Diagnostic Potential of Circulating Biomarkers in Adenosine Deaminase Diagnosed Pleural Tuberculosis Cases

Pleural tuberculosis accounts for nearly 20% of Extra pulmonary tuberculosis. Adenosine deaminase, commonly used biomarker for the diagnosis, is non specific and there is paucity of literature on its correlation with conventional or newer methods for the diagnosis of extra pulmonary forms of TB. The aim of the study was to assess diagnostic potential of T cell function markers [interferon (IFN-γ), interleukin (IL-2) and IFN-γ/IL-2 ratio]; macrophage activation marker [neopterin]; and oxidative stress markers [protein carbonyl and malondialdehyde (MDA)] in pleural tuberculosis. 26 pleural TB cases diagnosed on the basis of suggestive chest X-ray and raised serum ADA levels and healthy controls were included in the study. Pleural fluid specimens were subjected to Zeihl Neelsen staining and culture on Lowenstein Jensen medium. Serum IFN-γ, IL-2, neopterin and protein carbonyl levels detection were done by ELISA and MDA levels were determined by measuring the thiobarbituric acid reactive substances. Median serum levels of IFN-γ, IL-2, IFN-γ/IL-2 ratio, neopterin, protein carbonyl and MDA were significantly different between cases and controls. Levels of all biomarkers except IL-2 were significantly higher in cases with contact history. Mean levels of ADA and ESR were 46.27 U/L and 46.62 mm/hr in PTB cases. AUC for IFN-γ, IL-2, IFN-γ/IL-2 ratio, neopterin, protein carbonyl and MDA were significantly discriminative for cases and controls. IFN-γ/IL-2 ratio was best discriminatory biomarker with highest area under ROC curve. Though no correlation was seen between ADA and any of the six biomarkers, ESR levels correlated significantly with all biomarkers except IL-2 by spearman’s correlation coefficient. Though all the circulating biomarkers under study provide useful supportive evidence for the diagnosis of PTB, further studies involving diverse control groups particularly non-PTB effusion are needed to validate these results.     

Immunodiagnosis of tuberculosis: An update

Tuberculosis is still a major health problem in most developing countries and its incidence is rising in many developed countries. This resurgence has been attributed to the HIV epidemic and TB has been declared as a global health emergency by WHO in 1993. The diagnosis of tuberculosis mainly depends upon initial clinical suspicion and radiographic findings with subsequent bacteriological confirmation by sputum smear examination and culture. Lack of sensitivity in smear examination, non specificity of radiological findings, extended tum around time ofMycobacterium tuberculosis culture and difficulties in diagnosing paucibacillary, childhood and extrapulmonary tuberculosis has necessitated to explore the utility of immunodiagnosis of tuberculosis as a convenient and cost effective test to supplement clinical information for definite diagnosis. Many commercial tests are available in the market for diagnosis of TB. Most of these tests are based on the detection of IgG, IgA and IgM antibodies to specific mycobacterial antigen or mixture of antigens. Indigenous immunoassay systems have explored excretory-secretory ES-31 mycobacterial antigen for immunodiagnosis of TB. Many a time there is lack of consistent elevation in all the three Ig classes in active infection thus making it more important to determine the ideal antibody isotype assay for reliable diagnosis of tuberculosis and to save the costs of the patient for unnecessary investigations.     

Potential of mycobacterial excretory secretory protein antigens (sevatb ES-31, ES-43, EST-6 and ES-20) as biomarkers to detect <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> bacilli

