Abstract: Mycobacterium tuberculosis (M. tuberculosis) is an airborne contagious disease that is transmitted by coughing, sneezing or even talking. Once a person becomes infected, any condition that weakens the immune system can trigger the development of active M. tuberculosis. Exposure to smoke can increase the risk of M. tuberculosis by reducing resistance to initial infection or by promoting the development of active M. tuberculosis in already infected persons. Exposure to the pollutants contained in biomass smoke or other sources of air pollution can weaken the immune system, impair the lungs and make them more susceptible to infection and disease. The association between air pollution and respiratory infections is well understood but the relation between air pollution and tuberculosis is not. So, it is anticipated that the prevalence of M. tuberculosis is positively correlated with environmentally polluted areas. In order to compare the prevalence of M. tuberculosis in two different areas in Alexandria city, industrial area (showing petroleum oil, fertilizer and cement industries) and residential area, golden standard methods [acid fast staining and culture] and molecular assays [Polymerase Chain Reaction technique (PCR) and Enzyme-Linked Immunospot assay (ELISpot)] of M. tuberculosis detection were used. There was a significant increase in the prevalence of M. tuberculosis in the industrial district than the residential one. Molecular tool of M. tuberculosis diagnosis was found to be the best than immunological method. Air pollution produced by petroleum, fertilizer and cement industries most likely has major effect on increasing the prevalence of M. tuberculosis infection. On the other hand, the power of the molecular techniques was confirmed and showed to be more sensitive than the traditional methods and the PCR assay was more accurate than the ELISpot assay.
INTRODUCTION
It is well characterized that mammalian host defense against M. tuberculosis depends on two types of immunity: innate and adaptive (Kaufmann, 2001). On the other hand, several investigators have reported that industrial pollution in air (out and indoor), water and heavy metals can badly affect human and animals immune defense against infections (Chalupka, 2005).
Tuberculosis causes an estimated two million deaths per year (Maher and Raviglione, 2005), the overwhelming majorities occur in developing countries (Raviglione et al., 1995) and it infects approximately one third of the world’s population (Dye et al., 2005). The incidence of M. tuberculosis has stabilized or is on the decrease in many parts of the world, While the incidence rate is rising by approximately 350 cases per 100 1000 population in Africa (WHO, 2007).
The conventional methods for laboratory diagnosis of M. tuberculosis, including acid fast staining and culturing are either insensitive (Hajime et al., 2002) or time-consuming (Pfyffer et al., 2003). For enhancing treatment strategies, plus efficacy markers to compare them, new diagnostic tools for M. tuberculosis, were needed to help combat the epidemic disease, in which the use of nucleic acid amplification and detection in sputum, blood and body fluids may provide quick and specific results for identifying the M. tuberculosis complex (Wang et al., 2004; Piersimoni and Scarparo, 2003). Recent developments of immune-based assays to detect M. tuberculosis infection are a significant advance. ESAT-6 and CFP-10 are two proteins encoded by the RD1 genomic segment of M. tuberculosis, which is absent from all BCG strains and the vast majority of environmental mycobacteria. As a result, enzyme-linked immunospot (ELISPOT) assays that detect interferon-gamma (IFN-γ) release in response to these antigens differentiate between M. tuberculosis infection and immune sensitisation by BCG vaccination or exposure to environmental mycobacteria (Hill et al., 2004, 2005; Nicol et al., 2005). However, its performance in rapid diagnosis of active M. tuberculosis in disease-endemic areas is still unknown. In the present, we have two targets, the first was to compare between the different methods of Diagnosis on which the ELISpot (T SPOT- M. tuberculosis) assay in clinically suspected cases of M. tuberculosis was evaluated in comparison to the molecular detection using specific PCR system, in which blood samples were used. Both methods were carried out in comparison with the gold standard method of M. tuberculosis detection (acid fast staining and culturing). The second target was to compare between two environmentally different regions to study the effect of pollution on the M. tuberculosis infection.
