SARS-CoV-2 BA.1 and BA.2 coinfection detected by genomic surveillance in Brazil, January 2022.

In January 2022, our genomic surveillance network identified a SARS-CoV-2 BA.1 and BA.2 coinfection in a sample from a patient residing in Brazil. Our results suggest that the true number of SARS-CoV-2 coinfections remains largely underestimated.

Local Transmission of SARS-CoV-2 Lineage B.1.1.7, Brazil, December 2020.

In December 2020, research surveillance detected the B.1.1.7 lineage of severe acute respiratory syndrome coronavirus 2 in São Paulo, Brazil. Rapid genomic sequencing and phylogenetic analysis revealed 2 distinct introductions of the lineage. One patient reported no international travel. There may be more infections with this lineage in Brazil than reported.

Transmission between Archaic and Modern Human Ancestors during the Evolution of the Oncogenic Human Papillomavirus 16.

Every human suffers through life a number of papillomaviruses (PVs) infections, most of them asymptomatic. A notable exception are persistent infections by Human papillomavirus 16 (HPV16), the most oncogenic infectious agent for humans and responsible for most infection-driven anogenital cancers. Oncogenic potential is not homogeneous among HPV16 lineages, and genetic variation within HPV16 exhibits some geographic structure. However, an in-depth analysis of the HPV16 evolutionary history was still wanting. We have analyzed extant HPV16 diversity and compared the evolutionary and phylogeographical patterns of humans and of HPV16. We show that codivergence with modern humans explains at most 30% of the present viral geographical distribution. The most explanatory scenario suggests that ancestral HPV16 already infected ancestral human populations and that viral lineages co-diverged with the hosts in parallel with the split between archaic Neanderthal-Denisovans and ancestral modern human populations, generating the ancestral HPV16A and HPV16BCD viral lineages, respectively. We propose that after out-of-Africa migration of modern human ancestors, sexual transmission between human populations introduced HPV16A into modern human ancestor populations. We hypothesize that differential coevolution of HPV16 lineages with different but closely related ancestral human populations and subsequent host-switch events in parallel with introgression of archaic alleles into the genomes of modern human ancestors may be largely responsible for the present-day differential prevalence and association with cancers for HPV16 variants.

Clinical characteristics of women diagnosed with carcinoma who tested positive for cervical and anal high-risk human papillomavirus DNA and E6 RNA.

High-risk human papillomavirus (hrHPV) is an essential cause of cervical carcinoma and is also strongly related to anal cancer development. The hrHPV E6 oncoprotein plays a major role in carcinogenesis. We aimed to evaluate the frequency of hrHPV DNA and E6 oncoprotein in the anuses of women with cervical carcinoma. We analyzed 117 women with cervical cancer and 103 controls for hrHPV and the E6 oncogene. Positive test results for a cervical carcinoma included 66.7 % with hrHPV-16 and 7.7 % with hrHPV-18. One case tested positive for both HPV variants (0.9 %). The samples from the anal canal were positive for HPV-16 in 59.8 % of the cases. Simultaneous presence of HPV in the cervix and anal canal was found in 53.8 % of the cases. Regarding expression of E6 RNA, positivity for HPV-16 in the anal canal was found in 21.2 % of the cases, positivity for HPV-16 in the cervix was found in 75.0 %, and positivity for HPV-18 in the cervix was found in 1.9 %. E6 expression in both the cervix and anal canal was found in 19.2 % of the cases. In the controls, 1 % tested positive for HPV-16 and 0 % for HPV-18. Anal samples from the controls showed a hrHPV frequency of 4.9 % (only HPV16). The presence of hrHPV in the anal canal of women with cervical cancer was detected at a high frequency. We also detected E6 RNA expression in the anal canal of women with cervical cancer, suggesting that these women are at risk for anal hrHPV infection.

Human papillomavirus genotypes distribution in 175 invasive cervical cancer cases from Brazil.

