|Ahead of print publication
Evaluation of Gene-Xpert in paediatric tuberculous meningitis cases: A hospital-based study
Sweta Muni1, Anand Kumar Gupta2, Deepak Pankaj3, Rakesh Kumar1, Shailesh Kumar1, Namrata Kumari1
1 Department of Microbiology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India
2 Department of Paediatrics, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India
3 Department of General Surgery, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India
|Date of Submission||12-Apr-2022|
|Date of Acceptance||15-Jun-2022|
|Date of Web Publication||17-Oct-2022|
Department of Microbiology, Indira Gandhi Institute of Medical Sciences, Sheikhpura, Patna, Bihar
Source of Support: None, Conflict of Interest: None
Introduction: Tuberculous meningitis (TBM) is the infection of Mycobacterium tuberculosis (MTB) among extra-pulmonary organs. The diagnosis of TBM can be considered a double sword in low-resource settings. On one side, there is poor access to health-care services, limited diagnostic capacity, and poor affordability. These factors hamper early treatment initiation while in high-resource settings, clinical suspicion towards TBM is considered minimal, and this lack of recognition many times leads to treatment delay. Xpert MTB/RIF test has come up as the diagnostic rescue with overall sensitivities exceeding 80% and specificity up to 100%. Materials and Methods: An observational study was done on 368 children up to 14 years of either gender with suspected TBM in 18 months. This whole duration was consumed in the planning of the study, obtaining ethical clearance, data collection, data analysis and report writing. Cerebrospinal fluid was collected by lumbar puncture. Samples from all patients underwent testing based on GeneXpert, Ziehl–Neelsen (ZN) stain and mycobacteria growth indicator tube (MGIT) culture. IBM Statistical Package for the Social Sciences (SPSS version 22) was used for data analysis. Results: A total of 321 patients were included in the analysis. The male-to-female ratio was 1.55, thus showing a male preponderance. The majority belonged to Hinduism. The median age was 7.5 years. Based on clinical assessment and radiology, 48 patients were diagnosed with confirmed and probable TBM. Overall, the sensitivity of Gene Xpert, ZN smear and MGIT was 68.75%, 4.2% and 75.0%, respectively. Rifampicin sensitivity of the isolated organism was reported, and it was found that 18 out of 33 patients, implying 54.5% of patients were resistant to rifampicin. Conclusion: Till today, TBM poses a life-threatening situation despite advances made in the diagnosis have been achieved. In this context, Gene Xpert represents a step forward.
Keywords: Cerebrospinal fluid sample, mycobacterial growth indicator tube, nucleic acid amplification test, NaOH-N-Acetyl-L-cysteine method
|How to cite this URL:|
Muni S, Gupta AK, Pankaj D, Kumar R, Kumar S, Kumari N. Evaluation of Gene-Xpert in paediatric tuberculous meningitis cases: A hospital-based study. Adv Hum Biol [Epub ahead of print] [cited 2022 Dec 2]. Available from: https://www.aihbonline.com/preprintarticle.asp?id=358636
| Introduction|| |
Tuberculous meningitis (TBM) occurs as a consequence of extrapulmonary involvement of infection with Mycobacterium tuberculosis (MTB), and it is the most severe form of involvement that has been found to be associated with substantial mortality. Literature over the years has reported that not <30% of patients presenting with this condition succumb to death soon. Rest who could survive end up with compromised neurological sequelae., Dual infection of HIV and TBM comes with an even worse prognosis where mortality has been reported to exceed 60%. In light of such devastating consequences, the best predictor of survival remains early diagnosis and treatment for TBM.,,,, Despite this fact, early diagnosis of TBM remains a challenge for the health care system because of its very non-specific presentation. Moreover, there is a dearth of diagnostic test that is rapid and sensitive at the same time. There is a lack of suspicion towards TBM on admission, so patients are given broad-spectrum antibiotics, but as the condition deteriorates, the need for consideration of other differential diagnoses arises. Diagnosis of TBM can be considered a double sword in low-resource settings. On one side, there is poor access to health-care services, limited resources for aiding diagnosis, and poorer affordability. On the other end, we have high-resource settings where clinical suspicion towards TBM is minimal, and this lack of recognition many times leads to treatment delay.
