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   2012| June  | Volume 135 | Issue 6  
    Online since July 23, 2012

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Analysis of humoral responses to proteins encoded by region of difference 1 of Mycobacterium tuberculosis in sarcoidosis in a high tuberculosis prevalence country
Ritesh Agarwal, Dheeraj Gupta, Rajagopala Srinivas, Indu Verma, Ashutosh N Aggarwal, Suman Laal
June 2012, 135(6):920-923
  3 838 219
Personalized nutrition: Translating nutrigenetic/nutrigenomic research into dietary guidelines
Kamla Krishnaswamy
June 2012, 135(6):924-925
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Gender, women and primary health care renewal: A discussion paper
Ritu Priya
June 2012, 135(6):925-927
  - 607 177
Novel lymphoblastoid cell lines from primary neoplasms of the upper aero-digestive tract
Sanjay Katiyar, Sneha Balani, Virendra Singh
June 2012, 135(6):803-805
  - 602 1,177
Monitoring of substance abuse in India - Initiatives & experiences
Rajat Ray, Anita Chopra
June 2012, 135(6):806-808
  - 1,077 269
Role of the RT-PCR method in ante-mortem & post-mortem rabies diagnosis
Dan David
June 2012, 135(6):809-811
  - 801 258
A multiplex PCR assay for the simultaneous identification of virulent & avirulent Bacillus anthracis targeting genes of plasmids & chromosomal DNA
Sanjay Kumar, Urmil Tuteja, Kumari Sarika, Om Kumar
June 2012, 135(6):917-919
  - 1,001 223
The right medicine for World Drug Day
John H Halpern
June 2012, 135(6):801-802
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Establishment & characterization of lymphoblastoid cell lines from patients with multiple primary neoplasms in the upper aero-digestive tract & healthy individuals
Tabish Hussain, Ashwin Kotnis, Rajiv Sarin, Rita Mulherkar
June 2012, 135(6):820-829
Background & objectives: A major drawback for genetic studies as well as long-term genotype-phenotype correlation studies in cancer is lack of representative human cell lines providing a continuous source of basic biomolecules and a system to carry out various experimental investigations. This can be overcome to some extent by establishing lymphoblastoid cell lines (LCLs) by infecting peripheral blood lymphocytes with Epstein Barr virus (EBV) which is known to immortalize human resting B cells in vitro giving rise to actively proliferating B-lymphoblastoid cell lines. The present study involves preparation and characterization of LCLs generated from patients with multiple primary neoplasms (MPN) of upper aero-digestive tract (UADT). Methods: Thirty seven LCLs were established from UADT MPN patients and healthy age, sex and habit matched controls using EBV crude stock. Characterization was done with respect to expression of CD-19 (Pan B-cell marker), CD3 (T cell specific marker), CD56 (NK-cell specific marker), cell morphology, ploidy analysis, genotype and gene expression comparison with the parent lymphocytes. Results: LCLs showed rosette morphology with doubling time of approximately 24 h. Ploidy analysis showed diploid DNA content which was maintained for at least 30 population doublings. When compared with parent lymphocytes there appeared no change at genetic and gene expression level. Interpretation & conclusions: Our results show that lymphoblastoid cell lines are a good surrogate of isolated lymphocytes bearing their close resemblance at genetic and phenotypic level to parent lymphocytes and are a valuable resource for understanding genotype-phenotype interactions.
