Consensus Primers Research Articles

Genotyping and phylogenetic analysis of human Papillomaviruses from women with genital warts

Background and objectives. The pathophysiology of Human Papillomavirus (HPV) infections is dependent on the genotype of virus, the host immune system and the local environmental conditions. HPV infections are linked to a variety of benign and malignant disorders. Anogenital warts is a Sexually Transmitted Infection (STI) primarily caused by Low-Risk HPV (LR-HPV) genotypes 6 and 11. The aims were to Investigation of the prevalence and genotypic distribution of HPV in women with genital warts in Mosul/Iraq, HPV-DNA sequencing and Phylogenetic analysis including minimum spanning trees of amplified HPV L1 gene comparison the present sequences with similar genotypes in GenBank – National Center Biotechnology Information (NCBI) using mega-7-software. Materials and methods. A total of 150 samples were collected from women with genital wart infections. Polymerase Chain Reaction (PCR) was performed using GP+5/GP+6 and MY09/MY11 consensus primers. Results. About 84.6% prevalence of HPV genotypes. The highest frequency (32% and 28.6%) were observed in the 19-28 and 29-38 age groups. Conclusion. Women in Mosul, Iraq, who have genital warts are more likely to have different genotypes of HPV. HPV 11 type was found to be the most common, followed HPV6 and HPV10 as LR HPV, while HPV 16 and HPV45 were the most common as HR HPV. The analysis of the phylogenetic tree revealed a genetic relationship between our isolates and global isolates ranging from (10-100%) bootstrap ratio this indicating phylogenetic relationships (divergent and convergent evolution) this was meant the diversity in origin of the various genotypes common in Mosul, Iraq.

Abstract A112: The seroprevalence of nine human papillomavirus types included in the nonavalent vaccine in unvaccinated women living in Puerto Rico

Abstract Introduction Human papillomavirus (HPV) surveillance is lacking in Puerto Rico (PR). We aim to estimate the seroprevalence of the HPV types included in the nonavalent vaccine (9vHPV) in women and explore associations. Methodology A population-based cross-sectional study of socio-demographic, clinical [including, sexually transmitted infections (STIs)], and behavioral characteristics of sexually active women (16-64 years) was conducted in PR from 2010-2013. Blood samples from 524/566 (92.6%) HPV unvaccinated women were included in this sub- analysis. Antibody responses to 9vHPV types (HPV-6/11/16/18/31/33/45/52/58) were analyzed using M9ELISA by the U.S. Centers for Disease Control and Prevention (CDC). A self-collected cervical and anal sample from all 524 women were also tested using L1 consensus primer (MY09/MY11) PCR followed by dot hybridization to detect 40 HPV types. Statistical analyses included Pearson’s chi-square and logistic regression models. Adjusted odds ratios (aOR) and 95% confidence intervals (CI) were reported. Results The mean age of participants was 42.4 years ± 0.6 standard deviations (SD), 52.5% were married/cohabited, and 9.7% were uninsured. About half (51.3%) had between 3-9 lifetime sexual partners, 15.9% had history of STIs, and 40.3% had a current anogenital HPV infection. Overall, 65.8% were seropositive to at least one of the 9vHPV types (1.0% to all 9vHPV types), 59.9% to any of the seven oncogenic types (HPV-16/18/31/33/45/52/58) and 49.1% to any of the quadrivalent vaccine (4vHPV) types. The seroprevalence for specific HPV types ranged between 10.7%-28.1% [HPV-33 (10.7%), 11 (11.6%), 45 (11.8%), 58 (15.8%), 18 (18.7%), 31 (25.0%), 6 (26.2%), 52 (26.9%), and 16 (28.1%)]. In bivariate analysis, significant associations between 9vHPV types were observed with marital status, age at sexual debut, lifetime sexual partners, anal sex, history of STIs and anogenital HPV infection (p<0.05). In the multivariate model, divorced/separated/widowed women (aOR: 2.2, 95%CI: 1.2-4.3) had higher odds of seropositivity to any of the 9vHPV types than single women. Women with 3-9 (aOR: 2.4, 95%CI, 1.5-3.8) and with >10 (aOR: 3.1, 95%CI, 1.6-5.8) lifetime sexual partners had higher odds of seropositivity than women with 1-2 partners. Women with a history of STIs (aOR: 2.1, 95%CI, 1.1-4.0) and with an anogenital HPV infection (aOR: 1.9, 95%CI, 1.2-2.9) had higher odds of HPV seropositivity than their counterparts. In this model, no differences were observed by age, education, healthcare coverage, age at sexual debut or anal sex. Conclusion Before implementation of the HPV nonavalent vaccine, the seroprevalence of at least one of the 9vHPV types among women in PR was high (65.8%). However, less than 1% of women were seropositive for all nine HPV types, indicating this population would benefit from HPV vaccine implementation. Acknowledgements Study funded by the National Institute of Allergy and Infectious Diseases Grant (Grant #1SC2AI090922-01) of the National Institutes of Health. Citation Format: Angeline Cruz, Jeslie M. Ramos-Cartagena, Gitika Panicker, Elizabeth Unger, Ana P Ortiz. The seroprevalence of nine human papillomavirus types included in the nonavalent vaccine in unvaccinated women living in Puerto Rico [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr A112.