MATERIALS AND METHODS
Population Study
The study was conducted in the year 2006/2007 where, the population study
comprises 100 persons (suspected to be infected with M. tuberculosis)
simply random selected from those attendants to El-Maamora hospital for chest
disease in Alexandria for check up, 50 living in an industrial district matched
for age and sex with 50 individuals living in residential district both districts
are in Alexandria, Egypt. A written consent was taken from each person involved
in this study. On enrollment, each person was interviewed and their medical
records reviewed.
Blood samples were collected from each person for DNA extraction which used in the amplification by PCR and ELISpot tests.
Complete Medical History
Sex, age, residence, occupation, living conditions and personal habits such
as smoking were registered. Family history of Chronic Obstructive Pulmonary
Disease (C.O.P.D), bronchial asthma or M. tuberculosis was also recorded.
Medical Examination
Anthropometric measurement (weight, height) and calculation of body mass
index [wt (kg) ht-1 (m)2]
was recorded. In addition to general medical examinations including history,
symptoms, signs, radiologic, pathologic and microbiological results and follow-up
observations were carefully reviewed.
Routine Laboratory Evaluation
This examination was performed for each person according to his compliant
(as urine examination, stool examination and complete blood count). Sputum samples
were collected for 3 consecutive days then subjected routine work examination
by direct smear and culture examination.
Chest Radiography
Individuals who had cough and other complaints related to respiratory system
were subjected to chest radiography.
Methods for Detection of M. tuberculosis Traditional Assay
Ziehl Neelsen (Zn) Stain
The standard method for direct smear examination was carried out according
to Boyed (1995).
Culture Method
According to Metchock et al. (1999), a direct inoculum was cultured
on conventional solid medium (the egg-based Lowenstein- Jensen -LJ) and incubated
at 37°C for 28 days.
Molecular Assays
Total DNA Extraction of Blood Samples
Peripheral Blood Mononuclear Cell (PBMCs) were isolated from heparinized
blood sample using lymphocyte separation buffer and gradient centrifugation
at 2000 rpm for 30 min at room temperature (ficoll solution, density of 1.077
g mL-1, Seromed, Germany). Isolated cells were washed 2X by Phosphate
Buffered Saline (PBS). Each million cells were suspended in 100 μL proteinase
K buffer (50 mM Tris pH 8.0, 50 mM KCl, 2.5 mM MgCl2, 0.45% Tween
20, 0.45% Nonidet 40) containing 100 μg mL-1 proteinase K, followed
by incubation at 56°C for 3 h, then at 95°C for 15 min to inactivate
the enzyme, finally the DNA was purified by phenol/chloroform extraction and
ethanol precipitation (Mirza et al., 2003).
M. tuberculosis Detection by PCR Method
M. tuberculosis DNA was detected in the blood samples by amplification
of IS6110 insertion sequence. It was performed using sequence specific primers
in a nested PCR reaction as described (El Demellawy et al., 2006). Briefly,
The first PCR amplification of IS6110 insertion sequence was carried out in
a mixture containing DNA concentration 70-100 ng, 1X PCR buffer, 200 μM
dNTPs (Amersham Biosciences, Freiberg, Germany), 1 μM of each of the external
primers, the forward primer M. tuberculosis 1 with sequence 5’-GGA
CAA CGC CGA ATT GCG AAG GGC-3’ and the reverse primer M. tuberculosis
2 with sequence 5’-TAG GCG TCG GTG ACA AAG GCC ACG-3’, 15 mM MgCl2
and 1 U Taq DNA polymerase (Invitrogen Life Technologies, Scotland, UK). The
amplification was initiated by one cycle as follows: denaturation at 95°C
for 5 min, annealing at 65°C for 1 min, then extension at 72°C for 1.5
min followed by 30 cycles, each as follows: denaturation at 95°C for 1 min,
annealing at 65°C for 1 min and extension at 72°C for 1.5 min A final
extension at 72°C for 15 min was performed. Size of the amplified fragment
after this round was 580 bp. Nested PCR amplification was carried out using
5 μL of the first PCR amplified product in a PCR mixture containing 1X
PCR buffer, 200 μM dNTPs, 1 μM of each of the internal primers, the
forward primer M. tuberculosis 3 with sequence 5’-ACG
ACC ACA TCA ACC-3’and the reverse primer M. tuberculosis 4 with
sequence 5’-AGT TTG GTC ATC AGC C-3’, 15 mM MgCl2 and 1 U Taq
DNA polymerase for 20 cycles, each consists of denaturation at 95°C for
1 min, annealing at 48°C for 1 min and extension at 72°C for 1.5 min
followed by extra extension at 72°C for 15 min. This step will give amplified
fragment of 181 bp .
