Arati Mane

INCREASED EXPRESSION OF VIRULENCE ATTRIBUTES IN ORAL CANDIDA ALBICANS ISOLATES FROM HUMAN IMMUNODEFICIENCY VIRUS-POSITIVE INDIVIDUALS

Oral candidiasis caused by Candida albicans is recognized as one of the most frequent opportunistic infections in human immunodeficiency virus (HIV)-infected individuals. The overall severity and chronicity of oral candidiasis has been attributed exclusively to the HIV-induced immune deficiency of the affected individuals but not to the virulence factors of the pathogen, i.e. C. albicans. However, genotypic and phenotypic studies have suggested that HIV infection might be associated with preferential selection of C. albicans strains with altered virulence determinants, leading to colonization with Candida populations that are better able to cause disease in these immunologically compromised hosts. If this process of selection is indeed related to pathogenicity, it may be possible to measure alterations in different virulence factors produced by C. albicans in HIV-infected patients. To evaluate this hypothesis, the present work was undertaken to determine simultaneously the expression of five virulence factors in oral C. albicans isolates colonizing and infecting HIV-positive and -negative individuals. The significance of genotypes in the pathogenesis of oral candidiasis was also elucidated. Oral swabs were collected from 335 consecutive individuals (210 HIV-positive and 125 HIV-negative). Virulence factors and genotypes were determined for all the C. albicans strains isolated. The results showed significantly increased expression of proteinase, phospholipase and haemolytic activities, as well as a greater ability to adhere, in isolates from HIV-positive compared with HIV-negative individuals (P<0.05). However, no significant differences in virulence factor expression in isolates colonizing or infecting HIV-positive individuals were seen. Genotype A was the predominant type (71.3 %); however, a relationship could not be established between the genotypes and the virulence factors, or with clinical infection. These data support the concept of preferential C. albicans strain selection with altered virulence determinants in HIV-infected individuals and emphasize the need for further molecular genetic linkage studies that could be helpful in dissecting the molecular causes of preferential strain selection, which may lead to new approaches for therapeutic intervention.

HPV Genotype Distribution in Cervical Intraepithelial Neoplasia among HIV-Infected Women in Pune, India

Background The distribution of HPV genotypes, their association with rigorously confirmed cervical precancer endpoints, and factors associated with HPV infection have not been previously documented among HIV-infected women in India. We conducted an observational study to expand this evidence base in this population at high risk of cervical cancer. Methods HIV-infected women (N = 278) in Pune, India underwent HPV genotyping by Linear Array assay. Cervical intraepithelial neoplasia (CIN) disease ascertainment was maximized by detailed assessment using cytology, colposcopy, and histopathology and a composite endpoint. Results CIN2+ was detected in 11.2% while CIN3 was present in 4.7% participants. HPV genotypes were present in 52.5% (146/278) and ‘carcinogenic’ HPV genotypes were present in 35.3% (98/278) HIV-infected women. ‘Possibly carcinogenic’ and ‘non/unknown carcinogenic’ HPV genotypes were present in 14.7% and 29.5% participants respectively. Multiple (≥2) HPV genotypes were present in half (50.7%) of women with HPV, while multiple ‘carcinogenic’ HPV genotypes were present in just over a quarter (27.8%) of women with ‘carcinogenic’ HPV. HPV16 was the commonest genotype, present in 12% overall, as well as in 47% and 50% in CIN2+ and CIN3 lesions with a single carcinogenic HPV infection, respectively. The carcinogenic HPV genotypes in declining order of prevalence overall included HPV 16, 56, 18, 39, 35, 51, 31, 59, 33, 58, 68, 45 and 52. Factors independently associated with ‘carcinogenic’ HPV type detection were reporting ≥2 lifetime sexual partners and having lower CD4+ count. HPV16 detection was associated with lower CD4+ cell counts and currently receiving combination antiretroviral therapy. Conclusion HPV16 was the most common HPV genotype, although a wide diversity and high multiplicity of HPV genotypes was observed. Type-specific attribution of carcinogenic HPV genotypes in CIN3 lesions in HIV-infected women, and etiologic significance of concurrently present non/unknown carcinogenic HPV genotypes await larger studies.