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Changing pattern of substance abuse in patients attending a de-addiction centre in north India (1978-2008)
Debasish Basu, Munish Aggarwal, Partha Pratim Das, Surendra K Mattoo, Parmanand Kulhara, Vijoy K Varma
June 2012, 135(6):830-836
Background & objectives: The patterns of abused psychoactive substances change over time, and it is important to document such changes. The present retrospective study was carried out to document these changes in patients registered in a de-addiction centre in north India over three decades. Methods: Case notes of all patients registered in the centre from September 1978 till December 31, 2008 were reviewed. Comparisons were made among three decades (1978-1988, 1989-1998, and 1999-2008). Results: The number of registered subjects increased eight-fold over the decades, and age of the subjects presenting for the treatment decreased. The percentages of subjects presenting for the treatment with opioid dependence were 36.8 per cent (n=204), 42.9 per cent (n=809) and 53.2 per cent (n=2219), respectively for the three decades (P<0.001). The proportion of subjects using natural opioids decreased over the three decades (47.4, 26.5 and 18.3%; P<0.001), with a concomitant emergence and/or increase of newer and prescription opioids such as buprenorphine, codeine and dextropropoxyphene. Dependence on tobacco and sedative-hypnotics also increased, and inhalant abuse was reported especially in the third decade. Polysubstance dependence increased significantly over the decades (P<0.001). Interpretation & conclusions: Our results showed major shifts in the patterns of substance abuse in clinic-attending patients in north India over the three decades from 1978 till 2008. These have important implications for all the stakeholders concerned with combating the challenge of psychoactive substance abuse in our society.
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Role of reverse transcriptase polymerase chain reaction for the diagnosis of human rabies
Manisha Biswal, Radha Kanta Ratho, Baijayantimala Mishra
June 2012, 135(6):837-842
Background & objectives: Traditionally, rabies diagnosis is made by demonstration of rabies viral antigen by direct immunofluorescence (DIF) and mouse inoculation test (MIT). The present study was carried out to evaluate the role of reverse transcriptase-polymerase chain reaction (RT-PCR) in comparison with these conventional techniques for the diagnosis of rabies. Methods: Skin biopsies, corneal impression smears and saliva sample were collected ante-mortem and brain tissue and CSF were collected post-mortem from ten clinically suspected rabies patients. DIF, Seller staining, MIT and RT-PCR were performed on the patients' samples for the diagnosis of rabies. The ability of RT-PCR to detect rabies virus earlier as compared to other assays was tested both for reference virus as well as clinical isolates. Results: All samples taken ante-mortem were negative for DIF test. Six of 10 post-mortem brain tissues of the clinically suspected patients were positive both by RT-PCR and MIT, of these six, five were positive by DIF test and four were positive by Seller stain. RT-PCR could detect the rabies virus earlier as compared to DIF, both from clinical isolates and fixed rabies virus. Interpretation & conclusions: The present results showed 100 per cent sensitivity and specificity of RT-PCR as compared to 83.3 per cent of DIF and 66.7 per cent of Sellers stain for diagnosis of rabies. RT-PCR also detected rabies viral infection earlier as compared to conventional tests and can also be used on ante-mortem samples. Thus, the present study shows the usefulness of RT-PCR as an alternative to MIT for the confirmation of rabies diagnosis.
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Expression of androgen receptor in breast cancer & its correlation with other steroid receptors & growth factors
Ashwani K Mishra, Usha Agrawal, Shivani Negi, Anju Bansal, R Mohil, Chintamani Chintamani, Amar Bhatnagar, Dinesh Bhatnagar, Sunita Saxena
June 2012, 135(6):843-852
Background & objectives: Breast cancer is the second most common malignancy in Indian women. Among the members of the steroid receptor superfamily the role of estrogen and progesterone receptors (ER and PR) is well established in breast cancer in predicting the prognosis and management of therapy, however, little is known about the clinical significance of androgen receptor (AR) in breast carcinogenesis. The present study was aimed to evaluate the expression of AR in breast cancer and to elucidate its clinical significance by correlating it with clinicopathological parameters, other steroid receptors (ER and PR) and growth factors receptors (EGFR and CD105). Methods: Expression of AR, ER, PR, epidermal growth factor receptor (EGFR) and endoglin (CD105) was studied in 100 cases of breast cancer by immunohistochemistry (IHC). Risk ratio (RR) along with 95% confidence interval (CI) was estimated to assess the strength of association between the markers and clinicopathological characteristics. Categorical principal component analysis (CATPCA) was applied to obtain new sets of linearly combined expression, for their further evaluation with clinicopathological characteristics (n=100). Results: In 31 cases presenting with locally advanced breast cancer (LABC), the expression of AR, ER, PR, EGFR and CD105 was associated with response to neoadjuvant chemotherapy (NACT). The results indicated the association of AR+ (P=0.001) and AR+/EGFR- (P=0.001) with the therapeutic response to NACT in LABC patients. The AR expression exhibited maximum sensitivity, specificity and likelihood ratio of positive and negative test. The present results showed the benefit of adding AR, EGFR and CD105 to the existing panel of markers to be able to predict response to therapy. Interpretation & conclusions: More studies on the expression profiles of AR+, AR+/CD105+ and AR+/EGFR- in larger set of breast cancer patients may possibly help in confirming their predictive role for therapeutic response in LABC patients.
