Comparison of four different human papillomavirus genotyping methods in cervical samples: Addressing method-specific advantages and limitations.

Since human papillomavirus (HPV) is recognized as the causative agent of cervical cancer and associated with anogenital non-cervical and oropharyngeal cancers, the characterization of the HPV types circulating in different geographic regions is an important tool in screening and prevention. In this context, this study compared four methodologies for HPV detection and genotyping: real-time PCR (Cobas® HPV test), nested PCR followed by conventional Sanger sequencing, reverse hybridization (High + Low PapillomaStrip® kit) and next-generation sequencing (NGS) at an Illumina HiSeq2500 platform. Cervical samples from patients followed at the Family Health Strategy from Juiz de Fora, Minas Gerais, Brazil, were collected and subjected to the real-time PCR. Of those, 114 were included in this study according to the results obtained with the real-time PCR, considered herein as the gold standard method. For the 110 samples tested by at least one methodology in addition to real-time PCR, NGS showed the lowest concordance rates of HPV and high-risk HPV identification compared to the other three methods (67-75 %). Real-time PCR and Sanger sequencing showed the highest rates of concordance (97-100 %). All methods differed in their sensitivity and specificity. HPV genotyping contributes to individual risk stratification, therapeutic decisions, epidemiological studies and vaccine development, supporting approaches in prevention, healthcare and management of HPV infection.

Differential survival of Brazilian patients with diffuse large B-cell lymphoma with and without HIV infection.

OBJECTIVE: Combinatorial antiretroviral therapy provided improvement of HIV patients' immune function and a decrease in the incidence of non-Hodgkin lymphoma (NHL). Diffuse large B-cell lymphoma (DLBCL) is one of the most common NHL forms affecting HIV+ patients. The present study aimed to evaluate the impact of HIV infection on the prognosis of patients treated for DLBCL in a reference cancer treatment center in Brazil. METHODS: A retrospective case-control study was developed with patients followed-up at the Brazilian National Cancer Institute, in which 243 DLBCL patients (91 HIV+ and 152 HIV-) were enrolled. HIV- controls were matched to HIV+ according to date of cancer diagnosis, clinical staging, primary cancer treatment and date of birth. Sociodemographic and cancer treatment data were extracted from medical charts. Kaplan-Meier analyses were carried out to estimate survival, while univariate and multiple Cox regression analyses were used to determine factors associated with mortality. RESULTS: A total of 98 deaths were observed in a 5-year period after cancer diagnosis. A negative association of HIV infection with both overall and disease-specific survival 1 year after cancer diagnosis was observed [hazard ratio (HR) = 1.98 and 1.96, respectively]. The negative association with HIV infection with disease-specific survival remained significant for a 5-year period after cancer diagnosis (HR = 1.53). HIV viral load above 1000 copies/ml at study entry was also associated with shorter overall and cancer-specific survival. CONCLUSIONS: HIV infection negatively impacted prognosis and mortality of DLBCL patients irrespective of cancer-related clinical factors.

Survival in HIV+ and HIV- women with breast cancer treated at the National Cancer Institute in the city of Rio de Janeiro, Brazil, between 2000 and 2014.

BACKGROUND: The goal was to assess the survival of HIV+ women and HIV- women for breast cancer at a referral center for cancer treatment in Brazil. METHODS: A retrospective cohort study was performed. A total of 136 women patients with breast cancer were included, being 36 HIV+ women and 100 HIV- women. Controls (HIV-) were selected according to HIV status, matched by date of cancer diagnosis, clinical stage, breast cancer treatment, and date of birth. Sociodemographic and cancer treatment data, as well as clinical HIV data, were extracted from physical and electronic medical records and secondary Instituto Nacional of cancer databases. To estimate survival, the Kaplan-Meier method was used. To determine the factors associated with mortality, Cox regression were used. RESULTS: The mean age of patients at diagnosis of cancer was 52 years. Regarding marital status, HIV+ patients had a higher frequency of single status). There were 44.1% deaths that occurred during the study period. Among HIV+ patients, there were 16 deaths, 15 of which were due to cancer. In HIV- patients there were 44 deaths (44%), with 32 cancer as the cause of death and 12 due to other causes. For the analysis of Overall. Differences were found in overall survival at 60 months (p=0.026), 55% and 69% respectively. The increased risk of death at 60 months among HIV+ women was observed also, after adjusting for schooling and molecular subtype (HR=1.95; 95% CI 1.03 - 3.70; p=0.041). CONCLUSION: HIV infection influenced a worse prognosis for women with breast cancer regardless of tumor factors.