M. tuberculosis Detection by Elispot Assa
ELISpot was performed by using a commercial kit (R and D System, Inc. UK)
as previously described in Nicol et al. (2005). Briefly, 96-well polyvinylidene
fluoride–backed plates were coated with 15 μg mL-1 of monoclonal
antibody 1-D1K against IFN-γ. M. tuberculosis specific antigen (ESAT-6
or CFP-10) or mitogen was added followed by addition of purified PBMCs (250,000/well)
in duplicate wells. No antigen was added to the background control wells. After
incubation for 18 h, plates were washed, 100 μL (1 μg mL-1)
of biotinylated monoclonal antibody 7-B6-1-biotin against IFN-γ was added
and incubated for 2 h followed by washing with streptavidin-alkaline phosphatase
toxoid and incubated for 1.5 h; plates were washed again and 100 μL of
chromogenic alkaline phosphatase substrate was added. After 10-15 min, the plates
were washed and spots were enumerated with a stereomicroscope independently by 2 observers. Mean values determined by the 2 observers and both duplicate wells were used in all calculations. The number of spots in the background control wells was subtracted from the number in the test wells and a response was considered positive if the number of spots per test well was >10 and at least twice the value found in the background control wells.
Statistical Analyses
The data were subjected to statistical analysis using SPSS version 11 to
estimate Probability (P) value and Chi-square (χ2), where probability
will be significant when its values p≤0.005 and Chi-square (χ2)
is a statistical model used for comparison between distributions of patients
according to different environmental variables of the study.
RESULTS AND DISCUSSION
The environmental parameters effects on the prevalence of M. tuberculosis infection was detected by analyzing data of the M. tuberculosis patient statistically using Chi square (χ2) test according to different factors including age, sex, crowding index, family income, occupation, smoking and medical family history (Table 1). From this analysis it was concluded that, the major affected age is in the range of 20-30 years old in the residential area and 30-40 years old in the industrial area. Regarding the sex factor, men were more affected in industrial area than the women in which is understood due to their exposure to the industrial pollution more than female. The medical family history has great role in the M. tuberculosis infection, where, M. tuberculosis was detected clearly in the families that suffer from M. tuberculosis and asthma especially in the industrial area, this might be due to the fact that the chance of infection becomes optimum, when there is infectious disease within the family itself (Perez-Padilla et al., 2001; Thierry et al., 1994). On other hand, the smoking habits by either cigarettes or nargila smoking proved to be an effective reason for M. tuberculosis infection. The nargila smokers specially who are living in the industrial area were more susceptible to infection than the cigarette smokers which could be due to the increase in probability cross infection through the smoking apparatus. However, either methods of smoking proved to have significant influence on M. tuberculosis infection as shown in Table 1.
Crowded areas could be also considered as a significant factor on M. tuberculosis infection, especially in industrial area. This could be owing to the easily distribution of the infectious disease within a restricted area with a bad ventilation condition and overcrowded with infected people. In addition to the previous factors, the financial level has a great role in the disease handling and control. As the income of the family increase the disease control, consequently, the number of infected patients will decrease as reported here.
On studying the occupation effect on the M. tuberculosis infection, it was found that, the nearer to the risky areas, the more effectively M. tuberculosis infection occurs. This can be due to the more exposure to the most pollution sources that can affect their sensitivity and infection with M. tuberculosis . The current research reveled that, family history [χ2 = 5.47, p = 0.019] is the most effective parameter that influence the infection of M. tuberculosis . The incidence of M. tuberculosis percentage was jumped up to 46 in Industrial area, while dropped down to 16 in residential area. In other words, the incidence of M. tuberculosis in industrial area was more than 2.8 times the incidence of M. tuberculosis in residential district. This study emphasizes the important role of industrial pollution on the prevalence of the M. tuberculosis as we expected from our hypothesis and in agreement with previous reports by Mishra et al. (1999) and Gupta et al. (1997).