Adherence to buccal epithelial cells, enzymatic and hemolytic activities of Candida isolates from HIV-infected individuals

Candida spp., along with other microorganisms are usually found in the normal flora of the human oral mucosa. However in HIV-infected individuals these yeasts can become opportunistic pathogens and lead to mucosal infections. The transformation from a harmless commensal to a virulent pathogen under conditions of dysfunctional host defense system is attributable to an extensive repertoire of selectively expressed virulence determinants. The present study was undertaken to analyze the production of four important virulence factors viz. adherence to buccal epithelial cells (BEC), proteinase activity, phospholipase activity and hemolysis, and to evaluate the correlation between these virulence factors in 65 Candida isolates from HIV-infected individuals with oral candidiasis. A total of 95.3%, 67.7%, 41.5% and 100% of the Candida isolates showed adherence to BEC, and proteinase, phospholipase and hemolytic activities, respectively. Production of proteinase and phospholipase enzymes was seen in 89.7% and 59.0% of C. albicans isolates and 34.6% and 15.4% of non-Candida albicans Candida (NAC) isolates, respectively. C. albicans showed significantly greater level of virulence factor expression with regards to adherence to BEC (P < 0.001), phospholipase production (P < 0.044) and hemolysis (P = 0.037) as compared to NAC. A correlative relationship between proteinase activity and adherence to BEC, as well as phospholipase production was noted.

HIV-1 RNA shedding in the female genital tract is associated with reduced quantity of Lactobacilli in clinically asymptomatic HIV-positive women☆ , ☆☆

We quantified vaginal lactobacilli and determined their relationship with genital HIV-1 shedding and found a significant negative association between reduced quantity of lactobacilli and cervical HIV-1 viral load (r(2) = - 0.8900, P < 0.01), which may have implications of increased chances of sexual transmission of HIV-1 and genital infections.

Molecular mechanisms associated with Fluconazole resistance in clinical Candida albicans isolates from India

Resistance to azole antifungals is a significant problem in Candida albicans. An understanding of resistance at molecular level is essential for the development of strategies to tackle resistance and rationale design of newer antifungals and target‐based molecular approaches. This study presents the first evaluation of molecular mechanisms associated with fluconazole resistance in clinical C.albicans isolates from India. Target site (ERG11) alterations were determined by DNA sequencing, whereas real‐time PCRs were performed to quantify target and efflux pump genes (CDR1, CDR2, MDR1) in 87 [Fluconazole susceptible (n = 30), susceptible‐dose dependent (n = 30) and resistant (n = 27)] C.albicans isolates. Cross‐resistance to fluconazole, ketoconazole and itraconazole was observed in 74.1% isolates. Six amino acid substitutions were identified, including 4 (E116D, F145L, E226D, I437V) previously reported ones and 2 (P406L, Q474H) new ones. CDR1 over‐expression was seen in 77.7% resistant isolates. CDR2 was exclusively expressed with CDR1 and their concomitant over‐expression was associated with azole cross‐resistance. MDR1 and ERG11 over‐expression did not seem to be associated with resistance. Our results show that drug efflux mediated by Adenosine‐5′‐triphosphate (ATP)‐binding cassette transporters, especially CDR1 is the predominant mechanism of fluconazole resistance and azole cross‐resistance in C. albicans and indicate the need for research directed towards developing strategies to tackle efflux mediated resistance to salvage azoles.

Antimicrobial activity of plant extracts against sexually transmitted pathogens

Comprehensive management of sexually transmitted infections (STIs) using vaginal or rectal microbicide-based intervention is one of the strategies for prevention of HIV infection. Herbal products have been used for treating STIs traditionally. Herein, we present in vitro activity of 10 plant extracts and their 34 fractions against three sexually transmitted/reproductive tract pathogens – Neisseria gonorrhoeae, Haemophilus ducreyi and Candida albicans. The plant parts were selected; the extracts/fractions were prepared and screened by disc diffusion method. The minimum inhibitory and minimum cidal concentrations were determined. The qualitative phytochemical analysis of selected extracts/fractions showing activity was performed. Of the extracts/fractions tested, three inhibited C. albicans, ten inhibited N. gonorrhoeae and five inhibited H. ducreyi growth. Our study demonstrated that Terminalia paniculata Roth. extracts/fractions inhibited growth of all three organisms. The ethyl acetate fraction of Syzygium cumini Linn. and Bridelia retusa (L.) Spreng. extracts was found to inhibit N. gonorrhoeae at lowest concentrations.

Biofilm production in oral Candida isolates from HIV-positive individuals from Pune, India

Biofilm formation is implicated as a potential virulence factor in Candida species and carries important clinical repercussions because of their increased resistance to antifungal treatment, ability to withstand host defences and to serve as a reservoir for continuing infections. The present study was undertaken to determine the biofilm production among oral Candida isolates from HIV‐positive and HIV‐negative individuals from Pune, India. Biofilm formation was determined using the spectrophotometric or microtitre plate method in 182 Candida isolates, of which 154 were from HIV‐positive and 28 were from HIV‐negative individuals. A total of 63.2% of the Candida isolates were biofilm producers. Significantly increased biofilm forming abilities both qualitatively as well as quantitatively were observed in Candida isolates from HIV‐positive individuals (66.2%) compared to isolates from HIV‐negative ones (46.4%), (P– 0.041). Eighty‐one (59.6%) C. albicans isolates and 34 (73.9%) non –C. albicans Candida (NCAC) showed biofilm positivity. The NCAC showed significantly greater intensity of biofilm formation compared to the C. albicans, P– 0.032. Our results thus show the enhanced biofilm forming abilities of oral Candida isolates from HIV‐infected individuals compared to HIV‐uninfected ones and highlight the important role played by biofilm formation in the pathogenesis of NCAC isolates.

