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ORIGINAL ARTICLE
Year : 2014  |  Volume : 27  |  Issue : 4  |  Page : 733-739

The value of serum lipoarabinomannan in the diagnosis of pulmonary tuberculosis


1 Department of Chest Diseases and Tuberculosis, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Medical Biochemistry, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission08-Dec-2013
Date of Acceptance06-Feb-2014
Date of Web Publication22-Jan-2015

Correspondence Address:
Mai Mahmoud El-kalashy
Department of Chest Diseases and Tuberculosis, Faculty of Medicine, Menoufia University, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.149720

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  Abstract 

Objective
The aim of this study was to assess the value of serum lipoarabinomannan (LAM) in the diagnosis of pulmonary tuberculosis (TB).
Background
The accurate diagnosis of TB is difficult. Recent studies indicate that a LAM assay (ELISA) may have some utility in the diagnosis of TB.
Material and methods
Sera from 40 individuals with confirmed pulmonary TB and 20 healthy individuals were tested for the presence of LAM by means of ELISA assay. Culture positivity for Mycobacterium tuberculosis was used as the reference standard for TB diagnosis.
Results
The serum LAM ELISA test showed high positivity in confirmed TB patients; sputum smear combined with LAM diagnosed 95% of the confirmed cases; tuberculin skin test, elevated erythrocyte sedimentation rate, immunocompetent state, and radiological tests have shown significant differences from qualitative and quantitative serum LAM. Serum LAM sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were 90, 100, 93.3, 100, and 83.3%, respectively.
Conclusion
The LAM test is a simple and reliable test for the diagnosis of pulmonary TB.

Keywords: ELISA test, lipoarabinomannan, pulmonary tuberculosis, serum samples, Ziehl-Neelsen stain


How to cite this article:
Abd el-Atty HE, MohamadBakr R, El-Helbawy R, Fathyabbass H, El-kalashy MM. The value of serum lipoarabinomannan in the diagnosis of pulmonary tuberculosis. Menoufia Med J 2014;27:733-9

How to cite this URL:
Abd el-Atty HE, MohamadBakr R, El-Helbawy R, Fathyabbass H, El-kalashy MM. The value of serum lipoarabinomannan in the diagnosis of pulmonary tuberculosis. Menoufia Med J [serial online] 2014 [cited 2020 Feb 17];27:733-9. Available from: http://www.mmj.eg.net/text.asp?2014/27/4/733/149720


  Introduction Top


Tuberculosis (TB) has been a cause of significant morbidity and mortality in mankind throughout history [1]. More than two billion people are estimated to be infected with Mycobacterium tuberculosis. According to the WHO (2010), 8.8 million are new cases. Three million annual deaths are due to TB [2] [Figure 1].
Figure 1: Growth of TB in Africa and Eastern Europe (Richard 2007, The Johns Hopkins University and Richard Chaisson).

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In the last few years, several new diagnostic tools have been developed. Many of these are better tests compared with acid-fast bacilli (AFB) smears and are much faster than mycobacterial cultures in the diagnosis of pulmonary TB, but their costs and technical requirements have limited their use to developed countries. Isolation of M. tuberculosis remains the golden standard in the diagnosis of TB [3].

A number of alternative diagnostic tests that use molecular, chromatographic, and immunological methods have been developed to overcome the insensitivity of the smear method and the time required for culture. Some tests use the specific humoral or cellular responses of the host to infer the presence of infection or disease. They do not require a specimen from the site of infection. Numerous serological tests that use various antigens, such as secreted and heat shock proteins, lipopolysaccharides, and peptides, have been developed [4].

Lipoarabinomannan (LAM) [Figure 2] is among the two potential pathogenic lipopolysaccharides in mycobacterial diseases, the other being lipomannan [6], and it is currently considered by some authorities as a mycobacterial virulence factor [6]. The LAM test was noted as a simple and reliable test for the diagnosis of pulmonary TB infection [7].
Figure 2: Cartoon showing the basic structure of mycobacterial lipoarabinomannan (LAM) and its three main domains: (i) the glycophospholipid anchor binds the molecule to the plasma membrane of the organism. (ii) The mannan core is attached to this and is highly conserved across mycobacterial species. (iii) The variable branching arabinan side chains and the variable mannose capping of these side chains give rise to the diversity of LAM molecules [5].

