Estudo prospectivo para identificar a presença do papilomavírus humano em gestantes adolescentes por meio da captura híbrida II e correlacionar com colpocitologia oncótica convencional, em base líquida e colposcopia. O grupo constituído por 60 gestantes entre 12 a 18 anos e idade gestacional media de 23 semanas. A captura híbrida II foi positiva em 51,7%. A colpocitocologia oncótica convencional : normal em 90% e anormal em 10%. Os achados citológicos anormais: lesão intraepitelial escamosa de baixo grau em 8,3% e carcinoma invasor em 1,7%. A colpocitologia em base líquida: normal em 90% e anormal em 10%. Os achados citológicos anormais: lesão intraepitelial escamosa de baixo grau em 8,3% e lesão intraepitelial escamosa de alto grau em 1,7%. Os achados colposcópicos normais foram o epitélio escamoso normal em 20%, epitélio glandular em 18,3% e a zona de transformação normal em 40%. A zona de transformação anormal presente em 21,7% / Study delineated to identify human papillomavirus by hybrid capture II in pregnant teenagers and to correlate with conventional, liquid-based Pap test and colposcopic findings. The study group was constituted by 60 pregnant women aged between 12 and 18 years old; mean gestational age was 23 weeks. They were submitted to anamnese, Pap smear, hybrid capture technique and colposcopy. Hybrid capture II of human papillomavirus was positive in 51,7% of the cases. Conventional Pap test was normal in 90% and abnormal in 10% of the cases. Abnormal results: low-grade squamous intraepithelial lesion in 8,3% and Invasor carcinoma in 1,7%. Based-liquid Pap test was normal in 90% and abnormal in 10% of the cases. Abnormal results: 8,3% of low-grade squamous intraepithelial lesion and 1,7% of high-grade squamous intraepithelial lesion. Normal results: 20% of normal squamous epithelium, 18,3%of columnar epithelium and 40% of normal transformation zone. Abnormal transformation zone was seen in 21,7% of the cases

Made available in DSpace on 2016-08-19T18:15:58Z (GMT). No. of bitstreams: 1 ELIONORA DE JESUS CARNEIRO JANSEN DE MELLO.pdf: 1110156 bytes, checksum: 21ebd7627a73f6cf7dc192f934be47e1 (MD5) Previous issue date: 2009-11-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The human papillomavirus (HPV) is the agent of most common sexually transmitted infection in adolescence worldwide. The cervical cancer, related to this infection, is the leading cause of death in Maranhão. The biomolecular diagnosis used to HPV detection is the polymerase chain reaction (PCR). It is proposed to investigate the frequency of the HPV genotypes in high school students from a public school in Sao Luis by different diagnostic methods. We conducted a cross-sectional study, descriptive at adolescents in high school, totaling 39 female students aged 15 to 19 years in the period July 2008 to June 2009. A questionnaire covering socio-demographic and sexual behavior was used. It was carried out cytopathology, colposcopy, cervical biopsy and genotyping by Nested PCR in each sample. The inclusion criteria were presence of sexual activity and we excluded women who had no sexual activity, those with positivity for HIV and pregnant. The data were transferred to a spreadsheet in the software Excel 2007 and evaluated using Epi-Info 6.04. The socio-demographic data showed that most female adolescents studied was 15 to 17 years old, they lived in urban area and they were full-time students, they were white and had schooling from 12 to 14 years. In 25 (64.1%) adolescents, the first sexual intercourse occurred before age 16 (p = 0.240), half said to have among one and four sexual intercourses per week (p = 0.082), one partner (51.3%), and two to three partners (38.5%), 82% reported ever having had