|Year : 2020 | Volume
| Issue : 1 | Page : 21-25
Cartridge-based nucleic acid amplification test (Xpert Mycobacterium tuberculosis/Rifampicin Assay): An essential molecular diagnostic test for early diagnosis and initiation of treatment in childhood tuberculous meningitis and primary multidrug-resistant cases
Prasanta Kumar Das, Somtirtha B Ganguly, Bodhisatya Mandal
State TB Demonstration Cum Training Centre, Intermediate Reference Laboratory, Kolkata, West Bengal, India
|Date of Submission||02-Aug-2019|
|Date of Acceptance||24-Sep-2019|
|Date of Web Publication||17-Mar-2020|
Dr. Prasanta Kumar Das
State TB Demonstration Cum Training Centre, Intermediate Reference Laboratory, Kolkata, West Bengal
Source of Support: None, Conflict of Interest: None
Background: Tuberculosis (TB) still remains the major public health threat in India. Early diagnosis, so as to initiate early treatment is a priority as any delay, may complicate the prognosis further leading to the failure of an effective control. India accounts for 6% incidence of pediatric TB cases in a population that has 40% as estimated latent TB cases. Pediatric samples were considered as the key population in the study. TB remains the most common cause of childhood meningitis in high burden countries. India accounted for the highest number of TB deaths among children in the year 2015, followed by Nigeria and China. Of which, tuberculous meningitis (TBM) stands to be the most common cause of morbidity. Extrapulmonary TB accounts for 25% of the total TB cases reported among which the most severe form being TBM. Although severe and difficult to diagnose due to lack of specific tests to get confirmed, cartridge-based nucleic acid amplification test (CBNAAT) is the only solution for microbiological confirmation of the disease for early treatment initiation and reduction of morbidity and mortality. Previously, due to lower case detection rates owing to inadequate diagnostic facilities of higher analytical sensitivity, the mortality and morbidity continued to be in the higher range, and empirical treatment modality was the mainstay till the CBNAATs came into existence. The increase in the case detection for tuberculous meningitis at a manageable stage of the disease would help in decreasing the mortality rates. The study explores the increased case detection rates among pediatric population suffering from tuberculous meningitis from referral units to Intermediate Reference Laboratory (IRL), Kolkata. Cerebrospinal fluid (CSF) from the pediatric patients has not been very difficult procedure though invasive, especially in the pediatric age group, and can be practiced at peripheral unit having CBNAAT facility. Methods: CSF from pediatric patients showing obvious clinical signs of meningitis was collected from tertiary unit linked to IRL, Kolkata, and tested on CBNAAT by Xpert Mycobacterium tuberculosis/ rifampicin (RIF) (Cepheid) along with all the pulmonary and extrapulmonary samples based on the manufacturer's instruction. The data were captured in the Inbuilt software of the GeneXpert MTB/RIF (Xpert) and Genotype MTBDRplus (DRplus) (Cepheid , USA) of the cartridge based nucleic acid amplification test machines automatically during the tests and exported to the Microsoft Excel sheets for further analysis. A defined study design against each and every objective was setup to analyze the effective detection of tuberculous meningitis case and their resistance to RIF along with the other pulmonary and extrapulmonary samples. Results and Conclusions: The study concluded the confirmation of tuberculous meningitis cases by the help of CBNAAT. The detection of microbiologically confirmed TB by these specimens sent from the referral unit linked with IRL, Kolkata, for pulmonary and extrapulmonary cases along with tuberculous meningitis cases is significant and facilitates the early initiation of treatment for TB and more so to those who are newly detected as multidrug-resistant TB especially below the 6 months of age. About 5% prevalence and around 4.9% incidence are an alarming situation in the TB control scenario of West Bengal. The positive case detection among the CSF samples was found to be more in the 0.6–1-year group followed by the age group of 1–5 years. The percentage positivity of CSF is recorded as 2.38%, and the percentage of RIF resistance among positive cases from CSF was found to be 13.79% (n = 28).
