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ORIGINAL ARTICLE  
Year : 2020  |  Volume : 63  |  Issue : 2  |  Page : 210-213
Diagnosis of pulmonary tuberculosis from gastric aspirate samples in nonexpectorating pediatric patients in a tertiary care hospital


Department of Microbiology, ABVIMS, Dr. RML Hospital, New Delhi, India

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Date of Web Publication18-Apr-2020
 

   Abstract 


Objectives: The aim of this study was to assess the utility of Xpert assay, Ziehl–Neelsen (ZN) staining, and Mycobacteria Growth Indicator Tube (MGIT™) culture for diagnosis of pediatric pulmonary tuberculosis from gastric aspirate (GA) samples and to compare Xpert assay and ZN staining with MGIT rapid liquid culture. Materials and Methods: GA samples from 210 nonexpectorating children, aged between 6 months to 12 years, presenting to the pediatric out-patient department (OPD) with clinical suspicion of tuberculosis (TB) were collected. The samples were tested by GeneXpert, ZN staining, and MGIT liquid culture. Results: GeneXpert is a more sensitive method for rapid and early diagnosis of pediatric TB when compared with microscopy.

Keywords: GeneXpert, MGIT, pediatric TB, ZN staining

How to cite this article:
Sharma S, Shulania A, Achra A, Jeram H, Kansra S, Duggal N. Diagnosis of pulmonary tuberculosis from gastric aspirate samples in nonexpectorating pediatric patients in a tertiary care hospital. Indian J Pathol Microbiol 2020;63:210-3

How to cite this URL:
Sharma S, Shulania A, Achra A, Jeram H, Kansra S, Duggal N. Diagnosis of pulmonary tuberculosis from gastric aspirate samples in nonexpectorating pediatric patients in a tertiary care hospital. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 May 27];63:210-3. Available from: http://www.ijpmonline.org/text.asp?2020/63/2/210/282711





   Introduction Top


As per the World Health Organization's (WHO) global tuberculosis report 2018, every year millions of people continue to fall sick because of tuberculosis (TB), and worldwide it is the leading cause of death from a single infectious agent. Globally, approximately 10.0 million people (range, 9.0–11.1 million) developed TB disease in 2017 equivalent to 133 cases (range, 120–148) per 100 000 population, which included 5.8 million men, 3.2 million women, and 1.0 million children. In the same year, there were 1.3 million deaths because of TB (range, 1.2–1.4 million) among HIV-negative people; 300,000 deaths (range, 266,000–335,000) among HIV-positive people; and 233,000 children.[1] Globally, 10% of all the TB cases are manifested in children under 15 years of age; however, the actual burden of TB in children is likely higher given the challenges in diagnosing childhood TB.[2] The extent of childhood TB in India is unknown because of diagnostic difficulties; however, it is estimated to be 10.2% of the total adult incidence.[3] Pediatric TB is a neglected disease because of its variable presentation as compared with the adult population. Children, typically, are unable to expectorate sputum or produce small quantities. Few bacilli are present in the respiratory secretions, thus, causing limited detection by sputum smear microscopy. Alternate samples include gastric aspirate (GA), induced sputum (IS), nasopharyngeal aspirate, bronchioalveolar lavage (BAL), laryngeal swab, etc., Depending on the diagnostic test used, these samples have shown variable sensitivity and specificity. It has been reported that GA samples provide the highest (40–92%) detection rate, depending on the sensitivity of the laboratory test adopted. Other samples have shown poorer detection rates ranging from 4–43% for BAL, 24–30% for nasopharyngeal aspiration, 27–63% for a laryngeal swab, and 20–30% by using IS.[4],[5] Thus, GA is an important sample for the diagnosis of pediatric pulmonary TB. Smear microscopy by Ziehl–Neelsen (ZN) staining for acid-fast bacilli is rapid and inexpensive method for diagnosis of TB but lacks sensitivity.[6]

The Xpert® MTB/RIF assay (Cepheid, USA) is an automated nucleic acid amplification test for simultaneous detection of Mycobacterium TB complex (MTBC) and its resistance to rifampicin directly from clinical samples. The assay does not require sample processing but can be used on chemically inactivated specimen and results are available within 2 h. Therefore, it is simple, less time consuming, and does not require special technical expertise and biosafety requirements. It is the only rapid molecular test initially recommended by WHO in 2010 for diagnosing pulmonary TB in adults. Since 2013, it has also been recommended for use in children and to diagnose specific forms of extrapulmonary TB.[1]