STDs (p = 0.704). Three reported abortion (p = 0.463). One reported having given birth to 17 years. As for gynecologic complaints, the main were leukorrhea (79.5%, p = 0.016) and pruritus (23.1%, p = 0.928). Eight (20.5%) reported family history of cancer. At colposcopy, there were no vulvar or vaginal lesions and we diagnosed minor injury in seven adolescents (17.0%). The Pap smear result showed inflammation in 37 (94.9%) and LSIL / HPV in a test. Histopathology was performed in 15 students (38.5%), and seven had changes suggestive of HPV. Genotyping was positive in 29 students (74.3%), predominantly to 14 high-risk genotypes in 21 (72.4%) adolescents, namely: 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 82, distributed in multiple coexisting and simple infections. In assessing the accuracy of Nested PCR, positive predictive value was 85.7% (true positives), and negative predictive value was 12.5% (true negatives), Nested PCR provides useful diagnostic information to the study of human papillomavirus, as multiple and simple infections diagnosis. / O papilomavírus humano (HPV) é o agente da infecção sexualmente transmissível mais comum na adolescência em âmbito mundial. O câncer do colo do útero, relacionado a esta infecção, é a principal causa de morte no Maranhão. O diagnóstico biomolecular usado é a reação em cadeia de polimerase (PCR) para detecção do HPV. Propõe-se investigar a frequência dos genótipos de HPV em adolescentes do ensino médio de uma escola pública de São Luís-MA, por diferentes métodos diagnósticos. Realizou-se um estudo transversal, descritivo, em adolescentes, cursando o ensino médio, totalizando 39 alunas com idade entre 15 a 19 anos no período julho de 2008 a junho de 2009. Utilizou-se questionário abordando variáveis sócio-demográficas e comportamento sexual. Realizou-se citopatologia, colposcopia, colpobiópsia e genotipagem por PCR-Nested em toda amostra. O critério de inclusão compreendeu presença de atividade sexual e os de não inclusão foram mulheres que não tiveram atividade sexual, positividade para HIV e gravidez. Os dados foram avaliados e transferidos para uma planilha no software Excel 2007 e Epi-Info 6.04. Os dados sóciodemográficos mostraram que a maioria das adolescentes estudadas tinha de 15 a 17 anos, era de procedência urbana e estudantes em tempo integral, cor branca e escolaridade entre 12 e 14 anos. Em 25 (64,1%) adolescentes, a sexarca ocorreu antes dos 16 anos (p=0,240), a metade disse ter de uma a quatro relações sexuais semanais (p=0,082); um parceiro (51,3%), e dois a três parceiros (38,5%); 82% disseram nunca ter tido DST (p=0,704). Três relataram abortamento (p=0,463). Uma informou parto aos 17 anos. Quanto às queixas ginecológicas, as principais foram leucorréia (79,5%; p=0,016) e prurido (23,1%; p=0,928). Oito (20,5%) relataram antecedentes de câncer familiar. À colposcopia, nenhuma apresentou lesões vulvares ou vaginais, tendo sido diagnosticado lesão menor em sete adolescentes (17,0%). O exame citopatológico apresentou resultado inflamatório em 37 (94,9%) e LSIL/HPV em um exame. A histopatologia foi realizada em 15 alunas (38,5%), sendo que sete apresentaram alterações sugestivas de HPV. A genotipagem foi positiva em 29 alunas (74,3%), com predomínio para 14 genótipos de alto risco em 21 (72,4%) adolescentes, sendo eles: 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 82, distribuídos em infecções múltiplas coexistentes e simples. Na avaliação da acurácia por PCR - Nestad o valor preditivo positivo foi de 85,7% (verdadeiros positivos), e o valor preditivo negativo foi de 12,5% (verdadeiros negativos), técnica que fornece informações diagnósticas úteis ao estudo do papilomavírus como infecção múltipla e simples.