There is a need for a simple and reliable method to identify Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM). The utility of mycobacterial ES-31, ES-43, EST-6 or ES-20 antigen as a biomarker for differentiation of Mycobacterium tuberculosis bacilli from nontuberculous mycobacteria was explored using Fluorescein isothiocyanate conjugated antibodies against these antigens. Detection of these antigens was done from M.tb H₃₇Ra and H₃₇Rv DSS antigen. The presence of antigen in bacilli using FITC labelled antibody was indicated by green fluorescence on the cell surface while, its absence by no fluorescence under microscope. In M.tb H₃₇Ra and H₃₇Rv bacilli, fluorescence was observed on addition of FITC labelled anti ES-31 and anti ES-43 antibody; whereas no fluorescence was observed in case of EST-6 and ES-20 antibody conjugates. However all the antigens were detected in detergent soluble sonicate antigen of tubercle bacilli on addition of FITC conjugates. Fluorescence was not observed for ES-31, ES-43, EST-6 and ES-20 antigen in any of the tested NTM as well as in Escherichia coli. SEVA TB ES-31 and ES-43 may be used as biomarkers to distinguish M.tuberculosis bacilli from NTM.     

Serodiagnosis of tuberculosis using two ELISA systems

Serodiagnosis by ELISA has been widely explored over the years, in the diagnosis of tuberculosis. Two ELISA systems were evaluated for detection of mycobacterial antibodies in pulmonary and extra pulmonary tuberculosis. The two test assays explored were ERBA LISA (TB IgG) test (Anda Biologicals) which uses A60 antigen complex found in the cytosol of typical and atypical mycobacteria, and SEVA TB (IgG) ELISA, which uses a 31 kDa, glycoprotein antigen purified fromM. tb H₃₇Ra culture filtrate. Sera from 98 proven tuberculosis [pulmonary TB (48), tuberculous lymphadenopathy (30), tuberculous meningitis (15) & genitourinary TB (5)] were studied along with 32 healthy controls. The overall positivity obtained using ERBA LISA (TB IgG) test and SEVA TB (IgG) ELISA test was 72.9% and 91.6% in pulmonary tuberculosis, 43.3% and 76.6% in tuberculous lymphadenopathy respectively. The sensitivity of ERBA LISA test in tuberculous meningitis and genito-urinary TB was significantly low (26.6% & 40% respectively) compared to sensitivity obtained using SEVA TB ELISA (86.6% & 60% respectively) with overall specificity of 60% and 87.5%. Thus SEVA TB IgG ELISA test was found to be more sensitive than ERBA LISA in detecting IgG antibodies in tuberculous sera, in particular in extra pulmonary tuberculosis cases.     

Analysis of SEVA TB ES-31 antigen specific immunoglobulins IgM, IgA and IgG in sera of sputum and culture positive pulmonary tuberculosis

Tuberculosis remains major health problem in India and developing countries Immunodiagnosis has important role in screening, diagnosis and management of tuberculosis. SEVA TB ES-31 antigen has shown potential in detecting tuberculous IgG antibody in earlier studies from our laboratory. In the present study we have analysedSEVA TB ES-31 antigen specific immunoglobulinsIgM, IgA and IgG in clinically and bacteriologically confirmed pulmonary tuberculosis cases to determine the usefulness of specific immunoglobulin class in the diagnosis of patients attending the hospital. Of the 30 cases of pulmonary tuberculosis 25 (83.3%) were positive for IgG, 19 (63.3%) for IgM and 16 (53.3%) for IgA. On combining IgG and IgM positivity, sensitivity was increased to 93.3%. While combining IgG and IgA positivity, sensitivity increased to 90%. However specificity was decreased to 66.6% and 70% for both of these combinations respectively. It could be envisaged from this study that IgG antibody detection against ES-31 antigen showed acceptable sensitivity (83.3%) and specificity (86.6%) compared to IgM or IgA alone or in combination. When immune responses were analysed according to degree of sputum positivity, IgG response was observed to be predominant in all grades, compared to IgM or IgA antibody. The addition of IgM or IgA as an adjunct test increases the sensitivity but at the cost of specificity. Hence the detection of IgG alone is more useful compared to IgM or IgA assay, in detecting tuberculosis disease cases coming to the hospital.     