Prevalence of anal human papillomavirus infection among HIV-infected women from India.

acute lymphoblastic leukemia), incurable (eg, metastatic melanoma), or unknown (eg, carcinoma of unknown primary). Although widely used in cancer clinical discussions, clinical remission is also used to describe a wide range of other noncancer chronic diseases. The remission concept clearly denotes improvement but also underscores vigilance and uncertainty that require the need for careful follow-up. In addition, the remission concept can be further subdivided into partial and complete remissions, a helpful designation given the likely stepwise progression towards an HIV cure. Although the remission concept may be less familiar in lowincome country settings, it would be largely recognizable to HIV-infected individuals in a wide range of other settings. The remission concept has been used by leadership at the National Institutes of Health’s National Institute of Allergy and Infectious Diseases and the IAS. The concept of “HIV remission” may be particularly useful as more HIV-infected individuals enter clinical trials and physicians are discussing this concept outside the research settings. Embracing the concept of clinical remission does not mean that we have abandoned hope of finding therapies that could create a sterilizing cure nor are we advocating for scientific research and advocacy to halt the aspirational (towards an HIV cure) language that has ignited a new subfield of HIV research. Clinical discussions between physicians and patients require honesty about the uncertainty in this undertaking. Such careful communication may present waves of hope, misperception, and disappointment among HIVinfected individuals and the health professionals who serve them. Should effective HIV cures be developed, the downstream effects on health and well-being, risk of onward transmission, risk of reacquisition, and social stigma are unclear. Furthermore, these larger biological and social questions will not be addressed by nomenclature. But, nomenclature critically sets the bar for expectations as research advances and clinicians increasingly discuss cure research with their patients with HIV. Although only one individual in all of history seems to be cured of HIV infection, the concept of remission is both easier to explain and more consistent with the state of the science. We owe it to our patients with HIV to use caution and consider the conceptual framework of HIV remission.

Genotype-specific incidence and clearance rates of human papilloma virus (HPV) infection in HIV-infected women from Pune, India

Results A total of 215 eligible participants were followed for a median time of 11 months (range: 8-23 months) with a follow-up period of 223 person-years. Of the 104/215 (48.4 %) HIV-infected women who were HPV-negative at baseline, 12 women were newly detected with HPV at follow-up visit reflecting an incidence rate of 5.4 per 100 person-years. Type-specific incidence rates ranged between 0.45-3.42 per 100 person-years for carcinogenic HPV types and between 0.45-1.79 per 100 person-years for other HPV types. Of the 111/215 (51.6 %) women with HPV at baseline, 21 women cleared all types, reflecting a clearance rate of 9.4 per 100 person-years. Type-specific clearance rates ranged between 0.45-4.48 per 100 person-years for carcinogenic HPV types and between 0.45-4.04 for other HPV types. Conclusions This study adds to the scant global data of natural history of HPV infection in HIV-infected women. Knowledge of incidence and clearance rates can inform cost effectiveness and decision analysis models for estimating effectiveness of HPV vaccination and screening strategies for cervical cancer prevention for HIV-infected women.

Characterization of vaginal lactobacilli from HIV-negative and HIV-positive Indian women and their association with genital HIV-1 shedding

One of the crucial determinants for successful administration of lactobacilli to the vaginal niche is the use of appropriate Lactobacillus species. In this cross-sectional study 54 human immunodeficiency virus (HIV)-negative and 76 HIV-positive antiretroviral treatment-naïve women were evaluated for culturable vaginal lactobacilli and their association with genital HIV-1 shedding. Lactobacillus species were identified by 16S rDNA sequencing while cervical and plasma HIV-1 viral load was determined by Abbott real-time PCR. Lactobacilli were isolated in 77.8 % HIV-negative and 73.7 % HIV-positive women. The mean log10 plasma and cervical HIV-1 viral loads (RNA copies ml-1) were 3.73±1.02 and 2.85±0.32 respectively. We observed that presence of L. crispatus, L. gasseri or L. jensenii species was associated with undetectable cervical HIV-1 (P=0.046) and reduced genital HIV-1 shedding (P=0.048) compared to other species. Our findings endorse using Lactobacillus-based strategies to aid the prevention of HIV-1 transmission among Indian women, however confirmation by future prospective studies is indeed warranted.