abstract: Over the past several decades, there has been a growing interest in the use of fluorescent probes in low-cost diagnostic devices for resource-limited environments. This dissertation details the design, development, and deployment of an inexpensive, multiplexed, and quantitative, fluorescence-based lateral flow immunoassay platform, in light of the specific constraints associated with resource-limited settings. This effort grew out of the need to develop a highly sensitive, field-deployable platform to be used as a primary screening and early detection tool for serologic biomarkers for the high-risk human papillomavirus (hrHPV) infection. A hrHPV infection is a precursor for developing high-grade cervical intraepithelial neoplasia (CIN 2/3+). Early detection requires high sensitivity and a low limit-of-detection (LOD). To this end, the developed platform (DxArray) takes advantage of the specificity of immunoassays and the selectivity of fluorescence for early disease detection. The long term goal is to improve the quality of life for several hundred million women globally, at risk of being infected with hrHPV. The developed platform uses fluorescent labels over the gold-standard colorimetric labels in a compact, high-sensitivity lateral flow assay configuration. It is also compatible with POC settings as it substitutes expensive and bulky light sources for LEDs, low-light CMOS cameras, and photomultiplier tubes for photodiodes, in a transillumination architecture, and eliminates the need for expensive focusing/transfer optics. The platform uses high-quality interference filters at less than $1 each, enabling a rugged and robust design suitable for field use. The limit of detection (LOD) of the developed platform is within an order of magnitude of centralized laboratory diagnostic instruments. It enhances the LOD of absorbance or reflectometric and visual readout lateral flow assays by 2 - 3 orders of magnitude. This system could be applied toward any chemical or bioanalytical procedure that requires a high performance at low-cost. The knowledge and techniques developed in this effort is relevant to the community of researchers and industry developers looking to deploy inexpensive, quantitative, and highly sensitive diagnostic devices to resource-limited settings. / Dissertation/Thesis / Doctoral Dissertation Electrical Engineering 2018

Introduktion: Humant papillomvirus, HPV, är världens vanligaste sexuellt överförbara infektion. De flesta HPV-infektioner läker ut av sig själva men de kan också bli kroniska och på sikt leda till cellförändringar och cancer. Sedan 2012 erbjuds alla flickor i Sverige vaccination mot HPV för att förhindra livmoderhalscancer, som är den vanligaste HPV-relaterade cancerformen. Vaccinationen utförs av skolsköterskor i årskurs 5-6. Vaccinationstäckningen för HPV-vaccination är dock lägre än för övriga vaccinationer i det svenska barnvaccinationsprogrammet. Skolsköterskorna har en viktig roll när det gäller att bemöta tveksamma vårdnadshavare och att få upp vaccinationstäckningen för att på sikt minska antalet cancerfall orsakade av HPV. Syftet med studien var att beskriva hur skolsköterskor i Halland agerar när vårdnadshavare tackar nej till HPV-vaccination för flickor i årskurs 5-6 och vad de upplever är anledningen till nekande. Metoden som valdes var en webbaserad enkätundersökning bland skolsköterskor på mellanstadieskolor i de sex halländska kommunerna. Enkäten skickades ut till 101 respondenter. Svarsfrekvensen var 56 procent och resultatet visade att skolsköterskorna i Halland inte agerar alls när vårdnadshavare tackar nej till HPV-vaccination för flickor i årskurs 5-6. Det främsta motivet till detta är att skolsköterskorna erbjuder nytt vaccinationstillfälle i senare årskurser. Skolsköterskornas uppfattning är att desinformation på internet, rädsla för biverkningar och flickornas ringa ålder utgör de vanligaste anledningarna för vårdnadshavare att tacka nej. Slutsats:Fortsatt arbete för att stärka skolsköterskorna i sin roll som vaccinationsförespråkare är nödvändig för att öka vaccinationstäckningen för HPV. / Introduction: Human papillomavirus, HPV, is the most common sexually transmitted infection. Most HPV infections disappear without intervention but HPV infection can also become chronic and lead to cancer. Since 2012, all girls in Sweden are offered vaccination against HPV to prevent cervical cancer, the most common HPV-related form of cancer. The vaccination is provided by school nurses in grade 5 or 6. HPV vaccination compliance is lower than other vaccinations in the National Vaccine Program for children in Sweden. School nurses play an important role in encouraging guardians to vaccinate their children, especially those who hesitate, to increase the number of girls getting vaccinated. Increasing compliance via school nurses´ actions can reduce the number of HPV related cancers in the long term. The aim of the study was to describe how school nurses in Halland, a province on the southwest coast of Sweden, react to guardians´ rejection of the HPV vaccination for girls in grade 5 or 6. Also queried was what these nurses hear as the reasons for refusal. The assessment method selected was a web-based survey. The survey link was provided to 101 school nurses at schools teaching 5th and 6th grade in the six municipalities of Halland. The response rate was 56 percent and the results show that the school nurses in Halland do not react at all when guardians decline vaccination. The reasons given are that they believe that vaccinations are voluntary and/or that vaccinations are offered again in later grades. School nurses perceive that disinformation on the internet, fear of side effects and the young age of the girls are the main reasons for guardians´ declining HPV vaccination. Conclusion: Further motivation among school nurses in Halland is needed to increase HPV vaccination compliance.