Composite Analysis of the Virome and Bacteriome of HIV/HPV Co-Infected Women Reveals Proxies for Immunodeficiency.

The human cervical microbiome is complex, and its role in health and disease has just begun to be elucidated. In this study, 57 cervical swab samples from 19 HIV/HPV co-infected women were analyzed for both virome and bacteriome composition. Virome analysis focused on circular DNA viruses through rolling circle amplification followed by next-generation sequencing (NGS). Data were assigned to virus families and genera, and HPV types were identified. NGS data of bacterial 16S from a subset of 24 samples were assigned to operational taxonomic units and classified according to vaginal microbiome community state types (CSTs). Four viral families were found: Papillomaviridae, Anelloviridae, Genomoviridae, and Herpesviridae. Papillomavirus reads were more abundant in women with premalignant cervical lesions, which were also strongly associated with multiple (≥3) high-risk HPV infection. Anellovirus read abundance was negatively correlated with host CD4+ T-cell counts. The bacteriome revealed the presence of CST III and CST IV, and women with ≥1% frequency of genomovirus or herpesvirus reads displayed an increased risk of carrying CST IV. By characterizing the composition of the cervical circular DNA viruses and the bacteriome of HIV/HPV co-infected women, we identified putative interactions between these two microorganism communities and their associations with patients' clinical characteristics, notably immunodeficiency status.

Analysis of the cervical microbiome and potential biomarkers from postpartum HIV-positive women displaying cervical intraepithelial lesions.

The cervical microbiota composition and diversity of HIV-positive women in the postpartum period is unknown. Using a high-throughput bacterial 16S rRNA gene sequencing, we identified four community state types (CSTs). CST III (Lactobacillusdominant) and CST IV (IV-A, IV-B.1, IV-B.2; high-diversity) were found in 41% and 59% of samples, respectively. We did not find association of any CST to postpartum period (six or twelve months), HPV infection or cytology (normal or lesion). However, five bacterial genera were associated with cervical lesions (Gardnerella, Aerococcus, Schlegelella, Moryella and Bifidobacterium), with significant odds ratio (OR) of 40 (2.28-706) for the presence of Moryella and 3.5 (1.36-8.9) for Schlegelella. Longitudinal analysis of samples at postpartum that regressed (lesion to normal), progressed (normal to lesion) and maintained the cytology (lesion or normal) evidenced Gardnerella with a significantly higher abundance in regressing lesions. In the current study, we report the first data on the cervical microbiota of HIV-positive women in the postpartum period. Consistent with previous studies of HIV-negative cohorts, HIV-positive women present a stable cervical microbiota of high-diversity in the postpartum period. Our results highlight that specific microbiota species may serve as sensors for changes in the cervical microenvironment associated with cervical lesions.

Characterisation of complete high- and low-risk human papillomavirus genomes isolated from cervical specimens in southern Brazil

The classification of human papillomavirus (HPV) intratypic lineages by complete genome sequencing is a determinant in understanding biological differences in association with this disease. In this work, we have characterised complete HPV genomes from southern Brazil. Fifteen cervicovaginal Pap smear negative samples previously categorised as HPV-positive were sequenced using ultradeep sequencing, and 18 complete genomes from 13 different HPV types were assembled. Phylogenetic and genetic distance analyses were performed to classify the HPV genomes into lineages and sublineages. This is the first report describing the distribution of HPV intratype lineages of high and low oncogenic risk in asymptomatic women from southern Brazil.

Characterisation of complete high- and low-risk human papillomavirus genomes isolated from cervical specimens in southern Brazil.

The classification of human papillomavirus (HPV) intratypic lineages by complete genome sequencing is a determinant in understanding biological differences in association with this disease. In this work, we have characterised complete HPV genomes from southern Brazil. Fifteen cervicovaginal Pap smear negative samples previously categorised as HPV-positive were sequenced using ultradeep sequencing, and 18 complete genomes from 13 different HPV types were assembled. Phylogenetic and genetic distance analyses were performed to classify the HPV genomes into lineages and sublineages. This is the first report describing the distribution of HPV intratype lineages of high and low oncogenic risk in asymptomatic women from southern Brazil.

Outcomes of cervical cancer among HIV-infected and HIV-uninfected women treated at the Brazilian National Institute of Cancer.

