RESUMO
Trypanosomatids are obligate parasites of animals, predominantly insects and vertebrates, and flowering plants. Monoxenous species, representing the vast majority of trypanosomatid diversity, develop in a single host, whereas dixenous species cycle between two hosts, of which primarily insect serves as a vector. To explore in-depth the diversity of insect trypanosomatids including their co-infections, sequence profiling of their 18S rRNA gene was used for true bugs (Hemiptera; 18% infection rate) and flies (Diptera; 10%) in Cuba. Out of 48 species (molecular operational taxonomic units) belonging to the genera Vickermania (16 spp.), Blastocrithidia (7), Obscuromonas (4), Phytomonas (5), Leptomonas/Crithidia (5), Herpetomonas (5), Wallacemonas (2), Kentomonas (1), Angomonas (1) and two unnamed genera (1 + 1), 38 species have been encountered for the first time. The detected Wallacemonas and Angomonas species constitute the most basal lineages of their respective genera, while Vickermania emerged as the most diverse group. The finding of Leptomonas seymouri, which is known to rarely infect humans, confirms that Dysdercus bugs are its natural hosts. A clear association of Phytomonas with the heteropteran family Pentatomidae hints at its narrow host association with the insect rather than plant hosts. With a focus on multiple infections of a single fly host, using deep Nanopore sequencing of 18S rRNA, we have identified co-infections with up to 8 trypanosomatid species. The fly midgut was usually occupied by several Vickermania species, while Herpetomonas and/or Kentomonas species prevailed in the hindgut. Metabarcoding was instrumental for analysing extensive co-infections and also allowed the identification of trypanosomatid lineages and genera.
Assuntos
Coinfecção , Filogenia , RNA Ribossômico 18S , Trypanosomatina , Trypanosomatina/genética , Trypanosomatina/classificação , Trypanosomatina/isolamento & purificação , Animais , Cuba/epidemiologia , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/análise , Coinfecção/parasitologia , Dípteros/genética , Hemípteros/parasitologia , Hemípteros/genética , DNA de Protozoário/genética , DNA de Protozoário/análiseRESUMO
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.
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Cervical cancer is a serious public health problem in Brazil, especially in Manaus (Amazonas), the city with the highest incidence rate of cervical cancer in the country. Persistent infection with oncogenic human papillomavirus (HPV) genotypes is the cause of disease development. The aim of this study was to investigate the prevalence of oncogenic genotypes in women at high risk for cervical precancer examined in two policlinics in Manaus. One hundred and two patients who underwent colposcopy took part in the research. The DNA samples obtained from the cervical epithelium were analyzed by PCR with type-specific primers for the detection of eight oncogenic genotypes, which were chosen based on previous studies. The presence of HPV virus was detected in all samples. The most prevalent oncogenic genotypes were 18 (47.1%) and 16 (45.1%). Interestingly, HPV 18 was considered uncommon in this region. In addition to these, genotypes 31 (19.6%), 58 (19.6%), 33 (18.6%), and 45 (15.7%) also had a relatively high frequency in this population. Fifty-six women (54.9%) had multiple infections with up to five oncogenic types. Also, the presence of genotypes other than 16 and 18 was observed in most samples (57.8%), which also deserves attention since they are not covered by currently available vaccines against HPV in Brazil. The high prevalence and multiple infections with several oncogenic HPV genotypes in association with precursor lesions for cervical cancer highlighted the need to improve strategies to prevent this disease in Amazonas.