O Câncer Cervical é um importante problema de saúde pública, causando aproximadamente 270 milhões de mortes anuais. Atualmente, esse câncer é o segundo mais comum em mulheres no mundo, com os maiores índices de incidência ocorrendo nos países em desenvolvimento. O Câncer cervical tem como agente etiológico o Papilomavírus Humano (HPV). O vírus possui DNA circular duplo com genes que codificam oito proteínas, E1, E2, E4, E5, E6, E7 (não estruturais), e L1 e L2 (estruturais) que formam o capsídio viral. A proteína L1 quando produzida em sistema heterólgo de expressão tem a capacidade de auto-arranjo em partículas semelhantes ao vírus chamadas de VLPs (\"Vírus like Particles\"), que são a base das vacinas profiláticas disponíveis, Gardasil® e Cervarix®. Neste trabalho, foram produzidas VLPs da proteína L1 do HPV-16 na levedura metilotrófica Pichia pastoris utilizando dois tipos de vetores de expressão para leveduras, um epissomal e outro integrativo, para os trabalhos de purificação. Inicialmente as amostras obtidas ao final da purificação apresentaram degradação e agregação da proteína L1, além de contaminantes. O protocolo de purificação foi modificado e as amostras obtidas no final do processo de purificação não apresentaram degradação e nem agregação da proteína L1, embora ainda tenha sido detectada a presença de contaminantes e, portanto, as amostras obtidas estavam semi-purificadas. Uma segunda abordagem para o desenvolvimento de uma vacina alternativa foi realizada, na qual foram produzidos candidatos vacinais baseados na proteína L2 do Papilomavírus Humano (L2 do HPV-16, L2 do HPV-18 e L2 Multimérica), e também foram expressas proteínas fusionadas ao domínio ZZ de Staphylococcus aureus, utilizado neste caso como adjuvante. Em geral as proteínas de fusão, ainda que apresentando a fusão em sua forma funcional, geraram uma fraca indução de anticorpos IgG e de anticorpos neutralizantes. Pode-se observar uma produção de anticorpos neutralizantes contra pseudovírus homólogos e heterólogos que dependeu da combinação com os adjuvantes utilizados. Numa tentativa de maximizar a eficiência imunológica dos antígenos utilizados, um novo ensaio de imunização foi realizado com uma formulação conjunta contendo três proteínas (L2 do HPV-16, L2 do HPV-18 e L2 Multimérica), sendo também avaliadas as propriedades de três adjuvantes. A combinação da formulação vacinal com os adjuvantes gerou altos níveis de anticorpos L2-específicos, destacando-se as formulações com o hidróxido de alumínio/MPLA e com a vacina celular Pertussis low. Com a utilização desses adjuvantes, os níveis de anticorpos permaneceram elevados a longo termo. Pode-se observar a imunogenicidade de cada um dos antígenos dependeu de sua formulação com os adjuvantes testados. / Cervical cancer is one of the most important problems of public health, causing nearly 270 million deaths annually. Currently, cervical cancer is the second more common type in women worldwide and the highest incidences occuring in developing countries. Human papillomavirus (HPV) are the ethiologic agents of cervical cancer. HPV have a double-stranded circular DNA genome encoding eight viral proteins, E1, E2, E4, E5, E6, E7 (non-structural), and L1, L2 (structural), the last ones forming the viral capsid. L1 protein expressed in heterologous systems self-assembles into VLPs (\"Virus like Particles\"), the base of prophylactic vaccines available commercially, Gardasil and Cervarix. In this work, we produced VLPs of HPV-16 L1 protein in the methylotrophic yeast Pichia pastoris for purification assays. Initially, samples obtained from purification presented degradation and aggregation of L1 protein and also contaminants. The purification protocol was modified and samples obtained in the new process did not present degradation nor aggregation but the presence of some contaminants could still be detected. Therefore, samples obtained were semi-purified. A second approach to the development of an alternative vaccine was