This study aimed to estimate some haematological changes in patients who are infected with pulmonary tuberculosis in Sobi Specialist Hospital, Ilorin, Nigeria

EFFECT OF TUBERCULOSIS ON HEAMATOLOGICAL PARAMETRE OF INFECTED PATIENT AT SOBI SPECIALIST HOSPITAL

ABSTRACT

The aim of this study is to investigate the changes of some hematological parameters in patients affected with pulmonary tuberculosis in Kwara State Specialist Hospital, Sobi, Ilorin.  100 patients with pulmonary
tuberculosis (50males and 50females) and 40 healthy controls (20 males and 20 females) have included in present study. Patients have been classified into3 groups: group1includes newly diagnosed patients, group 2 includes patients after twomonths from starting treatment and group 3 includes Patients after six months from starting treatment. The mean of ages was 44± 2yearsfor patients and 42± 2 years for the control. This study founded that values of Hb, PCV, platelets and ESR for both sexes were significantly changed in group 1 in comparison with group 2. Values of Hb, PCV, platelets and ESR for both sexes were significantly changed in group 1 in comparison with group 3. This study has show that values of Hb, PCV, platelets and ESR values for both sexes were significantly changed in group 1 in comparison with healthy controls. Values of platelets and ESR for both sexes and values of PCV for males were insignificantly changed while values of Hb for both sexes and values of PCV for females were significantly changed in group 2 in comparison with group 3. This study showed that values of PCV for males were insignificantly changed while values of Hb, platelets and ESR for both sexes and PCV for females were significantly changed in group 2 in comparison with healthy controls. The results has proved that the values of platelets and ESR for both sexes and Hb for females and PCV for males were insignificantly changed while values of Hb for males and PCV for females were significantly changed in group 3 in comparison with healthy controls.

CHAPTER ONE

1.0      INTRODUCTION

Tuberculosis (TB), one of the oldest known diseases and still a major cause of mortality today, has many manifestations affecting the lung, the central nervous system and many other organ systems, but it is primarily a pulmonary disease. It is caused by a closely-related group of organisms, all of which form the Mycobacterium tuberculosis complex. These organisms include M. tuberculosis, M. bovis, M. africanum, M. microfti and M. Canetti .[Iseman, M.D 2000].

Pulmonary tuberculosis (PTB) is a lower respiratory tract infection that is initiated by the deposition of Mycobacterium tuberculosis (MTB) contained in aerosol droplets, onto lung alveolar surfaces. The active disease is characterized by a protracted cough, fatigue, loss of weight and appetite and night sweats. Haemoptysis, secondary infection by fungi and permanent lung damage are few of the complications of PTB . [Fraser R.S 1993].

Tuberculosis is a major public health problem in Nigeria with an estimated prevalence of 616 cases per100, 000. Nigeria ranks first in Africa and fourth among the 22 high TB burden countries in the world. No fewer than 460,000 cases of tuberculosis are reported annually in Nigeria. [WHO Report 2008] The co-infection of pulmonary tuberculosis with human immune deficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) has in the recent past compounded the epidemiology, clinical outcome, diagnosis and treatment of the disease worldwide . [Center for Disease Control and Prevention 1995]

This has led to the different reports made for this condition in Nigeria. Previous study has shown that anaemia, raised erythrocyte sedimentation rate (ESR), peripheral leukocyte count and neutrophilia are the most common haematological manifestations associated with PTB. [Devi, U, Mohan, R. C, Srivastava] Another study carried out in Kano, Northern Nigeria, reported mild anaemia, and raised ESR, leucocytosis, neutrophilia with toxic granulation, thrombocytosis, occasional stickiness of platelets and monocytosis.[Nwako, E. O. K Kwaru, A Ofulu, A. $ Babashani, M 2005].

In Benin, Southern Nigeria, a study showed significant increase in plasma viscosity and fibrinogen levels and a decrease in platelet count, total white cell count and packed cell volume when compared to values for apparently healthy subjects. [Awodun, O.A Ajayi, I.O. $ Famodu A.A. 2007]. This study was undertaken to analyses the hematological parameters in patients with Mycobacterium tuberculosis and to evaluate their diagnostic and prognostic significance, at Sobi Specialist Hospital, Ilorin, Nigeria..