Microbiological confirmation of TBM has remained a challenge for most laboratories. In light of the lack of a confirmatory test for TBM, Ziehl–Neelsen (ZN) microscopy staining of acid-fast bacilli (AFB) in cerebrospinal fluid (CSF) has long been applied as a rapid diagnostic technique; although, the sensitivity of ZN staining for TBM is limited up to 10%–20% in a most conducive environment. Achieving a sensitivity of 20% is a meticulous and time-consuming task. Practically, it becomes very tedious to invest so much time in reporting negative samples. In contrast to the diagnosis of TBM, examination of sputum smears in just 10 min yields a diagnostic accuracy of 62%–70%.,,
In May 2015, a meeting was held in Vietnam to discuss diagnostic advances in the field of TBM. This meeting was attended by laboratory scientists all across ten nations. These scientists were involved in pioneer studies on the use of Xpert MTB/Rif (Xpert, Cepheid, Sunnyvale, California) for TBM diagnosis., The meeting was concluded with the World Health Organization (WHO) recommendation for the use of this technique 'in preference to conventional microscopy and culture as the initial diagnostic test for CSF, if the sample volume is low or if additional specimens cannot be obtained to make a quick diagnosis.', There was a concurrence that this recommendation of the WHO needs to be used with caution due to undocumented limitations of the procedure in the implementation manual.
The GeneXpert MTB/RIF is a closed-cartridge-based system. It requires minimal manpower and only 2 h to give results. Based on projects across six nations to validate this method, this has been approved by the WHO since 2010. It is a real-time polymerase chain reaction test that detects the presence of M. tuberculosis complex bacilli. Along with the diagnosis, it also gives information on the susceptibility of the causative organism to rifampin. This is considered a surrogate marker for the presence of multidrug resistance (MDR) disease in the patient. The closed-cartridge system has many advantages: (a) assay can be done without a laboratory environment, (b) lesser biohazard risk than smear microscopy with less generation of waste, and (c) reduced risk of cross-contamination. The sensitivity exceeds 90% alongside a high specificity too with sputum samples. In HIV-TB coinfection, the sensitivity is over 80%.,, After approval for sputum smears, Xpert MTB/RIF test has come up as the diagnostic rescue for extrapulmonary samples, as well, with overall sensitivities exceeding 80% and specificity up to 100%.,,,,
In this light of concern, this prospective analytical study was designed to address the issue of difficulty in diagnosing TBM in the paediatric age group. The main objective of the current study was to generate evidence in support of a newer microbial diagnostic modality in detecting TB meningitis in the paediatric population to introduce this rapid method for diagnostic accuracy in TBM patients.
| Materials and Methods|| |
Study type and duration
An observational study was planned and conducted at the Indira Gandhi Institute of Medical Sciences, Patna, Bihar. The total duration of the study was 18 months from January 2020 to June 2021. This whole duration was consumed in the planning of the study, obtaining ethical clearance, data collection, data analysis and report writing. Prior Institutional Ethical Committee (820/IEC/IGIMS/2019) approval certificate was obtained before starting the data collection for the study.
The study population comprised children of either gender up to 14 years of age presenting with suspected TBM to outpatient department (OPD) of Pediatrics at IGIMS, Patna.
Children of either gender <14 years and suspected of having TBM on clinical evaluation.
The volume of CSF <0.1 ml, inability or unwillingness to give informed written consent and patients who were on TB therapy for a duration longer than 1-week
All the paediatric patients who met the inclusion criteria and none of the exclusion criteria were included in the study. A total of 368 paediatric patients were reviewed for inclusion in the study during the total duration, and after assessing all the criteria, data from 321 study participants were used for final analysis.
Duly signed informed written consent forms were obtained from the accompanying guardian of the patient selected before enrolling them on the study. The age and gender of the patients were recorded against their registration numbers that were used as the unique identification of the patient to maintain anonymity and confidentiality of the data.
Sample collection and testing procedure
CSF was collected by the lumbar puncture technique. This CSF sample was centrifuged between 3000 and 4000 g for 15 min. The supernatant was removed to leave the deposit. This was aliquoted into various samples to be tested for Mycobacterial Tuberculosis using Ziehl–Neelsen smear preparation, inoculation of mycobacteria growth indicator tube (MGIT) culture and Gene Xpert/RIF testing. All tests were done according to the standard operating procedure, guidelines and protocols as lined by the Department of Microbiology, IGIMS, Patna.
