Validity of Antimycolic Acids Antibodies in the Diagnosis of Pulmonary Tuberculosis in TB / HIV Co-infected Patients in Khartoum State , Sudan

This study aimed to determine antimycolic acid antibodies [IgG and IgM] among TB/HIV Co-infected patients in Khartoum State. Sputum and blood specimens were collected from patients attended Alsha'ab Teaching Hospital, Tuberculosis Reference Laboratory, Ibrahim Malik Hospital and Abu Anga Hospital; patients were all informed and consented. Direct smears from sputum samples of 90 suspected patients showed that 17 (18.9%) were acid fast bacilli positive while 73 (81.1%) were negative. The ninety sputum specimens were subjected to PCR to amplify IS6110 region specific for Mycobacterium tuberculosis complex. The result showed that 79 (87.8%) were positive for IS6110 while 11 (12.2%) were negative. The 90 serum samples were investigated for HIV using dot plot technique, 9 samples (10%) were found HIV positive and they were all TB positive by PCR. 80 Serum samples were analyzed by ELISA, 16 (20%) gave positive result for antimycolic acid IgG while 64(80%) were negative and 55 (68.8%) were positive for antimycolic IgM, while 25 (31.3%) were negative. When HIV infection was correlated with specific antibodies for antimycolic acids, in 6 HIV positive samples, one (17 %) was positive and 5 (83 %) were negative for IgG, while 2 (33.3 %) were positive and 4 (67 %) were negative for IgM. This study concluded that patients with TB/HIV Co-infection have less capability of producing antimycolic acids antibodies, however, IgM antibodies could be of more serodiagnostic value.


INTRODUCTION
Tuberculosis remains a major global public health problem.The actual global prevalence of Mycobacterium tuberculosis infection is 32% corresponding to approximately 1.9 billion people.According to the World Health Organization (WHO), there were 8.8 million estimated new cases (Case rate, 140/100,000) of pulmonary tuberculosis, including 3.9 million smearpositive cases.In 2003, an estimated 1.9 million people died of tuberculosis, including patients co-infected with human immunodeficiency virus (HIV; 229,000) (Patrick et al., 2007).
Immunological mechanisms against M. tuberculosis have mainly been investigated in murine models, often a low dose of bacilli are introduced through an aerosol, to resemble human infection.The immune resistance to M. tuberculosis relies mainly on cellular immunity, which is stimulated by proteins excreted by the bacilli.However, an appreciable number of B cells have been found in granulomas of TB infected mice.Little is known about the natural evaluation of antibiotics against M.tuberculosis in mouse models.Mycobacteria produce a wide variety of glycolipids that are located in the outermost layers of the cell.Several of these compounds (cord -factor (CF), diacetyl trehaloses (DAT), phenoglycolipids (PCL), sulpholipids (CL-1), and Lipoarabinomannan (LAM) have been studied as target molecules for serology in diagnosis of TB.These molecules also interact with host immune cells and contribute to pathogenesis.Their structures are based on very characteristics methyl-branched longchain acids and alcohols (Minnikin et al., 2002).
The first myocolic acid was isolated from the tubercle bacilli in 1929 by Anderson (Anderson, 1929).The name mycolic acid was proposed in 1935 for a portion of the lipid fraction from M. tuberculosis by Stodola.Alex Lusuk and Anderson (Stodola et al., 1938) structural characteristic was followed in 1950 by Asselineau (Asselineau and Lederer, 1950).Mycolic acids are the major constituents of the inner leaflet of cell wall, where they form an effective impermeable barrier to protect the mycobacteria from antimicrobial agents (Stodola et al., 1938).
Tuberculosis is a fatal disease.Direct microscopic detection by direct smear from clinical specimens gives quick results but with specificity not more than 33% and culture on LJ media is time consuming, therefore, there has been strong need for the development of reliable, rapid and less costly diagnostic methods for detection of pulmonary tuberculosis.On the other hand, HIV emerged as an important infection that co-exist with tuberculosis and may affect the immune response as well as efficiency of TB treatment.TB is a prominent pulmonary opportunistic infection in HIV co-infected patients with an importance by far super passing other opportunistic pathogens (Stodola et al., 1938).
This study aims to determine the antibodies levels of antimycolic acids surrogates (anti IgG and anti IgM) among TB/HIV co-infected patients by using ELISA.

MATERIALS AND METHODS
Ninety patients with symptoms of pulmonary tuberculosis attending different medical centers in Khartoum State were included in this study.Data were collected using a questionnaire with informed consent.Sputum and venous blood samples were collected from all study subjects.ZN stain was used to detect acid fast bacilli in the sputum samples.

