HIV-Co Opportunistic Infections - A Current Picture in Tropical Climatic Eastern Indian Seropositive Population

the presence of Cryptosporidium parvum , a zoonotic pathogen that causes chronic watery diarrhea. The staining technique used for staining of Cryptosporidium was modified Ziehl-Neelsen (AFB staining). Cryptosporidia and other coccidia stained pink-red. The isolated pure culture of Cryptosporidium spp. was compared with a referral strain Cryptosporidium (Microbiology QC slides, Himedia SL45-10; Himedia).Enteric bacterial flora from the stool samples of diarrhea patients were isolated using differential selective screening media such as UTI agar (urinary tract infection agar; Himedia) after documenting their clinical manifestations, including intestinal flu, inflammation-associated cramping, abdominal pain, nausea and vomiting. Different enteric pathogens that exhibit a particular colony color facilitate the identity of particular microorganisms such as Escherichia coli (pink-magenta), Proteus mirabilis (light green), Enterococcus fecalis (bluish green), Staphylococcus aureus (cream), Pseudomonas aureginosa (colorless) and C . albicans (pin point white). For isolation of Vibrio cholerae and other enteropathogenic Vibrio , TCBS agar (thiosulfate citrate bile salt sucrose; SRL, Mumbai, India) was used. Cryptococcal meningitis was confirmed by clinical symptoms and signs as well as the detection of cryptococcal capsular antigen ( Cryptococcus antigen latex agglutination Test; Remel, Lenexa, Kans., USA). Toxoplasmic encephalitis was diagnosed in the presence of at least two of the following findings: a history of neurological symptoms, neurological signs at admission or suggestive computed tomography scan or magnetic resonance imaging of the brain (Luft, & Remington,1988, 1992). A response to anti- Toxoplasma (IgM) antibodies, which was detected by a serological ELISA commercial kit gives a satisfactory result.


Introduction
HIV or Human Immunodeficiency Virus, a RNA virus. On exposure to this virus, it directly attacks certain human organs, such as the heart, brain and kidneys. It plays a very significant role in weakening the immune system of its host. The immune system forms the framework in protecting the body from attack by any foreign agent like the bacteria, fungus, virus or any type of infection and even from some cancers by reacting accordingly. The primary cells attacked by HIV are the CD4+ lymphocytes, which are glycoprotein expressed on the surface of T h cells, macrophages, monocytes, regulatory T cells and dendritic cells and plays a vital role in performing important immune responses in the body. It helps in direct immune function in the body. It is via this CD4 that HIV-1 enters into the host T-cells. A progressive reduction in the number of T cells expressing CD4 occurs on HIV infection. Since CD4+ plays a very important role in maintaining a proper immune system function, progressive loss of CD4+ lymphocytes as destroyed by HIV, the immune system barely works. With more active HIV infection or on prolonged exposure to HIV would deprive the host of its CD4 containing cells and this reduced CD4 count leading to reduced host immunity. Opportunistic infections take advantage of this weak host immune system and manifest their adverse effects. Many people especially during the advanced stage of HIV infection face problems resulting from opportunistic infections (OIs) and cancers. The HIV/AIDS is a global epidemic and approximately 40 million people are living with HIV/AIDS worldwide (Quinn, 1996). Of all HIV/AIDS infected people, 95% are living in developing countries. Africa is the worst affected of all the continents. It consists of 19 countries worldwide with the highest prevalence of reported infections with more than 24.5 million, and more than 60% of the HIV-infected population. South Africa holds the record of being the country with the largest population living with the disease, at well over 5 million people infected. South Africa is followed by followed by Nigeria in 2nd place and India being the 3rd largest population of HIV infected people. Currently India about 5.134 million HIV infected cases are present in India which comprises of 65% cases of Southeast Asia. In India, HIV/ AIDS pandemic no longer belongs to the high-risk groups but now it is common among the general population (Ran & Hemalatha, 2006;Solomon et al.2006). Exponential growth of this epidemic is now at the threshold for India. www.intechopen.com

OIs associated with HIV
As mentioned earlier, taking advantage of this weakened immune system of its host with advanced HIV infection several infections and malignancies called 'opportunistic infections' appear.Opportunistic Infections (OIs) have been recognized as common complications of HIV infection due to immune deficiency which detoriates both the standard of life and life expectancy of the these HIV infected people. One of the main reasons behind hospitalization and substantial morbidity in HIV infected patients due to these OIs. OIs have been recognized as common complications of HIV infection since the beginning of the HIV epidemic (Kanabus et al. 2006;CDC, 1982;Selik et al.1984). These OIs develop several complicacies which lead to substantial morbidity and hospitalization. Several toxic and expensive therapies are required as a part of the treatment procedure. These ultimately lead to shorten the survival of people with HIV infection OI (Moore & Chassion, 1996;Finkelstein et al.1996). The decrease in the CD4 count in these HIV infected people is not doubt partially responsible for these various OIs (Talib et al.1993). A dramatic reduction in the incidence of OI among HIV-positive people who have received ART has been observed on introduction of antiretroviral therapy (ART); however, HIV/AIDS patients all around the world cannot afford or are not exposed to ART. Millions of people living with HIV in resource-poor communities/ countries donot get access to ART and in these cases especially the occurrence of OIs is very common (Kanabus et al. 2006). Even where ART drugs are available, they do not entirely remove the need for preventing and treating OI. Because of poor adherence, drug resistance or other factors measures to prevent and treat OIs become essential if ART stops working. However, providing prevention and treatment of OIs is of utmost importance to these people as it not only helps HIV-positive persons to live longer, healthier lives, but it can also help to prevent tuberculosis (TB) and other transmissible OIs from spreading to others. At present, the absolute CD4+ count determines the initiation of primary prophylactic therapies for OIs, which is an excellent predictor of the short-term overall risk of developing AIDS among HIV-infected patients (Stein et al.1992). The spectrum and frequency of certain OIs highlight the urgency of studying HIV/AIDS in resource-limited countries like India where locally specific disease patterns may be observed. With the identification of such opportunistic pathogens in these HIV/AIDS patients, the HIV epidemic can be more effectively managed if physicians and health planners are aware of this information. The data may thus serve as a baseline, which can be implemented to the remote district level study as well and may further give an insight into the HIV related opportunistic infections would render help for subsequent HIV/AIDS care and management in a developing country like India.  (NACO,2000). For the diagnosis of OIs, routine microbiology smears, cultures and serology should be performed with utmost precaution. Different samples as directed by clinicians were collected depending on patient symptoms and clinical presentation under universal aseptic precautions in suitable sterile containers for the routine diagnosis. For the isolation, culture and identification of species of the pathogen in order to detect the OIs from blood samples, sputum and stool. OIs were diagnosed according to the criteria suggested by the Centers for Disease Control and Prevention (CDC) (CDC,1992). For isolation of Candida causing oral candidiasis to the HIV patients, Sabourand's dextrose agar (SDA) was used as transporting media. Pseudohyphae and budding yeast were characteristic findings. The appearance of the lesion and presence of yeast forms on microscopic examination of the oropharynix were sufficient evidence to confirm the diagnosis (Kolmer et al.1969). The most prevalent and obtained pure culture of yeast was compared with referral strain Saccharomyces cerevisiae ATCC 2601 and that of Candida albicans spp. was compared with referral strain C. albicans ATCC 10231. Cases of TB were classified as definite if the culture for Mycobacterium tuberculosis was positive for acid-fast bacilli. The decontaminated sputum sample from the diagnosed TB patients was further studied for obtaining a pure culture of M. tuberculosis growing on prepared LJ slants.The positive pure cultures grew yellow colonies on the slants. The isolated pure culture of M. tuberculosis spp. was compared with referral strain M. tuberculosis ATCC 25177. Stool specimens from all diarrhea patients (following WHO criteria of watery stool for at last 48 h prior to investigation) were processed (da Silva, & Pieniazek, 2003) and examined microscopically for the presence of Cryptosporidium parvum, a zoonotic pathogen that causes chronic watery diarrhea. The staining technique used for staining of Cryptosporidium was modified Ziehl-Neelsen (AFB staining). Cryptosporidia and other coccidia stained pink-red. The isolated pure culture of Cryptosporidium spp. was compared with a referral strain Cryptosporidium (Microbiology QC slides, Himedia SL45-10; Himedia).Enteric bacterial flora from the stool samples of diarrhea patients were isolated using differential selective screening media such as UTI agar (urinary tract infection agar; Himedia) after documenting their clinical manifestations, including intestinal flu, inflammation-associated cramping, abdominal pain, nausea and vomiting. Different enteric pathogens that exhibit a particular colony color facilitate the identity of particular microorganisms such as Escherichia coli (pink-magenta), Proteus mirabilis (light green), Enterococcus fecalis (bluish green), Staphylococcus aureus (cream), Pseudomonas aureginosa (colorless) and C. albicans (pin point white). For isolation of Vibrio cholerae and other enteropathogenic Vibrio, TCBS agar (thiosulfate citrate bile salt sucrose; SRL, Mumbai, India) was used. Cryptococcal meningitis was confirmed by clinical symptoms and signs as well as the detection of cryptococcal capsular antigen (Cryptococcus antigen latex agglutination Test; Remel, Lenexa, Kans., USA). Toxoplasmic encephalitis was diagnosed in the presence of at least two of the following findings: a history of neurological symptoms, neurological signs at admission or suggestive computed tomography scan or magnetic resonance imaging of the brain (Luft, & Remington,1988. A response to anti-Toxoplasma (IgM) antibodies, which was detected by a serological ELISA commercial kit gives a satisfactory result. In our study, 125 HIV (HIV-1 subtype) patients with OI were studied, of whom, 105 (84%) were male and 20 (16%) were female. The majority of patients (52%) were 31 -40 years old followed by 27, 19 and 1% for the age groups 21 -30, ≥41 and ≤20 years old, respectively. Heterosexual mode of transmission was obtained to be the predominant mode of transmission of HIV accounting for 64% of cases, followed by other routes (Table 1). According to CD4 cell count/cu mm of blood ,the distribution of the study population was a maximum 36% of the population with 101-200 CD4+ cell count, followed by 23, 22 and 17% of patients with ≥201, 51 -100 and ≤50 CD4 cell count, respectively. A significant finding from an epidemiological point of view was the complete absence of Yeast, HBV and venereal disease in female subjects. The follow-up of the case studies revealed that 7 cases (5.6%) out of 125 HIV-infected patients expired during the later period of their treatment. Ten (8%) of the study patients were receiving ART. Dermatological reactions were found as drug-related complications in patients with HIV infection. The most serious disorder, Steven-Johnson syndrome (SJS), was found to occur in 5 (4%) HIV-infected patients after intake of a combination of rifampicinisoniazid after beginning treatment for recurrent Pulmonary TB occurring as an OI (Pitche et al. 2005).

Prevalence of different OIs
Oral candidiasis (OC) emerged as the most frequent infection to be associated with HIV infection in patients across the total range of CD4+ as studied by us among the spectrum of OIs observed. OC infection was found to be prevalent among 88% of these patients. Oral yeast (Saccharomyces)-infection was found in 5.6% of patients. TB emerged as the second most prevalent infection amongst the population as studied by us, developing in 57% of subjects. The diagnosis of TB was definite for all the suspected patients. Both pulmonary (69.4%) and extra pulmonary (16.6%) types were found to be prevalent. Amongst the HIV positive patients extra pulmonary TB was found to be prevalent amongst patients with lower median CD4+ counts (46/μl blood). But amongst the HIV/AIDS patients pulmonary TB was found amongst the patients having median CD4+ of 105/μl blood -which are the most common location of the disease. HIV is driving the tuberculosis (TB) epidemic in many developing countries including India. During our study to determine the drug resistance pattern of pulmonary TB among HIV seropositive and HIV negative hospitalized patients from different states of Eastern India, (published as Drug susceptibility profile of Mycobacterium tuberculosis isolated from HIV infected and uninfected pulmonary tuberculosis patients in Eastern India in Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 195-201) the TB positive isolates were screened and characterized by conventional laboratory methods followed by first-and second-line drug susceptibility testing on Lowenstein-Jensen medium by the proportion method. The drug susceptibility testing showed 17.7% and 6.6% multidrug-resistant (MDR) TB for the HIV positive and HIV negative patients, respectively. 22.2% of the isolated MDR-TB cases could be classified as extensively drug-resistant (XDR) TB isolates. 88.8% of all the MDR-TB isolates and all XDR-TB isolates were screened from HIV patients. 27.7% of the MDR-TB isolates showed resistance to all the first-line drugs. Mortality rate among the XDR-TB isolates was as high as 75%. Patients with interrupted anti-TB drug treatment were the ones most affected. These findings are critical and the risk to public health is high, particularly with HIV infected patients. The pathogen most frequently isolated from the diarrheic patients was C. parvum (43.1%). In about 47% of the immunocompromised patients, various enteropathogenic species of Vibrio were found, while simultaneously the presence of bacterial pathogens from Enterobacteriacae was 40% and E. coli was found in 42% of cases. Among the viral OIs, CMV infection was the most predominant; 45% of subjects were positive for CMV infection among 125 HIV-seropositive study subjects. The other viral infections found in the study population were HSV (7.2%) and HBV (5.6%) which were much less in comparison to the prevalence of CMV infection. The absence of any specific clinical symptoms of CMV amongst the patients was an important characteristic of CMV infection and was often indistinguishable from other types of infection. The common unifying feature of CMV disease in the immunocompromised patient was the presence of fever, and approximately 65% of cases developed hearing defects and perceptual organ damage such as optic atrophy and blindness. The most common form of disseminated cryptococcosis was Meningitis, and it developed in 4% of the study patients. All cases were diagnosed by clinical and neurological symptoms with confirmation by laboratory investigations. Very few other OIs were found, viz., herpes zoster virus (HZV) infection, syphilis from T.pallidum, etc., among the patients investigated. Interestingly, not even a single case of malignancy was found to occur among the investigated patients.

Highlights of our study
Our study highlights that candidial infection is the most common OI, followed by TB and diarrhea, all of which are presumed to result from our geographic, climatic and socioeconomic conditions. Early diagnosis of OIs and prompt treatment definitely contributes to increased life expectancy among infected patients, delaying the progression to AIDS. Despite the moderate (n = 125) number of patients included in our study, we believe the data obtained here provide some important background information that can form the basis of future, more elaborative and systematic studies. Furthermore, the data shown can be a valuable means of determining the range and relative frequency of infectious diseases, and this can potentially have an immediate impact on patient care by suggesting appropriate interventions based on the results. The following table (2) represents the cases of dual infection in these HIV infected patients:

Number of cases
Percentage of Cases

Primary clinical symptoms for CMV infection (As confirmed by the clinicians)
For the diagnosis of active CMV infection, the primary clinical symptoms were retinitis (an infection of the eyes), pneumonia, blindness and gastrointestinal disease. The common clinical feature of CMV disease is retinitis. Retinitis is charecterised by painless, gradual loss of vision, floaters etc. Often, retinitis begins in one eye, but then progresses gradually to the other eye. If kept without treatment, progressive damage to the retina can lead to blindness in 4-6 months or less. Even with regular treatment, the disease can worsen to blindness. This may be because CMV may become resistant to the drugs so the drugs can no longer kill the virus, or because the patient's immune system has deteriorated further. Patients with CMV retinitis also have a chance of developing retinal detachment, in which the retina detaches from the nerves of the eye, causing blindness. Other symptoms following active CMV infection include oesophagitis. Cytomegalovirus (CMV) esophagitis is a viral infection of the esophagus, which is the muscular tube through which food travels from the mouth to the stomach. It is charecterised by dysphagiadifficulty in swallowing or Odynophagia -painful swallowing,low grade fever, mouth sores. Colitis is another clinical symptom due to active CMV infection. The clinical presentation ae as follows: pain abdomen, bloody diarrhea, fever, anorexia, malaise,dehydeation, weight loss, chronic watery diahorrea. HIV infected patients suffering from CMV colitis usually benefit from antiviral treatment. CMV Pneumonitis is charecterised by cough, breathlessness. Pneumonitis is a common manifestation of CMV infection. It is highily associated with immunocompromised patients with fever and dyspnea. Encephalitis caused by CMV is characterized by altered mental status, convulsion and headache. Radiculoneuropathy is characterized by weakness/paralysis of lower limbs, pain lower back, urinary retention etc.

Primary clinical symptoms for HSV infection (As confirmed by the clinicians)
Among HIV-1 infected individuals, HSV-1 and HSV-2 infections are common, with prevalences that approximate or exceed those in the general population. Primary symptoms of HSV included persistent vesicular and ulcerative lesions of the oral and anogenital areas, often with extensive or deep ulcerations and blisters on or around the genitals or rectum. The blisters left tender ulcers (sores) that took two to four weeks to heal the first time they occurred. Other symptoms included tender tonsils covered with a whitish substance that made swallowing difficult or blisters present in the mouth.

Primary clinical symptoms for EBV infection EBV+ and
HIVincreases by many fold the risk of non-Hodgkins lymphoma (NHL) over general population, though overall risk remains small. In HIV, EBV most highly associated with Lymphoma, Primary CNS (PCNSL). Primary diagnosis of EBV was based on the clinical symptoms of fever, sore throat and swollen lymph glands. The commonestclinical presentation of EBV disease in HIV positives is Oral hairy Leukoplakia (OHL).

Primary clinical symptoms for HSV infection
Patients with HIV disease are at risk for developing severe illness from either varicella or zoster. Progressive primary varicella, a syndrome with persistent new lesion formation and visceral dissemination, may occur in HIV-infected patients and may be lifethreatening. Zoster eruptions in HIV-infected patients can be extensive and locally destructive, and can become secondarily infected. Zoster may also disseminate cutaneously, and has been reported as the cause of encephalitis in patients with HIV disease (Quinnan et al. 1984;Cone & Schiffman, 1984 ;Sandor et al. 1984 ;Ryder et al.1986;Friedman-Kien et al.1986;Cohen et al.1988;Colebunders et al.1988;Cohen, & Grossman, 1989 ;Gilson et al. 1989;Eidelberg et al.1986) was primarily diagnosed based on the clinical manifestations of severe headaches, backache, general malaise and fever accompanied by the typical exanthem (rash) of chickenpox. Other symptoms of VZV included painful oral lesions, vesicular rash, facial numbness and loss of hearing/ ear pain. All the clinical manifestations were confirmed by ELISA test. The following diagnostic test kits were used for the assay of the opportunistic viruses:

Anti-CMV
Serum anti-CMV was determined by a commercially available test kit, CMV IgM, IgG ELISA Test kit was used to detect antibodies against the CMV.

Anti -EBV 1
Serum anti-EBV was determined by a commercially available test kit, EBV IgG, IgM ELISA Test kit was used to detect antibodies against the EBV 1.

Anti-HSV 2
Serum anti-HSVwas determined by a commercially available test kit, HSV type1 IgM ELISA Test kit supplied was used to detect antibodies against the affinity chromatographically purified recombinant antigen HSV-2.

Anti -VZV
Serum anti-VZV was determined by a commercially available test kit, VZV IgM, IgG, IgA Was used to detect antibodies against the Ellen Strain antigen (ATCC). Of these herpes viruses studied, our study within the studied population suggests that, the occurrence of CMV is the highest. It accounts to 49% of the total population studied across the range of CD4+. From the different tests performed, the results were as follows: The following table (3) represents the cases of viral OIs in these HIV infected patients: Study population= 200 We were also interested to study the trend of co-infection with CMV amongst these HIV patients. The Venn diagrams below to demonstrate the actual distribution of these viral infections amongst the immunocompromised patients under our study:   The incidence of CMV was higher among males than females 59/39. HSV was also found to be more predominant in the males 63/ 31. The incidence of VZV and EBV was also found to be dominant in the male population, the dominancy being 46/19 and 31/21 respectively. HSV infection is found to be dominating in the heterosexual individuals among the study group. There are no significant variations among the homophiles, the drug users and the blood transfusion patients.The incidence of the different viral OIs was also assessed with respect to the CD4+ cell count/μl of blood and patients with mean CD4+ cell count of 51-100 showed the highest prevalence of opportunistic viral antibodies. This was followed by the group with CD4+ count of 101-150, 51-100, 151-200 and >200. Age related prevalence of opportunistic viral antibodies in the serum of 200 HIV infected patient cohorts was assessed and results showed that individuals in the age group of 21-40 years had the highest incidence of viral opportunistic infections as evident from the Table below.

Type of Viral infection
The following table (5) represents the HIV-seropositive patient groups in various risk factors:  (Wiley & Nelson ,1988;Santosh et al.1998). The diagnosis poses difficulties because a 2-3 week period is mandatory for virus isolation. While IgM antibodies as detected by ELISA correlate poorly with the clinical status of CMV infection and facilities for culture are usually not available in most centers (Lanjewar et al. 1996). There are a few reports available of CMV infection in Indian patients with HIV/AIDS, which are based primarily on clinical or autopsy evaluation (Lanjewar et al. 1988;Becker et al. 1996;Nebuloni et al. 1998). We found CMV as the most incidental co infection in HIV/ AIDS patient with the overall incidence of 49%. Human immunodeficiency virus (HIV) infection is associated with an increased risk for human herpesviruses (HHVs) and their related diseases. The incidence of HSV in human immunodeficiency virus (HIV)-seropositive patients has not been focused, with reports generally focusing on individual infection (Mcclain et al. 1995). In this report, the serum prevalence of HSV is found to be higher in HIV seropositive patients; the overall incidence is around 47%. VZV infection, the overall incidence being 32.5% is the third most incidental coinfection in HIV seropositive patients. VZV is one of the common aetiological agents of viral retinitis. Neurological complications of the reactivation of VZV occur most frequently in elderly persons and immunocompromised patients (Sixbey et al. 1989). Gray et al.(1994) reported VZV infection of the CNS in more than 4% of patients with AIDS examined at autopsy. In AIDS patients, VZV tends to reactivate from multiple dorsal root ganglia levels, and the disease is often disseminated. EBV is the least prevalent among the four HHVs studied in our work which is 26% of the total. EBV has been identified as a co-factor in the pathogenesis of a significant proportion of HIV related lymphoproliferative disorders and in oral hairy leukoplakia (Sculley et al. 1988). However, only limited information exists on the status of EBV in the course of HIV infection and the extent of its interaction with HIV. There is also growing interest in the biological properties and pathogenic potential of the different EBV subtypes, EBV-1 and EBV-2. Serological findings and studies in saliva and blood have indicated a high incidence of EBVwww.intechopen.com 2 infection in the course of HIV disease, but there is limited information regarding its significance (Sixbey et al. 1989;Sculley et al. 1988). The analysis of the association of immunological status and the presence of viral OIs revealed that the CD4 count was significantly associated with the presence of viral OIs. An increasing CD4 count significantly protected patients from expressing HHVs in our patient cohorts as indicated by the correlation as in Figure 2. It has been detected clinically that more frequently virus infections are associated with the compromised immunity in HIVinfected patients (Kyaw et al.1992 infections can indeed be correlated with the clinical manifestation and thus is helpful in predicting disease progression. Figure 3 clearly shows that the patient group with CD4+ counts between 51 and 100 cells/μl is most susceptible to viral OI in HIV/AIDS patient. The groups with CD4+ count less than 50 cells/μl are showing least opportunistic viruses which could be due to the advanced HAART treatment. This study is aimed at providing baseline data on viral opportunistic infections in HIV seropositive population as part of the preliminary investigation on the dynamics of viral opportunistic infections in immunocompromised population of India. One of the major problems is the lack of specific investigations that can provide rapid and reliable confirmation of a clinical diagnosis. A high level of alertness is needed at both clinical and laboratory level and routine surveillance studies need to be undertaken. Institutions in India and other developing countries need to be equipped to face the emerging challenge, in the form of updating the present knowledge, by way of education and training of the personnel, acquisition of skills of improved procedures, and their implementation in appropriate settings with adequate administrative support.

Conclusion
This study is aimed at providing baseline data for different pathogenic opportunistic infections in HIV seropositive population as part of the preliminary investigation on the dynamics of viral opportunistic infections in immunocompromised population of Eastern India. As many HIV/AIDS patients in India cannot afford ART, so the detection and awareness towards OIs related to HIV is of immense importance especially in Indian perspective or with respect to other developing countries also. These data may also be of immense importance especially to those countries of the tropical region as the spread of OIs might be comparable to that of our study. A high level of alertness is needed at both laboratory and clinical level and routine surveillance studies need to be undertaken. Institutions in India and other developing countries need to be equipped to face the emerging challenge, in the form of updating the present knowledge, by way of education and training of the personnel, acquisition of skills of improved procedures, and their implementation in appropriate settings with adequate administrative support. Further investigations have to be undertaken with matched control samples as case control analysis with respect to all OIs in HIV seropositive individuals.

Acknowledgment
The authors thank the technical staffs Mr. Probal Kanti Ray and Mr Tapan Chakrabarti for their laboratory help and Mr. Avirup Chakraborty, PhD student for doing desk work. The work was supported by fund received from Indian Council of Medical Research, Government of India.