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Identificação de múltiplos marcadores polimórficos em pneumocystis jirovecii: relação com a evolução clínica
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Pneumocystis jirovecii é um importante agente infeccioso, causador de
pneumonia (PPc) em doentes com infecção vírus da imunodeficiência humana/síndroma
de imunodeficiência adquirida (VIH/sida), tendo sido, também, já descritos casos de
infecção em imunocompetentes ou em doentes com graus moderados de
imunodeficiência. Sequências polimórficas de ácido desoxirribonucleico (DNA) são
detectadas frequentemente em isolados de P. jirovecii, sugerindo que a variação
genética é comum neste microrganismo e que diversos subtipos genéticos podem
existir. Permanecem por esclarecer as razões pelas quais os doentes sofrem de diferentes
graus de severidade de PPc. Alguns estudos apontam para que determinados indicadores
do prognóstico clínico possam estar relacionados com a apresentação e a evolução da
infecção por P. jirovecii. Por outro lado, também é sugerido que polimorfismos
específicos possam determinar características epidemiológicas do microrganismo, como
a distribuição geográfica de determinados subtipos, os modos de transmissão, ou
mesmo, fenómenos de resistência a fármacos, ou diferentes graus de virulência.
Coloca-se a hipótese de que a virulência de um determinado subtipo de P. jirovecii
possa estar dependente de múltiplos genes e, portanto, da associação de polimorfismos
múltiplos que ocorrem em diversas regiões do genoma.
Com o objectivo de identificar genes potencialmente associados com factores da
infecção por P. jirovecii e com o propósito de desenvolver técnicas moleculares de alto
rendimento, efectuou-se o estudo da variabilidade genética de isolados deste
microrganismo, em Portugal. O trabalho envolveu a recolha de espécimes pulmonares
de doentes infectados por P. jirovecii com diferentes características clínicas e integrou
as metodologias de imunofluorescência indirecta com anticorpos monoclonais
(IFI/AcM), reacção de polimerização em cadeia (PCR), sequenciação directa, análise de
fragmentos de restrição (RFLP), PCR multiplex, genotipagem por reacção de
polimerização de base única (SBE), PCR em tempo real (RT-PCR) e DNA pooling.
Os genes candidatos considerados no presente estudo, foram seleccionados com
base numa extensa pesquisa bibliográfica: o gene da subunidade grande do rRNA
mitocondrial (mtLSU rRNA), o gene do citocromo b (CYB), o gene da superóxido
dismutase (SOD), o gene da -tubulina ( TUB), o gene da tiorredoxina reductase (TRR1), o gene da timidilato sintetase (TS), o gene da dihidrofolato reductase (DHFR),
o gene da dihidropteroato sintetase (DHPS), a região conservada (UCS) dos genes da
família das glicoproteínas major de superfície (MSG) e o gene da protease kexin-like
(KEX1). Este procedimento inicial, permitiu avaliar o interesse relativo destes 10 loci,
verificar a informação sobre as respectivas sequências, sua variabilidade, importância a
nível metabólico e a potencialidade para estas regiões poderem estar relacionadas com
características da infecção, como resistência a fármacos, ou factores de virulência.
A diversidade genética, as frequências de distribuição de genótipos e a relação
entre os genótipos observados e diversos parâmetros da infecção, foram investigadas
por PCR seguido de sequenciação directa, ou RFLP. A análise dos resultados permitiu
confirmar o declínio das mutações associadas a fenómenos de resistência aos fármacos
da família das sulfas, em Portugal, provavelmente como consequência do decréscimo no
uso de profilaxia anti-P. jirovecii com sulfas, após a introdução da terapêutica
anti-retrovírica potente (HAART). Nos 10 loci de P. jirovecii analisados, foram
identificados e caracterizados, um total de 23 polimorfismos, dos quais, quatro
(mt85, SOD110, SOD215, DHFR312) demonstraram estar relacionados com parâmetros
clínicos da infecção, e, por isso, foram considerados como relevantes no seguimento da
investigação. Verificou-se que um genótipo específico (SOD110C/SOD215T)
demonstrou estar associado a casos de PPc com maior severidade, enquanto que outros
dois genótipos (mt85C/SOD215C e SOD110T/SOD215C) foram associados a casos de
PPc com menor severidade. A análise cruzada das sequências permitiu detectar
correlações estatisticamente significativas entre diversos polimorfismos, sugerindo a
existência de potenciais haplótipos. Observou-se um elevado grau de recombinação
entre os genótipos multilocus (MLGs) de P. jirovecii, sendo que, um dos MLGs
(MLG E) se encontrava sobre-representado na população, sugerindo uma estrutura
epidémica, na qual o fenómeno de recombinação genética é frequente, mas onde
ocasionalmente, um clone bem sucedido, tende, rapidamente, a aumentar a sua
frequência originando um clone epidémico.