Keywords: Cartridge-based nucleic acid amplification test, cerebrospinal fluid, childhood pulmonary tuberculosis
|How to cite this article:|
Das PK, Ganguly SB, Mandal B. Cartridge-based nucleic acid amplification test (Xpert Mycobacterium tuberculosis/Rifampicin Assay): An essential molecular diagnostic test for early diagnosis and initiation of treatment in childhood tuberculous meningitis and primary multidrug-resistant cases. Biomed Biotechnol Res J 2020;4:21-5
|How to cite this URL:|
Das PK, Ganguly SB, Mandal B. Cartridge-based nucleic acid amplification test (Xpert Mycobacterium tuberculosis/Rifampicin Assay): An essential molecular diagnostic test for early diagnosis and initiation of treatment in childhood tuberculous meningitis and primary multidrug-resistant cases. Biomed Biotechnol Res J [serial online] 2020 [cited 2021 Jan 17];4:21-5. Available from: https://www.bmbtrj.org/text.asp?2020/4/1/21/280855
| Introduction|| |
It is estimated that India accounts for 25% of global tuberculosis (TB) burden of the world. The pediatric population accounted for 6% of the total burden. In West Bengal, India, before the availability of cartridge-based nucleic acid amplification test (CBNAAT), the pediatric TB was seen in 3.8% cases among the total population tested.
As the initial diagnosis was solely dependent on smear microscopy, paucibacillary samples were left undetected. It is estimated that India accounts for around 40% of the latent TB infection., The pediatric case detections were less owing to a dearth of a sensitive technology not being able to detect the underlying cause.,, The drug resistance cannot be detected by smear microscopy. Nucleic acid amplification methods are able to detect Mycobacterium tuberculosis complex (MTBC) categorically along with the point mutations at the rifampicin (RIF) resistance determining region. Moreover, the analytical sensitivity of smear microscopy is not sufficient enough as it accounts for a poor-positive predictive value owing to its nonspecific detections.
Among the extrapulmonary TB cases which contribute to 25% of the total caseload, the most severe form was seen in tuberculous meningitis cases [Chart 1].
The following study explored the increased case detection for the pediatric group showing clinical signs of meningitis after the availability of CBNAAT at the districts of West Bengal. The alarming data of more than 4% TB pediatric TB prompted in further collection of samples.
The detection of TB in the pediatric age group is a difficult task either pulmonary or extrapulmonary as the sample collection is critical in these cases and disease like tuberculous meningitis in this age group where the only specimen for confirmatory microbiological diagnosis is cerebrospinal fluid (CSF), though difficult but can even be done at the peripheral units with minimum expertise to collect but specific for diagnosis and lifesaving too which helps in early initiation of treatment.
| Methods|| |
The objectives included the most common referral sites sending samples to Intermediate Reference Laboratory (IRL), Kolkata, for the suspected cases of tuberculous meningitis (TBM) of the pediatric age group.
The CSF is subjected to CBNAAT for microbiological confirmation.
CSF for all the suspected tuberculous meningitis cases among the pediatric age group was tested, irrespective of the geographical location of the patients which came to IRL with a test request for CBNAAT, have been included in the study.
The methods involve prospective analysis of the CBNAAT data of the pediatric samples coming to IRL, Kolkata, from May 2014 to May 2017. The proposal was reviewed and approved by the Research and Ethical Committee, IRL, Kolkata, India (Ethics Committee Approval number: STDC/IRL – CBNAAT pediatric project no. 012/2014 dated January 5, 2014–2017).
CSF samples were collected by the pediatricians at the block-level/tertiary care hospitals and aseptically transferred to IRL, Kolkata.
Data were captured for the entire period from the recording and registration register; the study designed was worked out based on the available data using computational software.
Selective screening helped in tracking the contribution of the referral sites. The age- and sample-related propensity in the case detection was also studied. The probe comprised not only the detection of MTBC by CBNAAT but also incidence and concordance studies of the RIF-resistant cases.