Mycobacterial culture is considered as the gold standard, but it is time-consuming (2–6 weeks) and requires good laboratory infrastructure and technical expertise. BD BACTEC Mycobacteria Growth Indicator Tube (MGIT) automated mycobacterial detection system is an automated liquid culture system. It is designed and optimized for the rapid detection of mycobacteria from clinical specimens as compared with conventional solid culture on the Lowenstein–Jensen (LJ) medium.

The aim of this study was to assess the utility of Xpert assay, ZN staining, and MGIT culture for the diagnosis of pediatric pulmonary TB from GA samples and to compare Xpert assay and ZN staining with MGIT rapid liquid culture as the gold standard.


   Materials and Methods Top


GA samples from 210 nonexpectorating children, aged between 6 months to 12 years, presenting to the pediatric out-patient department (OPD), from April 2018 to March 2019 with strong clinical suspicion of pulmonary TB were included in the study. Early morning, empty stomach GA was collected by a pediatrician and then sent to the microbiology department. The sample was divided into two parts. The first part was centrifuged and two drops of sample pellet (approximately 200 μl) were used for smear preparation and stained by ZN method as per the Revised National Tuberculosis Program (RNTCP) guidelines. The slides that showed pink-colored acid-fast bacilli were taken as smear-positive.[7] For GeneXpert assay processing, 0.5 ml of the remaining sample deposit was treated with 1.5 ml of sample reagent and processed according to the manufacturer's standard operating procedure (SOP) (Cepheid, USA). The results were obtained electronically. Samples whose results were obtained as invalid or error were rerun and results were obtained accordingly. Lastly, the second part of the sample was processed using the N-acetyl-L cysteine-sodium hydroxide (NALC-NaOH) method as per the manufacturer's instructions, cultured on MGIT media, and incubated in MGIT BACTEC 960 liquid culture system. When flagged positive, ZN staining and culture on 5% sheep blood agar were performed from the tube directly to see any contamination as per the manufacturer's instructions. All the tubes were incubated for a period of 42 days until they were flagged negative by the system. Tubes that were positive for acid-fast bacilli by smear microscopy were further subjected to rapid immunochromatography test (ICT) kit for the detection of MPT 64 antigen. ICT positive tubes were marked as Mycobacterium TB complex and the ICT negative tubes were considered as nontuberculous Mycobacterium (NTM) species.[8]

All the results were entered into a Microsoft Excel sheet and correlation tables were made. The sensitivity and specificity were calculated by 2*2 tables for smear microscopy and Xpert taking the liquid culture as the gold standard.


   Results Top


The mean age of the children was obtained to be 5.6 years with a male to female ratio of 1.2:1.

Out of the 210 samples, 34 (16.19%) were positive by Xpert assay, 15 (7.14%) were positive for acid-fast bacilli in smear microscopy, and 35 (16.66%) were obtained to be culture positive [Table 1].
Table 1: Results obtained by the three modalities incorporated in the study

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Of all the 210 samples, 7 (3.33%) was obtained as error and 3 (1.4%) were invalid in Xpert assay. When rerun, all 10 samples showed MTB not detected.

Out of the 34 GeneXpert positive samples, 32 were obtained to also be culture positive. Whereas two cultures and smear-positive samples were detected negative by GeneXpert. In one sample, smear and GeneXpert were positive but no growth was obtained by MGIT culture. In addition, one sample that was smear and GeneXpert negative were later obtained to be culture positive. Moreover, one sample that was smear- and culture-negative was detected positive by GeneXpert [Table 2].
Table 2: Comparison of GeneXpert results with ZN stain and MGIT culture results

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Out of these 34 GeneXpert positive samples, 2 (5.88%) were positive for rifampicin resistance. This result was not compared with the Drug Susceptibility Testing (DST) of the MGIT culture-positive samples as that facility was not available at the time of commencement of the study. Out of the 35 culture-positive cases, 32 (91.42%) were MTB complex organisms, whereas 3 (8.57%) were obtained to be NTM species.