A técnica de PCR multiplex/SBE foi aplicada com sucesso na identificação de
polimorfismos genéticos de P. jirovecii em pools de DNA, demonstrando que é possível
uma análise num formato múltiplo, abrangendo um elevado número de amostras.
A metodologia proposta demonstrou ser uma ferramenta útil na caracterização do perfil genético de P. jirovecii, permitindo a amplificação e caracterização de diversos
fragmentos, de uma forma rápida, implicando redução de custos e de tempo de
operação, e possibilitando, a aplicação em larga escala. A associação das técnicas de
PCR multiplex/SBE e de DNA pooling por RT-PCR permitiu determinar a distribuição
das frequências relativas dos polimorfismos de base única (SNPs, do inglês single
nucleotide polymorphims) DHFR312, mt85, SOD215 e SOD110 em subgrupos de
doentes, com diferentes características clínicas. Esta abordagem, possibilitou a
identificação de SNPs presentes nos diferentes subgrupos, permitindo a classificação de
polimorfismos, aparentemente, não relevantes (ex. DHFR312), e, conseguindo
seleccionar outros polimorfismos, potencialmente relacionados com diferentes
parâmetros da infecção (ex. mt85, SOD215 e SOD110). Assim, os SNPs seleccionados
podem, de alguma forma, estar associados a haplótipos de P. jirovecii com
características distintas, ou seja, potencialmente implicados em diferentes graus de
severidade da doença.
Os resultados obtidos com o presente estudo, permitem colocar a hipótese de que
haplótipos de P. jirovecii com características distintas podem existir, e que, regiões
genéticas específicas podem funcionar como marcadores desses mesmos organismos.
A aplicação das metodologias de alto rendimento, propostas nesta investigação, pode
acelerar a identificação de polimorfismos de P. jirovecii relevantes a nível clínico,
permitindo uma aproximação baseada em haplótipos para caracterização clínica deste
microrganismo patogénico, auxiliando e adequando a escolha do tratamento mais
indicado, possibilitando assim, um melhor controlo da doença. A robustez, sensibilidade
e especificidade, demonstradas pelas técnicas de PCR multiplex/SBE e de DNA
pooling, fazem com que sejam indicadas, não só para o estudo de P. jirovecii, mas
também para o estudo de inúmeros microrganismos, em especial de microrganismos
não cultiváveis, nos quais, a limitação da amostra, faz com que sejam requeridas
técnicas de alta capacidade, para que seja possível a caracterização de isolados, tal como ficou demonstrado, no presente estudo, para o caso de P. jirovecii.
Pneumocystis jirovecii pneumonia (PcP) is a common and serious disease in persons infected with the human immunodeficiency virus (HIV). P. jirovecii has also been isolated from immunocompetent persons and patients with moderate degrees of immunodeficiency. Polymorphic desoxiribonucleic acid (DNA) sequences are frequently detected in P. jirovecii isolates suggesting that genetic variation is common in this organism and different genetic subtypes may exist. Several aspects of the disease remain unknown; particularly the reasons why some patients suffer from moderate PcP with favorable response to the anti-P. jirovecii drugs, while others suffer from severe PcP, which could be fatal, despite therapy. While several prognostic indicators have been associated with the outcome of PcP, advances in understanding the genetic diversity of P. jirovecii have shown that specific polymorphisms could determine epidemiological profiles of the pathogen, including geographical distribution, modes of transmission, drug resistance, and virulence of particular subtypes. Data suggests that the virulence of a specific P. jirovecii genetic subtype may be dependent on multiple polymorphisms that occur at several genomic regions. The aims of the present study were to: (i) investigate possible associations between relevant polymorphic sequences and P. jirovecii infection parameters; (ii) develop a novel molecular approach based on new high-throughput methodologies. A description of genetic diversity in P. jirovecii isolates, collected from Portuguese infected patients presenting different clinical parameters, based on the identification of multiple single nucleotide polymorphisms (SNPs), was achieved using indirect immunofluorescence with monoclonal antibodies (IFI/AcM), polymerase chain reaction (PCR), DNA sequencing, restriction fragment length polymorphism analysis (RFLP), multiplex-PCR, single base extension (SBE), real time PCR (RT-PCR), and DNA pooling. The candidate P. jirovecii DNA regions were selected based on literature research. A total of 10 distinct loci were considered the