Mapping of Human Polyomavirus in Renal Cell Carcinoma Tissues.

Worldwide, the incidence of renal cell carcinoma (RCC) is rising, accounting for approximately 2% of all cancer diagnoses and deaths. The etiology of RCC is still obscure. Here, we assessed the presence of HPyVs in paraffin-embedded tissue (FFPE) resected tissue from patients with RCC by using different molecular techniques. Fifty-five FFPE tissues from 11 RCC patients were included in this study. Consensus and HPyV-specific primers were used to screen for HPyVs. Both PCR approaches revealed that HPyV is frequently detected in the tissues of RCC kidney resections. A total of 78% (43/55) of the tissues tested were positive for at least one HPyV (i.e., MCPyV, HPyV6, HPyV7, BKPyV, JCPyV, or WUyV). Additionally, 25 tissues (45%) were positive for only one HPyV, 14 (25%) for two HPyVs, 3 (5%) for three HPyVs, and 1 one (1%) tissue specimen was positive for four HPyVs. Eleven (20%) RCC specimens were completely devoid of HPyV sequences. MCPyV was found in 24/55 RCC tissues, HPyV7 in 19, and HPyV6 in 8. The presence of MCPyV and HPyV6 was confirmed by specific FISH or RNA-ISH. In addition, we aimed to confirm HPyV gene expression by IHC. Our results strongly indicate that these HPyVs infect RCC and nontumor tissues, possibly indicating that kidney tissues serve as a reservoir for HPyV latency. Whether HPyVs possibly contribute to the etiopathogenesis of RCC remains to be elucidated.

Equus caballus papillomavirus Type 7 is a rare cause of equine penile squamous cell carcinomas

Penile squamous cell carcinomas (SCCs) are common, potentially life-threatening neoplasms of horses. They are well-recognized to be caused by Equus caballus papillomavirus (EcPV) type 2, although EcPV2 cannot be detected in all cases. A 23-year-old standardbred gelding developed multiple penile in situ and invasive SCCs that contained histological evidence of PV infection. By using both consensus and specific PCR primers, these lesions were found to contain EcPV7 DNA, but not DNA from EcPV2 or any other PV type. To determine how frequently EcPV7 is present in equine penile SCCs, specific primers were used to detect EcPV2 and EcPV7 in a series of 20 archived samples. EcPV7 was the only PV detected in one, both EcPV2 and 7 were detected in five, and only EcPV2 was detected in 14 SCCs. EcPV7 DNA was also detected in three of 10 archived oropharyngeal SCCs, although only as a co- infection with EcPV2. This is the first report of EcPV7 causing disease in horses. These results suggest EcPV7 could cause a subset of equine penile SCCs, and this is the first evidence that PV types other than EcPV2 can cause these neoplasms. The detection of EcPV7 in the oropharyngeal SCCs suggests a potential role of this PV type in the development of these SCCs. There were no clinical or histological features that differentiated lesions containing EcPV7 DNA from those containing EcPV2 DNA. If EcPV7 causes a proportion of equine penile SCCs, vaccines to prevent EcPV2 infection may not prevent all equine penile SCCs.

HPV Detection on Lung Interstitium Through Bronchoalveolar Lavage (BAL) in Patients from City Recife, Pernambuco-Brazil

Bronchoalveolar lavage (BAL) is a non-invasive tool that allows direct sampling of the lung interstitium, facilitating cytological, microbiological, and molecular analyses. The Human Papillomavirus (HPV), known for its oncogenic role mainly in the genital regions, also can be found in the lungs, but its pathological implications remain poorly elucidated. This study aims to identify the presence of HPV-DNA in patients with suspected lung diseases through BAL. A cross-sectional and analytical study was carried out involving 146 patients admitted to hospitals in the Metropolitan Region of Recife. To detect HPV DNA, 46 samples were processed using PCR technique with MY09/11 consensus primers, followed by Nested-PCR with GP5/6 primers. The presence of HPV-DNA was confirmed in 4 (8.69%) samples, all benign and suspected of interstitial diseases. These findings indicate the feasibility of detecting HPV in the lung interstitium by BAL using Nested-PCR and suggest a new perspective in understanding the presence of HPV in lung tissue. In addition, to corroborating the statement that HPV is not limited to tissue and malignant samples, offering a new methodological dimension to the HPV investigation on lung.