performed with the production of vaccine candidates based on the HPV L2 protein (HPV-16 L2, HPV-18 L2 and Multimeric L2) and we also expressed the same proteins fused to Staphyloccocus aureus ZZ domain, using it as an adjuvant. In general, the fused proteins induced low antibodies titer and low neutralizing antibodies titer when compared to other formulations. Neutralizing antibodies elicited against homologous and heterologous pseudovirus depended on the adjuvant used. In another approach, trying to enhance immunologic antigen efficiencies, an immunization assay was performed with a formulation containing L2 HPV- 16, L2 HPV-18 and Multimeric L2 and the properties of three adjuvants were assessed. The vaccine formulations adjuvanted with alum/MPLA and cellular Pertussis low vaccine induced the highest levels of L2-specific antibodies. In addition, the usage of adjuvants maintained the antibody levels in the long term. The immunogenicity of each antigen depends on the adjuvant combination used.

Genital human papillomavirus infection is the most common sexually transmitted virus in the United States, with almost 20 million Americans currently infected and an additional 6.2 million becoming newly infected each year. Women rely strongly on their health care providers to educate and comfort them regarding this distressing diagnosis. This study will use an online, self-completion questionnaire to obtain women’s opinions regarding their health care providers’ performances concerning the initial consultation after learning of their HPV diagnosis. Findings from this study provided insight as to how women prefer receiving information from their health care providers concerning their diagnosis of HPV. In addition, study findings provide suggestions for improving clinical practices regarding HPV consultations in the Tri-cities area of East Tennessee.

<p>Oncogenic human papillomaviruses (HPVs) are implicated in 99.7 % of cervical cancer cases but require the co-operation of other factors. To investigate potential genetic risk factors we have typed the HLA class II DRB1 and DQB1 loci in 478 women diagnosed with cervical carcinoma in situ and in 608 age-matched controls. Quantitative measurements of HPV 16, HPV 18/45 and HPV 31 were obtained. The DRB1*1501 and DQB1*0602 alleles were found to increase the risk of HPV 16 infection. Carriers of DRB1*1501 and DQB1*0602 were also shown to have an increased risk of a higher viral load compared to non-carriers. The DRB1*1301 and DQB1*0603 alleles were found to protect from HPV 18/45 and 31 infections as well as resulting in a lower viral load in carriers compared to non-carriers. Women with a high HPV 16, 18/45 or 31 viral load were more prone to long-term infections and women with a low HPV 16 viral load were more prone to short-term infections. Carriers of DRB1*1501 and DQB1*0602 alleles were also shown to have an increased risk of long-term infections compared to short-term infections. We also tested if an HPV susceptibility locus found for epidermodysplasia verruciformis (EV) was also linked to HPV susceptibility in cervical cancer. We did not find any linkage to this locus in a set of 77 families, each with at least three cases diagnosed with cervical carcinoma in situ. Other potential risk factors tested were HPV 16 E6 variants together with a p53 codon 72 polymorphism and HLA class II alleles. We found an association between the E6 L83V variant and the HLA DR4-DQ3 haplotype, as well as an increased frequency of Arg homozygosity of p53 in women infected with the L83V variant. These results show that alleles at HLA class II loci represents risk factors for persistent HPV infection and thereby also contribute to the risk of development of cervical carcinoma <i>in situ</i>.</p>

