Tuberculosis: Diagnostic Challenges in Rural Africa
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Tuberculosis (TB) remains among the top 10 infectious diseases with highest mortality globally since the 1990s despite effective chemotherapy. Among 10 million patients that fell ill with tuberculosis in the year 2017, 36 % were undiagnosed or detected and not reported; the number goes as high as 55 % in Tanzania, showing that the diagnosis of TB is a big challenge in the developing countries. There have been great advancements in TB diagnostics with introduction of the molecular tests such as Xpert MTB/RIF, loop-mediated isothermal amplification, lipoarabinomannan urine strip test, and molecular line-probe assays. However, most of the hospitals in Tanzania still rely on the TB score chart in children, the WHO screening questions in adults, acid-fast bacilli and chest x-ray for the diagnosis of TB. Xpert MTB/RIF has been rolled-out but remains a challenge in settings where the samples for testing must be transported over many kilometers. Imaging by sonography - nowadays widely available even in rural settings of Tanzania - has been shown to be a useful tool in the diagnosis of extrapulmonary tuberculosis. Despite all the efforts and new diagnostics, 30-50 % of patients in high-burden TB countries are still empirically treated for tuberculosis. More efforts need to be placed if we are to reduce the death toll by 90 % until 2030.Keywords:
Lipoarabinomannan
Tuberculosis diagnosis
Mycobacterium tuberculosis encodes two-component signal systems. Recently, it was established that the viability of the M. tuberculosis phoP mutant is attenuated in the mouse, suggesting the requirement of the phoP gene for M. tuberculosis intracellular growth. It is now largely acknowledged that M. tuberculosis mannosylated lipoarabinomannans (ManLAM) play a key role in M. tuberculosis intramacrophagic survival by altering the macrophage functions. So ManLAM were extracted and purified from the M. tuberculosis MT103 wild-type strain and from the M. tuberculosis phoP mutant. Their two major functional domains (i) the mannooligosaccharide caps and (ii) the mannosyl phosphatidylinositol anchor were here investigated. Using capillary electrophoresis, it is demonstrated that both mutant and wild-type M. tuberculosis strains share the same capping motifs: mono-, di- and trimannosyl α(1→2) units, with the same relative abundance. Using two-dimensional NMR spectroscopy, the same acyl forms were found to be shared by both strains. However, their relative abundance was quite different. Indeed, in the phoP mutant a decrease of the triacylated ManLAM and an increase of the monoacylated ManLAM were observed. The difference in the proportion of ManLAM acyl forms and the reduced virulence of the M. tuberculosis phoP mutant are discussed.
Lipoarabinomannan
Wild type
Mycobacterium tuberculosis complex
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BACKGROUND. Inadequate tuberculosis (TB) diagnostics are a major hurdle in the reduction of disease burden, and accurate point-of-care tests (POCTs) are urgently needed. We assessed the diagnostic accuracy of Fujifilm SILVAMP TB lipoarabinomannan (FujiLAM) POCT for TB diagnosis in HIV-negative outpatients and compared it with Alere Determine TB LAM Ag (AlereLAM) POCT and a laboratory-based ultrasensitive electrochemiluminescence LAM research assay (EclLAM).
Lipoarabinomannan
Point-of-Care Testing
Tuberculosis diagnosis
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Objective: In settings of high HIV prevalence, tuberculosis control and patient management are hindered by lack of accurate, rapid tuberculosis diagnostic tests that can be performed at point-of-care. The Determine TB LAM Ag (TB LAM) test is a lateral flow immunochromatographic test for detection of mycobacterial lipoarabinomannan (LAM) in urine. Our objective was to determine sensitivity and specificity of the TB LAM test for tuberculosis diagnosis. Design: Prospective diagnostic accuracy study. Setting: Hospital and outpatient settings in Uganda and South Africa. Participants: HIV-infected adults with tuberculosis symptoms and/or signs. Methods: Participants provided a fresh urine specimen for TB LAM testing, blood for mycobacterial culture, and 2 respiratory specimens for smear microscopy and mycobacterial culture. Main Outcome Measures: For the TB LAM test, sensitivity in participants with culture-positive tuberculosis and specificity in participants without tuberculosis. Results: A total of 1013 participants were enrolled. Among culture-positive tuberculosis patients, the TB LAM test identified 136/367 (37.1%) overall and 116/196 (59.2%) in the group with CD4 ≤100 cells per cubic millimeter. The test was specific in 559/573 (97.6%) patients without tuberculosis. Sensitivity of the urine TB LAM test plus sputum smear microscopy was 197/367 (53.7%) overall and 133/196 (67.9%) among those with CD4 ≤100. CD4 ≤50 [adjusted odds ratio (AOR), 6.2; P < 0.001] or 51–100 (AOR, 7.1; P < 0.001), mycobacteremia (AOR, 6.1; P < 0.01) and hospitalization (AOR, 2.6; P = 0.03) were independently associated with a positive TB LAM test. Conclusions: In HIV-positive adults with CD4 ≤100, the TB LAM urine test detected over half of culture-positive tuberculosis patients, in <30 minutes and without the need for equipment or reagents.