Direct diagnosis ofMycobacterium tuberculosis in blood samples of HIV infected patients by polymerase chain reaction

We have developed a simple, economical and reproducible method for processing blood samples from HIV infected patients for diagnosis of tuberculosis. The procedure was validated on 55 samples selected for tuberculosis based on clinical criteria. 52 patients had radiological changes indicative of pulmonary tuberculosis of which only 28 were positive for AFB in sputum (sensitivity 54%) and 27 for tuberculin (sensitivity 52%). 26 HIV positive patients who showed positive X-ray did not react to tuberculin. The genus PCR probe missed 3 samples (sensitivity 94%) compared to X-ray.M.tuberculosis was detected in the blood of all X-ray positive cases by PCR using TB400 probe (sensitivity 100%) and another probe forM. tuberculosis, IS6110, missed 6 of them (sensitivity 88% compared to X-ray and 89% compared to TB400). It is proposed that this simple sample processing method could be used to screen all blood samples quickly for mycobacteremia using the genus PCR and only those positive for mycobacteria need to be tested forM.tuberculosis. This would save the scarce resources and time by reducing significantly the number of samples to be screened for species confirmation.     

Diagnostic role of the antibody response to the 38kDa, 16kDa proteins and lipoarabinomannan of mycobacterium tuberculosis

The antibody response to the 38kDa, 16kDa and Lipoarabinomannan (LAM) antigens ofMycobacterium tuberculosis was evaluated using three different ELISAs based on these antigens. The study group included tuberculosis patients (n=52), patients with HIV and TB co-infection (n=10), other chest symptomatics (n=5), HIV infected individuals (n=10), leprosy cases (n=7) and healthy controls (n=75). The results indicate that the 38kDa and LAM based ELISA for IgM/IgG has a low specificity (ranging from 69–85%) and sensitivity (ranging from 55–78%). When three ELISAs are carried out on a single patient the probability of detection of tuberculosis was significantly increased to 95.2% indicating that a single ELISA test is of low sensitivity and that a combination of ELISA’s may be needed to be of any value as a diagnostic test for tuberculosis. Additionally, a western blot assay of the serum antibody response to protein fraction ofM. tuberculosis was analysed in 15 tuberculosis patients and five healthy controls. A multiple antibody response to various M.tuberculosis proteins was observed which varied from patient to patient as compared to controls who showed a single 38–39 kDa protein band positivity. These finding suggest that a western blot assay which determines the antibody response to a set of antigenic components ofM. tuberculosis could be a better serological test for the diagnosis of tuberculosis in our population.     

Assessment of Serum Calcium and Phosphorus in Pulmonary Tuberculosis Patients Before,During and After Chemotherapy

Our study was aimed to assess the levels of serum calcium and phosphorus in pulmonary tuberculosis patients. Blood samples were collected from 40 patients with pulmonary tuberculosis before treatment (PTB-0), at the end of 2 months of intensive phase of treatment (PTB-2) and after 6 months of treatment (PTB-6). Age and weight matched normal healthy volunteers (n = 37) served as normal controls. Serum was analyzed for calcium and phosphorus. Serum calcium significantly decreased to hypocalcemic levels and serum phosphorus significantly decreased but was within normophosphatemic limits in pulmonary tuberculosis. Chemotherapy for tuberculosis managed to raise serum levels of both the ions, with hypocalcemia still persisting in majority of patients during treatment but getting resolved in a significant percentage of patients at the end of 6 months of treatment. Results indicate the need for calcium and phosphorus supplements in tuberculosis patients during chemotherapy. This study also warrants the need for regular monitoring of serum calcium and phosphorus in patients undergoing anti-tuberculosis treatment.     

Utility of serum LDH isoforms in the assessment of mycobacterium tuberculosis induced pathology in TB patients of Sahariya tribe

The present study was carried out in the Sahariya tribe of Central India, which reportedly have high prevalence of pulmonary tuberculosis. Total serum LDH and its tissue specific isoforms were estimated in TB patients and matched healthy controls to test the utility of LDH as diagnostic marker for tuberculosis. About 210 sputum positive cases and 328 age and sex matched sputum negative controls were recruited. The spectrophotometeric and densitometric analysis of each LDH isoform was carried out in both cases and controls. The mean values of serum LDH were estimated and compared for each class by t-test. The statistical comparisons were made between sputum negative controls and sputum positive cases by Mann-Whitney’s U test. The spectrophotometric estimation of serum LDH revealed significant (P=0.0016) increase in its level in cases (290 IU/L) as compared to controls (248 IU/L). The densitometric analysis of individual LDH isoforms in cases and controls demonstrated significant elevation in LDH1 (P>0.05), LDH2 (P>0.05) and LDH3 (P<0.005) in sputum positive cases in comparison to sputum negative controls. Our study revealed a positive correlation between serum LDH level and the presence of mycobacteria and their load, suggesting utility of LDH as an important diagnostic marker of tuberculosis induced stress, at least in tribal areas lacking access to modern clinical tests.     

Molecular Mycobacteriology and Expansion in Disease Diagnosis

Molecular diagnostic tools for tuberculosis (TB) have evolved quickly with new innovations which can provide unprecedented opportunities for the rapid, sensitive and specific diagnosis of M. tuberculosis in clinical specimens and the status of its drug sensitivity. Microscopy and culture methods can not be replaced but the molecular assays can be applied in parallel with any new molecular tests for the diagnosis of TB. For extra pulmonary specimens, the use of the amplification methods is advocated, since rapid and accurate laboratory diagnosis is critical. Customization of the diagnostic usefulness of a molecular assay, according to the ease, reliability and need for health care sector is of immense value in a modern clinical mycobacteriology laboratory.     

科技助力结核病防控：现状、进展与对策

结核分枝杆菌(Mycobacterium tuberculosis)导致的结核病(Tuberculosis,TB)这一长期困扰人类的慢性传染病至今依然是全球面临的重大公共卫生问题之一,也是单一传染性病原体致死的主要原因。目前距世界卫生组织(WHO)和联合国(UN)提出的TB控制目标尚有很大差距,如不采取紧急行动并争取尽快实现防控科技上的突破(如获得新疫苗和新药)从而快速降低TB发病率,全球TB防治目标很可能无法实现。文章总结了全球及我国TB发展态势,并分析了科技在TB防治中的贡献。基于以上分析,针对TB科技防治对策进行了思考并提出了建议,包括加强TB的基础研究、研发新型TB疫苗和药物、发展新型TB诊断技术手段、健全体系支撑并加强保障措施等,以期推进我国科技防治TB的政策布局与实践创新,最终促进WHO提出的《终止结核病战略》目标的实现。     

Diagnosis of gastrointestinal tuberculosis: Using cytomorphological,microbiological, immunological and molecular techniques — A study from Central India

The present study included three groups: (A) age and gender matched control (n=24) with no previous signs of M. tuberculosis complex (MTBC) infection, (B) patients (n=28) diagnosed with gastro-intestinal TB (GITB), (C) patients (n=50) with clinical and histo-pathological signs of GITB, but were culture and AFB negative. Real time assay performed using fluorescence resonance energy transfer hybridization probes showed a positivity index of 36 % in group C, i.e. 18 were found reactive from the total 50 cases studied. In addition, immune characterization of these 18 cases showed depleted CD₄⁺ count and increased levels of IFN-γ and TNF-α cytokines. No positive case was found in group A, while in group B, out of total 28 cases studied 27 were found positive. A combinatorial diagnostic approach for rapid detection and characterization of GITB might provide specific therapeutic strategies for prevention and treatment of the infection in future.     