<p>Human papillomavirus type 16 (HPV-16) is the major cause of cervical cancer. HPV-16 gene expression is tightly linked to the differentiation programme of the infected epithelium. Expression of the late genes, L1 and L2, encoding the capsid proteins, is delayed until the more terminally differentiated cells. Successful inhibition of HPV-16 late gene expression early in the viral life cycle is essential for persistence of infection, the highest risk factor for cervical cancer.</p><p>The goal of this thesis was to identify regulatory RNA elements and cellular factors that influence RNA processing events, such as alternative splicing and polyadenylation, during late gene expression. For this purpose, transfection of plasmids containing almost the full-length HPV-16 genome into HeLa cells, followed by RNA analysis, was employed. An exonic splicing enhancer (ESE) was identified that firmly supported the use of the E4 3’ splice site. A key regulator of HPV-16 gene expression, the E4 ESE was required for early mRNA splicing and polyadenylation, as well as for inhibition of premature late gene expression. The early polyadenylation signal (pAE) is also an important block of premature late gene expression. An upstream polyadenylation element (USE) was identified in the early 3’ untranslated region that enhanced polyadenylation at pAE, and interacted specifically with the cellular factors CstF-64, hnRNP C1/C2, PTB and hFip1. With the help of adenoviral E4orf4, a protein which causes dephosphorylation of SR proteins, we found that overexpression of SRp30c activated HPV-16 late gene expression by an exon skipping mechanism, and that SRp30c may interfere with early mRNA terminal exon definition.</p><p>This work identified a crucial splicing enhancer, as well as a number of cellular proteins binding to an USE in the early region of HPV-16. Furthermore, the cellular splicing factor SRp30c was shown to play a role in the regulation of HPV-16 late gene expression.</p>

Oncogenic human papillomaviruses (HPVs) are implicated in 99.7 % of cervical cancer cases but require the co-operation of other factors. To investigate potential genetic risk factors we have typed the HLA class II DRB1 and DQB1 loci in 478 women diagnosed with cervical carcinoma in situ and in 608 age-matched controls. Quantitative measurements of HPV 16, HPV 18/45 and HPV 31 were obtained. The DRB1*1501 and DQB1*0602 alleles were found to increase the risk of HPV 16 infection. Carriers of DRB1*1501 and DQB1*0602 were also shown to have an increased risk of a higher viral load compared to non-carriers. The DRB1*1301 and DQB1*0603 alleles were found to protect from HPV 18/45 and 31 infections as well as resulting in a lower viral load in carriers compared to non-carriers. Women with a high HPV 16, 18/45 or 31 viral load were more prone to long-term infections and women with a low HPV 16 viral load were more prone to short-term infections. Carriers of DRB1*1501 and DQB1*0602 alleles were also shown to have an increased risk of long-term infections compared to short-term infections. We also tested if an HPV susceptibility locus found for epidermodysplasia verruciformis (EV) was also linked to HPV susceptibility in cervical cancer. We did not find any linkage to this locus in a set of 77 families, each with at least three cases diagnosed with cervical carcinoma in situ. Other potential risk factors tested were HPV 16 E6 variants together with a p53 codon 72 polymorphism and HLA class II alleles. We found an association between the E6 L83V variant and the HLA DR4-DQ3 haplotype, as well as an increased frequency of Arg homozygosity of p53 in women infected with the L83V variant. These results show that alleles at HLA class II loci represents risk factors for persistent HPV infection and thereby also contribute to the risk of development of cervical carcinoma in situ.

The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing. Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension. The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results. Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses. Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results. <b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis