Visual inspection of the cervix after application of 3-5% acetic acid (VIA) is a potential alternative to cytology for screening in low-resource countries. The present study evaluated the performance of VIA, magnified visual inspection after application of acetic acid (VIAM), and cytology in the detection of high-grade cervical cancer precursor lesions in Kolkata (Calcutta) and suburbs in eastern India. Trained health workers with college education concurrently screened 5881 women aged 30-64 years with VIA, VIAM, and conventional cervical cytology. Detection of well-defined, opaque acetowhite lesions close to the squamocolumnar junction; well-defined, circumorificial acetowhite lesions; or dense acetowhitening of ulceroproliferative growth on the cervix constituted a positive VIA or VIAM. Cytology was considered positive if reported as mild dysplasia or worse lesions. All screened women (N = 5881) were evaluated by colposcopy, and biopsies were directed in those with colposcopic abnormalities (N = 1052, 17.9%). The final diagnosis was based on histology (if biopsies had been taken) or colposcopic findings, which allowed direct estimation of sensitivity, specificity, and predictive values. Moderate or severe dysplasia or carcinoma in situ (CIN 2-3 disease) was considered as true positive disease for the calculation of sensitivity, specificity, and predictive values of screening tests. 18.7%, 17.7% and 8.2% of the women tested positive for VIA, VIAM, and cytology. One hundred twenty two women had a final diagnosis of CIN 2-3 lesions. The sensitivities of VIA and VIAM to detect CIN 2-3 lesions were 55.7% and 60.7%, respectively; the specificities were 82.1% and 83.2%, respectively. The sensitivity and specificity of cytology were 29.5% and 92.3%, respectively. All the tests were associated with negative predictive values above 98%. VIA and VIAM had significantly higher sensitivity than cytology in our study; the specificity of cytology was higher than that of VIA and VIAM.

India is experiencing a rapid spread of human immunodeficiency virus type 1 (HIV-1), primarily through heterosexual transmission of subtype C viruses. To delineate the molecular features of HIV-1 circulating in India, we sequenced the V3-V4 region of viral env from 21 individuals attending an HIV clinic in Calcutta, the most populous city in the eastern part of the country, and analyzed these and the other Indian sequences in the HIV database. Twenty individuals were infected with viruses having a subtype C env, and one had viruses with a subtype A env. Analyses of 192 subtype C sequences that included one sequence for each subject from this study and from the HIV database revealed that almost all sequences from India, along with a small number from other countries, form a phylogenetically distinct lineage within subtype C, which we designate C IN . Overall, C IN lineage sequences were more closely related to each other (level of diversity, 10.2%) than to subtype C sequences from Botswana, Burundi, South Africa, Tanzania, and Zimbabwe (range, 15.3 to 20.7%). Of the three positions identified as signature amino acid substitution sites for C IN sequences (K340E, K350A, and G429E), 56% of the C IN sequences contained all three amino acids while 87% of the sequences contained at least two of these substitutions. Among the non-C IN sequences, all three amino acids were present in 2%, while 22% contained two or more of these amino acids. These results suggest that much of the current Indian epidemic is descended from a single introduction into the country. Identification of conserved signature amino acid positions could assist epidemiologic tracking and has implications for the development of a vaccine against subtype C HIV-1 in 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-B 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.

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.

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.