Physical activity - an important preanalytical variable

The concentration of several biochemical and hematological biomarkers is strongly influenced by a number of preanalytical variables. Several lines of evidence attest that short, middle, and long-term exercise, as well as the relative intensity of physical effort (from mild to strenuous), may influence a broad array of laboratory variables. The amount of extracellular release and clearance from blood of most of these biomarkers is markedly influenced by the biological characteristics of the molecule(s), level of training, type, intensity and duration of exercise, and time of recovery after training. It is hence noteworthy that test results that fall outside the conventional reference ranges in athletes not only may reflect the presence of a given disease, but may frequently mirror an adaptation to regular training or changes that have occurred during and/or following strenuous exercise, and which should be clearly acknowledged to prevent misinterpretation of laboratory data. The aim of this narrative review is to provide an update about the most significant changes of some biochemical and hematological biomarkers in response to physical exercise, for appropriate interpretation of these changes in the context of physically active subjects.     

Preliminary study on circulating serum levels of tumor necrosis factor—Alpha in patients of tuberculosis

Tumor necrosis factor-alpha (TNF-α) has been implicated in the pathogenesis of several non-infectious and infectious diseases including tuberculosis. In a prospective longitudinal study, TNF-α level in blood was estimated by sandwich ELISA using anti human TNF-α antibody, in 22 patients with active pleuro-pulmonary and lymphnode tuberculosis before and after chemotherapy and 8 healthy controls. Six patients and six controls had detectable levels (> 5 pg/ml) of TNF-α in blood. The mean TNF-α levels in controls and cases before and after treatment were 182.4pg/ml, 896.7 pg/ml and 678.7pg/ml pg/ml respectively. Though not statistically significant, there was a trend towards younger age, shorter duration of symptoms, presence of fever and anorexia, and high ESR, in patient with high serum TNF-α levels.     

Diagnostic potential of fractionatedMycobacterium tuberculosis H37Ra excretory-secretory (EST-DE1) antigen in pulmonary tuberculosis

Tuberculosis is emerging as a major public health problem in developing and developed world. Early and precise diagnosis is of prime importance in successful control of infection. Indirect ELISA with penicillinase as marker was developed using purifiedM. tuberculosis excretory-secretory (EST-DE₁) antigen for detecting IgG antibodies in pulmonary tuberculosis. The assay System gave a overall sensitivity of 82% for both smear positive and smear negative pulmonary tuberculosis cases with a specificity of 84%. The positive and negative predictive values were 75% and 88% respectivaly. Further studies with EST-DE₁ antigen revealed that, it contains two of the active antigen fractions of Mtb EST antigen i.e. Mtb EST-4 (56–68 KDa) and Mtb EST-6 (37–45 KDa), as demonstrated by inhibition ELISA. Reactivity with monoclonal antibodies HGT 3a showed the presence of 38 KDa molecule in EST-DE₁ antigen.     

Urinary Urea,Uric Acid and Hippuric Acid as Potential Biomarkers in Multiple Sclerosis Patients

Urine is a proven source of metabolite biomarkers and has the potential to be a rapid, noninvasive, inexpensive, and efficient diagnostic tool for various human diseases. Despite these advantages, urine is an under-investigated source of biomarkers for multiple sclerosis (MS). The objective was to investigate the level of some urinary metabolites (urea, uric acid and hippuric acid) in patients with MS and correlate their levels to the severity of the disease, MS subtypes and MS treatment. The urine samples were collected from 73 MS patients-48 with RRMS and 25 with SPMS- and age matched 75 healthy controls. The values of urinary urea, uric acid and hippuric acid in MS patients were significantly decreased, and these metabolites in SPMS pattern showed significantly decrease than RRMS pattern. Also showed significant inverse correlation with expanded disability status scale and number of relapses. Accordingly, they may act as a potential urinary biomarkers for MS, and correlate to disease progression.     