ConsensusPrime—A Bioinformatic Pipeline for Efficient Consensus Primer Design—Detection of Various Resistance and Virulence Factors in MRSA—A Case Study

Background: The effectiveness and reliability of diagnostic tests that detect DNA sequences largely hinge on the quality of the used primers and probes. This importance is especially evident when considering the specific sample being analyzed, as it affects the molecular background and potential for cross-reactivity, ultimately determining the test’s performance. Methods: Predicting primers based on the consensus sequence of the target has multiple advantages, including high specificity, diagnostic reliability, broad applicability, and long-term validity. Automated curation of the input sequences ensures high-quality primers and probes. Results: Here, we present a use case for developing a set of consensus primers and probes to identify antibiotic resistance and virulence genes in Staphylococcus (S.) aureus using the ConsensusPrime pipeline. Extensive qPCR experiments with several S. aureus strains confirm the exceptional quality of the primers designed using the pipeline. Conclusions: By improving the quality of the input sequences and using the consensus sequence as a basis, the ConsensusPrime pipeline pipeline ensures high-quality primers and probes, which should be the basis of molecular assays.

Bovine Meat and Milk Factor-like Sequences Are Frequently Detected in Renal Cell Carcinoma Tissues.

Previous studies have indicated a potential role of diet in the pathogenesis of renal cell carcinoma (RCC). Recently, circular bovine meat and milk factor (BMMF) DNAs have been identified in peritumoral tissues of human colon and breast cancers. Here, we investigated the prevalence of the DNA of these novel human pathogenic infectious agents in RCC and adjacent peritumoral renal tissues. DNA was extracted from formalin-fixed and paraffin-embedded (FFPE) RCC and peritumoral kidney tissues, including a test (n = 11) and a validation (n = 152) collection. BMMF1 and BMMF2 consensus primers were designed to screen for the presence of BMMF1- and BMMF2-like DNA. In addition, BMMF-specific PCR was performed on selected cases to test for the presence of additional regions of BMMF1 and BMMF2 genomes. A reference collection of hepatocellular carcinomas (HCCs; n = 60) and adjacent peritumoral liver tissues (n = 50) was also included. Our results demonstrated that BMMF1 and BMMF2 DNAs are frequently found in human RCC tissues and are particularly more prevalent in peritumoral kidney tissues. Of note, BMMF1 and BMMF2 genotype heterogeneity was higher in peritumoral kidney tissues compared to RCC tissues. This is the first study to directly test human FFPE tissues for BMMF1- and BMMF2-like DNA using consensus PCR and demonstrate BMMF DNA in neoplastic and peritumoral kidney tissues. The findings are in line with the recently proposed indirect etiopathogenetic role of BMMFs in, e.g., colorectal carcinogenesis. Follow-up studies are needed to explore the potential role of BMMFs in the etiopathogenesis of RCC.

High-risk human papillomaviruses l1 gene isolates identified in Western Kazakhstan

Kazakhstani researchers reported a significant prevalence of highly carcinogenic human papillomavirus types in the country.<br /> The article <b>aimed</b> to present HPV L1 gene sequencing developments in women affected with cervical cancer throughout the western part of Kazakhstan with provided findings on the geographic pathways of obtained isolates.<br /> <b>Methods. </b>The HPV L1 gene was amplified using the consensus primers MY09HPV 5’-CGTCCMARRGGAWACTGATC-3’ and MY11HPV 5’ – GCMCAGGGWCATAAYAATGG-3’.  The purified DNA was used as the target for direct nucleotide sequencing. Phylogenetic analyses were conducted using the MegAlign program from the LASERGENE software package (version 6.0; DNA star, Madison, WI) and with MEGA version 5.0 software. A multiple alignment was created through Clustal W software, and the neighbor-joining method was used to construct the phylogenetic tree.<br /> <b>Results.</b> Of 70 HPV samples transported to the Astana shared laboratory for gene L1 sequencing, only ten appeared fit to obtain isolates (14.3%). The viral load of the samples ranged from 3.3 to 8.2, and the range of DNA concentration was from 8.16 to 69.6 ng/uL. HPV16 unique Kazakhstani isolate from Aktobe, having its own branch, and not yet registered in the world genebank, was revealed. An isolate of potentially carcinogenic HPV53 forming a remote cluster with KF436822/1, KU951264.1 - Southwest China, and 97% identity with EU056643.1 - Ireland, and acted as a single agent for invasive cervical cancer was identified.<br /> In general, the sequencing findings indicate the variety of ways for HPV pervasion into the western region of Kazakhstan: North and South America, Europe, and Asia.<br /> <i><i>The study was recorded in the ISRCTN registry, No. 7154910, 02/01/2018.</i></i>