In order to make successful comparison between the prevalence of M. tuberculosis
in industrial area and residential area, we have used traditional methods
(acid fast staining and culture) and molecular diagnostic tests (PCR and ELISpot).
As indicated in Table 2a and b, the overall
tests sensitivity, specificity, PPV, NPV and accuracy (%) were taken into account,
where the results revealed that the molecular diagnostic methods were more sensitive
than the traditional assays with higher NPV and accuracy (%).
| Table 1: | Socio-demographic data of the studied two group |
| *BMI: Body Mass Index,*p = 0.005, industrial vs residential (n = 50 for each group)- see text for more details. | |
| Table 2a: | Evaluation of different methods of assay for detection of M. tuberculosis in industrial area samples based on clinical findings |
| PPV: Positive Predictive Value, NPV: Negative predictive value | |
| Table 2b: | Evaluation of different methods of assay for detection of M. tuberculosis in residential area samples based on clinical findings |
| PPV: Positive Predictive Value, NPV: Negative predictive value | |
On the other hand, the specificity and the PPV (%) were the same for all methods. These results are in support to those reported by other researchers (Claudio and Claudio, 2003; Alexander et al., 2006), in which the diagnostic efficacy of the molecular methods is higher than the traditional assays. In this context, we evaluated the performance of the PCR using blood samples in comparison with ELISpot assay. Unsurprisingly, the efficacy of PCR assay was higher than the ELISpot assay, in a sense that the PCR technique is able to detect as little as 10 FG or the equivalent of 1 to 20 copies of M. tuberculosis complex genomic DNA. Accuracy of PCR in the present study reached up to 98% in both industrial and residential areas, while sensitivity of PCR detected in industrial area was 92 in comparison to 97.2% in residential area. The Accuracy of ELISpot assay dropped to 96% in industrial area in comparison to 92% in residential area, while sensitivity was 93.8% in industrial area and 88.9% in residential area. Indicating that ELISpot assay was as sensitive as PCR assay in detection of M. tuberculosis infection in industrial area, while in residential area PCR assay is more sensitive. These results were in agreement with previous data record by Jann-Yuan et al. (2007). Although previous studies demonstrated that the ELISpot assay for INF-γ is a powerful tool for detecting latent M. tuberculosis Infection (Alexander et al., 2006; Hadnagy et al., 1996) present results showed that in people who were previously vaccinated with BCG, the diagnostic value of this test in detecting active M. tuberculosis was less in percentage of sensitivity, NPV and accuracy, even in an area with expected high incidence of the disease. The performance of the PCR assay in this study was superior to that of the ELISpot assay because the probability of a wrong locus being mistakenly amplified twice is very low. Thus, false-positive results were decreased.
CONCLUSIONS
Air pollution, such as cement dust and smoke release from petroleum industries, has great impact on M. tuberculosis infection and spreading of the disease with the presence of other factors such as age, sex, family history and socioeconomic factors. Accurate diagnosis of M. tuberculosis is essential for disease control, however, the present study showed that detection of M. tuberculosis using PCR assay is more quick, less expensive, sensitive and can be used to detect M. tuberculosis DNA in sputum, blood and other body fluids, while ELISpot assay, although it is sensitive, is limited to detect presence of active M. tuberculosis infection in blood samples only. The present study recommend the use of PCR assay for M. tuberculosis detection and increasing the public awareness about air pollution and the positive importance of decreasing this type of pollution on human health. It is intend in the future to study the correlation between the degree of pollution and the corresponding infection.
ACKNOWLEDGMENTS
The author is appreciating very much the role of Dr. Somia Hussein (El-Maamora hospital for chest disease, Alexandria) for being helpful in providing the clinical cases and samples analyzed in the present study, also Dr. Maha Eldemellawy and Dr. Hala El-Adawi in Mubarak City for Scientific Research and Technology Applications for their kind help and cooperation.