Lipoarabinomannan
Tuberculosis diagnosis
Sputum culture
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ABSTRACT Lipomannan (LM) and lipoarabinomannan (LAM) are mycobacterial glycolipids containing a long mannose polymer. While they are implicated in immune modulations, the significance of LM and LAM as structural components of the mycobacterial cell wall remains unknown. We have previously reported that a branch-forming mannosyltransferase plays a critical role in controlling the sizes of LM and LAM and that deletion or overexpression of this enzyme results in gross changes in LM/LAM structures. Here, we show that such changes in LM/LAM structures have a significant impact on the cell wall integrity of mycobacteria. In Mycobacterium smegmatis , structural defects in LM and LAM resulted in loss of acid-fast staining, increased sensitivity to β-lactam antibiotics, and faster killing by THP-1 macrophages. Furthermore, equivalent Mycobacterium tuberculosis mutants became more sensitive to β-lactams, and one mutant showed attenuated virulence in mice. Our results revealed previously unknown structural roles for LM and LAM and further demonstrated that they are important for the pathogenesis of tuberculosis. IMPORTANCE Tuberculosis (TB) is a global burden, affecting millions of people worldwide. Mycobacterium tuberculosis is a causative agent of TB, and understanding the biology of M. tuberculosis is essential for tackling this devastating disease. The cell wall of M. tuberculosis is highly impermeable and plays a protective role in establishing infection. Among the cell wall components, LM and LAM are major glycolipids found in all Mycobacterium species, show various immunomodulatory activities, and have been thought to play roles in TB pathogenesis. However, the roles of LM and LAM as integral parts of the cell wall structure have not been elucidated. Here we show that LM and LAM play critical roles in the integrity of mycobacterial cell wall and the pathogenesis of TB. These findings will now allow us to seek the possibility that the LM/LAM biosynthetic pathway is a chemotherapeutic target.
Lipoarabinomannan
Mycobacterium smegmatis
Pathogenesis
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Abstract Objectives Even today, tuberculosis (TB) remains a leading public health problem; yet, the current diagnostic methods still have a few shortcomings. Lipoarabinomannan (LAM) provides an opportunity for TB diagnosis, and urine LAM detection seems to have a promising and widely applicable prospect. Design or methods Four databases were systematically searched for eligible studies, and the quality of the studies was evaluated using the quality assessment of diagnostic accuracy studies‐2 (QUADAS‐2). Graphs and tables were created to show sensitivity, specificity, likelihood ratios, diagnostic odds ratio (DOR), the area under the curve (AUC), and so on. Results Based on the included 67 studies, the pooled sensitivity of urine LAM was 48% and specificity was 89%. In the subgroup analyses, the FujiLAM test had higher sensitivity (69%) and specificity (92%). Furthermore, among patients infected with human immunodeficiency virus (HIV), 50% of TB patients were diagnosed using a urine LAM test. Besides, the CD4+ cell count was inversely proportional to the sensitivity. Conclusions Urine LAM is a promising diagnostic test for TB, particularly using the FujiLAM in HIV‐infected adults whose CD4+ cell count is ≤100 per μl. Besides, the urine LAM test shows various sensitivities and specificities in different subgroups in terms of age, HIV infection status, CD4+ cell count, and testing method.
Lipoarabinomannan
Diagnostic odds ratio
Tuberculosis diagnosis
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Estimates of the burden of childhood tuberculosis have been hampered by the lack of a reliable diagnostic test. Clinical scoring systems, radiological findings and tuberculin skin testing (the traditional methods used for diagnosis) are unreliable, particularly in the era of HIV. Microbiologic confirmation using induced sputum is feasible and has become increasingly important to define the burden of disease and to enable appropriate treatment. The availability of a rapid molecular diagnostic test (Xpert® MTB/RIF; Cepheid) is an important advance that can improve case detection in children and enable rapid detection of mycobacterial drug resistance. Xpert testing of two induced sputum specimens detected approximately 75% of children with culture-confirmed disease. Urine lipoarabinomannan has shown promise as a rapid diagnostic in a subgroup of HIV-infected severely immunocompromised adults, but there have been no data in children so far. Further research is needed to develop a rapid point-of-care, reliable and affordable diagnostic test for childhood tuberculosis that can be widely used.
