Oral Screening for Pre-cancerous Lesions Among Areca-nut Chewing Population from Rural India.

PURPOSE: To detect early oral premalignant lesions (OPLs) in a rural population chewing tobacco-free areca nut preparations, determine their awareness level of oral cancer and educate them about maintaining good oral health. MATERIALS AND METHODS: A total of 2175 18- to 65-year-old areca nut chewers (male:female ratio 2.5:1), without a history of consuming tobacco in any form, from the villages of two districts of the West Bengal state of India were screened clinically through oral examination for suspected OPLs. A pre-designed questionnaire was employed to record demographic data, information on tobacco-free areca-nut chewing habit and knowledge about oral diseases. Education on oral health was provided through distribution of printed leaflets, display of banner/posters and a public-announcement system. RESULTS: Chewing areca nut in the form of betel quid was more popular (90.7%) than chewing areca nut alone (9%) or tobacco-free packaged areca nut preparation sold as 'pan masala' (0.3%). OPLs were detected in 7.3% of the subjects, more among the males. An increasing incidence of OPLs could be observed with an increase in age as well as with duration and frequency of areca-nut chewing, while decreasing incidence was observed with an increase in educational level. Oral submucous fibrosis showed the highest prevalence (2.7%) among the various OPLs detected. CONCLUSIONS: Tobacco-free areca-nut chewing is an independent risk factor for the development of OPL and a large rural population still practices such high risk behaviour. In rural areas with limited health care resources, screening by visual oral examination involving minimum cost may prove useful to reduce oral cancer mortality.

Reduced expression of LIMD1 in ulcerative oral epithelium associated with tobacco and areca nut.

PURPOSE: The aim of this study was to cast light on initiating molecular events associated with the development of premalignant oral lesions induced by tobacco and/or areca nut. METHOD: Immunohistochemical analyses of cell cycle regulatory proteins (LIMD1, RBSP3, p16, RB, phosphorylated RB, p53), EGFR and SH3GL2 (EGFR associated protein) were performed with inflammatory/ ulcerative epithelium and adjacent hyperplastic/mild dysplastic lesions. RESULTS: No change in expression of the proteins was seen in inflammatory epithelium. Reduced nuclear expression of LIMD1 was evident in ulcerative epithelium. In hyperplastic lesions, reduced expression of RBSP3, p16, SH3GL2 and overexpression of p-RB and EGFR were apparent. Reduced nuclear expression of p53 was observed in mild dysplastic lesions. CONCLUSION: Our data suggest that inactivation of LIMD1 in ulcerative epithelium might predispose the tissues to alterations of other cell cycle regulatory and EGFR signaling proteins needed for the development of premalignant oral lesions.

Genetic characterization of HIV-1 from semen and blood from clade C-infected subjects from India and effect of therapy in these body compartments.

Biologic and genetic differences between HIV-1 clade C in India and clade B in US suggest that the effect of anti-viral therapy in various body compartments may differ between these two clades. We examined the effect of therapy on viral loads in semen and blood of HIV-1-clade C infected subjects from India and evaluated whether HIV-1 in the semen is different from that in blood in these subjects. HIV-1 RNA was detected in semen and blood at all stages of the disease. Viral loads in semen and blood were strongly correlated with each other, but not with the CD4+ T cell count. Anti-viral treatment reduced viral load drastically in blood and semen within one month of post therapy. Genetic characterization of HIV-1 in the semen and blood demonstrated that they were highly compartmentalized. These data have important implications of sexual transmission of HIV-1 in clade C HIV-1 infected subjects.

Estimation of the predictive role of plasma viral load on CD4 decline in HIV-1 subtype C-infected subjects in India.

BACKGROUND: Plasma viral load has been shown to be a meaningful prognostic marker for disease progression in untreated, HIV-1 subtype B-infected subjects in United States and Western Europe and therefore used as a prognostic marker for disease progression. Because of high expenses of commercially available viral load assays, the role of viral load in disease progression has not been evaluated in HIV-1 subtype C-infected patients in India. METHODS: We developed an inexpensive real-time reverse transcriptase-polymerase chain reaction assay to quantify viral load in plasma of HIV-1 subtype C-infected subjects from India and used it in a longitudinal analysis of viral load and CD4 cell number in HIV-infected subjects from Calcutta, India. RESULTS: The real-time reverse transcriptase-polymerase chain reaction assay can quantify plasma viral load with a linear range of detection from 10 to 10 HIV-1 RNA copies per input. Longitudinal analysis of viral load in a cohort of 39 subjects over an average period of approximately 3 years indicates that 1-log increase in HIV-1 RNA level was associated with a decline of 67 CD4 cell count. Furthermore, HIV-1 RNA level between 500 and 50,000 copies per milliliter would predict a 12.9% decrease in CD4 cell count per year, whereas HIV-1 RNA levels above 50,000 copies HIV-1 RNA per milliliter would predict a 25.3% decrease in CD4 cells per year. In addition, we estimated that the mean incubation period of disease development, as defined by the loss of CD4 below 200, is 8.2 years. CONCLUSION: Our report on the level of viral load on predicting CD4 decline in Indian subjects with HIV-1 provides an additional important tool to the physicians for treating and planning a therapeutic strategy to control HIV-1 infection in India.