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A novel autologous stem cell procedure for the treatment of aplastic anaemia using reprogrammed mature adult cells: A pilot study
Ilham Saleh Abuljadayel, Dipika Mohanty, Rajendar K Suri
June 2012, 135(6):853-872
Background & objectives: Aplastic anaemia is a life threatening rare bone marrow failure disorder. The underlying haematopoietic cellular deficit leads to haemorrhage, infection and severe anaemia. The treatment of choice for this haematological condition is allogeneic bone marrow transplantation from fully matched HLA sibling. Though this procedure is curative in the majority of young patients with aplastic anaemia, extending this benefit to older patients or those lacking a family donor remains a major challenge. Herein, the safety and efficacy of infusing autologous retrodifferentiated haematopoietic stem cells (RHSC) into four patients with aplastic anaemia without the use of any pre- or post-conditioning regimen including immunosuppression is described. Methods: Un-mobilized, mononuclear cells were harvested from four patients with acquired aplastic anaemia by aphaeresis. Mononuclear cells of patients were cultured with purified monoclonal antibody against the monomorphic regions of the beta chain of MHC class II antigens (Clone CR3/43) for 3 h, to obtain autologous RHSC. Autologous RHSC were washed and infused into the four patients without the use of any pre- or post-conditioning regimen. Thereafter, the efficacy (engraftment) of autologous RHSC was assessed in these patients. Results: Following single infusion of the autologous RHSC, two of the four patients with aplastic anaemia become transfusion independent for more than seven years. Karyotyping and G-banding analysis prior and post-procedure in all patients remained the same. Interpretation & conclusions: The findings of this pilot study demonstrated the functional utility of reprogrammed fully differentiated adult cells into pluripotent stem cells with extensive repopulation potentials in a human setting and without any pre- or post-conditioning regimen, including immunosuppression. This autologous approach of stem cell creation may broaden the curative potentials of stem cell therapy to a wider population of patients with aplastic anaemia, including many patients suffering from other haematological and non-haematological disorders.
  - 2,308 365
Prevalence of dental fluorosis & dental caries in association with high levels of drinking water fluoride content in a district of Gujarat, India
PV Kotecha, SV Patel, KD Bhalani, D Shah, VS Shah, KG Mehta
June 2012, 135(6):873-877
Background & objectives: Endemic fluorosis resulting from high fluoride concentration in groundwater is a major public health problem in India. This study was carried out to measure and compare the prevalence of dental fluorosis and dental caries in the population residing in high and normal level of fluoride in their drinking water in Vadodara district, Gujarat, India. Methods: A cross-sectional study was conducted in Vadodara district, six of the 261 villages with high fluoride level and five of 1490 with normal fluoride level in drinking water were selected. The data collection was made by house-to-house visits twice during the study period. Results: The dental fluorosis prevalence in high fluoride area was 59.31 per cent while in normal fluoride area it was 39.21 per cent. The prevalence of dental caries in high fluoride area was 39.53 per cent and in normal fluoride area was 48.21 per cent with CI 6.16 to 11.18. Dental fluorosis prevalence was more among males as compared to females. Highest prevalence of dental fluorosis was seen in 12-24 yr age group. Interpretation & conclusions: The risk of dental fluorosis was higher in the areas showing more fluoride content in drinking water and to a lesser degree of dental caries in the same area. High fluoride content is a risk factor for dental fluorosis and problem of dental fluorosis increased with passage of time suggesting that the fluoride content in the water has perhaps increased over time. Longitudinal studies should be conducted to confirm the findings.