OBJECTIVE: We assessed mortality, treatment response, and relapse among HIV-infected and HIV-uninfected women with cervical cancer in Rio de Janeiro, Brazil. DESIGN: Cohort study of 87 HIV-infected and 336 HIV-uninfected women with cervical cancer. METHODS: Patients at the Brazilian National Institute of Cancer (2001-2013) were matched on age, calendar year of diagnosis, clinical stage, and tumor histology. Staging and treatment with surgery, radiotherapy, and/or chemotherapy followed international guidelines. We used a Markov model to assess responses to initial therapy, and Cox models for mortality and relapse after complete response (CR). RESULTS: Among 234 deaths, most were from cancer (82% in HIV-infected vs. 93% in HIV-uninfected women); only 9% of HIV-infected women died from AIDS. HIV was not associated with mortality during initial follow-up but was associated more than 1-2 years after diagnosis [overall mortality: stage-adjusted hazard ratio 2.02, 95% confidence interval (CI) 1.27-3.22; cancer-specific mortality: 4.35, 1.86-10.2]. Among 222 patients treated with radiotherapy, HIV-infected had similar response rates to initial cancer therapy as HIV-uninfected women (hazard ratio 0.98, 95% CI 0.58-1.66). However, among women who were treated and had a CR, HIV was associated with elevated risk of subsequent relapse (hazard ratio 3.60, 95% CI 1.86-6.98, adjusted for clinical stage). CONCLUSION: Among women with cervical cancer, HIV infection was not associated with initial treatment response or early mortality, but relapse after attaining a CR and late mortality were increased in those with HIV. These results point to a role for an intact immune system in control of residual tumor burden among treated cervical cancer patients.

Identification of novel human papillomavirus lineages and sublineages in HIV/HPV-coinfected pregnant women by next-generation sequencing.

Infection by human papillomavirus (HPV) is a necessary condition for development of cervical cancer, and has also been associated with malignancies of other body anatomical sites. Specific HPV types have been associated with premalignant lesions and invasive carcinoma, but mounting evidence suggests that within-type lineages and sublineages also display distinct biological characteristics associated with persistent infections and evolution to cervical cancer. In the present study, we have assessed HPV multiple infection and variation from a cohort of highly susceptible, HIV(+) pregnant women using next-generation sequencing and an in-house pipeline for HPV full-length genome assembly. Seventy-two consensus sequences representing complete or near-complete (>97%) HPV genomes were assembled, spanning 28 different types. Genetic distance and phylogenetic analyses allowed us to propose the classification of novel HPV lineages and sublineages across nine HPV types, including two high-risk types. HPV diversity may be a hallmark of immunosuppressed patients upon HIV infection and AIDS progression.

Bonafide, type-specific human papillomavirus persistence among HIV-positive pregnant women: predictive value for cytological abnormalities, a longitudinal cohort study.

This study investigated the rate of human papillomavirus (HPV) persistence, associated risk factors, and predictors of cytological alteration outcomes in a cohort of human immunodeficiency virus-infected pregnant women over an 18-month period. HPV was typed through L1 gene sequencing in cervical smears collected during gestation and at 12 months after delivery. Outcomes were defined as nonpersistence (clearance of the HPV in the 2nd sample), re-infection (detection of different types of HPV in the 2 samples), and type-specific HPV persistence (the same HPV type found in both samples). An unfavourable cytological outcome was considered when the second exam showed progression to squamous intraepithelial lesion or high squamous intraepithelial lesion. Ninety patients were studied. HPV DNA persistence occurred in 50% of the cases composed of type-specific persistence (30%) or re-infection (20%). A low CD4+T-cell count at entry was a risk factor for type-specific, re-infection, or HPV DNA persistence. The odds ratio (OR) was almost three times higher in the type-specific group when compared with the re-infection group (OR = 2.8; 95% confidence interval: 0.43-22.79). Our findings show that bonafide (type-specific) HPV persistence is a stronger predictor for the development of cytological abnormalities, highlighting the need for HPV typing as opposed to HPV DNA testing in the clinical setting.