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  Patients and methods Top


This study was carried out in the Chest and Medical Biochemistry Department, Faculty of Medicine, Menoufyia University Hospitals.

The study was conducted on two groups: the case group and the control group. The case group comprised 40 individuals diagnosed as having active pulmonary TB [based on positive sputum smear, positive bronchoalveolar lavage (BAL) smear, and positive culture for M. tuberculosis]. The control group comprised 20 normal healthy individuals. After providing informed consent, each patient underwent the following:

  1. Detailed history taking: this included personal history, present history, past history of chest diseases or other diseases, family history, and history of contact with TB patient and traveling details.
  2. Clinical examination.
  3. Full routine laboratory investigations: this included complete blood picture, erythrocyte sedimentation rate (ESR), fasting serum glucose level, kidney function tests (urea and creatinine), liver function tests (serum albumin, AST, and ALT), serum LDH, and coagulation profile (prothrombin time and concentration). The patients underwent measurement of oxygen saturation (arterial blood gas sampling) and ECG. They underwent roentgenographic examinations, including plain chest radiograph in posteroanterior and lateral views, as well as CT scanning of the chest (if needed). Tuberculin skin testing (TST) was carried out using 5 tuberculin units injected intradermally and interpreted 48-72 h later. Sputum smear (spontaneously expectrated sputum or induced sputum) and BAL, using fibreoptic bronchoscopy [Figure 3], were examined for the presence of AFB by means of Ziehl-Neelsen stain and cultured on Löwenstein-Jensen medium. All patients were instructed to fast for 4-6 h before the fiberoptic bronchoscopic examination, which was conducted using the trans-nasal approach, except in one patient with nasal obstruction, in whom the trans-oral approach was adopted. The patient's written consent was obtained. Supplemental oxygen was given through an oxygen mask. The patients were premedicated with 0.5-1 mg atropine intramuscular half an hour before the procedure, and topical anesthesia was induced with 2% lidocaine solution sprayed directly into the patient's mouth. Sedation with 2-5 mg Midazolam was given by slow intravenous injection over 30 s and then the fiberoptic bronchoscope was passed in with the shaft lubricated with lidocaine gel, and it was advanced into the nostril under direct vision and then flexed downward. When the glottis and larynx came into view, the mobility of the vocal cords was assessed, and 2 ml of lidocaine was injected through FOB on the vocal cords. The bronchoscope was advanced through the opening with no force. Once the vocal cords had been passed, an additional 2 ml liquid of lidocaine was instilled through the suction channel to suppress coughing. The patient was then subjected to a bronchial wash and bronchial brushing, and postbronchoscopic sputum was collected for Ziehl-Neelsen staining.
Figure 3: The fiberoptic bronchoscopy used in the present work (Brand: Pentax Model: FB-19H, 1990).

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LAM ELISA test: LAM was measured in the serum samples of the patients and controls using ELISA. Test procedure: All reagents and patients' sera were brought to room temperature. The concentrated wash buffer was diluted 1 : 5 with distilled water. The number of teeth required (sample and controls) was determined. Three drops (0.15 ml) of sample diluents were added into each numbered micro test well. In the next row of micro test wells, four drops of 0.2 ml of colloidal gold reagent were added. A positive reaction generated by the Mycodot test was recorded as an intense colored spot developed on the teeth of the comb. The concentration of serum LAM in the samples was then determined by comparing the optical density (OD) of the samples with the standard curve.

Statistical analysis

Data were collected, tabulated, and statistically analyzed using SPSS, version 20. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated using Bay's formula to compare the efficiency of LAM serum value in the diagnosis of pulmonary TB. The ROC curve was used to evaluate LAM as a screening test; sensitivity, specificity, PPV, NPV, and accuracy were calculated.