Investigation of the Presence of Bovine Papillomavirus in Blood Leukocytes of Cattle in West Mediterranean of Turkey

One hundred Holstein cattle, varying in age from one to ten years, who had warts on different parts of its bodies and had previously had Bovine papillomavirus (BPV) typing had blood samples taken from them from various managements in Burdur-Center and its surroundings. DNA was extracted from leukocytes using the DNeasy Blood & Tissue Kit (Qiagen, Germany). RT-PCR test was performed using consensus and type-specific primers for the detection of BPV types. As a result of the study, RT-PCR test and consensus primers were not detected in blood leukocyte samples. Through the use of type-specific primers in an RT-PCR assay, the viral genome components of BPV-10 (56), BPV-11 (1), BPV-10+BPV-11 (2), BPV-10+BPV-12, and BPV-10+BPV-14 (2) were detected in these samples. BPV-12 was detected for the first time in blood leucocytes. In blood samples taken from animals classified according to the anatomical regions where the warts are located the highest BPV positivity (22,2%) was detected in animals with warts on the teats. Also, the most BPV-10 type was determined in the blood samples of animals with wart lesions on the teat and lobes. When the distribution of BPV PCR positive samples according to age groups was made, BPV PCR positive was found mostly in the between 1 and 47 month age group (71,4%). It was determined that the prevalence of BPV was high in the between 1 and 47 age group, while the rates of 48-83 months and later decreased with increasing age. In this investigation, it was found that BPV type 10 had been prevalent in blood leukocyte samples from cattle with warts in various areas of the body and with variable macroscopic appearance.

Molecular detection and identification of Plasmodium spp. isolated from captive-bred cynomolgus monkeys in Bogor, Indonesia

Background and Aim: Asian macaques are natural hosts of several Plasmodium species. Some monkey malaria parasites may infect humans and cause zoonotic infections. This study was conducted to estimate the prevalence of monkey malaria parasites in Bogor, Indonesia, based on molecular detection and identification, particularly in cynomolgus monkeys, which have a wide geographic distribution and share extensive habitats with humans. These data are needed to evaluate the status of simian malaria among macaques in Bogor and to study the potential risks to human health. These updated data will provide sufficient information for implementing malaria control strategies in the future and for developing a potential malaria vaccine using monkeys as an animal model. Materials and Methods: Blood samples of 274 cynomolgus monkeys (Macaca fascicularis) were collected and identified using microscopy. DNA was extracted from positive blood samples and analyzed using polymerase chain reaction (PCR) to amplify the small subunit ribosomal RNA (SSU rRNA) target gene using consensus primers for Plasmodium species. The PCR-positive samples were then nucleotide-sequenced using commercial sequencing services, analyzed using the BioEdit program, and aligned using Basic Local Alignment Search Tool from the National Center for Biotechnology Information. Phylogenetic trees were constructed using MEGA 11.0 and the neighbor-joining (NJ) method to determine the kinship of Plasmodium. Bootstrapping was performed using 500 replicates to assess the robustness of tree topologies. Results: Thirty-eight of the 274 microscopically positive samples for Plasmodium spp. were also positive using PCR, resulting in a 1640 bp amplicon. Further, analysis using nucleotide sequencing confirmed that these positive samples were Plasmodium inui with more than 99% nucleotide identity compared to GenBank sequences. Phylogenetic tree analysis of the SSU rRNA partial gene showed that all our isolates clustered and were closely related to a P. inui strain isolated from cynomolgus macaques in South China in 2011. Conclusion: P. inui is the predominant malaria parasite in cynomolgus monkeys from Bogor. Keywords: malaria, phylogenetic tree, Plasmodium inui, small subunit ribosomal RNA.

Prenatal Transmission of High- Risk HPV 16 And HPV 18 Among Antenatal Mothers In Western Nigeria

Several reported evidences do exist that the transmission of human papilloma (HP) viral infection may occur via non-sexual routes while others show the possibility of detecting HP-viral-DNA in reproductive cells, cord blood, and placental cells. However, there is paucity of background information on HP-viral infection transmission among mothers and newborns which necessitates study. The study aims at determining prenatal transmission of high-risk human papillomavirus genotypes HPV 16 and HPV 18 among antenatal mothers and maternal cervical cells in Western Nigeria. We investigated 113 pregnant women from whom cervical samples were collected during the antenatal clinic and their newborns at the University of Ilorin Teaching Hospital. HP-viral screening, DNA extraction and molecular detection was carried out using MY09/MY11 and GP5+/GP6+ consensus primers while HP-viral high risk genotypes were detected using type-specific primers. HP-viral DNA was detected among 54 (47.8%) cord blood samples and 11 (9.7%) cervical specimens with nine specimens from mothers while newborns samples showed a corresponding HP-viral-DNA results. The genotypic characterization/identification of HP-virus type 16 and 18 from both specimens showed an overall prevalence of 15.0% (7/113) for HPV 16 and 8.0% (9/113) for HPV-18, while only three specimens from corresponding mothers and newborns had HPV-16. Such observation indicates/suggests that mothers are the most probable transmission source of HP-viral-DNA to newborns. Furthermore, the presence of HPV-16 (a high-risk genotype) could portend HP-viral diseases in newborns. In order to avoid transmission during pregnancy, routine monitoring and surveillance are advised for mothers-to-be and those who have recently given birth. HPV vaccinations are required during family planning prior to pregnancy.

Diversity of classic and novel human astrovirus in outpatient children with acute gastroenteritis in Shanghai, China.

Human astrovirus (HAstV) is an important pathogen of acute gastroenteritis (AGE) in children. This study was aimed at investigating the diversity and epidemiology of classic and novel HAstV in outpatient children aged 0-16 years old with AGE in Shanghai. From May 2020 to December 2022, a total of 1,482 stool samples were collected from children diagnosed as AGE from the Children's Hospital of Fudan University. HAstV was identified using pan-astrovirus consensus primers by Reverse transcription PCR. During the study period, 3.3% (49/1,482) of specimens were identified as HAstV, with a detection rate of 2.5% (37/1,482) for classic HAstV and 0.8% (12/1,482) for novel HAstV. Among the 12 novel HAstV strains, 11 (91.7%) belonged to the HAstV-MLB and 1 (8.3%) was HAstV-VA. Genotyping revealed six circulating genotypes. Strain HAstV-1 was predominant in the study population with a detection rate of 1.8% (26/1,482) followed by HAstV-MLB1 (0.7%, 10/1,482) and HAstV-4 (0.6%, 9/1,482). Of note, all the HAstV-4 strains detected in this study were close to one astrovirus strain isolated from Bactrian camels with 99.0-100.0% amino acid sequences identity. In this study, HAstV was detected in all age groups with the highest detection rate of HAstV-positive specimens observed in children older than 73 months (5.7%, 12/209). This study provided useful information and contributed to the molecular epidemiology of both classic and novel HAstV, which were simultaneously characterized and reported for the first time in Shanghai.

Human Papillomavirus genotypes distribution and associated risk factors among women living in Southern Morocco

PurposeThe aim of this work was to investigate potential risk factors associated with HPV infection and to determine the HPV genotype prevalence among women from the Southerns areas of Morocco. MethodsA total of n = 308 sexually active women provided their written consent to participate in this study. A detailed questionnaire was used to collect data, related to the age and life style of participants. HPV L1 gene detection was performed by a nested PCR method using consensus primers. HPV genotypes were determined using direct Sanger sequencing method. Statistical analysis of the results obtained was carried out using R software. ResultsWe detected HPV in 42.5 % of the total investigated samples. HPV infection was significantly associated with the following risk factors: age of the first intercourse (OR = 2.27, 95 % CI: 1.03–5.10, p = 0.044), and previous history of STIs (OR = 3.13, 95 % CI: 1.12–9.63, p = 0.034). High risk-HPV and Low risk-HPV genotypes were found in 26.6 % and 5.5 % of the participants, respectively. The most prevalent genotypes were HPV16 (22 %), HPV18 (2.6 %), HPV11 (1.6 %) and HPV83 (1.3 %). Multiple infections were found in 3.2 %. The genotypes covered by the bivalent, quadrivalent and nonavalent HPV vaccines account for 57.5 %, 62.8 % and 66.6 %, respectively. ConclusionHPV infection prevalence reported in this study among women from the Southerns areas of Morocco is one of the highest reported in the country so far. Our finding confirm that the current HPV vaccines directed against the most prevalent HPV16 genotype, recently introduced in the country, should offer a good protection to the most vulnerable population if implemented properly. These results highlight the importance of HPV screening and vaccination programs in this region.

Single-Step IGHV Next-Generation Sequencing Detects Clonality and Somatic Hypermutation in Lymphoid Malignancies: A Phase III Diagnostic Accuracy Study.

Multiplex PCR based on consensus primers followed by capillary electrophoresis and Sanger sequencing are considered as the gold standard method for the evaluation of clonality and somatic hypermutation in lymphoid malignancies. As an alternative, the next-generation sequencing (NGS) of immune receptor genes has recently been proposed as a solution, due to being highly effective and sensitive. Here, we designed a phase III diagnostic accuracy study intended to compare the current gold standard methods versus the first commercially available NGS approaches for testing immunoglobulin heavy chain gene rearrangements. We assessed IGH rearrangements in 68 samples by means of both the NGS approach (LymphoTrack® IGH assay, and LymphoTrack® IGH somatic hypermutation assay, run on Illumina MiSeq) and capillary electrophoresis/Sanger sequencing to assess clonality and somatic hypermutations (SHM). In comparison to the routine capillary-based analysis, the NGS clonality assay had an overall diagnostic accuracy of 96% (63/66 cases). Other studied criteria included sensitivity (95%), specificity (100%), positive predictive value (100%) and negative predictive value (75%). In discrepant cases, the NGS results were confirmed by a different set of primers that provided coverage of the IGH leader sequence. Furthermore, there was excellent agreement of the SHM determination with both the LymphoTrack® FR1 and leader assays when compared to the Sanger sequencing analysis (84%), with NGS able to assess the SHM rate even in cases where the conventional approach failed. Overall, conventional Sanger sequencing and next-generation-sequencing-based clonality and somatic hypermutation analyses gave comparable results. For future use in a routine diagnostic workflow, NGS-based approaches should be evaluated prospectively and an analysis of cost-effectiveness should be performed.

Detection of novel coronaviruses from dusky fruit bat (Penthetor lucasi) in Sarawak, Malaysian Borneo.

Sarawak has one of the highest diversity of fruit bats species (family Pteropodidae) in Malaysia, with 19 species described. Most coronavirus (CoV) studies have mainly focused on insectivorous bats, resulting in a lack of information on CoVs present in frugivorous bats. In addition, bat CoV surveillance activities are lacking in Malaysia. Our study focuses on determining the presence of bat CoVs in dusky fruit bat (Penthetor lucasi). Guano samples belonging to P. lucasi were collected from Wind Cave Nature Reserve. The samples were screened for the presence of CoVs using validated hemi-nested consensus RNA-dependent RNA polymerase consensus primers. The bat CoV positivity rate was 38.5% (n=15/39), with the viruses belonging to two subgenera: Alphacoronavirus (α-CoV) and Betacoronavirus (β-CoV). Phylogenetic analysis revealed that CoVs from 14 samples of P. lucasi belong to the genus α-CoV and may represent previously described genetic lineages in insectivorous bats in Wind Cave. However, only one sample of P. lucasi was detected with β-CoV which is closely related to subgenus Nobecovirus, which is commonly seen in frugivorous bats. This study provides the first available data on CoVs circulating in P. lucasi.

The first identification of Tula orthohantavirus in forest dormice (Rodentia: Gliridae) from Iran

Abstract Orthohantaviruses are a group of zoonotic pathogens that can cause severe and potentially fatal syndromes in humans. Although orthohantaviruses have been documented in Iran, little is known about their potential reservoirs in the country. The present study constitutes the first attempt to detect orthohantavirus infections in rodents in Iran. Sampling was performed in the East Azerbaijan province, during October 2017 and June 2018. To detect orthohantavirus infection, we performed a Pan-hantavirus nested reverse transcription-polymerase chain reaction targeting the large segment of the viral genome using consensus primers on lung tissue samples. We identified Tula orthohantavirus for the first time in Iran and from Dryomys nitedula sensu lato (s.l.) (Gliridae), a non-muroid host. There are two possible interpretations for this finding: an interspecies spillover or the presence of a broader primary host/reservoir list. However, at present, we do not advocate for either interpretation, but instead, argue for further accumulation of relevant data. Phylogenetic analysis revealed a close relationship between the Tula orthohantavirus identified in this study and those detected in Turkey. Nevertheless, comprehensive field studies, as well as full-length sequence analyses, are required to gain a better understanding of the potential reservoirs, ecology, and epidemiological cycle of the virus in Iran.