Bonafide, type-specific human papillomavirus persistence among HIV-positive pregnant women: predictive value for cytological abnormalities, a longitudinal cohort study

This study investigated the rate of human papillomavirus (HPV) persistence, associated risk factors, and predictors of cytological alteration outcomes in a cohort of human immunodeficiency virus-infected pregnant women over an 18-month period. HPV was typed through L1 gene sequencing in cervical smears collected during gestation and at 12 months after delivery. Outcomes were defined as nonpersistence (clearance of the HPV in the 2nd sample), re-infection (detection of different types of HPV in the 2 samples), and type-specific HPV persistence (the same HPV type found in both samples). An unfavourable cytological outcome was considered when the second exam showed progression to squamous intraepithelial lesion or high squamous intraepithelial lesion. Ninety patients were studied. HPV DNA persistence occurred in 50% of the cases composed of type-specific persistence (30%) or re-infection (20%). A low CD4+T-cell count at entry was a risk factor for type-specific, re-infection, or HPV DNA persistence. The odds ratio (OR) was almost three times higher in the type-specific group when compared with the re-infection group (OR = 2.8; 95% confidence interval: 0.43-22.79). Our findings show that bonafide (type-specific) HPV persistence is a stronger predictor for the development of cytological abnormalities, highlighting the need for HPV typing as opposed to HPV DNA testing in the clinical setting.

The effect of human leukocyte antigen G alleles on human papillomavirus infection and persistence in a cohort of HIV-positive pregnant women from Brazil.

Patients with compromised immune systems have more severe intraepithelial lesions and more rapid disease progression, in addition to increased risk for cervical cancer. Persistent infection by the human papillomavirus (HPV) is a necessary step in that process. By inducing expression of inhibitory ligands of natural killer cells, like HLA-G, HPV avoids the elimination of infected cells. Recent studies have investigated polymorphisms in HLA-G that may be associated with susceptibility to HPV infection and persistence. One hundred-forty HIV(+) pregnant women from Brazil had a DNA fragment comprising HLA-G exons 2-4 PCR-amplified, cloned, sequenced and analyzed for allele determination. Altogether, 22 alleles comprising 52 different genotypes were found. Four novel HLA-G alleles were characterized. We have not observed association of specific HLA-G alleles with HPV infection, but found a protective effect of the G:01:01:02 allele against the occurrence of intraepithelial lesions. In addition to describing new HLA-G alleles and defining new reference sequences, our data provide a better understanding of the impact of HLA-G alleles on HPV-related disease.

Identification and characterization of highly divergent simian foamy viruses in a wide range of new world primates from Brazil.

Foamy viruses naturally infect a wide range of mammals, including Old World (OWP) and New World primates (NWP), which are collectively called simian foamy viruses (SFV). While NWP species in Central and South America are highly diverse, only SFV from captive marmoset, spider monkey, and squirrel monkey have been genetically characterized and the molecular epidemiology of SFV infection in NWPs remains unknown. We tested a large collection of genomic DNA (n = 332) comprising 14 genera of NWP species for the presence of SFV polymerase (pol) sequences using generic PCR primers. Further molecular characterization of positive samples was carried out by LTR-gag and larger pol sequence analysis. We identified novel SFVs infecting nine NWP genera. Prevalence rates varied between 14-30% in different species for which at least 10 specimens were tested. High SFV genetic diversity among NWP up to 50% in LTR-gag and 40% in pol was revealed by intragenus and intrafamilial comparisons. Two different SFV strains infecting two captive yellow-breasted capuchins did not group in species-specific lineages but rather clustered with SFVs from marmoset and spider monkeys, indicating independent cross-species transmission events. We describe the first SFV epidemiology study of NWP, and the first evidence of SFV infection in wild NWPs. We also document a wide distribution of distinct SFVs in 14 NWP genera, including two novel co-speciating SFVs in capuchins and howler monkeys, suggestive of an ancient evolutionary history in NWPs for at least 28 million years. A high SFV genetic diversity was seen among NWP, yet these viruses seem able to jump between NWP species and even genera. Our results raise concerns for the risk of zoonotic transmission of NWP SFV to humans as these primates are regularly hunted for food or kept as pets in forest regions of South America.

HIV/HPV co-infection during pregnancy in southeastern Brazil: prevalence, HPV types, cytological abnormalities and risk factors.

OBJECTIVE: HIV(+) pregnant women are at a higher risk of HPV infection and development of cervical cancer. Our objectives were to assess the prevalence and HPV types in HIV(+) pregnant women and to identify risk factors for HPV infection and cytological abnormalities. METHODS: Cervicovaginal smears were collected during pregnancy from 140 women. Partial HPV L1 gene and the exon 4 of the human TP53 gene (containing codon 72) were PCR-amplified and sequenced. Amplified products indicating multiple HPV infection were further cloned and sequenced. The association of demographic, obstetric and HIV-related clinical variables with HPV infection and cervical lesions was tested by univariate analyses, and significant factors were subsequently tested by logistic regression multivariate analysis. RESULTS: HPV DNA tested positive for 118 patients and HPV types were identified in 104 samples. Twenty-eight different types were found, HPV-16 and HPV-58 being the most prevalent. High-risk types were present in 79.8% of samples and multiple infections in 16.3%. Abnormal cervical smears were found in 44 patients (31.4%). Absolute CD4(+) T-cell counts below 350 were associated with HPV infection. Younger age was associated with cervical abnormalities and higher CD4(+) T-cell count was an apparent protective factor. CONCLUSIONS: We found a high prevalence of HPV infection and high-risk types in this cohort. Our results highlighted the relevance of immune system integrity rather than TP53 variants for protecting this highly vulnerable population to HPV infection and carcinogenesis.