High replication fitness and transmission efficiency of HIV-1 subtype C from India: Implications for subtype C predominance.

HIV-1 subtype C has been the predominant subtype throughout the course of the HIV-1 epidemic in India regardless of the geographic region of the country. In an effort to understand the mechanism of subtype C predominance in this country, we have investigated the in vitro replication fitness and transmission efficiency of HIV-1 subtypes A and C from India. Using a dual infection growth competition assay, we found that primary HIV-1 subtype C isolates had higher overall relative fitness in PBMC than subtype A primary isolates. Moreover, in an ex vivo cervical tissue derived organ culture, subtype C isolates displayed higher transmission efficiency across cervical mucosa than subtype A isolates. We found that higher fitness of subtype C was not due to a trans effect exerted by subtype C infected PBMC. A half genome A/C recombinant clone in which the 3' half of the viral genome of subtype A was replaced with the corresponding subtype C3' half, had similar replicative fitness as the parental subtype A. These results suggest that the higher replication fitness and transmission efficiency of subtype C virus compared to subtype A virus from India is most probably not due to the envelope gene alone and may be due to genes present within the 5' half of the viral genome or to a more complex interaction between the genes located within the two halves of the viral genome. These data provide a model to explain the asymmetric distribution of subtype C over other subtypes in India.

Cytokine levels in HIV infected and uninfected Indian women: correlation with other STAs.

Host immune status is an important determinant of disease progression. Infections in the genital tract may alter the immunity in the particular site and hence affect the production of local cytokines. We performed this study to determine whether HIV in association with cervical HPV and CT/GC infections influences the production of local cytokines. Cervical secretions from 100 women with or without HIV infection were collected for measuring IL-1 beta, -6, -10 and -12 concentrations by ELISA. Cervical HPV and CT/GC DNA were detected by HCII test. Significant elevations of IL-6 and IL-10 were observed in patients having HIV infection. Although cervical HPV infection increased the concentrations of both IL-6 and IL-1 beta but HPV induced abnormal cervical smear was associated only with increased IL-6 concentrations significantly. Double infection had marked relation with IL-6 and IL-10. CT/GC had no direct effect on any of these cytokines but in association with HIV and HPV, these bacterial pathogens elevated the concentrations of IL-6 significantly. Thus, our results suggest that the presence of HIV and other STAs in the genital tract can cause imbalance of local cytokine levels which in turn may facilitate other opportunistic infections.

Genetic and functional characterization of the LTR of HIV-1 subtypes A and C circulating in India.

Genetic analysis of HIV-1 sequences circulating in different parts of India have shown that the predominant proportion of HIV-1 subtypes circulating in India is type C and a small fraction are subtypes A, B, E, and CRFs. We sequenced the HIV-1 LTR promoter region of seven subtype C and five subtype A isolates obtained from two major cities in India. Sequence analysis of the complete promoter and TAR regions revealed conserved subtype-specific variability in several major binding sites. Three NF-kappaB sites were present in all subtype C isolates and two isolates contained an insertion in the MFNLP. The transcriptional activity of one of these isolates may have been hindered due to this insertion. Despite the apparent variability between the LTRs we did not observe any significant difference in the transcriptional activity between subtype C and subtype A. To our knowledge, this is the first study characterizing the genetic structure and functional attributes of subtype A LTRs from India.

Construction and characterization of an infectious molecular clone of HIV-1 subtype A of Indian origin.

India has the second highest number of HIV-1 infected people next to South Africa. The predominant proportion of HIV-1 circulating in India is of subtype C origin, with a small fraction made up of subtypes A and B. In this report, we describe the construction and characterization of the first full-length infectious molecular clone p1579A-1 HIV-1, from an HIV-1 subtype A infected person from India, using long PCR and successive ligation of the amplimers. Phylogenetic analysis of the sequence of the entire proviral DNA and LTR confirmed p1579A-1 to be an HIV-1 subtype A. Analysis of the env gene of p1579A-1 showed a conserved GPGQ motif and the absence of basic amino acids at positions 11 and 25 suggesting CCR5 coreceptor usage. Analysis of env N-linked glycosylation sites revealed fewer sites in the V1 region of envelope compared to other subtype A. Transcription factor binding site analysis of the LTR sequences identified conserved as well as unique transcription factor binding sites (TFBS) in p1579A-1. This infectious clone of HIV-1 can be useful to study the molecular mechanism of dominance of subtype C in India.