Reversible peripheral blood abnormalities are commonly associated with pulmonary TB. Insight into the relationship between hematological abnormalities and mycobacterial infection has come from an understanding of the immunology of mycobacterial infection. Little is known about the prevalence of these hematological abnormalities and the effect of anti-tuberculosis treatment on the various hematological parameters in the Indian subcontinent . [Vijayan V.K Das S. Pulmonary tuberculosis. In Suredra Shrma [ed]]. Tuberculosis 1^st edition. New Delhi, Jaypee Publishers, 2009, 217-27.

CHAPTER TWO

2.0      LITERATURE REVIEW

Pulmonary tuberculosis is a highly infectious disease [San Java. N $ Mark J 2004 Detention of tuberculosis. Public health and the law. M.JA 180 [11] 573-576] in which single patient may have devastating effects on tuberculosis control program by infecting large number of people. [Peter et al 1994] Tuberculosis (TB) caused by infection with Mycobacterium tuberculosis, which is part of a complex of organisms including Mycobacterium bovis (reservoir cattle) and Mycobacterium africanum (reservoir human). [College et al 2010]

In 2009, there were an estimated 9.4 million incident cases of TB globally (equivalent to 137 cases per 100 000 population), most of the estimated number of cases in 2009 occurred in Asia (55%) and Africa (30%). [WHO, Report, 2010]. Accurate and rapid diagnosis is the key to control the disease but, the current routine diagnostic tests for TB (chest x-ray, culture, tuberculin skin test and acid-fast staining) all have their limitations, in which chest x-ray alone is inconclusive, culture take too long to produce a result, the tuberculin skin test lacks specificity and reliability, serological tests using different TB antigens to detect Mycobacterium tuberculosis infection are fast but lack the desired sensitivity. [Al-zamel F. 2009].

The primary method for diagnosing pulmonary tuberculosis in low-income and middle-income countries is direct sputum smear microscopy, which is fast, inexpensive, and specific for Mycobacterium tuberculosis but, it has low sensitivity because of variable quality of the test in program conditions. [Karen, et al. 2006]New methods have been developed, such as nucleic acid amplification technology which, although specific, can yield false-positive results, as well as immunologic tests which have their disadvantages and unanswered questions, so we still need accurate and rapid method of diagnosing both active and latent TB infection .[Chales, et al. 1989]. In pulmonary tuberculosis many hematological and biochemical abnormalities are common and they are valuable aids to diagnosis. [Southern, et al 2006]. In pulmonary tuberculosis patients rise in hemoglobin and haematocrit levels are used as  markers reflecting response to treatment. [Al Umor, et al. 2009]

The Erythrocyte Sedimentation Rate (ESR) is usually elevated in such conditions: infections, collagen diseases, metastatic malignant tumors and renal disease (said to be the leading causes of elevated values ≥ 100 mm/h.). [Metz, J. 2007] Reduction in ESR were regarded as good indicator to observe drug response and disease progress or regress. [Mohan, et al. 2003]. Anaemia is a cardinal feature in patients with bacterial infections, particularly infections lasting longer than a month, including pulmonary tuberculosis [Eyshi, et al 2009] in which the precise mechanism of anaemia in pulmonary tuberculosis is not clearly known. [Nsal, et al 2005]

The occurrence of anaemia among patients diagnosed as active pulmonary tuberculosis was very high and it was contributed to anaemia of chronic disease. [Lewis, et al. 2006]

Reactive thrombocytosis is found in a number of clinical situations including infectious diseases such as pulmonary tuberculosis. [Markarem, et al 1974].

Tuberculosis (TB), one of the oldest known diseases and still a major cause of mortality today, has many manifestations affecting the bone, the central nervous system and many other organ systems, but it is primarily a pulmonary disease. It is caused by a closely-related group of organisms, all of which form the Mycobacterium tuberculosis complex. These organisms include M. tuberculosis, M. bovis, M. africanum, M. microfti and M. Canetti. [Iseman, et al 2000] Pulmonary tuberculosis (PTB) is a lower respiratory tract infection that is initiated by the deposition of Mycobacterium tuberculosis (MTB) contained in aerosol droplets, onto lung alveolar surfaces. The active disease is characterized by a protracted cough, fatigue, loss of weight and appetite and night sweats. Haemoptysis, secondary infection by fungi and permanent lung damage are few of the complications of PTB. [Freser R.S 1993]