BACKGROUND: Invasive cervical cancer is the second most common malignant tumor affecting Brazilian women. Knowledge on Human Papillomavirus (HPV) genotypes in invasive cervical cancer cases is crucial to guide the introduction and further evaluate the impact of new preventive strategies based on HPV. We aimed to provide updated comprehensive data about the HPV types' distribution in patients with invasive cervical cancer. METHODS: Fresh tumor tissue samples of histologically confirmed invasive cervical cancer were collected from 175 women attending two cancer reference hospitals from São Paulo State: ICESP and Hospital de Câncer de Barretos. HPV detection and genotyping were performed by the Linear Array HPV Genotyping Test (Roche Molecular Diagnostics, Pleasanton,USA). RESULTS: 170 out of 172 valid samples (99%) were HPV DNA positive. The most frequent types were HPV16 (77.6%), HPV18 (12.3%), HPV31 (8.8%), HPV33 (7.1%) and HPV35 (5.9%). Most infections (75%) were caused by individual HPV types. Women with adenocarcinoma were not younger than those with squamous cell carcinoma, as well, as women infected with HPV33 were older than those infected by other HPV types. Some differences between results obtained in the two hospitals were observed: higher overall prevalence of HPV16, absence of single infection by HPV31 and HPV45 was verified in HC-Barretos in comparison to ICESP patients. CONCLUSIONS: To our knowledge, this is one of the largest studies made with fresh tumor tissues of invasive cervical cancer cases in Brazil. This study depicted a distinct HPV genotype distribution between two centers that may reflect the local epidemiology of HPV transmission among these populations. Due to the impact of these findings on cervical cancer preventive strategies, extension of this investigation to routine screening populations is warranted.

Early Emergence and Dispersal of Delta SARS-CoV-2 Lineage AY.99.2 in Brazil.

The year of 2021 was marked by the emergence and dispersal of a number of SARS-CoV-2 lineages, resulting in the "third wave" of COVID-19 in several countries despite the level of vaccine coverage. Soon after the first confirmed cases of COVID-19 by the Delta variant in Brazil, at least seven Delta sub-lineages emerged, including the globally spread AY.101 and AY.99.2. In this study we performed a detailed analysis of the COVID-19 scenario in Brazil from April to December 2021 by using data collected by the largest private medical diagnostic company in Latin America (Dasa), and SARS-CoV-2 genomic sequences generated by its SARS-CoV-2 genomic surveillance project (GENOV). For phylogenetic and Bayesian analysis, SARS-CoV-2 genomes available at GISAID public database were also retrieved. We confirmed that the Brazilian AY.99.2 and AY.101 were the most prevalent lineages during this period, overpassing the Gamma variant in July/August. We also estimated that AY.99.2 likely emerged a few weeks after the entry of the B.1.617.2 in the country, at some point between late April and May and rapidly spread to other countries. Despite no increased fitness described for the AY.99.2 lineage, a rapid shift in the composition of Delta SARS-CoV-2 lineages prevalence in Brazil took place. Understanding the reasons leading the AY.99.2 to become the dominant lineage in the country is important to understand the process of lineage competitions that may inform future control measures.

Molecular characterization of a new SARS-CoV-2 recombinant cluster XAG identified in Brazil.

Recombination events have been described in the Coronaviridae family. Since the beginning of the SARS-CoV-2 pandemic, a variable degree of selection pressure has acted upon the virus, generating new strains with increased fitness in terms of viral transmission and antibody scape. Most of the SC2 variants of concern (VOC) detected so far carry a combination of key amino acid changes and indels. Recombination may also reshuffle existing genetic profiles of distinct strains, potentially giving origin to recombinant strains with altered phenotypes. However, co-infection and recombination events are challenging to detect and require in-depth curation of assembled genomes and sequencing reds. Here, we present the molecular characterization of a new SARS-CoV-2 recombinant between BA.1.1 and BA.2.23 Omicron lineages identified in Brazil. We characterized four mutations that had not been previously described in any of the recombinants already identified worldwide and described the likely breaking points. Moreover, through phylogenetic analysis, we showed that the newly named XAG lineage groups in a highly supported monophyletic clade confirmed its common evolutionary history from parental Omicron lineages and other recombinants already described. These observations were only possible thanks to the joint effort of bioinformatics tools auxiliary in genomic surveillance and the manual curation of experienced personnel, demonstrating the importance of genetic, and bioinformatic knowledge in genomics.

Monitoring of group C rotavirus in children with acute gastroenteritis in Brazil: an emergent epidemiological issue after rotavirus vaccine?

Group C rotavirus (GpCRV) has a worldwide distribution; however, its epidemiology and ecology are still unclear. Evidence for a possible zoonotic role has been postulated recently for Brazilian children strains. The aim of this study was to monitor GpCRV in children ≤15 years with acute gastroenteritis during the 2007-2010 national Brazilian rotavirus surveillance, and to undertake the molecular characterization of the major VP6 capsid protein. A total of 3,019 fecal samples were first screened for Group A rotavirus (GpARV). A total of 2,205 GpARV ELISA negative samples were tested further for the presence of GpCRV by SDS-PAGE, electronic microscopy, and RT-PCR for the VP6 gene. The genetic diversity of GpCRV was carried out by sequencing the VP6 gene. GpARV and GpCRV infections were detected in 24.6% (742/3,019) and 0.3% (8/3,019), respectively. The GpCRV detection rate increased from 0.2% (1/422) in 2007 to 1% (7/708) in 2008, and GpCRV cases were not detected in 2009 and 2010. The phylogenetic analysis indicated that the strains belonged to the human lineage, and showed a genetic relationship with the GpCRV strain from Japan isolated in 2009. None of the study sequences was related closely to animal GpCRV strains. This study provides further evidence that GpCRV is a minor cause of acute childhood gastroenteritis in Brazil, and does not suggest that GpCRV may assume epidemiological importance in the future, even after the introduction of a GpARV vaccine. In addition, the molecular analyses of the GpCRV samples in this study do not support the zoonotic hypothesis.

SARS-CoV-2 saliva testing is a useful tool for Covid-19 diagnosis.

SARS-CoV-2 is the etiologic agent of coronavirus disease 2019 (COVID-19) and is mainly detected by RT-PCR methods from upper respiratory specimens, as recommended by the World Health Organization. Oro/nasopharyngeal swabbing can be discomfortable to the patients, requires trained healthcare personnel and may generate aerosol, increasing the risk of nosocomial infections. In this study, we describe two SARS-CoV-2 RNA extraction-free single RT-PCR protocols on saliva samples and compared the results with the paired oro/nasopharyngeal swab specimens from 400 patients. The two saliva protocols demonstrated a substantial agreement when compared to the oro/nasopharyngeal swab protocol. Moreover, the positivity rate of saliva protocols increased according to the disease period. The 95 % limit of detection of one of the therefore implemented saliva protocol was determined as 9441 copies/mL. Our results support the conclusion that RNA extraction-free RT-PCR using self-collected saliva specimens is an alternative to nasopharyngeal swabs, especially in the early phase of symptom onset.

Dynamics of SARS-CoV-2 Variants of Concern in Brazil, Early 2021.

Brazil is the country with the second-largest number of deaths due to the coronavirus disease-2019 (COVID-19). Two variants of concern (VOCs), Alpha (B.1.1.7) and Gamma (P.1), were first detected in December 2020. While Alpha expanded within an expected rate in January and February 2021, its prevalence among new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases started to decrease in March, which coincided with the explosion of Gamma variant incidence all over the country, being responsible for more than 95% of the new cases over the following months. A significantly higher viral load [i.e., mean cycle threshold (Ct) values] for Gamma in comparison to non-VOC samples was verified by the analysis of a large data set of routine reverse transcription-PCR (RT-PCR) exams. Moreover, the rate of reinfections greatly increased from March 2021 onward, reinforcing the enhanced ability of Gamma to escape the immune response. It is difficult to predict the outcomes of competition between variants since local factors like frequency of introduction and vaccine coverage play a key role. Genomic surveillance is of uttermost importance for the mitigation of the pandemic.

DNA Recovery Using Ethanol-Based Liquid Medium from FTA Card-Stored Samples for HPV Detection.

