Comparison of DNA Extraction Protocols and Molecular Targets to Diagnose Tuberculous Meningitis

Tuberculous meningitis (TBM) is a severe form of extrapulmonary tuberculosis. The aims of this study were to evaluate in-house molecular diagnostic protocols of DNA extraction directly from CSF samples and the targets amplified by qPCR as an accurate and fast diagnosis of TBM. One hundred CSF samples from 68 patients suspected of TBM were studied. Four DNA extraction techniques (phenol-chloroform-thiocyanate guanidine, silica thiocyanate guanidine, resin, and resin with ethanol) were compared and CSF samples were used to determine the best target (IS6110, MPB64, and hsp65 KDa) by qPCR. The extraction protocol using the phenol-chloroform-thiocyanate guanidine showed the best results in terms of quantification and sensitivity of PCR amplification, presenting up to 10 times more DNA than the second best protocol, the silica guanidine thiocyanate. The target that showed the best result for TBM diagnosis was the IS6110. This target showed 91% sensitivity and 97% specificity when we analyzed the results by sample and showed 100% sensitivity and 98% specificity when we analyzed the results by patient. The DNA extraction with phenol-chloroform-thiocyanate guanidine followed by IS6110 target amplification has been shown to be suitable for diagnosis of TBM in our clinical setting

The relative test performance characteristics of two commercial assays for the detection of Mycobacterium tuberculosis complex in paraffin-fixed human biopsy specimens

The Seeplex™ TB Detection-2 assay (Rockville, MD) is a nested endpoint PCR for the Mycobacterium tuberculosis complex (MTBC) targets IS6110 and MPB64 that utilizes dual priming oligonucleotide technology. When used to detect the presence of MTBC DNA in formalin-fixed paraffin-embedded tissue specimens, the sensitivity and specificity of this assay is equivalent to a labor-intensive traditional endpoint PCR assay and is more sensitive than a commercial real-time PCR assay

A systematic review and meta-analysis of the diagnostic accuracy of nucleic acid amplification tests in cerebrospinal fluid for tuberculous meningitis

Introduction: Diagnosis of tuberculous meningitis (TBM) is difficult and poses a significant challenge to physicians worldwide. Recently, nucleic acid amplification (NAA) tests have shown promise for diagnosis of TBM, although performance has been variable. We undertook a systematic review and meta-analysis to evaluate the diagnostic accuracy of NAA tests in cerebrospinal fluid (CSF) samples against culture as the reference standard or a combined reference standard (CRS) for TBM.Methods: We searched Embase, PubMed, Web of Science and the Cochrane library for the relevant records. QUADS-2 tool was used to assess the quality assessment of the studies. Diagnostic accuracy measures (i.e. sensitivity and specificity) were pooled with a random effects model. All Statistical analyses were performed with STATA version 14 (Stata Corporation, College Station, TX, USA), Meta-DiSc version 1.4 (Cochrane Colloquium, Barcelona, Spain) and RveMan version 5.3 (Copenhagen: The Nordic Cochrane Centre, the Cochrane Collaboration).Results: Sixty-three studies were included in final analysis, comprising 1381cases of confirmed TBM and 5712 non-TBM controls. These 63 studies were divided into two groups comprising 71 datasets (43 in-house tests and 28 commercial tests) that used culture as the reference standard and 24 datasets (21 in-house tests and 3 commercial tests) that used a CRS. Studies which used a culture reference standard had better pooled summary estimates compared to studies which used CRS. The overall pooled estimates of sensitivity, specificity, positive likelihood ratio (PLR) and negative likelihood ratio (NLR) of NAA tests against culture were 82% (95% CI: 75-87), 99% (95% CI: 98-99), 58.6 (35.3-97.3) and 0.19 (0.14-0.25), respectively. The pooled sensitivity, specificity, PLR and NLR of NAA tests against CRS were 68% (95% CI: 41-87), 98% (95% CI: 95-99), 36.5 (15.6-85.3) and 0.32 (0.15-0.70), respectively.Conclusion: The analysis has demonstrated that the diagnostic accuracy of NAA tests is currently insufficient to replace culture as a lone diagnostic test. NAA tests may be used in combination with culture due to the advantage of time to result and in scenarios where culture tests are not feasible. Further work to improve NAA tests would benefit from standardized reference standards and the methodology

The PCR-Based Diagnosis of Central Nervous System Tuberculosis: Up to Date

Central nervous system (CNS) tuberculosis, particularly tuberculous meningitis (TBM), is the severest form of Mycobacterium tuberculosis (M.Tb) infection, causing death or severe neurological defects in more than half of those affected, in spite of recent advancements in available anti-tuberculosis treatment. The definitive diagnosis of CNS tuberculosis depends upon the detection of M.Tb bacilli in the cerebrospinal fluid (CSF). At present, the diagnosis of CNS tuberculosis remains a complex issue because the most widely used conventional “gold standard” based on bacteriological detection methods, such as direct smear and culture identification, cannot rapidly detect M.Tb in CSF specimens with sufficient sensitivity in the acute phase of TBM. Recently, instead of the conventional “gold standard”, the various molecular-based methods including nucleic acid amplification (NAA) assay technique, particularly polymerase chain reaction (PCR) assay, has emerged as a promising new method for the diagnosis of CNS tuberculosis because of its rapidity, sensitivity and specificity. In addition, the innovation of nested PCR assay technique is worthy of note given its contribution to improve the diagnosis of CNS tuberculosis. In this review, an overview of recent progress of the NAA methods, mainly highlighting the PCR assay technique, was presented

Penyengatan Meningeal Sisterna Basalis Meningitis TB pada Computed Tomography Scanning: Sebuah Ulasan Bergambar

Tuberculosis (TB) is one of the major worldwide threat and global burden in Indonesia. CNS tuberculosis is the most severe form of TB infection. CT evaluation on diagnosing tuberculosis meningitis (TBM) with triad of hydrocephalus, basal meningeal enhancement and infarction was reported to be sensitive. PCR cerebrospinal fluid (CSF) is known to be specific, however negative results have been reported due to presence of PCR’s inhibitors, poor lysis of mycobacteria, and the uneven distribution in specimens. CT Scan plays the vital role in diagnosing TBM patient with the presence of TBM CT triad, especially basal cistern enhancement (BME) which is the specific enhancement pattern in MTB patient. There are 9 must know BME pattern, such as contrast filling the cistern, double and triple line, linear enhancement at MCA cistern, Y sign, posterior infundibular recess enhancement, ill defined border, join the dots sign, nodular enhancement, and asymmetry off all pattern. Familiarizing BME criteria is essential to provide confident diagnosis and reduce morbidity and mortalit

Molecular diagnostic methods for rapid diagnosis of central nervous system infections

Central nervous system infections (CNSI) are serious life-threatening conditions caused by bacteria, viruses, fungi, and parasites and lead to high morbidity and mortality worldwide. Therefore, rapid identification of causative organisms and appropriate treatment are important. The traditional identification methods are time-consuming and lack sensitivity and specificity. Although culture method is gold standard for CNSI, it is time-consuming and microbiology reporting requires several days. Multiplex PCR assays can detect multiple pathogens simultaneously in clinical samples and overcome the limitations of conventional identification techniques. Despite the availability of several commercial molecular-based platforms for the detection of pathogens causing CNSI, there are still limitations in terms of cost, false positive results, and false negative results, which are limited to targeted pathogens in the panel. Moreover, validation of many commercially available and in-house laboratory-developed molecular assays is still lacking. In addition, molecular diagnostic tests need to be used in correlation with the clinical context to ensure better diagnosis and management of infections

A point-of-care diagnostic for tuberculosis

Mycobacterium tuberculosis is a significant global health concern, requiring rapid and accurate detection methods for effective diagnosis and prompt treatment. This research builds on previous studies to advance the development of a point-of-care (POC) assay for the rapid identification of M. tuberculosis, employing the BCG strain of M. bovis as a surrogate model. A microwave-assisted DNA extraction technique was optimised to rapidly release DNA from M. bovis suspended in water and in simulated sputum containing methylcellulose to mimic the viscosity of respiratory samples. DNA probes specific for the IS6110 and IS1081 gene targets were developed, attached to magnetic nanoparticles, and assessed for their ability to capture target DNA in different sample volumes. The sensitivity and specificity of the assay were optimised, resulting in a reduction in the time taken to identify M. bovis positively. The sensitivity of the approach was further enhanced by increasing the sample volume from 200 μL to 10,000 μL. This microwave-based method yielded faster results than conventional methods, such as culture and PCR. Our results suggest that there is potential to further increase assay sensitivity and reduce detection time, ultimately leading to the development of a rapid, sensitive assay for the detection of M. bovis and M. tuberculosis

Missed Window, Lost Life: The Deadly Course of TB Meningitis in a Pregnant Woman

Tuberculosis meningitis is the most severe form of extrapulmonary TB. TB meningitis has an insidious onset and atypical clinical manifestation. Thus, it is usually in its advanced stage when it is diagnosed resulting in poor therapeutic efficacy and often causing severe extrapulmonary tuberculosis with high mortality. Despite the presence of different diagnostic and treatment modalities, mortality remains unacceptably high. A pregnant female patient was referred to our hospital’s emergency with suspected meningitis with a history of fever and recurrent vomiting. She was being managed conservatively and several tests were done (Complete Hemogram, Metabolic Profile, and Infection Panel). After various neurological investigations, CT scan of the brain revealed a lesion, suspicious of TB meningitis, though not confirmed. She gave birth to a baby girl, after which she felt drowsy. ATT was initiated 5 days post admission, on the day following her delivery. Her condition was still deteriorating. The patient died at last, following which the final diagnosis of TB meningitis came. The following case illustrates the clinical presentation and diagnostic approach in a pregnant lady. TBM diagnosis has many intricacies though the disease is completely curable. 

Challenges in diagnosis and treatment of extrapulmonary tuberculosis

The infectious disease tuberculosis (TB) is a global health problem. Diagnosis of extrapulmonary TB is often challenging due to non-specific symptoms and findings, and the lack of sensitive diagnostic tests. This leads to both under- and overdiagnosis and extensive use of empirical TB treatment, which increases the risk of placing individuals without TB or individuals with multi-drug resistant TB on prolonged, incorrect treatment. New tools for diagnosis and assessment of treatment response of extrapulmonary TB are needed to improve TB care. The overall aim of this thesis was to investigate new methods for diagnosis and treatment monitoring of extrapulmonary TB. Specific aims included to assess the performance of the MPT64 antigen detection test (MPT64 test) and the Xpert Ultra assay for the diagnosis of extrapulmonary TB in a low TB prevalent setting. Further, to reproduce an anti-MPT64 antibody for large-scale use of the MPT64 test. Finally, to study the potential of IP-10 measured in dried plasma or blood spots as a biomarker for treatment response in patients with extrapulmonary TB. Extrapulmonary samples received for TB diagnostics at the Microbiology and/or Pathology laboratories in a clinical setting in Norway were collected and subjected to the MPT64 test and/or Xpert Ultra. The performance of the new tests was compared to results of routine TB diagnostics. New anti-MPT64 antibodies were developed in mice and rabbits by use of different antigen-adjuvant combinations and screening in immunohistochemistry. The best performing individual antibodies were pooled, and the performance of the final pooled anti-MPT64 antibody was evaluated in patients samples. IP-10 dynamics during TB treatment were studied in extrapulmonary TB patients at a tertiary care hospital on Zanzibar, Tanzania. Plasma, dried plasma spots and dried blood spots were collected at baseline, 2 months of treatment and end of treatment. IP-10 levels were measured by ELISA and compared to clinical improvement. In paper I, we found that the sensitivity of the MPT64 test for diagnosing extrapulmonary TB was lower compared to results obtained in previous studies conducted in TB endemic low-resource settings. The best test performance in our setting was demonstrated in formalin-fixed biopsies, where the MPT64 test showed an excellent specificity and a sensitivity in the same range as PCR-based tests, but lower than culture. The results of paper II indicate that it is possible to reproduce a functional polyclonal anti-MPT64 antibody for large-scale use of the MPT64 test, but that careful selection of the antigen-adjuvant combination for immunisation and comprehensive screening strategies are nessecary to obtain high-performance antibodies. In paper III, we found that the overall sensitivity and specificty of Xpert Ultra for diagnosing extrapulmonary TB in low-TB prevalent high-income setting were high and close to the performance of culture. In paper IV, we report that a significant decline in IP-10 levels in plasma, dried plasma spots and dried blood spots during treatment was observed in extrapulmonary TB patients, and the decline was significant already after two months in HIV-negative patients. The correlation between IP-10 measured in plasma and dried plasma spots or dried blood spots was high. To conclude, potential new tools for improved diagnosis and treatment monitoring of extrapulmonary TB have been identified. Our results indicate that Xpert Ultra can contribute to a more rapid diagnosis of extrapulmonary TB in our setting, and that IP-10 measured in dried blood spots is a promising marker for response to treatment in extrapulmonary TB patients. However, further studies with larger sample sizes that include relevant negative controls and longer follow-up of patients to evaluate the clinical impact of these new tools are needed. The utility of the MPT64 test is likely limited in settings with well-functioning culture facilities, but the test can contribute to strengthen the TB diagnosis in the event that samples have not been subjected to culture.Doktorgradsavhandlin

Classic and New Diagnostic Approaches to Childhood Tuberculosis

Tuberculosis in childhood differs from the adult clinical form and even has been suggested that it is a different disease due to its differential signs. However, prevention, diagnostics, and therapeutic efforts have been biased toward adult clinical care. Sensibility and specificity of new diagnostic approaches as GeneXpert, electronic nose (E-nose), infrared spectroscopy, accelerated mycobacterial growth induced by magnetism, and flow lateral devices in children populations are needed. Adequate and timely assessment of tuberculosis infection in childhood could diminish epidemiological burden because underdiagnosed pediatric patients can evolve to an active state and have the potential to disseminate the etiological agent Mycobacterium tuberculosis, notably increasing this worldwide public health problem