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Introducción: El significado biológico de las infecciones múltiples con virus del papiloma humano de alto riesgo oncogénico (VPH-AR), pertenecientes a la familia Alphapapillomavirus, en la carcinogénesis cervical aún es controversial. Objetivo: Proporcionar información sobre la circulación del VPH-AR del género Alphapapillomavirus-especie 9, e infecciones múltiples en mujeres ecuatorianas con lesiones intraepiteliales y cáncer cervicouterino (CaCU). Métodos: Se estudiaron 300 mujeres, residentes en la región Litoral del Ecuador. Se detectó la infección viral en muestras cervicales, mediante PCR anidada con cebadores genéricos MY09/11 y GP5/GP6. Los genotipos virales fueron identificados con el sistema comercial ANYPLEX II VPH28. La razón de prevalencia (RP) fue utilizada como medida de asociación entre las lesiones citológicas y las infecciones simples, múltiples o combinaciones de genotipos. Resultados: Se detectó VPH en el 92,00 % (276/300) de las mujeres, con frecuencias altas de infección por genotipos individuales, principalmente de alto riesgo oncogénico. Los VPH-AR más frecuentes fueron VPH58 (18,17 por ciento), 70 (8,64 por ciento), 53 (8,34 por ciento), 35 (7,45 por ciento), 16 (7,37 por ciento), 33 (6,55 por ciento), 31 (5,58 por ciento) y 18 (4,24 por ciento). En el 91,66 por ciento (253/276) de las muestras se detectaron infecciones múltiples, hasta con 13 tipos en una misma paciente, incluyendo varias especies del género Alphapapillomavirus. La combinación VPH16/VPH58 fue la más frecuente en lesiones de alto grado (RP = 2,9; p = 0,000), y la coinfección triple VPH16/VPH58/VPH70 predominó en las mujeres con CaCU (RP = 3,5; p = 0,007). Conclusión: Los resultados demuestran que la combinación VPH16/VPH58 del género Alphapapillomavirus, especie 9, podría ser un factor clave en la aparición de lesiones premalignas y su progresión hacia el CaCU(AU)
Introduction: It is still controversial the biological connotation of multiple infections with high-risk human papillomaviruses (hrHPV), that belong to the genus Alphapapillomavirus, for the cervical carcinogenesis. Objective: To provide information on the circulation of hrHPV, genus Alphapapillomavirus, specie 9, and the multiple infections in Ecuadorian women with intraepithelial lesions and cervical cancer. Methods: 300 women, from the coastal region of Ecuador, were screened. Viral infection was detected in cervix samples by nested PCR with MY09/11 and GP5/GP6 generic primers. Viral genotypes were identified using the commercial kit ANYPLEX II VPH28. The prevalence ratio (PR) was used to measure the association between cytological lesions and the simple, multiple or combined genotype infections. Results: Ninety-two percent of women (276/300) tested positive for HPV. Frequency of infection for single genotypes was high, mainly those of high oncogenic risk. The most frequent hrHPV genotypes were HPV58 (18.17 percent), 70 (8.64 percent), 53 (8.34 percent), 35 (7.45 percent), 16 (7.37 percent), 33 (6.55 percent), 31 (5.58 percent) and 18 (4.24 percent). In 91.66 percent (253/300) of the samples, multiple infections were detected, with up to 13 types in a single patient, including various species from the genus Alphapapillomavirus. The combination HPV16/HPV58 was the most frequent on high-grade lesions (PR = 2.9; p = 0,000), and HPV16/HPV58/HPV70 triple co-infection prevailed in women with cervical cancer (PR = 3.5; p = 0.007). Conclusions: The results evidence that the combination HPV16/HPV58, genus Alphapapillomavirus, specie 9, could be a key factor in the occurrence of premalignant lesions and their evolution into cervical cancer(AU)
Assuntos
Humanos , Feminino , Adulto , Pessoa de Meia-Idade , EquadorRESUMO
Background: The human papillomavirus (HPV) is the most frequent etiological agent driving development of cervical cancer (CC); therefore typing and classifying the status of these infections are of great importance for treatment. The frequency of the various HPV types may change in relation to low-grade lesions and have the potential to cause more severe lesions. Objective: The purpose of this study was the identification and typing of HPV in a rural population in Mexico. Methods: Detection and typing were determined by PCR-RFLPs and confirmed by viral DNA sequencing. Results: HPV was detected in 17.28% of the samples, this was 3.58% higher than had been determined in a rural population in Central Mexico. Viral types 16, 18 and 52 were found most frequently. Analysis of all HPV-positive samples revealed that 14.3% had a single infection; 57.1% had a double infection; and 28.6% had a triple infection. Thus, 85.7% of positive cases presented with multiple infections with HPV16 being the most prevalent. Only the lifetime number of sexual partners was found to have an association with the colposcopic diagnoses (OR = 7.08; 95% CI: 1.68-29.8; p > 0.008). Conclusion: A higher frequency of multiple HPV infections was found among our test population compared to other rural populations in Durango and Central Mexico. HPV type 16 was the most frequent infection.
Assuntos
Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Adulto , Alphapapillomavirus/genética , Alphapapillomavirus/patogenicidade , DNA Viral/genética , Feminino , Genótipo , Humanos , México/epidemiologia , Pessoa de Meia-Idade , Infecções por Papillomavirus/metabolismo , População Rural , Neoplasias do Colo do Útero/genéticaRESUMO
A modified pan-PV consensus-degenerate hybrid oligonucleotide primer (CODEHOP) PCR was developed for generic and sensitive detection of a broad-spectrum of human papillomaviruses (HPVs) infecting the cutaneous epithelium. To test the analytical sensitivity of the assay we examined 149 eyebrow hair follicle specimens from immunocompetent male patients. HPV DNA was detected in 60â% (89/149) of analysed eyebrow samples with a total of 48 different HPV sequences, representing 21 previously described HPVs and 27 putative novel HPV types. Evidence for ten novel HPV subtypes and seven viral variants, clustering to three out of five genera containing cutaneous HPVs, was also obtained. Thus, we have shown that the modified pan-PV CODEHOP PCR assay is able to identify multiple HPV types, even from different genera, in the same clinical sample. Overall, these results demonstrate that the pan-PV CODEHOP PCR is an excellent tool for screening and identification of novel cutaneous HPVs, even in samples with low viral loads.
Assuntos
Betapapillomavirus/isolamento & purificação , Primers do DNA/química , DNA Viral/genética , Gammapapillomavirus/isolamento & purificação , Genótipo , Infecções por Papillomavirus/diagnóstico , Reação em Cadeia da Polimerase/métodos , Adulto , Sequência de Bases , Betapapillomavirus/classificação , Betapapillomavirus/genética , Primers do DNA/metabolismo , Sobrancelhas/virologia , Gammapapillomavirus/classificação , Gammapapillomavirus/genética , Folículo Piloso/virologia , Humanos , Masculino , Tipagem Molecular/métodos , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/virologia , Filogenia , Prevalência , Sensibilidade e Especificidade , Eslovênia/epidemiologiaRESUMO
BACKGROUND AND OBJETIVE: Human papilloma virus (HPV) is one of the main risk factors associated with the development of cervical cancer and its precursor lesions. It has been reported that HPV16 and 18 types cover approximately 70% of cervical cancer worldwide; however, significant variation in percentages of HPV infections could be related to specific populations. MATERIALS AND METHODS: Purified DNA of 67 cervical samples were analyzed by Linear Array® HPV genotyping kit. These analyzed samples correspond to 19 cervical tumors, 15 high-grade squamous intraepithelial lesions, 20 low-grade squamous intraepithelial lesions, and 13 cervical samples without injury were studied, all of them previously diagnosed. RESULTS: In general, 16 different HPV types were found with differences in their frequencies, cervical invasive cancer being the richest in HPV sequences, followed by the low-grade squamous intraepithelial lesions and then high-grade lesions. HPV16 was the most frequently distributed type in neoplastic lesions of the cervix, followed by the HPV52, suggesting viral type variability, probably associated to the geographical region studied. CONCLUSIONS: The results could indicate variability in HPV presence in Mexico, underlining the important role for HPV52 among others in the Mexican population. This would also potentially have an impact on the current anti-HPV vaccination schemes.