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Analysis of calpain-3 protein in muscle biopsies of different muscular dystrophies from India
R Renjini, N Gayathri, A Nalini, MM Srinivas Bharath
June 2012, 135(6):878-886
Background & objectives: Calpain-3, a Ca [2]+ -dependent protease has been implicated in the pathology of neuromuscular disorders (NMDs). The current study aimed to analyze calpain-3 expression in cases diagnosed as muscular dystrophy from the Indian population. Methods: Calpain-3 Western blot analysis in muscle biopsies of immunohistochemically confirmed cases of Duchenne muscular dystrophy (DMD) (n=10), dysferlinopathy (n=30) and sarcoglycanopathy (n=8) was carried out. Calpain-3 Western blotting was also used in a blinded study to identify cases of calpain-3 deficiency in 28 NMD patients with potential muscular dystrophy. Results: Calpain-3 appeared as a full length 94 kDa band with an autolytic product (~60 kDa) on Western blots with antibody NCL-CALP-12A2 (Ab-2). Eight of the 10 DMD samples showed absence of 94 kDa band but presence of 60 kDa band while one case of sarcoglycanopathy showed absence of both. Twenty one of the 30 dysferlinopathy samples showed both bands while six showed only the 60 kDa band and three showed absence of both. In the blinded study, five NMD cases with potential muscular dystrophy that showed complete absence of both bands in retrospect exhibited clinical features of limb girdle muscular dystrophy 2A (LGMD2A). Interpretation & conclusions: While the study revealed a consistent pattern of calpain-3 in DMD, one sarcoglycanopathy and three dysferlinopathy samples exhibited secondary reduction in calpain-3. It was recognized that both calpain-3 bands should be considered to confirm calpain deficiency. Further, western blot offers an economical and fast preliminary screening method for LGMD2A especially in cases of complete absence of calpain-3 prior to conclusive diagnosis by genetic testing.
  - 1,614 317
A human corneal endothelium equivalent constructed with acellular porcine corneal matrix
Chengqun Ju, Li Gao, Xinyi Wu, Kunpeng Pang
June 2012, 135(6):887-894
Background & objectives: Artificial corneal endothelium equivalents can not only be used as in vitro model for biomedical research including toxicological screening of drugs and investigation of pathological corneal endothelium conditions, but also as potential sources of grafts for corneal keratoplasty. This study was aimed to demonstrate the feasibility of constructing human corneal endothelium equivalents using human corneal endothelial cells and acellular porcine corneal matrix. Methods: Porcine corneas were decellularized with sodium dodecyl sulphate (SDS) solution. Human corneal endothelial cells B4G12 were cultured with leaching liquid extracted from the acellular porcine corneal matrix, and then cell proliferative ability was evaluated by MTT assay. B4G12 cells were transplanted to a rat corneal endothelial deficiency model to analyze their in vivo bio-safety and pump function, and then seeded and cultured on acellular porcine corneal matrix for two wk. Corneal endothelium equivalents were analyzed using HE staining, trypan blue and alizarin red S co-staining, immunofluorescence and corneal swelling assay. Results: The leaching liquid from acellular porcine corneal matrix had little influence on the proliferation ability of B4G12 cells. Animal transplantation of B4G12 cells showed that these cells had similar function to the native cells without causing a detectable immunological reaction and neoplasm in vivo. These formed a monolayer covering the surface of the acellular porcine corneal matrix. Trypan blue and alizarin red S co-staining showed that B4G12 cells were alive after two wk in organ culture and cell boundaries were clearly delineated. Proper localizations of ZO-1 and Na+/K+ ATPase were detected by immunofluorescence assay. Functional experiments were conducted to show that the Na+/K+ ATPase inhibitor ouabain could block the ionic-pumping function of this protein, leading to persistent swelling of 51.7 per cent as compared to the control. Interpretation & conclusions: Our findings showed that B4G12 cells served as a good model for native corneal endothelial cells in vivo. Corneal endothelium equivalents had properties similar to those of native corneal endothelium and could serve as a good model for in vitro study of human corneal endothelium.