Tuberculosis is a major public health problem in Nigeria with an estimated prevalence of 616 cases per100,000. Nigeria ranks first in Africa and fourth among the 22 high TB burden countries in the world. No fewer than 460,000 cases of tuberculosis are reported annually in Nigeria. [WHO, Repor. 2008] The co-infection of pulmonary tuberculosis with human immune deficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) has in the recent past compounded the epidemiology, clinical outcome, diagnosis and treatment of the disease worldwide. [Center for Disease Control and prevention 1995]. This has led to the different reports made for this condition in Nigeria. Previous study has shown that anaemia, raised erythrocyte sedimentation rate (ESR), peripheral leukocyte count and neutrophilia are the most common haematological manifestations associated with Pulmonary tuberculosis (PTB). [Devis, et al 2003]. Another study carried out in Kano, Northern Nigeria, reported mild anaemia, and raised ESR, leucocytosis, neutrophilia with toxic granulation, thrombocytosis, occasional stickiness of platelets and monocytosis. [Nkwako, et al. 2005]

In Benin, Southern Nigeria, a study showed significant increase in plasma viscosity and fibrinogen levels and a decrease in platelet count, total white cell count and packed cell volume when compared to values for apparently healthy subjects. [Awodu, et al.2007] It is our aim to study the pattern of some haematological parameters in PTB infected subjects in Calabar, Nigeria. It is believed that the findings of this study may be useful as indicators of disease progression, cardiovascular risk factors and response to therapy in pulmonary tuberculosis.

2.1      AIM OF THE STUDY

This study aimed to estimate some haematological changes in patients who are infected with pulmonary tuberculosis in Sobi Specialist Hospital, Ilorin, Nigeria. To help the Medical staff in the early and precise detection and follow up of this disease.

This study has designed to determine the following:

1. Measurement of hemoglobin concentration.

2. Packed cell volume.

3. Platelets count.

4. Erythrocyte sedimentation rate

CHAPTER THREE

3.0      MATERIALS AND METHODS

3.1      STUDY AREA

Kwara State Specialist Hospital, Sobi, Ilorin. It is located in Ilorin South Local Government Area. It is a secondary Health care institution with about 250 inpatient and 450 outpatient capacities.  Ilorin is the Kwara State capital and is situated 360KM North of Lagos, at 8⁰30N and 4⁰30N and 482KM South West from Abuja, the federal capital. It is the gate way between the Northern and Southern part of the country.

The climate is tropical, consisting of two main seasons; the dry season and long wet season (March to November) with an intervening cold and dry harmattan period usually experienced from December to January with an average rain fall of 1,279mm per annum.

3.2      ETHICAL CONSIDERATION

Ethical clearance was obtained from the ethical committee of the Hospital management, before the commencement of this study.

3.3      STUDY DESIGN

The study was a cross sectional laboratory based analysis.

3.4      INCLUSSION CRITERIAL

All pulmonary tuberculosis bacilli positive patient at the Kwara state Specialist Hospital. Sobi Ilorin

3.5      EXCLUSSION CRITERIAL

1.     Extra pulmonary tuberculosis.

2.     Non-tuberculosis pulmonary infections.

3.     Chronic liver disease

4.     Chronic renal disease.

5.     Diabetes mellitus.

6.     Smoking.

7.     Pregnancy.

8.     Chronic cardiovascular diseases.

3.6      SAMPLES SIZE

Sample size was calculated as100, with 50 clinical positive and 50 clinical negative pulmonary tuberculosis bacilli, using Fisher’s formula (Araoye, 2003), N = t²*  p (1- p )  / m².Where N represents the required sample size, t is the confidence interval at 95% (standard value of 1.96), P represents estimated prevalence of Gram negative bacilli in Sobi Specialist Hospital, which is put at 0.22 from previous study (Irjm, 2012), and m is margin of error at 5% (Standard value of 0.05). N = 1.96² * 0.22 (1- 0.22) / 0.05² N= 3.8416 * 0.22 (0.78) / 0.0025, N = 0.6592/0.0025, N= 100.