Molecular Identification
Polymerase chain reaction (PCR) was used to confirm the results of conventional method.DNA was extracted from sputum using isopropanol extraction method; briefly, 100 µl of sputum was put into eppendorff tube and 400 µl of Lysis buffer were added and vortexed, then 300 µl of Isopropanol was added and mixed gently then centrifuged at 1200 rpm for 10 minutes, supernatant was decanted by gently inverting the tubes then the pellet was washed 3 times using 70% ethanol (by adding 1ml, mixing gently and centrifuging at 12000 rpm for 5 min each time).Finally, ethanol was poured completely and the tubes were inverted on tissue papers to dry then the pellet was resuspended in 50 µl TE buffer and kept in -20°C till use.

Monitoring of Antimycolic Acid Antibodies
Indirect enzyme-linked immunosorbent assay (ELISA) was to detect antimycolic acid profiles.Mycolic acids originating from Mycobacterium tuberculosis were isolated as described by Minnikin et al. (1988) and were used as the antigen for this assay.To prepare the coating solutions, the antigen was heated in PBS buffer for 20 min at 85°C.The solution was kept at the -80°C till used.The wells of flatbottom ELISA plates were coated overnight at 4°C with 100 μl/well of antigen solution.The coating solution was flicked out of the plates and replaced with 100 μl/well blocking buffer for 2 hours at room temperature.The blocking solution was aspirated and 100 μl/well from sera diluted 1:50 in dilution buffer were introduced in duplicates.The plates were incubated at room temperature for 1 hour.The sera were flicked out and the wells were washed three times by washing buffer.100 μl/well of Peroxidase-conjugated anti-human IgG was added and the plates were incubated for one hour at room temperature.After removal of the conjugate, the wells were washed three times with the washing buffer and then emptied by aspiration, 100 μl/ well of substrate solution (TMP) were added and the plates were kept in a dark place for 30 minutes at room temperature and the color development was monitored, 100 μl/well of stop solution (20% H 2 SO 4 ) were added and the results were read at 450 nm.Anti-IgM antibodies were detected in a similar way but anti-human IgM was used instead of anti-human IgG.

HIV Detection
HIV was detected by ICT.The test device was removed from the foil pouch, and placed on a flat, dry surface and blood specimen, serum or plasma (0.2 ml) was added into the sample well, 4 drops (about 0.12 ml) of assay diluents were added to the sample well.As the test begins to work, purple color across the result window in the center of the test device, will indicate a positive result.

RESULTS AND DISCUSSION
Direct smear showed acid fast bacilli positive in 17/90 while PCR results showed that 79/90 were positive for IS 6110 (Fig. 1).ICT Screening showed that 9 (10%) were HIV positive.When antimycolic IgG and IgM among TB/HIV Co-infected patients was monitored, the results showed that only one patient was positive for IgG. (Fig. 2).and two patients were positive for IgM (Fig. 3), while the rest were negative.
Regarding direct smear in this study, it reflected similar results to those reported by Aftab et al., (2008) and Sharma et al., 2011, who reported the low sensitivity of the direct smear in detecting M tuberculosis infection, this may be due to poor reagents in ZN stain or poor decolorization.
In this study, an attempt was made to show if anti-MA antibodies can be used as a marker for the diagnosis of active tuberculosis infection, especially in HIV endemic areas.
The results of PCR technique targeting IS6110 (87.8%) reflect that there was a high prevalence rate of TB among the total number of patients, as well as the higher sensitivity of PCR over direct smear detection.Similar findings were observed by Noord et al., (1994).
Patients with active pulmonary tuberculosis have elevated levels of specific antibodies to M. tuberculosis mycolic acids compared to negative controls as detected by ELISA.
Among TB/HIV co-infected patients, more than 80% of HIV positive showed no antibodies in their sera due to immunodeficiency.These findings were in agreement with those reported by Thanyani, (2008).
Antibodies to mycolic acids (MA) antigens can be detected as a marker of active tuberculosis with ELISA.In this technique, the lipid antigen is not encapsulated, but was directly adsorbed to the well bottoms of the microtiter plates.Thus, it doesn't yield the required sensitivity and specificity for accurate diagnosis of TB.One reason for this is the cross reactivity of natural anticholestrol antibodies with anti-mycolic acid antibodies (Benadie et al., 2008).
In conclusion, the fact that anti-MA antibodies still recognize MA in HIV seropositive patients with active pulmonary TB warrants further investigation into the use of anti MA antibodies as a possible serodiagnostic test for TB.