- Ziehl–Neelsen smear: ZN smear was prepared using standard methods. Two drops of CSF deposit were layered on the smear. It was then stained as per the standard procedures. Stained smears were meticulously examined under an oil immersion microscope. Any presence of acid-fast bacillus was considered a positive smear for TBM
- MGIT culture: MGIT tube containing 0.8 ml MGIT supplement (PANTA antibiotics-polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin) and growth supplements were used. These tubes were inoculated with a 100 microlitre portion of the deposit. Inoculated tubes were then incubated in an MGIT 960 machine. Results were obtained automatically after 56 days. Obtained positive samples were tested for susceptibility to rifampin, isoniazid, streptomycin, and ethambutol
- Xpert MTB/RIF: A 200 μl portion of the deposit was resuspended in phosphate-buffered saline to a 500 μl volume. The sample reagent supplied with the test (1.5 ml) was then added. This final obtained mixture was vortexed for 30 s. This step ensures that all bacteria remain resuspended. This was then left to stand for another 15 min. The mixture was shaken intermittently. Using a Pasteur pipette, the solution was transferred to the Xpert cartridge. These cartridges were then loaded for analysis on the Xpert machine. Results were then reported. Rifampin resistance was checked.
For this study, TBM was made as an exclusion diagnosis. Later on, patients with a confirmed diagnosis of TBM were classified as definite, probable, or possible TBM based on standardized case definition.
'Definite TBM was defined as a clinical syndrome consistent with TBM, with acid-fast bacilli seen on CSF smear or M. tuberculosis isolated in CSF MGIT culture. Patients in the 'probable TBM' group had a diagnostic score of 10 or more without cerebral imaging (MRI or CT scan) or 12 or more with cerebral imaging, with at least 2 points from CSF or cerebral imaging criteria.'
'Possible TBM were patients with a diagnostic score of between 6 and 9 if cerebral imaging was not performed or between 6 and 11 if cerebral imaging was performed.'
'No TBM were all patients who did not meet the criteria or did not receive treatment for TBM and received an alternative discharge diagnosis were classified as not having TBM.'
Results were reported as positive or negative for M. tuberculosis. Rifampicin resistance was reported as susceptible or resistant. IBM Statistical Package for Social Sciences (SPSS version 22, IBM, Chicago) was used for data analysis. Schedules that were completely filled up were considered for data analysis. Descriptive analysis was performed and reported in the form of numbers and percentages to show the distribution of the study participants according to their background characteristics.
| Results|| |
A total of 368 patients were approached for the study based on the inclusion and exclusion criteria mentioned above. Out of those included, patients for whom no final diagnosis could be made or the data recorded were found to be incomplete were excluded from the final analysis. Based on this, a total of 321 patients were included in the analysis [Figure 1].
Out of 321 total patients, the male female ratio was 1.55, thus showing a male preponderance. The majority (87.9%) belonged to Hinduism; the rest followed Islam. The majority of the OPD attendees came from the rural household, 225 out of 321. Age ranged from 1-month to 14 years. Infants comprised 15.3% of the total study population. Among the other 84.7% of the children above the age of 1 year, the mean age was 7.1 years, with a standard deviation of 4.85 years. The median age was 7.5 years.
The most common presentation among the patients was fever along with headache, vomiting and altered sensorium. Focal neurological signs presenting most commonly as weakness of a limb is an advanced presentation seen among 2.2% of patients. Another 4.9% had diplopia or cranial nerve paresis. Yet another 2.8% had presented with slurred speech.
On applying the case definition, 33 were classified as definite TBM, 15 had probable or possible TBM and 273 did not have TBM. Those who did not have TBM based on the case definitions were later followed up and found to be diagnosed with viral meningoencephalitis, bacterial meningitis, eosinophilic meningitis, cerebral vascular event, cryptococcal meningitis, sepsis, pneumonia, cerebral toxoplasmosis, psychiatric disorder, cerebral tumour, prolonged fever of unknown origin, progressive multifocal leucoencephalopathy, dengue and cerebral abscess. Samples from all patients underwent testing based on GeneXpert, ZN stain and MGIT culture. Diagnostic accuracy for TBM was estimated separately for all the methods [Table 1].