most promising sequences: the mitochondrial large-subunit rRNA (mtLSU rRNA), the cytochrome b (CYB), the superoxide dismutase (SOD), the -tubulin ( TUB), the thioredoxin reductase (TRR1), the thymidylate synthase (TS), the dihydrofolate reductase (DHFR),the dihydropteroate synthase (DHPS), the upstream conserved region (UCS) of the major surface glycoprotein gene (MSG), and the kexin-like serine protease (KEX1). The importance of the candidate loci were evaluated based on the bibliographic research. This initial step provided substantial information about sequence variation, metabolic pathways, and the relationship between these sequences and parameters of infection, such as drug resistance or virulence. The genetic diversity, the frequency distribution of genotypes and the relationship between observed genotypes and different parameters of infection were investigated using PCR followed by DNA sequencing or RFLP. The results confirmed a decline of P. jirovecii mutations associated with resistance to sulfa drugs in Portugal, probably as a result of the decreased use of sulfa prophylaxis after the introduction of the highly active antiretroviral therapy (HAART). Twenty three SNPs were detected and characterized among the 10 loci studied, of which four (mt85, SOD110, SOD215, DHFR312) were demonstrated to be related to clinical parameters of infection, and therefore were considered as relevant sequence variations to be analyzed in the subsequent investigation. It was found that a particular genotype (SOD110C/SOD215T) showed to be associated with more virulent PcP episodes, whereas two other genotypes (mt85C/SOD215C and SOD110T/SOD215C) were associated with less virulent PcP episodes. The study of associated variations and cross-genetic analysis demonstrated significant statistical associations between several SNPs, suggesting the existence of P. jirovecii haplotypes. The results also suggested large recombination between most P. jirovecii multilocus genotypes (MLGs). However, one specific MLG (MLG E) occurred at a higher frequency than would be expected according to panmitic expectations, suggesting linkage disequilibrium and clonal propagation. The persistence of this specific MLG may be a consequence of clonal reproduction of this successful genotypic array in a P. jirovecii population with epidemic structure. Multiplex amplification of genomic DNA associated with single base extension genotyping showed to be a suitable high-throughput methodology for large-scale P. jirovecii SNPs screening. The association of multiplex-PCR/SBE and DNA pooling by RT-PCR showed to be a reliable methodology to estimate P. jirovecii SNPs allele frequencies in P. jirovecii DNA pools. The proposed methodology proved to be a useful tool to study the P. jirovecii genetic profile, providing the characterization of multiplefragments in a cost-saving fast format, in which equal amounts of DNA from a large number of individual samples are pooled and then genotyped. In the present study, the relative frequencies of DHFR312, mt85, SOD215, and SOD110 were calculated in several subgroups of patients with different clinical features. This strategy provided a final selection of effectively important polymorphisms, allowing the exclusion of P. jirovecii polymorphisms apparently without clinical relevance (e.g. DHFR312), and the classification of other polymorphisms, correlated with specific parameters of PcP (e.g. mt85, SOD215 and SOD110). These SNPs may be associated with specific P. jirovecii haplotypes potentially involved with different degrees of virulence in PcP episodes. These results suggest that P. jirovecii haplotypes with distinct characteristics may exist and that specific genetic regions are expected to be suitable molecular markers for epidemiological and clinical applications. The high-throughput methodologies proposed in the present research were designed to accelerate the identification of clinical relevant P. jirovecii polymorphisms. Further application of these techniques will supply a haplotype based approach for clinical characterization of this pathogenic microorganism, leading to tailored treatment choices, and better management of the disease. The robustness and sensitivity shown by multiplex-PCR/SBE and DNA pooling demonstrated that these techniques are indicated for the study of P. jirovecii SNPs with clinical relevance, and for the study of SNPs of other microorganisms, especially those without an in vitro culture system, in which high-sensitivity techniques are required due to sample limitation, as demonstrated in the present study for P. jirovecii.