INTRODUCTION: Alternative methods of dry storage and transportation may be a viable alternative to the use of liquid storage medium for cervical samples, especially for screening programs in places with few resources. OBJECTIVE: The objective of this study is to verify the viability and efficacy of human papillomavirus DNA (HPV-DNA) detection in cervical cell samples collected and stored on a Flinders Technology Associates (FTA) card (Whatman Indicating FTA® Elute Micro Card) and subsequently recovered in ethanol-based liquid medium and to compare the results to those obtained using samples stored directly in ethanol-based liquid medium. STUDY DESIGN: Thirty-four women submitted to ETZ (excision of the transformation zone of the cervix) were included in this study. Before ETZ, 2 samples of exfoliated cervical cells were collected from each woman by a doctor and stored in ethanol-based liquid medium and on an FTA card. DNA recovery from FTA samples was performed using ethanol-based liquid medium. Detection of HPV-DNA in the samples was performed using the Cobas® 4800 HPV Test Platform. RESULTS AND CONCLUSIONS: The HPV-DNA detection positivity rates were 70.6% for the samples collected directly in liquid medium and 64.7% for the samples stored on the FTA card, with high detection accuracy in the DNA samples recovered from the FTA card (area under the curve = 0.958; 95% confidence interval = 0.890-1.000). The concordance between the results obtained using the 2 storage media was 94.1% (Kappa = 0.866). These preliminary results suggest that collection of cervical material on an FTA card may be an alternative to storage in liquid medium since the liquid medium has some limitations. In addition, DNA recovery from the card using ethanol-based liquid medium streamlines the workflow in the laboratory and reduces the cost associated with reagents, thereby facilitating access to the HPV test in places with few resources and potentially improving cervical cancer screening.

Detection of HPV E6 oncoprotein from urine via a novel immunochromatographic assay.

Cervical cancer is a significant public health problem, especially in low- and middle-income countries, where women have little access to cervical cancer screening; consequently 80% of cervical cancer related mortality occurs in these regions. The development of screening methods that need less infrastructure thus represents an urgent medical need. The study aims to compare the detection rates of high-risk human papillomavirus 16 and 18 E6 oncoprotein in urine, vaginal self-collected, and cervical scrapes of women using the OncoE6™ Cervical Test and compare the HPV16 and/or HPV18 E6 detection rates with the HPV DNA testing. Paired urine, vaginal self-collected and cervical specimens were collected from 124 women who participated in cervical cancer screening or treatment in this proof-of-concept study and underwent to HPV16/18-E6 testing and high-risk HPV DNA testing prior to treatment of cervical neoplasia or cancer. Concordance between urinary, vaginal and cervical HPV16/18-E6 and HPV-DNA testing was evaluated for patients classified as negative group (

HPV Vaccine: Updates and Highlights.

HPV is the most common sexually transmitted biological agent and is the cause of many conditions in men and women, including precancer lesions and cancer. Three prophylactic HPV vaccines targeting high-risk HPV types are available in many countries worldwide: 2-, 4- and 9-valent vaccines. All the 3 vaccines use recombinant DNA technology and are prepared from the purified L1 protein that self-assembles to form HPV type-specific empty shells. This non-systematic review aims to summarize the HPV epidemiology and the vaccine development to review the landmark trials of HPV vaccine, to present to most remarkable results from clinical trials and the real world, and to stress the challenges and the barriers for HPV vaccine implementation.

Establishment, molecular and biological characterization of HCB-514: a novel human cervical cancer cell line.

Cervical cancer is the fourth most common cancer in women. Although cure rates are high for early stage disease, clinical outcomes for advanced, metastatic, or recurrent disease remain poor. To change this panorama, a deeper understanding of cervical cancer biology and novel study models are needed. Immortalized human cancer cell lines such as HeLa constitute crucial scientific tools, but there are few other cervical cancer cell lines available, limiting our understanding of a disease known for its molecular heterogeneity. This study aimed to establish novel cervical cancer cell lines derived from Brazilian patients. We successfully established one (HCB-514) out of 35 cervical tumors biopsied. We confirmed the phenotype of HCB-514 by verifying its' epithelial and tumor origin through cytokeratins, EpCAM and p16 staining. It was also HPV-16 positive. Whole-exome sequencing (WES) showed relevant somatic mutations in several genes including BRCA2, TGFBR1 and IRX2. A copy number variation (CNV) analysis by nanostring and WES revealed amplification of genes mainly related to kinases proteins involved in proliferation, migration and cell differentiation, such as EGFR, PIK3CA, and MAPK7. Overexpression of EGFR was confirmed by phospho RTK-array and validated by western blot analysis. Furthermore, the HCB-514 cell line was sensitive to cisplatin. In summary, this novel Brazilian cervical cancer cell line exhibits relevant key molecular features and constitutes a new biological model for pre-clinical studies.