3.7      SAMPLE COLLECTION

One Hundred (100) clinically positive and negative pulmonary tuberculosis bacilli patient from the Medical Microbiology Laboratory of Sobi Specialist Hospital, Ilorin from January, 2015 to June, 2015, were identified and confirmed using standard microbiology identification techniques (CLSI, 2006).

3.8      SAMPLE TECHNICS

A purposive sampling method was employed for this study.

3.9      LABORATORY PROCEDURE

The collection of blood done in Sobi Specialist Hospital Consultant Clinic for Chest And Pulmonary Diseases, and Medical Microbiology Laboratory of Sobi Specialist Hospital, from 8.30 A.M. to 11.30 A.M. 5 ml. of blood drawn for each haematological study in tubes contain EDTA as anti-coagulants to prevent clotting of blood. Each sample was labeled and given a serial number together with the patient name. [Daniel, et al. 1999].

A cyanomethemoglobin method was used to estimate the haemoglobin contents of the blood. The method was based on Drabkin’s cyanide-ferriccyanide solution. Twenty micro liter (μL) of blood was added for 5 ml of Drabkin’s solution mixing, and incubated for at least 5 minutes at 37°C and then the results were estimated by using Hb meter at 540 nanometer (nm) wave length. [Eyshi, A. et al. 2009]

Microhaematocrit method was used to determine PCV.

Heparinized capillary tubes used, and blood was filled to approximately three quarters of their lengths then the unmarked end is closed with modeling clay and put in the microhaematocrit centrifuge. After centrifugation for 15 minutes, the red blood cells were separated from plasma and remain a band of buffy coat at the interface between them consisting of leukocytes and blood platelets. [Daniel, w. 1999].

This is done by taking 0.38 ml of diluting fluid (1% ammonium oxalate which hydrolyzes the RBCs,) and added to it 0.02 ml of blood taken by using haemoglobin pipette after having wiped the tip of the pipette ,and then the counting chamber charged with the help of a Pasteur's pipette and placing the counting chamber in a Petri dish containing a moistened filter paper and let stand for 15 minutes to give time to platelets to settle ,then counted under high power (40 x objective ). [Daniel W. 1999].

ERYTHROCYTE SEDIMENTATION RATE

A Westergreen tube: length-300mm (open at both ends), diameter 2.5mm were used. One part of anticoagulant (3.8% trisodium citrate solution) was added to 4 parts of blood (0.5 ml of anticoagulant is added to 2 ml of blood). The mixture was drawn into a Westergreen tube up to the zero mark and the tube was set upright in a stand with a spring clip on the top and rubber at the bottom. The level of the top of the red cell column was read at the end of 1 hour. [Daniel W. 1999].

All values were expressed as mean ± SE. The data were analyzed by using of computer SPSS statistics 11 program.

The differences were considered significant when the probability (P) was less than 0.05 (P >0.05) and highly significant when the probability (P) was less than 0.01 (P >0.01).ANOVA test was used to examine the differences between different groups. [Al – Omar I. Al – shban and Shah, A. 2009].

CHAPTER FOUR

4.0      RESULTS

4.1      HAEMOGLOBIN (HB)

Group 1 values for both males and females are highly significantly decreased (p < 0.01) in comparison with group 2, group 3 and healthy controls.

Group 2 values of both males and females are highly significantly decreased (p < 0.01) in comparison with group 3 and healthy controls. Group 3 values of males are significantly decreased (p < 0.05) for females they are insignificant (p > 0.05) in comparison with healthy controls.

2.2      PACKED CELLS VOLUME (PCV)

Group 1 values for both males and females are highly significantly decreased (p < 0.01) in comparison with group 2, group 3 and healthy controls.

Group 2 values for both males and female show insignificant changes (p > 0.05) in comparison with group 3. While group 2 values for males are insignificant (p > 0.05) but for females they are significantly decreased (p < 0.05) in comparison with healthy controls. On the other hand group (3) values for both males and female show insignificant changes (p > 0.05) in comparison with healthy controls.

Table 1

The changes in Haemoglobin (Hb) and pack cell volume (PCV) for pulmonary tuberculosis patients groups (Group 1 included newly diagnosed patients before starting treatment, group 2 included patients after two months from starting treatment and group 3 included patients after six months from starting treatment and controls.

-Values are mean ± SE.

-Different small letters indicates (p < 0.05).

-Different capital letters indicates (p < 0.001).

-Similar letters (capital & small) indicates no significant difference.

4.3      PLATELETS COUNT

The values of platelets count for all the patient groups (1, 2 and 3) for male pulmonary tuberculosis patients are: 401.2 ± 9.294; 312.5 ± 4.609 and226.7 ± 3.8 Í103/mm³ respectively , and for females are: 325.0 ± 3.162; 280.2 ± 3.246 and 192.3 ± 5.144 Í103/mm³ respectively. Group 1 values of both males and females are highly significantly increased (p < 0.01) in comparison with group 2, group 3 and healthy controls. Group 2 values of both males and females are highly significantly increased (p < 0.01) in comparison with group 3 and healthy controls. Group 3 values of both males and females are insignificant (p > 0.05) in comparison with healthy controls.

4.4      ERYTHROCYTE SEDIMENTATION RATE

The values of ESR for all the patient groups (1, 2 and 3) for male pulmonary tuberculosis patients are: 102 ± 5.241; 56.6± 6.12 and 24.6 ± 3.982 mm/hr respectively, and for females are: 113 ± 3.91; 62.5 ± 6.7 and 27.8 ± 4.234 mm/hr respectively. Group 1 values for males and females are highly significantly increased (p < 0.01) in comparison with group 2, group 3 and healthy controls. Group 2 values of both males and females are highly significantly increased (p < 0.01) in comparison with group 3 and healthy controls. Group 3 values of males are highly significantly increased (p < 0.01) while for the females they are insignificant (p > 0.05) in comparison with healthy controls.

CHAPTER FIVE

5.0      DISCUSSION

1. Haemoglobin HB and Packed cells volume PCV

There was a high significant (p < 0.01) decrease in the values of Hb and PCV values of both males and females in newly diagnosed pulmonary tuberculosis patients in comparison with healthy controls as shown in table 1 and this is in agreement with [Jemikalajab, J. and Okogun, G.2009.] who states that occurrence of anemia among patients that were diagnosed as active pulmonary tuberculosis was very high and it was contributed to anaemia of chronic disease. As treatment with chemotherapy goes on there was a respectable gradual improvement in the levels of Hbb and PCV towards control levels (table 1), this rise in hemoglobin and haematocrit levels can be used as a markers reflecting response to treatment as [Weiss, G.2002] and [30][Means, RT.2003]. focused on this point saying that in pulmonary tuberculosis PCV is an essential haematological index and it is a regular feature in assessment of prognosis of the disease, [Nemeth E.., Rivera, S. and Gabayan, v. 2004] [Karyadi, E.., Dolmans, W.., West, C.., Crevel, R..,Nelwan R.., Amin, Z. and Gros, R. 2007]and[Nsal, E.., Aksaray, S…, Ksal, D. and Ipit, T. 2005] explain the mechanism behind the occurrence of anaemia in pulmonary tuberculosis patients saying that the invasion of bacteria leads to activation of T-lymphocytes and macrophages, which induce the production of the cytokines like interferon gamma (INF gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and interleukin-6 (IL-6)which with their products will cause diversion of iron into iron stores in the reticulo-endothelial system resulting in decreased iron concentration in the plasma thus limiting its availability to red cells for haemoglobin synthesis, inhibition of erythroid progenitor cell proliferation and in appropriate production and activity of erythropoietin, the first leads to anaemia and the latter two result in suboptimal response of the bone marrow to the ana emia. Cytokines also impair red cell production in the marrow. IL-1 and TNF-alpha inhibit the production of erythropoietin, and together with INF-gamma impair responsiveness of progenitor cells to erythropoietin. An inhibitory effect of TNF-alpha on red cell production in the bone marrow has been demonstrated by both in vitro and in vivo studies. INF-gamma directly suppresses the proliferation of erythroid progenitor cells, in this way cytokines impair the physiological erythropoietin response to the anemia. In addition, TNF-alpha directly damages erythrocytes and decreases red cell life span. So the anemia of infection is therefore basically an underproduction anemia due to iron restriction, combined with inability of erythropoiesis to compensate adequately for the anaemia. Another study in Indonesia has done by [Awodu, O.., Ajayi, I. Famodu, I. 2007]also found an elevated levels of IL-6 and IL-1ra in the circulation and in unstimulated whole blood cultures from tuberculosis patients suffering from anaemia.

2. THE PLATELETS COUNT

From this study resulting data (figure1)showed that values of both male and females of platelets count of group 1 tuberculosis patients were highly significantly increased (p < 0.01)in comparison with values of group 2, group 3 and healthy controls. These results may be attributed to the reactive thrombocytosis which is found in a number of clinical situations including infectious diseases such as pulmonary tuberculosis. [Ksal, A.., Brandacher, G Steurer. 2001] Also the increase in platelet count was noticed in Saudi pulmonary TB patients as compared with the normal Saudi persons. [Mohan,C. and Srivastava, V. 2003]

The results have in contrast to [Kuashansay, K. 2003] who found that there was significantly lower platelet count in African pulmonary tuberculosis patients. In our study we notice that the values of platelets count of both males and females were changing from thrombocytosis to equalize the controls values and this was very clear because of the insignificancy (p > 0.05) between the values of group 3 and the values of healthy controls (table4.2) and this is compatible with [Mohan,C. and Srivastava, V. 2003]

 who founded in their study that platelet count were elevated in pulmonary TB which is well influenced and corrected by using different combinations of antituberculosis drugs[Means, RT.2003].  are on the same wave length in their study in which they states that: in pulmonary tuberculosis thrombocytosis is an essential hematological index and it is a regular feature in assessment  of prognosis of the disease .

            The explanation for these results returns to An elevated levels of IL-6 and IL-1ra in patients with pulmonary tuberculosis suffering from anemia as has been founded in a study in Indonesia. [Awodu, O.., Ajayi, I. Famodu, I. 2007]IL-6–induced thrombocytosis is accompanied by enhanced hepatic thrombopoietin production and elevated thrombopoietin plasma levels [Olaniyi, J. and Aken’Ova, Y.2003] and although first proposed to be the primary regulator of platelet production 45 years ago, the gene for thrombopoietin was cloned only within the last decade. Since then, understanding of platelet production has increased considerably. [Mohan,C. and Srivastava, V. 2003]

 This regulatory pathway might be of relevance for the understanding of reactive thrombocytosis. [Olaniyi, J. and Aken’Ova, Y.2003]

3. ERYTHROCYTE SEDIMENTATION RATE

When an inflammatory process is present, the high proportion of fibrinogen and other acute-phase proteins (haptoglobin, ceruloplasmin and CRP) and immunoglobulins in the blood causes red blood cells to stick to each other, these jammed red cells form what is called rouleaux which will settle and sediment faster. [Chales, M…, Arthur, V. and Neel, H. 1989]

In this work it has been found that the values of ESR for both males and females of pulmonary tuberculosis patients are highly significantly increased (p < 0.01) in comparison with group 2, group 3 and healthy controls and there were no significant difference (p > 0.05) between group 3 values and healthy control values (figure2). These findings are fully supported by different reports and studies specifying high erythrocyte sedimentation rate (ESR) levels in newly diagnosed and normal levels at the end of treatment of pulmonary tuberculosis patients like Al-Omar et al.[10] who stated that: erythrocyte sedimentation rate were elevated in pulmonary TB which is well influenced and corrected by using different combinations of antituberculosis drugs, other study says: erythrocyte sedimentation rate was significantly higher in pulmonary tuberculosis patients than controls, there was a significant reduction in the erythrocyte sedimentation rate from the 4th. week of therapy [Eyshi, A.., Rahimi, E. and Gharabaghi, N. 2009] and other one says: statistically significant haematologic abnormalities like high erythrocyte sedimentation rate founded in pulmonary tuberculosis patients in Ibadan, Nigeria. [Weiss, G. 2002]

CHAPTER SIX

6.0      CONCLUSIONS

Some haematological abnormalities are quite common in patients with pulmonary TB and physicians must maintain a high index of suspicion for diagnosis of pulmonary TB in patients with these abnormalities. As well as, these parameters can be used as indicators in the assessment of response to chemotherapy. Furthermore during this study our understanding of anemia and it’s mechanism of occurrence in pulmonary tuberculosis has increased considerably. Hopefully, this will perhaps yield an insight on the pathophysiology of this disease.

However, many more questions remain about hematopoietic during course of TB waiting to be studied and the field remains ready and open for rich investigations and researches.