Molecular evolution of α4 integrin binding site to lentiviral envelope proteins in new world primates.

Integrin epitopes encoded by ITGA4 exons 5 and 6 encompass the α4ß7 binding site to natural ligands and HIV-1 gp120. Functional assays of α4 variants of new world primates (NWP) showed reduced binding of several ligands, including the HIV-1 envelope, probably accounting for restriction phenotypes conferring resistance to lentiviral infection (Darc et al., 2011). In this paper, we have analyzed, by cloning and sequencing, the α4 domain polymorphisms present in 10 NWP species and four old world primates (including human). Analyses of differential selection at codon sites and along evolutionary lineages were carried out. We identified codons under positive selection, including polymorphic variations at codon 201, presumably convergent during NWP radiation and significant positive selection leading to a single allele (SagVar2).

Clonal analysis of hepatitis B viruses among blood donors from Joinville, Brazil: evidence of dual infections, intragenotype recombination and markers of risk for hepatocellular carcinoma.

Hepatitis B virus (HBV) is classified into seven major genotypes (A-H). Brazil, a country of continental proportions, has three prevailing lineages of HBV genotypes A, D, and F. Distinct HBV genotypes have been associated with differential risk of disease progression. Pre-S gene deletions and single nucleotide polymorphisms have also been linked to progression to liver diseases. In this study, the molecular epidemiology of HBV was examined in Southern Brazil. The occurrence of multiple HBV infections, HBV recombination, and genetic markers of disease progression were also evaluated. Seventy-eight persons infected with HBV had their viruses characterized molecularly by nested PCR, DNA sequencing, and phylogenetic inference. Multiple infections and recombinant viruses were evaluated by clonal and bootscanning analyses. The vast majority (96%) of the strains belonged to different D subgenotypes. Three of the four strains with unresolved genotypic classification showed evidence of dual infections with distinct D subgenotypes by clonal analysis. There was also evidence of intragenotype mosaic viruses. While four viruses had pre-S deletions as major variants, another two displayed minor variants with such characteristics. One strain carried the F141L mutation, associated recently with increased risk of hepatocellular carcinoma. These results emphasize the need for monitoring HBV genotype distribution around South America, as well as for the presence of genetic markers of disease progression in subjects diagnosed with HBV recently.

Determinants of HIV-1 mother-to-child transmission in Southern Brazil

Diferentes subtipos do virus da imunodeficiência humana do tipo 1 (HIV-1) podem ter propriedades biológicas, imunológicas e patogênicas distintas. A eficiência da transmissão materno-infantil (TMI) pode estar entre estas propriedades, porém resultados escassos e controversos foram descritos até o momento. Neste estudo, 102 crianças nascidas de mães infectadas pelo HIV-1 entre 1998 e 2004 na cidade do Rio Grande, Brasil, foram analisadas para fatores de risco potenciais associados à TMI. Aquela região geográfica é caracterizada por uma alta proporção de indivíduos infectados pelo subtipo C do HIV-1, permitindo a comparação entre os subtipos B e C e sua influência na transmissão vertical do vírus. A análise também incluiu parâmetros clínicos, obstétricos e imunológicos. Análises de regressão multivariada foram conduzidas para avaliar a influência daqueles parâmetros na TMI, e as razões de prevalência (RP) e intervalos de confiança de 95% (IC95) foram também calculados. Um prevalência surpreendentemente alta do subtipo C acima dos 70% foi encontrada. Somente a carga viral do HIV e o uso de protocolo ACGT 076 maternos forma preditivos de TMI. O subtipo do HIV-1 e a contagem de células T CD4+ não foram associados a um risco aumentado de transmissão. Embora uma clara expansão do subtipo C seja evidente no sul do Brasil, esta não parece estar correlacionada com risco aumentado de transmissão vertical.