The sensitivity of ZN staining for detection of MTB in CSF is very less. AFB could be stained in only two CSF samples. Both patients had TBM. Hence, though sensitivity was low for ZN staining to be a screening tool, it has high specificity [Table 2].
|Table 2: Diagnostic accuracy of Ziehl-Neelsen staining with various parameters|
Click here to view
Overall, the sensitivity of Xpert was 68.75%, and specificity was 100%. Positive predictive value (PPV) for Xpert was 100%, and negative predictive value (NPV) was calculated to be 78.62% [Table 3].
MGIT culture was 75%, and specificity of 100%. PPV was 100% and NPV 95.83% [Table 4].
|Table 4: Diagnostic accuracy of mycobacteria growth indicator tube with various parameters|
Click here to view
Rifampicin sensitivity of the isolated organism was reported, and it was found that 18 out of 33 patients, which comprises 54.55% of patients found positive for Xpert MTB, were resistant to rifampicin, thus showing the presence of multidrug-resistant tuberculosis in the study population.
Among patients with TBM, the mean CSF cell count was 312.6 cells/mm3. Lymphocytic pleocytosis was noted. In this group, the mean protein and glucose in CSF samples were 183.2 mg/dl and 32.8 mg/dl, respectively. CSF protein (>100 mg%), cells (>20/mm3) and sugar (<0.5) were compared between both group, and the results were statistically significant.
On computed tomography scan chest, bilateral infiltrates and hilar adenopathy along with miliary mottling were noted among 13 out of 48 cases (27.1%). This feature was suggestive of an active tuberculosis state.
Out of 48 TBM cases, there were 3 cases of extrapulmonary TB: 1 case each of spinal TB, renal TB and Central nervous system TB. Another 10.4% of patients had concomitant pulmonary TB. Mortality on follow-up for 8 months was 4.16%.
| Discussion|| |
The current study envisages the demographic characteristics of the population under consideration. The mean age of the patients was 7.1 years, with a standard deviation of 4.85 years. There was a male preponderance noted with a male-to-female ratio of 1.55:1. There has been a mixed report on gender dominance among TBM patients globally. While some report, it is more common among males, there are studies stating females are more affected.
For smear microscopy, the essential prerequisite is the presence of at least 5000–10,000 organisms per mL of sample. In this context, many similar studies, concluded a low sensitivity of smear microscopy in the diagnosis of TBM. The reported ranges from 2% to 30% for CSF. At the same time, the specificity of microscopy is always on the higher side, generally ≥90%., Hence, reports of the current that the sensitivity of ZN stating and microscopy for diagnosing TBM from CSF samples is 4.2% along with a specificity of 100% is not surprising. One of the underlying reasons for this is the usual scant bacillary concentration in the CSF. Besides, many times, tapped CSF volume is also too small to run tests and the field visible through an oil immersion lens is minuscule. It has been established the fact that factors that independently affect the reporting of smear microscopy are the volume of CSF tapped and time dedicated to the microscopic evaluation of the smear. In this context, the accepted norm is a microscopic examination of a minimum of 6 ml of CSF sample for at least 30 min is supposed to improve the accuracy of testing and the result. Another study by Kennedy et al. also gave similar speculation when demonstrating that acid-fast bacilli from CSF samples results are dependent on CSF volume, quick delivery of the sample to the laboratory from the point of collection without any delay and expertise of the technician involved.
Since time immemorial, the gold standard for diagnosis of acid-fast bacilli in any body fluid has been culture. Among various available culture media, liquid culture yields result twice faster as compared as solid culture, along with better sensitivity and remote chances of contamination. One of the strengths of the study was the incorporation of MGIT for all the obtained samples. The current study reported a sensitivity of 75% for MGIT with a 100% specificity. The associated disadvantage is the reporting time of culture is generally in days that delay the initiation of treatment and sometimes lead to loss to follow-up in poor set-ups. Another associated disadvantage is a requirement of a larger CSF volume to meet the required sensitivity, just like smear microscopy.
Xpert MTB/RIF has been implied to be a rapid as well as a specific test for diagnosis of TBM. While smear microscopy has been an age-old sensitive test in the field, Xpert MTB/RIF has emerged as a better candidate for implication as a screening tool with higher sensitivity, as demonstrated in various set-ups. This has been a landmark change in the diagnosis of paediatric TB after WHO recommendation supporting the use of Xpert MTB/RIF assay as the first step for diagnosis of clinically suspected paediatric cases. Reaching a sensitivity near to that of MGIT culture is reason enough to advocate a test to be made the diagnostic test as it yields results faster than the culture.