Lipoarabinomannan
Tuberculosis diagnosis
Point-of-Care Testing
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To the Editors: We read with interest the article by Drain et al1 reporting on the diagnostic accuracy of a urine lipoarabinomannan (LAM) lateral-flow point-of-care diagnostic assay for HIV-associated tuberculosis (TB). The ease of obtaining urine samples and the simplicity of this assay format make this a very attractive prospective tool.2 With a growing number of evaluations of this assay having been published, the evidence base concerning the use of this assay is to be reviewed by a WHO expert panel in 2015. Although studies agree that the sensitivity of the assay is only moderate at best, a number of features potentially compensate for this.2 First, as a point-of-care assay, treatment decisions can be made at a single clinical encounter, greatly increasing the chances of treatment being quickly started after a positive test result. Second, sensitivity is highest among those patients who are sickest and have the highest mortality risk, thereby benefitting those with the greatest clinical need.3 Third, it provides useful incremental sensitivity to that provided by existing diagnostic tests, such as sputum smear microscopy.4 For this assay to be useful in practice, however, specificity must be very high such that positive results can be acted on with confidence. It has been suggested that the specificity of any new TB diagnostic assay should be at least 95% in comparison with culture.5,6 It is therefore of concern that the assay evaluation by Drain et al7 found an assay specificity of 92%,1 similar to a specificity of 90% reported by the same authors in a previous evaluation. Several published studies have reported specificities of between 97% and 99%,8–10 although some have also reported substantially lower specificities when using nonoptimized cutoffs on the reference reading card.9–11 There are 2 key methodological determinants of the assessed specificity of the urine LAM assay. The first is which of the reference card cutoffs is used and how the test strips are interpreted—issues which Drain et al explored in their study. Suggested guidelines on reading the test strips were published in 201312 and have since been widely accepted and also adopted by the assay manufacturer, leading to modification of the reference card since January 2014. The second critical issue affecting assessed specificity is the use of an appropriate microbiological reference standard. Specificity can only be reliably assessed if all participants included in a study are correctly classified as having TB or being TB free after microbiological investigation. To accurately determine the TB status of HIV-infected patients can be difficult. HIV-associated TB is frequently extrapulmonary and is often challenging to either detect or exclude, especially in patients with advanced immunodeficiency.13 In their studies, Drain and colleagues used culture of a single sputum sample as the reference standard. As they themselves suggest, reliance on just 1 sputum sample may be insufficient. This could easily have given rise some false-negative reference standard tests, resulting in some true TB cases being misclassified as TB free. This, in turn, could have given rise to some true-positive LAM results being classified as false positives, directly resulting in underestimation of the specificity of the urine LAM assay. It was notable that assay specificity reported by Drain et al was only 80% among those with CD4 cell counts <100 cells per microliter; we suggest that this is because these are the very patients in whom extrapulmonary TB is most likely and in whom a single sputum reference standard is not likely to be adequate. Thus, we believe that differences in the robustness of the reference standard may well contribute to the substantial variation in the specificities for urine LAM assays reported by studies published to date. In our previous studies of the use of urine LAM assays among ambulatory outpatients, we found that liquid culture of 2 carefully obtained sputum samples (with the assistance of a respiratory nurse and at least one of the samples being obtained by sputum induction) provided a very adequate reference standard. In both of these studies, the LAM assays were found to have excellent specificity (both ≥99%).8,14 However, in our more recent study of HIV-infected patients requiring acute hospital admission, we used a much more comprehensive reference standard, as we suspected that sputum alone would likely provide an inadequate assessment in these very sick patients with very advanced immunodeficiency.15 We used a comprehensive sampling strategy as part of a larger study, obtaining 1745 respiratory and nonrespiratory samples from the 427 study patients (mean, 4.1 samples per patient). The samples represented a median of 3 anatomic compartments per patient, such as sputum, blood, urine, pleural fluid, etc. TB was defined by at least 1 positive culture or Xpert test on any clinical sample. In an exploratory analysis, we assessed the specificity of the LAM assay in one of the 2 ways. First, using the data generated from the comprehensive reference standard (ie, results from all respiratory and nonrespiratory specimens), specificity was found to be 98.9% (95% confidence interval: 96.9 to 99.8). However, we then calculated what the revised specificity would have been if only respiratory samples were taken into account; this resulted in an assessed specificity of 89.6% (95% confidence interval: 86.0 to 92.5). Thus, we found that reliance on respiratory samples alone for the reference standard would have resulted in a substantial underestimation of the urine LAM assay specificity (by 9.3%) and that this then fell below the suggested acceptable threshold for a new diagnostic test of >95%.5,6 It can be very challenging to obtain good quality sputum specimens from acutely ill HIV-positive hospital inpatients despite the assistance of a respiratory nurse and use of sputum induction in those in whom there is no contraindication. Drain and colleagues studied outpatients, in whom it can also be difficult to obtain good samples in the typically overcrowded clinics in sub-Saharan Africa where space and facilities are all too often limited. In contrast, good quality nonrespiratory samples, such as urine and mycobacterial blood cultures, can be readily obtained. Thus, we strongly advise that evaluations of the diagnostic accuracy of non–sputum-based diagnostic assays for TB (especially for HIV-associated TB) should use a more comprehensive reference standard that includes nonrespiratory samples in addition to sputum.
