ABSTRACT
AIMS: To analyse the V1 region of the 16S rDNA gene by a universal pyrosequencing protocol to identify and subtype Francisella in 31 strains from a repository collection and 96 patient isolates. METHODS AND RESULTS: Pyrosequencing was used to determine the nucleotide sequence of PCR amplification products of the variable region (V1) of the 16S rDNA from 31 repository strains and 96 isolates from Swedish patients with ulceroglandular tularaemia. Pyrosequencing resulted in a 37 nucleotide sequence, specific for Francisella sp., for all repository strains and patient samples analysed. In addition, the isolates could be divided into two groups based on the analysis of a single nucleotide polymorphism in the sequence: one group included Francisella tularensis ssp. tularensis, ssp. holarctica and ssp. mediasiatica, whereas the other group included Francisella tularensis ssp. novicida and other species of Francisella. The analysis of samples taken from patients suffering from ulceroglandular tularaemia revealed that all isolates belonged to the first group comprising subspecies of F. tularensis virulent for humans. CONCLUSIONS: The pyrosequencing analysis of the 16S rDNA V1 is a useful molecular tool for the rapid identification of suspected isolates of Francisella sp. in clinical or environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Virulent F. tularensis ssp. causing ulceroglandular tularaemia, or those with a potential to be used in a bioterrorism event, could rapidly be discriminated from subspecies less virulent for humans.
Subject(s)
Francisella/classification , Francisella/genetics , Molecular Typing/methods , Tularemia/microbiology , Base Sequence , DNA, Ribosomal/genetics , Female , Francisella/isolation & purification , Francisella/pathogenicity , Francisella tularensis/genetics , Francisella tularensis/isolation & purification , Francisella tularensis/pathogenicity , Humans , Male , Polymerase Chain Reaction , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/geneticsABSTRACT
Most human infections with the protozoan parasite Toxoplasma gondii are asymptomatic, but severe symptoms can occur in immunocompromised patients, in developing foetuses, and in ocular infections in immunocompetent individuals. The majority of T. gondii strains can be divided into three main lineages, denoted types I, II and III, which are known to cause different clinical presentations. Simple molecular methods with the capacity to discriminate rapidly among strains may help to predict the course of infection and influence the choice of treatment. In the present study, real-time PCR followed by pyrosequencing was used to discriminate among types I, II and III by analysis of two single nucleotide polymorphisms in the GRA6 gene. Twenty-one isolates of T. gondii characterised previously were analysed. Three different GRA6 alleles detected by the pyrosequencing technique identified types I, II and III isolates correctly, while four atypical isolates possessed either the GRA6 allele 1 or the GRA6 allele 3. Reproducibility was 100%, and typeability, when including atypical strains, was 81%. It was also possible to discriminate a mixture of two genotypes. The method was used to identify GRA6 type II in blood and lung tissue from an allogeneic transplant recipient with toxoplasmosis.
Subject(s)
Polymorphism, Single Nucleotide , Sequence Analysis, DNA/methods , Toxoplasma/classification , Animals , Antigens, Protozoan/genetics , DNA, Protozoan/analysis , Female , Genotype , Humans , Middle Aged , Polymerase Chain Reaction , Protozoan Proteins/genetics , Toxoplasma/geneticsABSTRACT
The aim of the study presented here was to evaluate the use of PCR for improving the diagnosis of Toxoplasma gondii infection in immunocompromised hosts. Three hundred thirty-two bronchoalveolar lavage (BAL) fluid samples were analyzed by real-time PCR targeting a 529 bp element of T. gondii. In positive samples, the genotype of the parasite was determined by sequence analysis of the GRA6 gene. Positive results were achieved for 2% (7/332) of the samples tested. Genotyping was possible in two samples and revealed GRA6 type II T. gondii. PCR for detecting T. gondii in BAL samples should be performed in all immunosuppressed HIV-positive patients with symptoms of a systemic infection of unknown etiology. Trimethoprim-sulfamethoxazole prophylaxis does not exclude concomitant infection with T. gondii.
Subject(s)
AIDS-Related Opportunistic Infections/diagnosis , Lung Diseases, Parasitic/diagnosis , Toxoplasmosis/diagnosis , AIDS-Related Opportunistic Infections/parasitology , Animals , Bronchoalveolar Lavage Fluid/parasitology , Genotype , Humans , Lung Diseases, Parasitic/parasitology , Parasitology/methods , Polymerase Chain Reaction/methods , Toxoplasma/geneticsABSTRACT
Sensitive and rapid detection of infection with Toxoplasma gondii in transplanted immunocompromised patients is crucial for a good prognosis. Two DNA fragments are used currently for detecting T. gondii infection by PCR, i.e., the B1 gene and a 529-bp repeat element that exists in 200-300 copies/genome. This study investigated whether targeting the 529-bp repeat element gives better sensitivity and accuracy than can be obtained when targeting the B1 gene (35 copies) when concentrations of T. gondii DNA are low. The results demonstrated that detection of the 529-bp repeat element increased diagnostic sensitivity and accuracy. Addition of an internal amplification control did not affect the PCR performance and was useful in order to monitor PCR inhibition by non-specific DNA in the LightCycler instrument. The real-time PCR was used successfully in a clinical context to monitor parasitaemia in the blood of a transplant recipient suffering from toxoplasmosis.