A meta-analysis by Detjen et al. concluded that the sensitivity of Xpert differs broadly based on a specimen collected, but the specificity ranged from 93% to 100%. In the current study also, the specificity has been reported to be 100%, while the sensitivity came to 68.75% for CSF, as the study prospects only TBM among all extra-pulmonary TB. This may be lower than the sensitivity reported from other types of samples like gastric aspirate as an adequate amount of CSF sample is crucial to achieving a good level of sensitivity, low sensitivity of Xpert in CSF in our study could be because of the low volume of CSF (<2 ml) tested with the assay. A pioneering study from South Africa also had a similar sensitivity to Gene Xpert.
In the 2013 meta-analysis conducted by the WHO, both the above-mentioned studies were included among the number of studies with few cases of meningitis among extrapulmonary tuberculous cohorts (a total of 117 cases in 16 studies) to evaluate the diagnostic accuracy of Gene Xpert for TBM. In their report, pooled sensitivity was 79.5%, with culture being the reference gold standard. When the diagnostic gold standard was clinical evaluation, sensitivity dropped down to 55% and had an NPV of 84%.
A similar study from Uganda, though, had a smaller study cohort that showed centrifugation of larger CSF volumes (median: 6 mL, interquartile range: 4–10 mL) resulted in 72% sensitivity, which was equivalent to culture (71%). A cross-sectional study by Sekadde et al. showed that Xpert is twice more accurate in diagnosing childhood pulmonary TB when compared to smear microscopy. In another study, Bates et al. also gave similar results that the Xpert has better diagnostic capacity than smear microscopy. In the current study also, Xpert detected ten such cases of TB, which were not detected with direct smear microscopy. However, there was no such case where direct smear microscopy revealed acid-fast bacilli, while Xpert showed negative results. Thus, the specificity of Xpert came to 100%.
Jenkins et al. conducted a systematic review where they included 97 studies covering approximately 1 lakh paediatric patients who developed TB in the year 2010. 3.2% of these cases had MDR disease. While in a similar study by Arora et al. in the national capital showed that MDR TB was confirmed in 24.7% of participating children with suspicion of MDR TB. In the current study, MDR TB was detected in more than 50% of the samples that tested positive by Gene Xpert. Here, in this study, MDR cases, by definition, were cases of primary drug resistance as the children never received an anti-tubercular regimen as given in their medical history and records. The resistance profile of the source cases with pulmonary TB was not a part of the study as tracing was out of bound of the research methodology. However, at the same time, it can be assumed that the source cases possibly transmitted the MDR strains of M. tuberculosis to these affected children.
Overall, the performance of the GeneXpert has been noteworthy as per the reports from globally. It greatly accelerated the time-to-results with a median time of <1-day as compared to more duration taken to generate reports of culture. Hence, this will help yield faster confirmation of TBM cases along with resistance profiles. This will fasten the treatment initiation and hence come up with better effectiveness of the therapy, hence improved outcome, survival and decreased neurological sequelae.
| Conclusion|| |
TBM remains a life-threatening situation even after advances have been made for a better and quicker diagnosis. Emphasis on a more efficient diagnostic modality for TBM in the paediatric age group is indispensable. Here, we conclude that Gene Xpert has a sensitivity of 68% and a specificity of 100%. Both these are higher than the conventional ZN smear and comparable to the MGIT culture, which is the gold standard for the diagnosis. Regarding antibiotic resistance, 54.5% of patients found positive for Xpert MTB were resistant to rifampicin, thus showing the presence of multidrug-resistant tuberculosis in the study population.
Limitation of the study
The current study envisages testing of a single specimen from suspected cases. It has been documented earlier than taking a repeat specimen has enhanced benefit on sensitivities of Xpert and culture. The researcher wishes to continue the study with a larger population and also include tracing of the resistant cases to find out the origin in future.
Taken from the Institutional Ethics Committee. Other ethical aspects like anonymity and confidentiality of data and patient autonomy were duly considered and taken care of during the conduct of the study.
We are thankful to the healthcare workers (faculty members) of IGIMS, Patna for their support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]