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:
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
Exclusion criteria:
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)
Materials required for decontamination procedure: 8
Modified Petroff’s Method: 8
The decontamination procedure was carried out as follows:
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
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).
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.
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:
Future Directions:
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.