COMBINED USE OF MINIMAL RESIDUAL DISEASE MONITORING AND FDG‐PET FOR OUTCOME PREDICTION IN FOLLICULAR LYMPHOMA: RESULTS FROM THE FONDAZIONE ITALIANA LINFOMI (FIL) FOLL12 TRIAL

Introduction: In follicular lymphoma (FL), FDG-PET is routinely employed to evaluate response at the end of induction (EoI), while minimal residual disease (MRD) analysis is highly reliable in predicting relapse over the entire disease history. Nonetheless, scant data are available describing the integration of these powerful prognostic tools for response evaluation in FL. This issue was investigated in the phase III FIL “FOLL12” trial [Luminari, JCO 2022]. Methods: Only patients with an available molecular marker for MRD and centrally reviewed EoI PET were included. MRD was systematically assessed by RQ-PCR with BCL2::IGH consensus primers in peripheral blood (PB) and bone marrow (BM), as previously reported with inferior follow-up [Ladetto ASH 2021]. PET scans were classified according to Deauville score (DS) criteria: DS1–3 was negative and DS4–5 positive. Results: Overall, 394 patients out of 780 were included (51%), as expected based on the MRD marker availability. These patients had a slightly better outcome than excluded cases (CR rate 82% vs. 73%, p = 0.003, 5y PFS 66% vs. 61%, p = 0.046). At EoI, both BM MRD and PET positivity were independent predictors of poor PFS in multivariate analysis (HR 1.66, CI: 1.01–2.71 and HR 2.03, CI: 1.30–3.18). The 68 discordant cases (i.e., MRD+/PET− or MRD−/PET+) showed an intermediate outcome (PFS HR 1.93, CI: 1.28–2.92) between the 314 double negative (reference) and the 10 double positive ones (HR 6.22, CI: 2.99–12.9), Figure 1A. Moreover, both EoI PET+ and the persistence/reappearance of MRD positivity in BM during the 18 months of rituximab maintenance/observation were independently associated to a higher risk of POD24 (HR 5.61, CI: 2.59–12.1, and HR 2.37, CI: 1.15–4.84, respectively). On the other hand, MRD kinetic analysis by non-invasive PB sampling during the rituximab maintenance/observation period substantially updated the risk status over PET results. Indeed, the marginal PFS HR for an MRD+ after an EoI PET negative result increased from 1.36 (CI: 0.47–3.96) at 6 months, to 2.09 (CI: 1.01–4.18) at 12 months, up to 3.60 (CI: 2.30–5.64) at 24 months (Figure 1B, blue curve). This means that patients scoring PET- at EoI but showing a persistent or reappearing MRD+ signal during the following 24 months had a dismal PFS, superimposable to that of PET+ patients (Figure 1C). Accordingly, in landmark multivariate analysis PB MRD+ either at 12 or 24 months predicted adverse PFS (HR 3.79, CI: 1.60–8.96 and HR 5.21, CI: 1.88–14.4, respectively), independently from EoI PET result. Keywords: indolent non-Hodgkin lymphoma, minimal residual disease, PET-CT Conflicts of interests pertinent to the abstract S. Ferrero Consultant or advisory role: Janssen, EUSA Pharma, Abbvie, Incyte, Clinigen, Sandoz, Italfarmaco Honoraria: Janssen, EUSA Pharma, Servier, Gentili Research funding: Morphosys, Janssen, Gilead, Beigene Educational grants: Takeda, Janssen, EUSA Pharma

Northern and central Chile still free of emerging flaviviruses in mosquitoes (Diptera: Culicidae).

Geographic isolation and strict control limits in border areas have kept Chile free from various pathogens, including Flavivirus. However, the scenario is changing mainly due to climate change, the reintroduction of more aggressive mosquitoes, and the great wave of migration of people from endemic countries in recent years. Hence, it is necessary to surveillance mosquitoes to anticipate a possible outbreak in the population and take action to control it. This study aimed to investigate the presence of Flavivirus RNA by molecular tools with consensus primers in mosquitoes collected in the extreme north and central Chile. From 2019 to 2021, a prospective study was carried out in localities of Northern and part of Central Chile. Larvae, pupae, and adults of mosquitoes were collected in rural and urban sites in each locality. The collected samples were pooled by species and geographical location and tested using RT-PCR and RT-qPCR to determine presence of Flavivirus. 3085 specimens were collected, the most abundant specie Culex quinquefasciatus in the North and Aedes (Ochlerotatus) albifasciatus in the Center of Chile. Both genera are associated with Flavivirus transmission. However, PCR and RT-PCR did not detect Flavivirus RNA in the mosquitoes studied. These negative results indicate we are still a free Flavivirus country, which is reaffirmed by the non-existence of endemic human cases. Despite this, routine surveillance of mosquitoes and the pathogens they carry is highly recommended to evaluate each area-specific risk of vector-borne transmission.

Exploring the Astrovirome of Shellfish Matrices Using Nanopore Sequencing.

Astroviruses are important human enteric pathogens transmissible with contaminated food and water. Astroviruses have also been identified in mammals, birds, lower vertebrates and invertebrates. The genetic diversity of human and animal astroviruses poses a challenge for diagnostics and taxonomy. As a proof of concept, we used a panastrovirus consensus primer set, able to amplify in a nested RT-PCR protocol a 400-nt-long fragment of the RNA-dependent RNA polymerase of most members of the Astroviridae family, in conjunction with a nanopore sequencing platform, to generate information on the astrovirome in filter-feeding mollusks. Amplicons generated from bivalve samples were used to generate libraries for deep sequencing. In three samples, only one unique RdRp sequence type was obtained. However, in seven samples and in three barcodes with eleven pooled samples, we identified a variety of known and unknown RdRp sequence types, in most cases distantly related to astrovirus sequences available in the databases. In total, 37 different sequence contigs were generated. Avian-origin astrovirus sequences were predominant, likely due to contamination of shellfish harvesting waters by marine birds. Astroviruses of the aquatic eco-system were also identified, whereas human astroviruses were not detected.

Demographic profile and risk factors of head-and-neck squamous cell carcinoma in west Tamil Nadu: A cross-sectional observational study

ABSTRACT JOURNAL/crsat/04.03/02201859-202306020-00007/figure1/v/2023-08-03T140821Z/r/image-tiff JOURNAL/crsat/04.03/02201859-202306020-00007/figure2/v/2023-08-03T140821Z/r/image-tiff Background: Head-and-neck squamous cell carcinomas (HNSCC) comprise 30% of all cancer cases in India. The risk factors for HNSCC include tobacco and alcohol consumption. Objectives: Our primary objective was to evaluate the epidemiological profile of patients with HNSCC in the western Tamil Nadu population. Our secondary objective was to assess the association of the epidemiological profile with different anatomical sites and risk factors. Materials and Methods: This was a cross-sectional study of data compiled from the clinical records of Sri Ramakrishna Institute of Oncology and Research Department, Sri Ramakrishna Hospital, Coimbatore (Tamil Nadu, India) from January 2018 to December 2021. Clinicodemographic data like age, sex, primary tumor site, clinical staging, family history, and exposure to risk factors (smoking, tobacco chewing, alcohol) were collected from patients with histologically confirmed HNSCC using a specially designed questionnaire. Human papillomavirus (HPV) status was determined by p16 immunohistochemistry and validated using polymerase chain reaction (PCR) by employing consensus primers for HPV deoxyribonucleic acid (DNA). Results: We enrolled 150 patients. The male-to-female ratio was 3:1. The mean age was 54.4 ± 10.2 years; majority of patients (59 [39.3%]) were aged between 51 and 60 years. The most frequently affected site was the oral cavity (58 [38.7%]). Tobacco chewing was the most common and the only risk factor observed among female patients (23 [62.2%]), while smoking along with alcohol consumption was commonly observed among male patients (24 [21.2%]). Smoking in combination with other risk factors (44 [29.3%]) was more common than smoking alone (9 [8%]). Tobacco chewing was a predominant risk factor for nasopharyngeal (4 [63.6%]) and oral cavity cancer (19 [32.8%]), while smoking was a predominant risk factor for laryngeal cancer (3 [30%]). Smoking with alcohol consumption was largely seen in oropharyngeal cancer (5 [33.3%]), while all three risk factors were observed in hypopharyngeal cancer (4 [14.3%]). Out of 38 samples tested, only four were positive for HPV (10.5%). Conclusions: The most affected demographic group with regard to HNSCC is middle-aged men (51–60 years), and the oral cavity is the most commonly affected site. Tobacco chewing is the most prevalent risk factor among women while smoking and alcohol consumption are the most common risk factors among men. Spreading awareness about the risk factors of HNSCC and their prevention is crucial to optimizing disease control.