  Results Top


  1. Case/control groups were well matched across demographic characteristics and showed the expected significant differences in ESR, TST, smear, and culture positivity (independent variables) [Table 1] and [Table 2].
  2. Group comparison for key outcome (dependent variables - qualitative and quantitative serum LAM levels) revealed statistically significant difference between the cases (PTB) and controls [Table 3].
  3. The present study revealed that the cutoff point of serum LAM was 0.375, with area under the curve of 0.819. The sensitivity, specificity, PPV, NPV, and accuracy were 90, 100, 100, 83.3, and 93.3%, respectively. The combination of serum LAM testing and sputum smear identified 95% of confirmed TB cases [Table 4] [Figure 4] and [Figure 5].
  4. In the present work, febrile illness, hemoptysis, immunocompetent state, TST, smear and culture positivity, extensive radiological lesions, and raised ESR were significant predictors associated with positive qualitative serum LAM (P < 0.05) [Table 5].
  5. The present study revealed that patients with advanced age (> 60 years), febrile illness, hemoptysis, extensive radiological lesions, raised ESR, and TST, smear, and culture positivity had significantly higher quantitative serum LAM levels compared with the control group [Table 6] and [Table 7].
Figure 4: Serum lipoarabinomannan in tuberculosis cases.

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Figure 5: ROC curve showing sensitivity and specificity of serum lipoarabinomannan. Area under the curve = 0.819. Cutoff point = 0.375.

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Table 1: Demographic data of the case and control groups

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Table 2: Diagnostic tools in tuberculosis cases

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Table 3: Cases and control groups on the basis of different parameters

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Table 4: Validity of serum lipoarabinomannan test, Ziehl-Neelsen for acid-fast bacilli smear, and both methods in detecting positive cases of tuberculosis in confirmed tuberculosis cases

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Table 5: Predictors of positive serum lipoarabinomannan in pulmonary tuberculosis cases

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Table 6: Mean and SD of lipoarabinomannan in pulmonary tuberculosis cases with respect to different variables

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Table 7: Relation of serum lipoarabinomannan test with sputum smear and culture

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  Discussion Top


Lawn et al. [8] explored pathogen and host factors potentially impacting LAM detection. They reported that 32/199 (16.1%) patients tested with LAM ELISA were positive. LAM was associated with positive sputum smear, culture, and declined host immune factors with low CD4 cell counts [8]. Shah et al. [9] also reported that HIV infection, mycobacteremia, and positive sputum smear were risk factors for a positive LAM test.

Similar results were reported by Shah et al. [9] - that HIV infection, mycobacteremia, and positive sputum smear were risk factors for a positive LAM test. Moreover, they also concluded that HIV-infected TB suspects with advanced immunosuppression, a group in which sputum microscopy is of low yield, may be a target population for whom the LAM test would be particularly useful, as they found that, among HIV-infected patients, individuals with CD4 counts less than 50 cells/ml had an average OD that was 1.05 OD units higher than that for individuals with CD4 counts greater than 150 (P < 0.0001) [9]. In addition, Lawn [5] postulated that this assay has good sensitivity for the diagnosis of HIV-associated TB in patients with advanced immunodeficiency.

Lawn et al. [8] stated that LAM is related to host immune factors. This was not surprising as the levels of TB bacteria in individuals with advanced immunodeficiency were likely to be higher, resulting in increased LAM antigen in biological fluid [8].

In the present work an increased sensitivity of the LAM test was found in the immunocompetent state, a finding that was not consistent with previous studies but which agrees with those of Tessema et al. [10] who showed that the immunocompromised status lowers the sensitivity of LAM antibody detection assay (ELISA) compared with the immunocompetent one. This was also reported by Sada et al. [11], Da Costa et al. [12], Ratanasuwan et al. [13], and Somi et al. [14], and was consistent with the results of the recent work.

The negative results obtained might be related to immunosuppression and to the inability of patients to degrade mycobacteria and to thus release antigen into circulation.