Lipoarabinomannan
Point of care
Point-of-Care Testing
Tuberculosis diagnosis
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Understanding the molecular components of immune recognition of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, can help designing novel strategies to combat TB. Here, we identify collectin CL-LK as a novel soluble C-type lectin able to bind M. tuberculosis, and characterize mycobacterial mannose-capped lipoarabinomannan as a primary ligand for CL-LK. Mice deficient in CL-K1, one of the CL-LK subunits, do not display altered susceptibility to M. tuberculosis. However, we found that the amount of CL-LK in the serum of patients with active TB is reduced, compared to that in controls, and correlates inversely to the magnitude of the immune response to the pathogen. These findings indicate that CL-LK might be of interest for future diagnostic and treatment monitoring purposes.
Lipoarabinomannan
Collectin
Mycobacterium tuberculosis complex
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Abstract Tuberculosis (TB) remains a leading cause of deaths among patients with acquired immunodeficiency syndrome patients. Early diagnosis of TB is essential for administering timely anti‐TB therapy and improving health outcomes, particularly in the people living with HIV. However, conventional techniques used to detect Mycobacterium tuberculosis have significant drawbacks: for example, sputum smear microscopy has low sensitivity, and liquid culture is time‐consuming in patients with HIV‐TB co‐infection due to low sputum production. In addition, while immunological‐based methods involving tuberculin skin testing and interferon gamma release assays are commonly used for auxiliary TB diagnosis, they are often inaccurate in immunodeficient patients. Molecular techniques such as line probe assays, Xpert MTB, and lipoarabinomannan assay are recommended for early diagnosis by World Health Organization. However, no single technique is sufficicent for diagnosing HIV/TB co‐infection, suggesting that multiple diagnostic tests should be used to detect TB. Here, we summarize the drawbacks and advantages of existing TB‐diagnostic methods, as well as their applications to diagnosing HIV/TB co‐infection. We describe newly emerging technologies such as whole genome sequencing and mass spectrometry, with the aim of providing updated guidelines and alternative strategies for TB diagnosis, and particularly HIV/TB diagnosis.
Lipoarabinomannan
Tuberculosis diagnosis
Nontuberculous Mycobacteria
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Mycobacterium tuberculosis bacilli is a causative organism for tuberculosis disease. The lungs are the most commonly affected organ with this infection but it can infect any vital organ of the body. The person with signs and symptoms of tuberculosis disease was identified with detection of acid-fast bacilli in the biological specimen and with rapid molecular testing. However, the limiting factor for the early tuberculosis (Tb) diagnosis is the running cost of the diagnostic test. It is the common reason preventing the advancement of diagnostic laboratories in low and middle-income countries. The person and community both suffered from this diagnostic delay. This delay causes significant morbidity and mortality in the patient. An alternate test and an alternate sample that can be easily obtained would be beneficial to prevent these diagnostic delay issues. Lipoarabinomannan (LAM) antigen detection in the urine sample is one such promising diagnostic test. Mycobacterium tuberculosis’s inner layer is made up of glycolipids. LAM is derived from phosphatidylinositol. LAM is a heat-stable amphiphilic cell wall component. It is the precursor of phosphatidyl-inositol-mannosidase and lipomannan. An extra mannose cap is a characteristic feature of a virulent strain of Mycobacterium. LAM has immunogenic and immunomodulatory properties. It is a promising diagnostic test because it is simple to do, the assay can be performed at the patient’s bedside and takes a while to perform. This assay sensitivity varies from 56% to 85% and it has greater than 88% specificity. In HIV seropositive patients, use of LAM assay can reduce 8-week mortality. LAM detection is also a very good assay for the detection of tuberculosis in renal failure and disseminated tuberculosis patients. Lipoarabinomannan detection in the urine is a possible test that can prevent delay in diagnosis. This is a promising test because it's easy to perform, the test can be done with a card test besides the patient's bed. It can be used in HIV seropositive patients and various other forms of extra pulmonary tuberculosis. It can be a very useful test for diagnosing a critically ill patient who is not able to produce a target sample.
Lipoarabinomannan
Tuberculosis diagnosis
Rapid diagnostic test
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