High frequency of BF mosaic genomes among HIV-1-infected children from Sao Paulo, Brazil.

HIV-1 genetic diversity information from a pediatric population is scarce. This study enrolled 128 children living with HIV/AIDS, 103 antiretroviral-treated and 25 naive, from the Sao Paulo metropolitan area. Gag, pol and env regions were amplified, and drug resistance mutations, V3 loop, tropism and viral clades were evaluated. Drug resistance mutations among naïve children infected by vertical transmission were uncommon (4.2%), whereas most ARV-experienced children showed extensive mutation patterns. Clade B predominated at the pol region, but the analysis of the three regions concatenated showed 28% with BF mosaic structures. The most common V3 motif was GPGR, followed by GWGR in clade B samples and GPGQ in clade F samples. A predicted X4 phenotype was observed in 27%, without correlation to HIV clade. These findings expand the limited information on molecular characteristics of HIV-1 among children living with HIV/AIDS in the area and may provide information useful for monitoring the epidemic.

Molecular characterisation of newly identified HIV-1 infections in Curitiba, Brazil: preponderance of clade C among males with recent infections.

As in many areas of Brazil, the AIDS epidemic in Curitiba is relatively stable, but surveillance is important to support public policy. The molecular characteristics of HIV may be instrumental for monitoring epidemic trends. We evaluated plasma HIV-1 RNA (n = 37) from 38 cases presenting with positive serology, who were among 820 consenting volunteers visiting the downtown counselling and serology testing centre. Seroprevalence was 4.6% (CI 95% 3.2-6.3) and the estimated HIV incidence, as defined by the BED assay, was 2.86 persons/years (CI 95% 1.04-4.68). An additional set of contemporaneous, anonymous samples from a local laboratory was also analysed (n = 20). Regions of the HIV-1 polymerase (n = 57) and envelope (n = 34) were evaluated for subtyping, determination of mosaic structure, primary drug resistance mutations (pDRM), envelope V3 loop motifs and amino acid signatures related to viral tropism. HIV-1 clade B was observed in 53% of cases; HIV-1C in 30% and BC mosaics in 14%, with one F genome and one CF mosaic. Clade C infection was associated with recent infections among males (p < 0.03). Stanford surveillance pDRM was observed in 8.8% of sequences, with 7% showing high level resistance to at least one antiretroviral drug. Tropism for CXCR4 co-receptor was predicted in 18% of envelope sequences, which were exclusively among clade B genomes and cases with serological reactivity to chronic infection.

Loss of Raf kinase inhibitor protein expression is associated with human papillomavirus 16 infection in anal tumors.

There has been an increase in the incidence of anal cancer in the past two decades, with squamous cell carcinoma (SCC) being the most frequent histological type identified. Among the risk factors, high-risk human papillomavirus (HPV) infection is the most pervasive. Raf kinase inhibitor protein (RKIP) is expressed in a number of normal human tissues and previous studies have demonstrated the prognostic value of the loss of RKIP expression in several gastrointestinal tumors. Therefore, the present study aimed to evaluate the clinical implications of RKIP expression in a series of neoplastic lesions of the anal canal. The resected tumors of 48 patients [8 high-grade intraepithelial lesions (HSILs), 14 adenocarcinomas and 26 squamous cell carcinomas (SCCs)] were immunohistochemically evaluated for RKIP expression, and the results were correlated with clinicopathological data. The results identified a decreased 5-year overall survival rate in patients with adenocarcinoma (40.8%) compared with patients with SCC (76.7%), and a decreased 5-year disease-free survival rate in patients at clinical stages III/IV (37.3 vs. 62.5 and 82.6% for clinical stages 0 and I/II, respectively). Low RKIP expression was revealed in 62.5% of HSILs, 88.5% of SCCs and 100.0% of the adenocarcinomas. High RKIP expression was associated with patient ethnicity (37.5% in non-Caucasians vs. 7.5% in Caucasians) and patient age (33.3% in younger patients vs. 0.0% in older patients). Finally, high RKIP expression was correlated with HPV16 infection status (40% in HPV- vs. 5.3% in HPV+ patients). A correlation was identified between high RKIP expression and lesions with a generally improved prognosis, such as those diagnosed in younger patients, in situ lesions and lesions of lower clinical grades; there was also a negative correlation between high RKIP expression and HPV16 positivity in patients.