Before the processing of the sample for GeneXpert, around 20 μl was taken for making smears on grease-free slides.
These slides were stained and examined under a fluorescent microscope.
The samples were processed as per the standard operating procedures led down by the GeneXpert MTB/RIF guidelines. For Xpert, around 2–5 ml of sample was taken. In case of CSF, the test was run with at least of 1 ml of sample. The samples were mixed with double amount of buffered solution. The sample along with the buffer solution was made to stand for 15 min with intermittent gentle shaking in between for effective mixing. The tests were then run on CBNAAT machines as per the manufacturer's user manual.
A 100 μl of the buffer sample deposits (after centrifugation at 3000 g for 20 min) was sent for line probe assay (Genotype MTBDRplus, V2 of Hain Lifesciences to see the concordance). Gastric lavage has been seen as a productive sample for tuberculosis diagnosis in the pediatric group.
Data were captured on Microsoft Excel worksheets, and age- and sample-wise propensity results were expressed in terms of percentage. A Student's t-test was done between the paired data of the quarterly point, and the prevalence showing P < 0.05 was obtained. Odds ratio between the smear results and the CBNAAT positivity was calculated out using MedCalc software (https://www.medcalc.org/calc/odds_ratio.php).
| Results|| |
CSF contributed to 8.68% of the pediatric samples in the study. Among the total number of pediatric cases that came for TB testing, extrapulmonary cases accounted for 16%. Of which, pediatric samples tested for TBM were 53.3% of the total extrapulmonary cases. The positivity within this group was 2.38% [Table 1], [Table 2], [Table 3].,, When seen in terms of absolute figures, it was 29 pediatric patients showing positivity in CSF of 1223 tested cases of meningitis (percentage positivity of 2.38%). Among which, RIF resistant was found to be 13.7% among the tested positives.
|Table 2: The age-wise contribution toward positive case detection by cartridge-based nucleic acid amplification tests|
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|Table 3: Sample-type contribution toward Mycobacterium tuberculosis detection and rifampicin resistance detection|
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| Discussion|| |
Among the top ten causes of death worldwide, TB accounts for one of the most important factors; approximately, 1.2 million children became ill and 0.17 million died with it., Five-hundred children died due to TB, and over three-quarters of a million children fall ill with TB each year.
The study was on multidimensional approach of pulmonary and extrapulmonary samples of the pediatric age group where the test was microbiologically confirmed by CBNAAT. Among the all samples received, 1233 were CSF in clinically suspected cases of meningitis.
As of now, prior to the era of CBNAAT, the suspected case of tuberculous meningitis was diagnosed by the supportive or ancillary tests available and treatment was based on high index of clinical suspicion, now introduction of CBNAAT enhanced the microbiologically confirmed disease and a concrete evidence before the initiation of treatment.
The percentage positivity among this group was found to be 2.38% (n = 1233) which is quite suggestive that this population remained unaddressed earlier before the introduction of CBNAAT owing to dearth of adequate technology.
Smear microscopy, biochemical test, and cytology have remained the sole diagnostic support in the block-level facilities earlier, but CBNAAT has been introduced at the peripheral level which increases the microbiologically confirmed tuberculous meningitis in this era of evidence-based medicine.,,,
| Conclusions|| |
The tuberculous meningitis cases were microbiologically confirmed by the help of CBNAAT.
The specimen sent from the referral unit directly linked with IRL Kolkata for all pulmonary and extrapulmonary cases for all clinically suspected cases of tuberculous meningitis cases. The yield of the test is significant which facilitates the early initiation of the treatment of TB and more so to those who are newly detected as multidrug-resistant TB as the primary case, especially below 6 months of age.
The positive case detection among the CSF samples was found to be more in the 0.6–1-year group, followed by the age group of 1–5 years. The percentage positivity of CSF is 2.38%, and the percentage of RIF resistance among positive cases from CSF was found to be 13.79% (n = 28).
We are thankful to all staff at IRL, West Bengal, India, for providing technical inputs and all patients suffering and the staffs contributing for their dedication.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]