Advances in urinary protein biomarkers for urogenital and non-urogenital pathologies

The discovery of protein biomarkers that reflect the biological state of the body is of vital importance to disease management. Urine is an ideal source of biomarkers that provides a non-invasive approach to diagnosis, prognosis and prediction of diseases. Consequently, the study of the human urinary proteome has increased dramatically over the last 10 years, with many studies being published. This review focuses on urinary protein biomarkers that have shown potential, in initial studies, for diseases affecting the urogenital tract, specifically chronic kidney disease and prostate cancer, as well as other non-urogenital pathologies such as breast cancer, diabetes, atherosclerosis and osteoarthritis. PubMed was searched for peer-reviewed literature on the subject, published in the last 10 years. The keywords used were “urine, biomarker, protein, and/or prostate cancer/breast cancer/chronic kidney disease/diabetes/atherosclerosis/osteoarthritis”. Original studies on the subject, as well as a small number of reviews, were analysed including the strengths and weaknesses, and we summarized the performance of biomarkers that demonstrated potential. One of the biggest challenges found is that biomarkers are often shared by several pathologies so are not specific to one disease. Therefore, the trend is shifting towards implementing a panel of biomarkers, which may increase specificity. Although there have been many advances in urinary proteomics, these have not resulted in similar advancements in clinical practice due to high costs and the lack of large data sets. In order to translate these potential biomarkers to clinical practice, vigorous validation is needed, with input from industry or large collaborative studies.Key words: urine, protein, biomarker     

A multiplexed immunoaggregation biomarker assay using a two-stage micro resistive pulse sensor

We present an immunoaggregation assay chip for multiplexed biomarkers detection. This chip is based on immunoaggregation of antibody functionalized microparticles (Ab-MPs) to quantify concentrations of multiple biomarkers simultaneously. A mixture of multiple types of Ab-MPs probes with different sizes and magnetic properties, which were functionalized by different antibodies, was used for the multiplexed assay. The interactions between biomarkers and their specific Ab-MPs probes caused the immunoaggregation of Ab-MPs. A two-stage micro resistive pulse sensor was used to differentiate and count the Ab-MP aggregates triggered by different biomarkers via size and magnetic property for multiplexed detection. The volume fraction of each type of Ab-MP aggregates indicates the concentration of the corresponding target biomarker. In our study, we demonstrated multiplexed detection of two model biomarkers (human ferritin and mouse anti-rabbit IgG) in 10% fetal bovine serum, using anti-ferritin Ab and anti-mouse IgG Ab functionalized MPs. We found that the volume fraction of Ab-MP aggregates increased with the increased biomarker concentrations. The detection ranges from 5.2 ng/ml to 208 ng/ml and 3.1 ng/ml to 5.12 × 10⁴ng/ml were achieved for human ferritin and mouse anti-rabbit IgG. This bioassay chip is able to quantitatively detect multiple biomarkers in a single test without fluorescence or enzymatic labeling process and hence is promising to serve as a useful tool for rapid detection of multiple biomarkers in biomedical research and clinical applications.     

<Emphasis Type="Italic">In vitro</Emphasis> released antigens in diagnosis and immunomonitoring of filaria and tuberculosis

In vitro released antigens by living parasites or bacteria underin vitro maintenance or short term culture showing specific humoral immune response have been explored in development of immunodiagnostics for infectious diseases such as filariasis and tuberculosis in our laboratory. ELISA usingB. malayi mf ES antigen has been explored for detecting IgG antibody by Indirect ELISA and antigen by Inhibition ELISA and in immunomonitoring of carriers as well as clinical filarial cases. A ten year follow up of mf carriers with DEC therapy showed disapperance of antigen and antibody followed by reappearance in few cases in an endemic area. None of the cases followed developed clinical symptoms suggesting the need for long term monitoring and treatment of microfilaraemic carriers. Further immunomonitoring was found to be useful in confirming filaria aetiology in the absence of microfilaremia and determining appropriate period of treatment of acute, early clinical and occult filarial infections for clinical relief and cure.Indirect Stick Penicillinase ELISA system using Mtb EST-6 antigen for detecting tuberculous IgG antibody and a Sandwich Penicillinase ELISA system using affinity purified antibody for detecting circulating antigen were explored in tuberculosis. A combination of both the assay systems with a sensitivity of 70% and specificity of 98% was found to be promising in the precise diagnosis of pulmonary tuberculosis. Further antigen detection was found to be useful in bone and joint tuberculosis.