Self-sampling coupled to the detection of HPV 16 and 18 E6 protein: A promising option for detection of cervical malignancies in remote areas.

OBJECTIVE: To evaluate both the performance and acceptability of a method coupling self-sampling with detection of cervical malignancy via elevated HPV 16 and 18 E6 oncoproteins (OncoE6™ Cervical Test) in remote areas in Brazil. METHODS: Women living in rural villages in proximity to Coari city, Amazonas, Brazil were invited to participate in a cervical cancer screening study. 412 subjects were enrolled; there were no refusals. In addition to E6 protein detection, DNA was extracted from the brushes and evaluated for HPV genotypes by PCR (PGMY09/11), followed by typing by the Papillocheck™ if positive. Subjects who were found to be positive for OncoE6 or HPV-DNA were referred for colposcopy. RESULTS: For 110 subjects (27%) this was the first cervical cancer exam. Overall the HPV-DNA prevalence was 19.1% (n = 79); 1.4% (n = 6) were positive by the OncoE6 Test. Fifty-six women attended the invitation for colposcopy where nine had an abnormal cervix and were subsequently biopsied. Histopathological analysis revealed 2 CIN3, 2 carcinomas and 5 CIN1. OncoE6 called two out of the three HPV 16 or 18 associated CIN3+ lesions. CONCLUSIONS: The findings suggest that self-administered sample collection in combination with OncoE6 Test is feasible in this population. This could enable expanded screening coverage while ensuring a high specificity which is imperative given the remote geographic location, since women bearing abnormal test results would necessitate travel and logistical burden to access colposcopy and treatment.

Characterization of topoisomerase II α and minichromosome maintenance protein 2 expression in anal carcinoma.

The present study aimed to ascertain the significance of topoisomerase II α (TOP2A) and minichromosome maintenance protein (MCM) 2 expression in anal carcinoma. A total of 75 anal lesions were retrieved from the files of the Department of Pathology of Barretos Cancer Hospital (Barretos, Brazil) in order to verify the human papillomavirus (HPV) statuses of these lesions and characterize the immunohistochemical expression levels of TOP2A and MCM2 in anal carcinoma, as these are important markers for cervical HPV-induced lesions; their expression was also compared with respect to p16 and Ki-67. The vast majority of the cases tested positive for HPV16 (84%); 1 case tested positive for both HPV16 and HPV18. Positive HPV16 status was more frequent in early stages than in advanced stages (P=0.008). Positive immunohistochemical reactivity for MCM2 and TOP2A protein was observed in 71.6 and 100% of cases, respectively. Positive reactivity for p16 was significantly associated (P=0.001) with histological grade, and was more commonly expressed in squamous cell carcinoma than adenocarcinomas. HPV16 was strongly associated with positive p16 protein expression (76.6%). However, the high expression of Ki-67 combined with the high expression of p16 was predominantly observed in Stage III-IV cases. MCM2, TOP2A, p16 and Ki-67 exhibited intense positive staining in the anal lesions, indicating that these markers were significantly and constantly expressed in anal carcinoma.

Angiosarcoma in HIV-negative patients is not associated with HHV-8.

BACKGROUND:: Angiosarcoma is an aggressive, malignant neoplasm of vascular or lymphatic origin. Herpes virus 8 (HHV-8) is a member of the herpes family with a tropism for endothelial cells and it has been proven to induce vascular neoplasms, such as Kaposi's sarcoma. The role of HHV-8 in the pathogenesis of angiosarcoma has not been well defined. OBJECTIVE:: To investigate the relationship between the presence of HHV-8 and angiosarcoma. METHODS:: In this study, the team investigated the relationship between the presence of HHV-8, as determined by polymerase chain reaction, and angiosarcoma, using samples from patients with epidemic Kaposi's sarcoma as controls. RESULTS:: While all control cases with epidemic Kaposi's sarcoma were positive for HHV-8, none of the angiosarcoma cases was. CONCLUSION:: These findings support most previous studies that found no association between HHV-8 and angiosarcoma.