The present data revealed that patients with smear-positive specimens had a higher serum LAM level (10.11 ng/ml) than that of smear-negative patients (4.15 ng/ml) (P < 0.001). Quantitative serum LAM test results positively correlate with the degree of bacillary burden in patients with microbiologically confirmed TB; it was higher in high-inoculum specimens (28.25 ng/ml). Similarly, Shah et al. [9] stated that there was a trend toward higher OD with increasing grades of smear positivity (median ODs of 0.13, 0.18, 0.26, and 0.38 for smear-negative, smear-positive grade 1+, smear-positive grade 2++, and smear-positive grade 3+++ cases, respectively), and individuals with smear-positive pulmonary TB had an average OD that was 0.33 OD units higher than that for individuals with smear-negative pulmonary T. The accuracy of serum LAM detection is higher in positive-AFB smear pulmonary TB patients than in negative-smear patients as described by Tessema et al. [10]. In the present work, positive TST was a significant predictor that was associated with positive qualitative serum LAM (P < 0.05). Moreover, patients with positive TST had higher significant quantitative serum LAM levels compared with those with negative TST. This agrees with a study by Hassan and Haridi [15] who reported the correlation between TST and anti-LAM tests. TST was positive in 20 (25%) patients and in 12 (30%) controls. Anti-LAM was positive in 32 (40%) patients and in four (10%) controls. Significant correlation was found between TST and anti-LAM ELISA tests in the patient group, whereas a highly significant difference was found between the patients and controls with respect to the anti-LAM antibody test. A significant correlation was found between anti-LAM test and pulmonary symptoms, whereas no significant relationship was found between the test and the other clinical or radiological findings [15]. Some authors reported that the percentage of TST positivity in their subjects varied between 16 and 54.7% [16],[17],[18],[19].

The present data revealed that, at the cutoff point of serum LAM (0.375) with area under the curve of 0.819, the sensitivity, specificity, PPV, NPV, and accuracy were 90, 100, 100, 83.3, and 93.3%, respectively. Many studies evaluated serum anti-LAM antibody detection in different situations and reported excellent specificity and variable sensitivity.[14],[20],[21],[22]

In another study, the sensitivity and specificity of a positive LAM for culture-confirmed TB were 65 and 86%, respectively, compared with the sensitivity and specificity of sputum smear for culture-confirmed TB, which was 36 and 98%, respectively. LAM sensitivity was higher and specificity was lower. The LAM test characteristics that were identified in the present study were consistent with those described in a systematic review of seven studies that assessed test accuracy using only microbiologically confirmed cases (such as the present study): sensitivity was 13-93% and specificity was 87-99% [23].

The LAM test was reported as simple and reliable, with a positivity of 100% in extrapulmonary and 88% in pulmonary TB infection [7].

Differences in LAM test characteristics may relate to either different LAM testing methodologies or different patient populations. Pooled sensitivity estimates from the two studies that evaluated the early prototype version of the test were significantly higher than estimates from studies that used either of the two commercial assays. Serum collection and processing may influence accuracy, although analysis of subgroups in which the blood used in the assay was either fresh or previously frozen found no statistically significant differences between these groups. In this study, most assays were conducted on serum frozen samples and very few tests were carried out on fresh samples for comparison.

A recent publication from the Los Alamos Biosensor Team described the association of a key TB virulence factor, LAM, with human high-density lipoproteins in blood as an implication of the diagnosis of TB. The team's efforts have focused on using the LAM virulence factor as a sensitive indicator for TB. The problem was that the biomarker, although being a reliable early indicator of TB, is very difficult to detect, especially in blood. Previously, the team had developed strategies for the detection of this biomarker in urine [24]. Subsequently, they developed a strategy for the ultrasensitive detection of the biomarker using a novel method called membrane insertion [24].

We recommend that this detection method could be of practical value in TB endemic areas and might be useful in routine tests for the early and supportive diagnosis of pulmonary TB and a valuable supplemental tool in TB control in low income countries. It is necessary to combine serum LAM with sputum smear microscopy to improve TB diagnosis. It will be valuable to conduct LAM assay in other body biological fluids such as BAL, sputum, and pleural fluid, and it needs further investigation. Quantitative and qualitative assay of LAM could be used as a biomarker reflecting response to treatment.


  Conclusion Top


A test capable of detecting active TB irrespective of its focus of infection in the body would be a valuable addition to effective and efficient diagnosis, and the LAM Mycodot test meets this need.

The LAM serum assay test is unlikely to be used alone for definitive TB diagnostic testing. It is useful as a rapid diagnostic modality that complements smear microscopy and has many characteristics that make it potentially useful in TB diagnosis.

Positive quantitative LAM detection results increased progressively with bacillary burden. The usage of the quantitative serum LAM test may thus offer additional clinical insight into the degree of TB disease severity that cannot be gleaned from qualitative results alone.


  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]


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