Considering culture as the gold standard, the sensitivity and specificity of the ZN stain and GeneXpert were calculated. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of GeneXpert was obtained to be 91.42%, 98.85%, 94.11%, and 98.29%, respectively. On the other hand, the sensitivity, specificity, PPV, and NPV of ZN stain was 42.85%, 100%, 100%, and 89.74%, respectively.


   Discussion Top


Diagnosing pediatric pulmonary TB is still a challenge because of its varied presentation. Thus, laboratory confirmation of TB is crucial for the management of the disease, thereby curtailing the transmission of infection. For a sample to be positive, a bacterial load of 104/ml organisms is required for smear microscopy, whereas 131CFU/ml of bacilli is required by GeneXpert to flag the sample as positive.[9],[10] In our study, we were able to diagnose more than twice the number of cases by GeneXpert than by ZN staining [Table 1], which is similar to the findings of other studies.[11],[12]

GeneXpert was also obtained to be positive in two smear and MGIT culture-negative cases, such MGIT negative cases usually indicate toward the dead bacillary load in the samples and may point toward inactive infection, which can still be picked up by the GeneXpert because of the excretion of the residual DNA from the dead bacilli after the use of antitubercular drugs.[13] Out of the 15 smear-positive cases, two samples were not detected by the GeneXpert, whereas growth was observed in the automated culture. Further identification by the rapid card test revealed them as the NTM species. This highlights the importance of screening the samples by ZN staining and then confirming the diagnosis by culture, as GeneXpert cannot detect the NTM species. NTMs may not be pathogenic in all the cases, but whenever they are associated with disease, they are more difficult to treat. In smear-negative samples, one MGIT positive sample was not detected by GeneXpert, which was again diagnosed as NTM species.

Out of the 20 smear-negative cases, GeneXpert was able to diagnose 19 cases, which were also culture positive. These results were comparable to other studies from north India.[9] One culture positive, smear-negative sample was also flagged negative by the GeneXpert. This sample later showed the growth of NTM species.

The sensitivity, specificity, PPV, and NPV of GeneXpert were obtained to be 91.42%, 98.85%, 94.11%, and 98.29%, respectively. The findings are similar to the results obtained by the other studies.[14],[15],[16],[17],[18] The sensitivity, specificity, PPV, and NPV of ZN stain were 42.85%, 100%, 100%, and 89.74%, respectively. The sensitivity of the GeneXpert was much better when compared with ZN stain. The specificity of smear microscopy was comparable with that of the GeneXpert (98.85% vs 100%) and is in concordance to the findings of other studies.[18] Studies have shown that when compared with ZN stain, GeneXpert increased TB detection rate by 23 to 60% among culture-confirmed cases while as in our study it was 54.28%.[14]

In this study, the prevalence of rifampicin resistance by Xpert assay was found to be 5.88%. The findings are similar to the percentages observed by other studies.[9],[10]


   Conclusion Top


Although GeneXpert and smear microscopy are comparable in specificity, GeneXpert is a much more sensitive assay for rapid and accurate diagnosis of TB. Our study advocates the use of GeneXpert in GA samples in the diagnosis of pulmonary TB in nonexpectorating pediatric patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Global burden TB WHO. 2018. Available from: https://www.who.int/tb/publications/global_report/en/. [Last accessed on 2019 Sep 01].  Back to cited text no. 1
    
2.
Child and adolescent TB. Available from: https://www.who.int/tb/areas-of-work/children/en/. [Last accessed on 2019 Sep 01].  Back to cited text no. 2
    
3.
Kumar A, Gupta D, Nagaraja SB, Singh V, Sethi GR, Prasad J. Updated national guidelines for pediatric tuberculosis in India, 2012. Indian Pediatr 2013;50:301-6.  Back to cited text no. 3
    
4.
Swaminathan S, Rekha B. Pediatric tuberculosis: Global overview and challenges. Clin Infect Dis 2010;50(Suppl 3):S184-94.  Back to cited text no. 4
    
5.
Marais BJ, Pai M. New approaches and emerging technologies in the diagnosis of childhood tuberculosis. Paediatr Respir Rev 2007;8:124-33.  Back to cited text no. 5
    