Pneumocystis jirovecii pneumonia (PcP) is a common and serious disease in persons infected with the human immunodeficiency virus (HIV). P. jirovecii has also been isolated from immunocompetent persons and patients with moderate degrees of immunodeficiency. Polymorphic desoxiribonucleic acid (DNA) sequences are frequently detected in P. jirovecii isolates suggesting that genetic variation is common in this organism and different genetic subtypes may exist. Several aspects of the disease remain unknown; particularly the reasons why some patients suffer from moderate PcP with favorable response to the anti-P. jirovecii drugs, while others suffer from severe PcP, which could be fatal, despite therapy. While several prognostic indicators have been associated with the outcome of PcP, advances in understanding the genetic diversity of P. jirovecii have shown that specific polymorphisms could determine epidemiological profiles of the pathogen, including geographical distribution, modes of transmission, drug resistance, and virulence of particular subtypes. Data suggests that the virulence of a specific P. jirovecii genetic subtype may be dependent on multiple polymorphisms that occur at several genomic regions. The aims of the present study were to: (i) investigate possible associations between relevant polymorphic sequences and P. jirovecii infection parameters; (ii) develop a novel molecular approach based on new high-throughput methodologies. A description of genetic diversity in P. jirovecii isolates, collected from Portuguese infected patients presenting different clinical parameters, based on the identification of multiple single nucleotide polymorphisms (SNPs), was achieved using indirect immunofluorescence with monoclonal antibodies (IFI/AcM), polymerase chain reaction (PCR), DNA sequencing, restriction fragment length polymorphism analysis (RFLP), multiplex-PCR, single base extension (SBE), real time PCR (RT-PCR), and DNA pooling. The candidate P. jirovecii DNA regions were selected based on literature research. A total of 10 distinct loci were considered the most promising sequences: the mitochondrial large-subunit rRNA (mtLSU rRNA), the cytochrome b (CYB), the superoxide dismutase (SOD), the -tubulin ( TUB), the thioredoxin reductase (TRR1), the thymidylate synthase (TS), the dihydrofolate reductase (DHFR),the dihydropteroate synthase (DHPS), the upstream conserved region (UCS) of the major surface glycoprotein gene (MSG), and the kexin-like serine protease (KEX1). The importance of the candidate loci were evaluated based on the bibliographic research. This initial step provided substantial information about sequence variation, metabolic pathways, and the relationship between these sequences and parameters of infection, such as drug resistance or virulence. The genetic diversity, the frequency distribution of genotypes and the relationship between observed genotypes and different parameters of infection were investigated using PCR followed by DNA sequencing or RFLP. The results confirmed a decline of P. jirovecii mutations associated with resistance to sulfa drugs in Portugal, probably as a result of the decreased use of sulfa prophylaxis after the introduction of the highly active antiretroviral therapy (HAART). Twenty three SNPs were detected and characterized among the 10 loci studied, of which four (mt85, SOD110, SOD215, DHFR312) were demonstrated to be related to clinical parameters of infection, and therefore were considered as relevant sequence variations to be analyzed in the subsequent investigation. It was found that a particular genotype (SOD110C/SOD215T) showed to be associated with more virulent PcP episodes, whereas two other genotypes (mt85C/SOD215C and SOD110T/SOD215C) were associated with less virulent PcP episodes. The study of associated variations and cross-genetic analysis demonstrated significant statistical associations between several SNPs, suggesting the existence of P. jirovecii haplotypes. The results also suggested large recombination between most P. jirovecii multilocus genotypes (MLGs). However, one specific MLG (MLG E) occurred at a higher frequency than would be expected according to panmitic expectations, suggesting linkage disequilibrium and clonal propagation. The persistence of this specific MLG may be a consequence of clonal reproduction of this successful genotypic array in a P. jirovecii population with epidemic structure. Multiplex amplification of genomic DNA associated with single base extension genotyping showed to be a suitable high-throughput methodology for large-scale P. jirovecii SNPs screening. The association of multiplex-PCR/SBE and DNA pooling by RT-PCR showed to be a reliable methodology to estimate P. jirovecii SNPs allele frequencies in P. jirovecii DNA pools. The proposed methodology proved to be a useful tool to study the P. jirovecii genetic profile, providing the characterization of multiplefragments in a cost-saving fast format, in which equal amounts of DNA from a large number of individual samples are pooled and then genotyped. In the present study, the relative frequencies of DHFR312, mt85, SOD215, and SOD110 were calculated in several subgroups of patients with different clinical features. This strategy provided a final selection of effectively important polymorphisms, allowing the exclusion of P. jirovecii polymorphisms apparently without clinical relevance (e.g. DHFR312), and the classification of other polymorphisms, correlated with specific parameters of PcP (e.g. mt85, SOD215 and SOD110). These SNPs may be associated with specific P. jirovecii haplotypes potentially involved with different degrees of virulence in PcP episodes. These results suggest that P. jirovecii haplotypes with distinct characteristics may exist and that specific genetic regions are expected to be suitable molecular markers for epidemiological and clinical applications. The high-throughput methodologies proposed in the present research were designed to accelerate the identification of clinical relevant P. jirovecii polymorphisms. Further application of these techniques will supply a haplotype based approach for clinical characterization of this pathogenic microorganism, leading to tailored treatment choices, and better management of the disease. The robustness and sensitivity shown by multiplex-PCR/SBE and DNA pooling demonstrated that these techniques are indicated for the study of P. jirovecii SNPs with clinical relevance, and for the study of SNPs of other microorganisms, especially those without an in vitro culture system, in which high-sensitivity techniques are required due to sample limitation, as demonstrated in the present study for P. jirovecii.
Descrição
Palavras-chave
Parasitologia médica Pneumocystis jirovecii Polimorfismo Doenças infeciosas Sida Imunodeficiência Resistência a fármacos Portugal
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical