Cartridge-based Nucleic Acid Amplification Research Articles

Disseminated Intravascular Coagulation during a Course of Miliary Tuberculosis with Acute Respiratory Distress Syndrome

Abstract Miliary tuberculosis (TB) can occasionally lead to acute respiratory distress syndrome (ARDS) and disseminated intravascular coagulation (DIC). In this case report, we present the case of an 18-year-old male who was diagnosed with miliary TB based on miliary shadows on X-ray and computed tomography of the chest, as well as positivity for mycobacterium TB in endotracheal aspirate by cartridge-based nucleic acid amplification. The patient’s hospital stay was complicated by ARDS and DIC, which was successfully managed with ventilatory support, administration of antitubercular treatment, systemic corticosteroids, and blood products.

Diagnostic Accuracy of Cartridge Based Nucleic Acid Amplification Test (CBNAAT) in Stool Samples in Pediatric Tuberculosis.

To find out the diagnostic accuracy of stool Cartridge-based nucleic acid amplification test (CBNAAT) as an alternate method as compared to CBNAAT in gastric aspirate (GA) samples in pediatric tuberculosis (TB). This cross-sectional study was performed at Department of Pediatrics of a tertiary care hospital. Children aged 0-18 y diagnosed as presumptive tuberculosis were consecutively enrolled. Gastric aspirate and corresponding stool sample was subjected to CBNAAT and its performance was compared in both samples using appropriate statistical tests. Total 100 patients were enrolled in the study. Diagnostic accuracy of CBNAAT was 81% and 80% in gastric aspirate and stool sample respectively. On comparing gastric aspirate with corresponding stool sample there was 97% agreement, with Cohen's kappa value of 0.94. There was a statistically significant association observed between gastric aspirate CBNAAT and stool CBNAAT p <0.001 using chi square test. Sensitivity of gastric aspirate CBNAAT and stool CBNAAT was 75% and 73% respectively and specificity was 100% for both the samples compared against Composite Reference Standard (CRS). The diagnostic accuracy of stool CBNAAT is comparable to GA CBNAAT in children and can be used as a good alternative to gastric aspirate for diagnosis of pulmonary and disseminated tuberculosis in children.

Detection of drug-resistant Mycobacterium tuberculosis in pericardial fluid culture and its correlation with cartridge based nucleic acid amplification test and adenosine deaminase activity

BackgroundPericardial effusion is the accumulation of fluid in the pericardial cavity. In nations with high tuberculosis (TB) load, TB is the most common cause of pericardial effusion. 1–2% of patients with pulmonary TB develop Pericardial TB worldwide. Multi-drug-resistant (MDR) TB, including extrapulmonary TB (EPTB) cases, are rising in number. Adenosine Deaminase (ADA) is an enzyme in lymphocytes and myeloid cells, which has certain immune functions in the body. ADA levels are increased in inflammatory conditions, like pleural, pericardial, or joint effusions, of bacterial etiology, granulomatous conditions, neoplasms, and autoimmune pathologies. TB is the only lymphocytosis involving disease with increased ADA levels. MDR EPTB is rare, but cases are on the rise, and tuberculous pericardial effusion is one such example. Hence, it is important to know the percentage of cases detected by a culture that can be identified by cartridge-based nucleic acid amplification test (CBNAAT), their resistance patterns, and to identify potential markers like ADA, which can help in early identification of cases. The objectives of this study were to identify the Mycobacterium tuberculosis (MTB) bacilli in culture, and correlate them with cartridge-based nucleic acid amplification test (CBNAAT) results and their drug-resistance, in the Pericardial tubercular effusion, and to find if Adenosine Deaminase (ADA) levels can be used as a predictor of the presence of MTB in pericardial fluid. MethodologyWe enrolled 52 patients with moderate to large tuberculous pericardial effusion, based on pericardial fluid analysis, CBNAAT, and culture methods, between January 2021 and December 2021. ResultsThe mean age of the patients was 41.85 + 17.88 years, with a median of 38 years. Males made up 57.7% of the total patients. MTB was detected in 16 (30.8%) patients in the CBNAAT evaluations. 14 (87.5%) of the CBNAAT-positive TB patients were sensitive to Rifampicin, whereas the remaining 2 (12.5%) were resistant to Rifampicin on CBNAAT. MTB was found to be growing in 8 (15.38%) drug sensitivity test cultures. Out of these 8, 6 were sensitive to first-line drugs, whereas 2 were resistant to both Isoniazid and Rifampicin. The presence of cough was found to have a significant difference between CBNAAT-detected MTB positive and negative patients (p = 0.020), whereas an insignificant difference was found for the presence of hypertension, diabetes mellitus, obesity, dyspnea, or fever. There was also an insignificant difference between the number of patients positive for the Tuberculin skin test, between the two groups. ADA was significantly higher in the MTB-detected CBNAAT group (85.91 + 37.60U/L vs 39.78 + 24.31U/L, p = 0.005), whereas the total leukocyte count, lymphocytes, neutrophils, random blood sugar levels, and serum protein levels had no significant difference. The area under the Receiver Operator Curve (CBNAAT positive: dependent variable; ADA: test result variable) was 0.854 (null hypothesis rejected), with a standard error of 0.078. ConclusionsCulture is the gold standard method to diagnose tuberculosis. Detection of MTB on pericardial fluid culture is very uncommon, though in our study, culture came out positive in 16% of patients, and 4% were resistant to rifampicin and isoniazid. Higher ADA levels in pericardial fluid are an indicator of tuberculous pericardial effusion.

Role of line probe assay in detection of Mycobacterium tuberculosis in children with pulmonary tuberculosis

BackgroundIndia is the highest TB burden country in the world with almost 27 lakh cases reported in 2019. Pediatric tuberculosis in India accounts for almost 31% of global TB burden. Despite such high mortality and morbidity in children, diagnosis of pulmonary TB in children still remains very challenging. Material and methodsThis cross-sectional study was conducted in a tertiary care hospital in Delhi, India. Children between 1 and 12 months with clinical suspicion of pulmonary tuberculosis who had not previously taken ATT were included.Early morning gastric aspirate samples were collected after overnight fasting, on two days. Both days sputum sample were subjected to sputum smear microscopy and one of the two samples was subjected to line probe assay (LPA), cartridge based nucleic acid amplification (CBNAAT) and mycobacterium growth in tube (MGIT-960). Results84 children with pulmonary tuberculosis were enrolled. The most common presenting complaint was fever seen in 83 patients (98.8%). Only 17 (20.24%) were sputum smear positive by Ziehl- Neelsen (ZN) staining. LPA was positive in 47 (55.95%) samples and among these positive samples both INH and RIF resistance was detected in 2 (4.26%) samples. CBNAAT was positive in 53 patients (63%). Growth in liquid culture media (MGIT 960) was observed in 44 (52.38%) samples. Among 17 smear positive samples, LPA was detected in 14 (82.35%) samples and among 67 smear negative sample LPA was detected in 33 (49.25%) samples. LPA had 63.46% sensitivity, 100% specificity in detecting mycobacterium tuberculosis. DiscussionWHO's recommendation for using LPAs has been limited to culture isolates or smear-positive sputum specimens. New data has since been generated on the use of LPAs as newer versions of LPA have been developed over past few years.Previous studies conducted using LPA version 1.0 reported much lower detection rate of mycobacterium tuberculosis in smear negative specimens.With the availability of newer versions such as Hain GenoTypeMTBDRplusversion 2 and Nipro NTM + MDRTB detection kit 2, the diagnostic utility of LPA may be enhanced.

Clinical profile and outcome in patients with rifampicin-resistant tuberculosis taking shorter multidrug-resistant tuberculosis treatment regimen at a tertiary care hospital in South Gujarat

Background: India remains one of the six countries with an enormous multidrug-resistant tuberculosis (MDR TB) burden. Shorter MDR regimen is more economical/cheaper, shorter and having an improved adherence for Rifampicin resistant (RR)-TB. There were not many studies on this shorter MDR regimen (containing high-dose moxifloxacin) in India, we had carried out this study to find out outcomes and adverse drug reactions (ADRs) of this Shorter MDR regimen. Aims and Objectives: To study the clinical profile, interim and final outcome, and ADRs in RR-TB patients taking shorter MDR regimens. Materials and Methods: Prospective observational longitudinal study which was carried out in the Department of Respiratory Medicine Department, New Civil Hospital, Surat from January 2019 to June 2020. All patients who were diagnosed as RR-TB by cartridge-based nucleic acid amplification were enrolled in the study. Eligible Patients (n = 60) were started on shorter MDR regimen and were followed up. Results: Out of these 60 patients 35 (58.3%) were male and 25 (41.6%) were female. The age group mainly included was between 20 and 40 years. Out of 60 patients, 55 (91.6%) had pulmonary and 5 (8.3%) had extrapulmonary TB. Interim Outcome in the form of culture conversion at the end of 4th, 5th, and 6th month was seen in 21 (45.65%), 37 (80.43%), and 45 (97.83%), respectively. The final outcome among 60 patients were 33 (55%) cured, 12 (20%) treatment completed, 6 (10%) lost to follow-up, 5 (8.33%) regimen changed, 3 (5%) died, and failure in 1 (1.66%). These results were matching with most of the other studies. Conclusion: This study showed successful outcome (cured and treatment completed) in 45 patients (75%) and adverse outcome (died, lost to follow-up, and failure) in 10 patients (16.67%). A shorter MDR regimen is shorter, cheaper, effective in most of the RR-TB patients and is well tolerated with mild side effects.

Role of cartridge-based nucleic acid amplification test in diagnosing extrapulmonary tuberculosis

Background: Tuberculosis (TB) is a major health concern globally. Extrapulmonary tuberculosis (EPTB) accounts for around 20% of the total (EPTB) cases which are difficult to diagnose due to the lack of availability of diagnostic tools. Although pulmonary TB is most common presentation, EPTB is also an important problem clinically. Cartridge-based nucleic acid amplification (CBNAAT) test has a well-documented role in diagnosing pulmonary tuberculosis. The potency of CBNAAT in the diagnosis of EPTB cases is still debated. Aim: To establish the importance and role of diagnosing EPTB by CBNAAT. Settings: Conducted in Dr. D Y Patil Medical College Hospital and Research Centre, Dr. D Y Patil Vidyapeeth, Pimpri, Pune-411018. Design: Retrospective cross-sectional study of suspected EPTB patients in a tertiary care center area from April 2020 to September 2020. Materials and Methods: Data of suspected EPTB patients were retrieved. Potency of CBNAAT in diagnosing EPTB was assessed. Statistical Analysis: All the statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS) version 20. Results: Samples collected from 556 suspected EPTB patients were subjected to CBNAAT. The collected samples included pus, pleural fluid, cerebrospinal fluid, gastric aspirate, peritoneal fluid, tissue biopsy, pericardial fluid, ascitic fluid, synovial fluid, drain fluid, para-spinal fluid. In CBNAAT results, 67 cases were positive for TB bacilli, rifampicin resistance detected in 4 samples. Conclusion: Xpert®MTB/RIF assay is a rapid and effective method for early detection of EPTB and can be used in conjunction with routine culture methods for detection of the same.

Adverse Drug Reaction Monitoring in Multidrug-Resistant Tuberculosis Patients Receiving Bedaquiline and Delamanid-Based Regimen.

Background and objectives Tuberculosis (TB) is an airborne contagious illness caused byMycobacterium tuberculosis.Ineffective anti-TB medication prolongs and exasperates illness, promotes disease spread,increases the probability of developing resistance to treatment, and increases death rate.Bedaquiline (BDQ) and Delamanid (DLM) were conditionally made available in the treatment of multidrug-resistant TB (MDR TB).In drug-resistantTB patients, adverse drug reactions (ADR) management is essential to improve medicationcompliance. In addition, we performed this study since there are very few studies published on the analysis ofADR monitoring of BDQ and DLM-based regimen. This study was performed tostudy the spectrum of ADR in drug-resistant TB patients receiving BDQ andDLM-based regimen. Methodology The study was conducted over a period of 26 months, in a hospital's departments of pharmacology and pulmonary medicine.Pre-extensively drug-resistant (Pre-XDR) and XDR TBwere established on the basis of cartridge-based nucleic acid amplification (CB-NAAT), line probe assay (LPA), drug susceptibility testing (DST), and bacteriological culture. Patients were prescribed with appropriate medicines at the initial visit and any adverse reactions to medication were assessed in the subsequent visit. The statistical analysis was done using frequency distribution procedure, chi-square test of independence. The significance level was set at p<0.05. Results It was revealed that there were as many as 24 types of ADRs manifested in different patients.The most frequent ADR was QTcF (corrected heart rate) prolongation. Conclusion The maximum number of patients had some form of ADR and the percentage was slightly higher in the BDQ group than in the DLM group.

Association of cycle threshold values of CBNAAT with severity and outcome in COVID-19

Determination of viral load through cycle threshold (Ct) values may act as a predictor of severity and outcomes in patients with corona virus disease 2019 (COVID-19). However, variable literature is available regarding this relationship. Our study attempted to explore this association and the effect of various socio-demographic and clinical parameters on severity and outcome of COVID-19. Retrospective analysis of records of 731 patients whose nasopharyngeal/ oropharyngeal swabs were subjected to cartridge based nucleic acid amplification (CBNAAT) on Cepheid Xpert Xpress SARS-Cov-2 was done. Cycle threshold (Ct) values of N2 and E genes were studied in relation to severity and outcome of COVID-19. The viral load as determined by Ct values was classified as high (&lt;25), medium (25.1-32) and low (&gt;32). Association of socio-demographic and clinical parameters with respect to severity and outcome was also studied. Severity and mortality were significantly more in elder individuals, those belonging to the rural background, those with symptoms &gt;7 days in duration before presentation and those with increasing number of co-morbidities (Severity: p&lt;0.001, mortality: p &lt;0.001, 0.005, 0.006 and &lt;0.001 respectively). The Ct values of gene N2 and E inversely correlated with severity and mortality from the disease (N2 gene: p=0.001 for both severity and mortality, E gene: severity: p&lt;0.001, mortality: p=0.016 respectively). The severity of the illness and chances of mortality were significantly lesser when the CT value of N2 gene was &gt;32, in comparison when it was upto 25, and when between 25.1 and 32 (severity: p=0.032 and 0.003 respectively, mortality: p=0.018 and &lt;0.001 respectively). Almost similar trends were seen with respect to E gene (severity: p&lt;0.001 and 0.067 respectively, mortality p=0.175 and 0.005 respectively). Viral load as determined by Ct values of N2 and E genes can act as surrogate markers for prediction of severity and disease outcomes in COVID-19.

A cross sectional study of in-hospital cases of Pediatric Tuberculosis detected by CBNAAT at a tertiary care teaching hospital of Central India.

Background: Childhood tuberculosis is difficult in the identification of the organism due to impropersampling as well as low sensitivity of the smear. Newer diagnostic methods like Cartridge basednucleic acid amplification tests (CBNAAT) can rapidly identify Mycobacterium tuberculosis withimproved sensitivity over the smear testing. Material &amp; Method:This observational record basedcross-sectional study was undertaken to identify the epidemiology of tubercular infection in childrendiagnosed with CBNAAT. The study was carried out by analyzing the data of children from six monthsto 18 years who were diagnosed with Tuberculosis and treated with anti-tuberculosis therapy (ATT)from April 2018 to March 2020. Results: Data of a total of 166 patients was analyzed. 42% ofoverall collected samples were reactive to CBNAAT testing for tuberculosis. 40 gastric aspiratesamples were collected and only four (10%) turned reactive for tuberculosis by CBNAAT. None of thepediatric samples was positive for MDR TB. 66% of children completed treatment and 33% weredeclared cured. Conclusion: 42% positivity after CBNAAT testing for tuberculosis infection incollected samples of sputum and gastric aspirate where only 10% yield in GA samples.

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

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).