6.
Hepple P, Ford N, McNerney R. Microscopy compared to culture for the diagnosis of tuberculosis in induced sputum samples: A systematic review. Int J Tuberc Lung Dis 2012;16:579-88.  Back to cited text no. 6
    
7.
TB India Report 2018: Ministry of Health and Family Welfare. Available from: https://tbcindia.gov.in/showfile.php?lid=2987. [Last accessed on 2019 Sep 01].  Back to cited text no. 7
    
8.
MGIT TM Procedure Manual. 2006. Available from: http://www.finddx.org/wp-content/uploads/2016/02/mgit_manual_nov2006.pdf. [Last accessed on 2019 Sep 01].  Back to cited text no. 8
    
9.
Pinyopornpanish K, Chaiwarith R, Pantip C, Keawvichit R, Wongworapat K, Khamnoi P, et al. Comparison of Xpert MTB/RIF assay and the conventional sputum microscopy in detecting mycobacterium tuberculosis in Northern Thailand. Tuberc Res Treat 2015;2015:571782.  Back to cited text no. 9
    
10.
Iram S, Zeenat A, Hussain S, Wasim Yusuf N, Aslam M. Rapid diagnosis of tuberculosis using Xpert MTB/RIF assay-Report from a developing country. Pak J Med Sci 2014;31:105-10.  Back to cited text no. 10
    
11.
Sekadde MP, Wobudeya E, Joloba ML, Ssengooba W, Kisembo H, Bakeera-Kitaka S et al. Evaluation of the Xpert MTB/RIF test for the diagnosis of childhood pulmonary tuberculosis in Uganda: A cross-sectional diagnostic study. BMC Infect Dis 2013;13:133.  Back to cited text no. 11
    
12.
Sandhya V, Prabhavathi R, Govindaraj M. Comparison of geneXpert versus sputum/gastric aspirate smear for AFB for the diagnosis of pulmonary tuberculosis in children. Int J Pediatr Res 2018;5:521-6.  Back to cited text no. 12
    
13.
Somily AM, Barry MA, Habib HA, Alotaibi FE, AlZamil FA, Khan MA, et al. Evaluation of GeneXpert MTB/RIF for detection of Mycobacterium tuberculosis complex and RPO B gene in respiratory and non-respiratory clinical specimens at a tertiary care teaching hospital in Saudi Arabia. Saudi Med J 2016;37:1395-8.  Back to cited text no. 13
    
14.
Zahoor D, Farhana A, Kanth F, Manzoor M. Evaluation of smear microscopy and geneXpert for the rapid diagnosis of pulmonary and extrapulmonary tuberculosis in a tertiary care hospital in North India: A descriptive prospective study. Int J Res Med Sci 2018;6:1756-60.  Back to cited text no. 14
    
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Singh S, Singh A, Prajapati S, Kabra SK, Lodha R, Mukherjee A, et al. Xpert MTB/RIF assay can be used on archived gastric aspirate and induced sputum samples for sensitive diagnosis of paediatric tuberculosis. BMC Microbiol 2015;15:191.  Back to cited text no. 15
    
16.
Bates M, O'Grady J, Maeurer M, Tembo J, Chilukutu L, Chabala C, et al. Assessment of the Xpert MTB/RIF assay for diagnosis of tuberculosis with gastric lavage aspirates in children in sub-Saharan Africa: A prospective descriptive study. Lancet Infect Dis 2013;13:36-42.  Back to cited text no. 16
    
17.
Sekadde MP, Wobudeya E, Joloba ML, Ssengooba W, Kisembo H, Bakeera-Kitaka S, et al. Evaluation of the Xpert MTB/RIF test for the diagnosis of childhood pulmonary tuberculosis in Uganda: A cross-sectional diagnostic study. BMC Infect Dis 2013;13:133.  Back to cited text no. 17
    
18.
Das A, Anupurba S, Mishra OP, Banerjee T, Tripathi R. Evaluation of Xpert MTB/RIF assay for diagnosis of tuberculosis in children. J Trop Pediatr 2018;65:14-20.  Back to cited text no. 18
    

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Correspondence Address:
Anuradha Shulania
Associate Professor, Department of Microbiology, ABVIMS, Dr. RML Hospital, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_694_19

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