Identification of subtype C human immunodeficiency virus type 1 by subtype-specific PCR and its use in the characterization of viruses circulating in the southern parts of India.

Human immunodeficiency virus type 1 (HIV-1) subtype C viruses are associated with nearly half of worldwide HIV-1 infections and are most predominant in India and the southern and eastern parts of Africa. Earlier reports from India identified the preponderance of subtype C and a small proportion of subtype A viruses. Subsequent reports identifying multiple subtypes suggest new introductions and/or their detection due to extended screening. The southern parts of India constitute emerging areas of the epidemic, but it is not known whether HIV-1 infection in these areas is associated with subtype C viruses or is due to the potential new introduction of non-subtype C viruses. Here, we describe the development of a specific and sensitive PCR-based strategy to identify subtype C-viruses (C-PCR). The strategy is based on amplifying a region encompassing a long terminal repeat and gag in the first round, followed by two sets of nested primers; one amplifies multiple subtypes, while the other is specific to subtype C. The common HIV and subtype C-specific fragments are distinguishable by length differences in agarose gels and by the difference in the numbers of NF-kappaB sites encoded in the subtype C-specific fragment. We implemented this method to screen 256 HIV-1-infected individuals from 35 towns and cities in four states in the south and a city in the east. With the exception of single samples of subtypes A and B and a B/C recombinant, we found all to be infected with subtype C viruses, and the subtype assignments were confirmed in a subset by using heteroduplex mobility assays and phylogenetic analysis of sequences. We propose the use of C-PCR to facilitate rapid molecular epidemiologic characterization to aid vaccine and therapeutic strategies.

Human papillomavirus infection among Indian mothers and their infants.

OBJECTIVE: Several studies have demonstrated that infants can acquire human papillomavirus (HPV) infection at birth from their mothers. The aim of the present investigation was to determine prevalence of HPV infection among pregnant women and evaluate the extent of perinatal transmission of HPVs to infants. METHODS: The study included 135 pregnant women and their infants. The polymerase chain reaction (PCR) was performed to detect HPV DNA in cervical cells of the women and buccal cells of the infants. RESULTS: HPVs detected were genotyped by PCR using type specific primers. HPV DNA was identified in 38 mothers (28.14%, 38/135) and 14 babies (10.37%, 14/135). The prevalence rate of HPV type 16 was highest both in HPV positive maternal (63.15%, 24/38) and baby samples (85.71%, 12/14). At birth, the frequency of HPV transmission from infected mothers to their infants was 18.42% (7/38). The proportion of infants with HPV infection delivered by cesarean section was 78.57% (11/14). CONCLUSION: Cesarean section was not found protective for infants against perinatal HPV transmission. Infection in the infants was cleared within one year. This is the first report of its kind from India.

High incidence and prevalence of HIV-1 infection in high risk population in Calcutta, India.

HIV-1 infection in India has been increasing steadily over the last decade. In the absence of potent antiviral therapy, estimates of HIV infection are needed to monitor the epidemic, institute prevention strategies in target populations and determine the suitable populations for vaccine studies. In this report we present the HIV-1 seroprevalence and annual estimates of seroincidence in a high risk population from Calcutta, the most populous city in the eastern part of India. In 1206 high risk subjects tested over two years between February of 1999 and December 2000, we have determined an overall seroprevalence of 40.1% using enzyme-linked immunosorbent assay followed by a confirmatory Western blot testing. Furthermore, using a newly described Standardized Testing Algorithm for Recent HIV-1 Seroconversion (STARHS), we have estimated an annual seroincidence rate of about 7% in this population during this two-year study. Such a high annual seroincidence rate makes this population well suited for studies of HIV-1 prevention, including vaccine trials.

Detection of HPV DNA in cervical carcinomas by PCR and hybrid capture assay.

HPV DNA was detected in exfoliated cervical cells of 73% (85/116) cervical cancer patients by PCR using HPV consensus primers and by hybrid capture assay (HC II) (Digene Corp., USA) in 77 of the 85 cases found HPV positive by PCR. Presence of HPV 16/18 DNA were investigated in the 79 cases by PCR using type specific primers. HPV 16 was detected in 31 (39%) patients, HPV 18 in 7 (8.8%), both HPV 16 and 18 in 19 (24%) and HPVs other than 16/18 in 22 (27.8%) cases. Age and clinical stages had no significant effect on HPV prevalence. Double infection of HPV 16 and 18 was significantly (p<0.05) high in the older patients (56 years or more) compared to younger group. Results indicated that cervical cancers in India are strongly associated with high-risk type HPV infection. HC II assays and PCR results for detection of HPV in cervical smears were comparable.