RESUMO
The emergence of chloroquine resistance in Plasmodium falciparum is a significant public health problem where malaria is endemic. We aimed to evaluate the efficacy of pyrosequencing to assess chloroquine resistance among P. falciparum isolates from the southwestern region of Saudi Arabia by analyzing the K76T and N86Y mutations in the P. falciparum chloroquine resistance transporter (PfCRT) and P. falciparum multidrug resistance 1 (PfMDR1) genes, respectively. Blood samples (n = 121) from microscopically positive P. falciparum cases were collected. DNA was extracted, and fragments from each of the genes were amplified by PCR using new sets of primers. The amplicons were sequenced using a pyrosequencer. All of the 121 samples were amplified for assessment of the PfCRT K76T and PfMDR1 N86Y mutations. All of the samples amplified for the PfCRT 76T mutation harbored the ACA codon (121/121; 100%), indicating the presence of the 76T mutation. For the PfMDR1 N86Y mutation, 72/121 samples (59.5%) had the sequence AAT at that position, indicating the presence of the wild-type allele (86N). However, 49/121 samples (40.5%) had a TAT codon, indicating the mutant allele (Y) at position 86. This study shows that pyrosequencing could be useful as a high throughput, rapid, and sensitive assay for the detection of specific single nucleotide polymorphisms in drug-resistant P. falciparum strains. This will help health authorities in malaria-endemic regions to adopt new malaria control strategies that will be applicable for diagnostic and drug resistance assays for malaria and other life-threatening pathogens that are endemic in their respective countries.
Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação de Sentido Incorreto , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Substituição de Aminoácidos/genética , Cloroquina/farmacologia , Primers do DNA/genética , DNA de Protozoário/química , DNA de Protozoário/genética , Humanos , Testes de Sensibilidade Parasitária/métodos , Parasitologia/métodos , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase , Arábia Saudita , Análise de Sequência de DNA/métodosRESUMO
Although it is desirable to identify the interactions between endotoxin/LPS and the innate immune mechanism, it is often not possible to isolate these interactions from other cell wall-related structures of protein or polysaccharide origin. There is no universally accepted method to extract different LPSs from different bacteria, and their natural state will be influenced by their interactions with the associated molecules in the bacterial outer membrane. It is now believed that Toll-like receptor (TLR) 4 is the main signal transducer of classical LPS (i.e. Escherichia coli LPS), while TLR2 is used by certain non-classical LPSs. There are contradictory reports as to whether Bacteroides fragilis LPS, a non-classical LPS, signals primarily through TLR2 or TLR4. This study was designed to address this problem. Different non-purified and purified B. fragilis LPSs extracted by different methods together with different heat-killed, whole-cell populations of B. fragilis were used to elucidate the TLR specificity. All of these B. fragilis preparations showed a significant signalling specificity for TLR2 but not for TLR4. This indicates that changing the extraction methods, with or without applying a repurification procedure, and varying the cell populations do not alter the TLR specificity of B. fragilis LPS.