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Absolute lymphocyte count as a surrogate marker for CD4 counts after six months of HAART initiation in a resource-limited setting in India
S Srirangaraj, D Venkatesha
June 2012, 135(6):895-900
Background & objectives: Owing to the ever-expanding access to HAART (highly active anti-retroviral therapy) in resource-limited settings, there is a need to evaluate alternate markers like absolute lymphocyte count (ALC) as a surrogate for CD4 counts. This study was done to assess the usefulness of ALC as a surrogate marker for CD4 counts in monitoring HIV-infected patients after HAART initiation. Methods: In this study, 108 HIV-positive adult patients of both sexes fulfilling the inclusion criteria were included. CD4 and ALC were recorded at baseline. After initiation on HAART, these patients were followed up at three month intervals. Results: ALC and CD4 counts were positively correlated (Spearman correlation coefficient= 0.553). After six months of HAART, the sensitivity of an ALC increase as a marker for CD4 count increase at six months was 82 per cent, specificity was 100 per cent, PPV was 100 per cent and NPV was 31 per cent. Area under the corresponding ROC curve for CD4 increase of >100 cells/μl was 0. 825 ± 0.053. Interpretation & conclusions: ALC may be a useful surrogate marker in predicting an increase in CD4 counts as a response to HAART, but of questionable value in predicting a decrease in CD4 counts.
  - 1,609 200
Evaluation of an immunochromatographic test for discrimination between Mycobacterium tuberculosis complex & non tuberculous mycobacteria in clinical isolates from extra-pulmonary tuberculosis
Anand Kumar Maurya, Vijaya Lakshmi Nag, Surya Kant, Ram Aawadh Singh Kushwaha, Manoj Kumar, Vikas Mishra, W Rahman, Tapan N Dhole
June 2012, 135(6):901-906
Background & objectives: Accurate diagnosis of tuberculosis (TB) is crucial to facilitate early treatment of the patients, and to reduce its spread. Clinical presentation of Mycobacterium tuberculosis complex (MTBC) and non tuberculous mycobacteria (NTM) may or may not be the same, but the treatment regimen is always different for both the infections. Differentiation between MTBC and NTM by routine laboratory methods is time consuming and cumbersome. This study was aimed to evaluate an immunochromatographic test (ICT), based on mouse monoclonal anti-MPT64, for simple and rapid discrimination between MTBC and NTM in clinical isolates from extra-pulmonary tuberculosis cases. Methods: A total of 800 clinical samples were collected from patients suspected to have extra-pulmonary tuberculosis. Preliminary diagnosis has been done by direct Ziehl-Neelsen (ZN) staining followed by culture in BACTEC system. A total of 150 clinical isolates, which were found positive in BD 460 TB system during September 2009 to September 2010 were selected for the screening by ICT test. p-nitro-α-acetylamino- β-hydroxy propiophenone (NAP) test was performed for differentiation of MTBC and NTM. M. tuberculosis complex was further confirmed by IS6110 PCR of BACTEC culture positive isolates, this served as the reference method for MTBC identification and comparative evaluation of the ICT kit. Results: Of the 150 BACTEC culture positive isolates tested by ICT kit, 101 (67.3%) were found positive for MTBC and remaining 49 (32.7%) were considered as NTM. These results were further confirmed by IS6110 PCR that served as the reference method for detection of MTBC. H 37 Rv reference strain was taken as a control for ICT test and IS6110 PCR. The reference strain showed the presence of MPT64 antigen band in the ICT test. Similar bands were formed in 101 of 102 MTBC isolates tested, proving 99.1 per cent sensitivity and no bands were detected in 48 (100%) NTM isolates tested, proving 100 per cent specificity of the ICT kit. Interpretation & conclusions: Our findings show that ICT test can be used on direct culture positive specimens. It does not require any special equipment, is simple and less time consuming. It can easily discriminate between MTBC and NTM and thus can help in appropriate management of tuberculosis.
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A ten year analysis of multi-drug resistant blood stream infections caused by Escherichia coli & Klebsiella pneumoniae in a tertiary care hospital
Sanghamitra Datta, Chand Wattal, Neeraj Goel, Jaswinder K Oberoi, Reena Raveendran, KJ Prasad
June 2012, 135(6):907-912
Background & objectives: Extensive use of antibiotics has added to the escalation of antibiotic resistance. This study was undertaken to evaluate the association, if any between antibiotic use and resistance in a hospital setting, and also detect the predominant mechanism of antibiotic resistance in Escherichia coli and Klebsiella pneumoniae over a period of 10 years. Methods: In a retrospective study of 10 years, a total of 77,618 blood culture samples from 2000 to 2009 from indoor patients were screened and those yielding E. coli and K. pneumoniae were included in the study. Antibiotic susceptibility records as well as the percentage of ESBL producers were noted. A total of 423 isolates of 2009 were also screened for AmpC and carbapenemase production. Antibiotic consumption data of 10 years were analysed. Results: ESBL producing E. coli increased from 40 per cent in 2002 to 61 per cent in 2009, similarly there was a significant (P<0.05) rise in resistance to cefotaxime (75 to 97%), piperacillin-tazobactum (55- 84%) and carbapenem (2.4-52%) in K. pneumoniae. A significant (P<0.05) association was observed between resistance and consumption of carbapenem and piperacillin and tazobactum consumption in K. pneumonia. Interpretation & conclusions: Our study demonstrated a rise in consumption and resistance to broad spectrum antimicrobial agents and also established an association between consumption and resistance to these antibiotics. Over a period of 10 years, the emergence of pan-resistance in K. pneumoniae could be due to the production of carbapenemases whereas ESBL production was the common mechanism of resistance in E. coli. This study warrants a directed effort towards continued surveillance and antibiotic stewardship to minimize selection pressure and spread.
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Antibacterial & antitoxic effects of the cardiovascular drug lacidipine in an animal model
Asish Dasgupta, Sujata G Dastidar
June 2012, 135(6):913-916
Background & objectives: Vibrio cholerae produces acute infection by liberating potent enterotoxin, called cholera toxin in human intestine. Cardiovascular drug lacidipine possessing powerful in vitro action against V. cholerae was tested to determine its possible activity against a toxigenic V. cholerae strain in an established animal model. Methods: In the rabbit intestine four loops were constructed, 3 of which were injected with over night grown V. cholerae 569B culture. Of these, two loops were simultaneously given graded doses (100, 200 μg) of lacidipine, one was left as such for a positive control. The first loop received sterile medium (negative control). After 18 h, contents of all the loops were examined for accumulation of fluid and number of viable cells. Results: Lacidipine when administrated with live V. cholerae 569B, caused a reduction in the number of viable bacteria along with amount of fluid in the loops. The amount of fluid and number of viable cells were much reduced in the loop that had 200 μg of lacidipine than the loop that received 100 μg of the drug. Interpretation & conclusions: Lacidipine has distinct inhibitory action against V. cholerae 569B with respect to both viability and production of cholera toxin in the rabbit ileum. Structural modifications of this compound may possibly lead to procurement of new potent antimicrobial drugs.
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Stem cells in the light of evolution
Chiranjib Chakraborty, Govindasamy Agoramoorthy
June 2012, 135(6):813-819
All organisms depend on stem cells for their survival. As a result, stem cells may be a prerequisite for the evolution of specific characteristics in organisms that include regeneration, multicellularity and coloniality. Stem cells have attracted the attention of biologists and medical scientists for a long time. These provide materials for regenerative medicine. We review in this paper, the link between modern stem cell research and early studies in ancient organisms. It also outlines details on stem cells in the light of evolution with an emphasis on their regeneration potential, coloniality and multicellularity. The information provided might be of use to molecular biologists, medical scientists and developmental biologists who are engaged in integrated research involving the stem cells.
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