Genotyping of Toxoplasma gondii by multiplex PCR and peptide-based serological testing of samples from infants in Poland diagnosed with congenital toxoplasmosis

Toxoplasma gondii has a clonal population genetic structure with three (I, II, and III) lineages that predominate in North America and Europe. Type II strains cause most cases of symptomatic human infections in France and the United States, although few other regions have been adequately sampled. Here we determined the parasite genotype in amniotic fluid and cerebrospinal fluid samples from congenital toxoplasmosis cases in Poland. Nineteen confirmed congenital cases of toxoplasmosis were analyzed, including both severe and asymptomatic cases. The genotype of parasite strains causing congenital infection was determined by direct PCR amplification and restriction fragment length polymorphism analysis. Nested multiplex PCR analysis was used to type four independent polymorphic markers. The sensitivity of multiplex nested PCR was >/=25 parasites/ml in amniotic fluid and cerebral spinal fluid samples. Parasite DNA was successfully amplified in 9 of 19 samples (eight severely affected and one asymptomatic fetus). Only genotype II parasites were identified as the source of T. gondii infection based on restriction fragment length polymorphism analysis. Strains causing congenital infections were also typed indirectly based on detection of antibodies to strain-specific peptides. Serotyping indicated that 12 of 15 cases tested were caused by type II strains and these positives included both symptomatic and asymptomatic infections. Overall, the combined analysis indicated that 14 of the cases were caused by type II strains. Our results are consistent with the hypothesis that parasite burden is associated with severity of congenital toxoplasmosis and indicate that serological testing provides a promising method for genotypic analysis of toxoplasmosis.     

Genotyping of Toxoplasma gondii strains from immunocompromised patients

The genotypes of Toxoplasma gondii strains isolated from HIV and non-HIV immunocompromised patients with cerebral and extracerebral toxoplasmosis were determined and compared to those of strains isolated from non-immunocompromised patients in order to identify the possible relationships between parasite genotype and morbidity of toxoplasmosis. One hundred and ten strains of T. gondii were obtained, either by cell culture (n = 73), brain biopsy (n = 17) or mouse inoculation (n = 20). Ninety strains isolated from immunocompromised patients (74 HIV+ and 16 non-HIV patients) were compared to 20 strains isolated from immunocompetent patients (17 cases of congenital toxoplasmosis, and three cases of primary acquired infection). Genotyping was performed by PCR/RFLP on locus SAG2, and T. gondii strains were classified as Type I, II or III. Ninety out of 110 strains were successfully genotyped, including 20 strains that had been maintained in mice, 69/73 strains maintained in cell cultures, but only 1/17 strains from formalin-fixed paraffin-embedded brain biopsies. 76.7% of the strains in the study population were of type II, 15.6% were type I and 7.7% were type III. The distribution of strain genotypes in immunocompromised and non-immunocompromised patients was comparable: 14.1% and 21% for type I, 76.1% and 79% for type II and 9.8% and 0% for type III, respectively; no correlation could be established between genotype and clinical presentation, i.e., cerebral or extracerebral toxoplasmosis. These results suggest that the type of infecting parasitic strain does not predominantly influence the pathogenesis of toxoplasmosis in immunocompromised patients and fully supports the need for specific prophylaxis in patients infected by T. gondii, regardless of the strain genotype.     

Genotypic characterization of Toxoplasma gondii strains associated with human toxoplasmosis in Spain: direct analysis from clinical samples

Genetic analysis of the SAG2 locus was performed to determine the prevalence of the different genotypes of Toxoplasma gondii (strain types I, II, and III) associated with human toxoplasmosis in Spain. This determination was made directly from primary clinical samples, obviating the previous process of isolation in mice or cell culture. A total of 34 isolates of T. gondii, collected from immunocompromised patients and congenital infection cases, were analyzed. Restriction fragment length polymorphism in PCR-amplified SAG2 products was used to group strains into one of the three genotypes of T. gondii. Complete characterization of the SAG2 gene was successful in 76.5% of the cases, demonstrating the feasibility of direct genotype analysis from clinical samples of different origins. Strains of T. gondii type II were the most prevalent in immunocompromised patients, with 52% of cases, while strains of type I were present in 75% of the congenital infection cases. These data differ from previous reports that show type II strains to be mostly associated with all kinds of human toxoplasmosis. These differences might be an effect of selection in the process of culture and isolation of the samples performed by other researchers prior to strain characterization.     

Acute virulence in mice is associated with markers on chromosome VIII in Toxoplasma gondii.

Toxoplasma gondii has an unusual population structure consisting of three widely distributed clonal lineages. Acute virulence in mice is strictly observed in type I strains, indicating that a genetic determinant(s) unique to this lineage controls acute pathogenesis. We have analyzed several naturally occurring recombinant strains of T. gondii that carry allele 1 at the SAG1 locus; this allele is characteristic of the type I strains and was previously found to be 100% correlated with the acute virulence phenotype. Recombinant strains G622-M and ROD both had a predominantly type III genotype, with the significant exception of allele 1 at the SAG1 locus. Although these two strains had virtually identical multilocus genotypes, they differed in their virulence in mice. Strain ROD was virulent whereas strain G622-M was nonvirulent, thus demonstrating that the presence of allele 1 at SAG1 is not alone sufficient to confer acute virulence. Several sequence polymorphisms upstream of SAG1 were found to be highly correlated with the acutely virulent lineages. Collectively, these results suggest that acute virulence is regulated by a region linked to the SAG1 locus on chromosome VIII in T. gondii.     

Population structure and genetic diversity of Actinobacillus actinomycetemcomitans strains isolated from localized juvenile periodontitis patients

The phylogeny of 20 Actinobacillus actinomycetemcomitans strains isolated from patients with localized juvenile periodontitis (LJP) was investigated by using partial sequence analysis of 16S rRNA genes, arbitrarily primed PCR (AP-PCR), and four additional PCR assays that amplified polymorphic regions in the leukotoxin (lkt), cytolethal distending toxin (cdt), major fimbrial subunit (flp-1), and serotype-specific O polysaccharide gene clusters. Our analysis also included four strains isolated from healthy subjects and nine reference strains. We found that A. actinomycetemcomitans strains comprised three major phylogenetic lineages. One lineage consisted of serotype b strains, a second lineage consisted of serotype c strains, and a third lineage consisted of serotype a, d, e, and f strains. 16S rRNA sequences within each lineage were highly conserved (<1% base substitutions), whereas sequences between lineages were exceptionally divergent (1.9 to 5.0% substitutions). Two strains exhibited 16S rRNA sequences that were even more distantly related to those of the three major lineages (2.7 to 6.7% substitutions), indicating that additional minor lineages or variants exist. The distribution of 16S rRNA sequences and lkt, cdt, flp-1, and AP-PCR genotypes was consistent with a clonal population structure, with little evidence of assortative recombination between strains of different serotypes. Strains from all three major lineages were recovered from LJP patients, suggesting that phylogenetically diverse strains of A. actinomycetemcomitans carry pathogenic potential.     

Direct genotyping of Toxoplasma gondii in ocular fluid samples from 20 patients with ocular toxoplasmosis: predominance of type II in France

We report the direct genotyping analysis of Toxoplasma gondii in ocular samples collected from 20 patients, as well as associated clinical and epidemiological data. This work was aimed at better understanding the impact of genotypes of Toxoplasma gondii strains on toxoplasmic retinochoroiditis. For this purpose, we studied the aqueous humor (AH) or vitreous humor (VH) of 20 patients presenting with ocular toxoplasmosis (OT) in 2 hospitals in France. Genetic characterization was obtained with microsatellite markers in a multiplex PCR assay. In contrast to the results of previous studies, we found no association between atypical Toxoplasma gondii genotypes and the occurrence of OT. Considering the local epidemiological data, our OT patients seemed to be infected more frequently by ordinary type II strains found in the environment. In conclusion, direct genotyping of Toxoplasma gondii strains from aqueous or vitreous humor showed a predominance of the type II genotype in ocular toxoplasmosis; this may be due to a high exposure rate of this genotype in humans.     

Prospective study of congenital toxoplasmosis screening with use of IgG avidity and multiplex nested PCR methods

Acute infection with Toxoplasma gondii during pregnancy can cause congenital toxoplasmosis. The aim of this study was to evaluate whether screening with the use of IgG avidity and multiplex nested PCR methods was effective to detect a high-risk pregnancy. In a prospective study, serum T. gondii IgG avidity was measured in consecutive 146 pregnant women testing positive for T. gondii antibody and either positive or equivocal for IgM. Multiplex nested PCR for T. gondii DNA on amniotic fluid, maternal blood, and umbilical cord blood were performed with informed consent. A total of 51 (34.9%) women presented with low IgG avidity (<30%), 15 (10.3%) presented with borderline avidity (30 to 35%), and 80 (54.8%) presented with high avidity (>35%) indices. Amniotic fluid obtained at amniocentesis or birth yielded positive PCR results in nine women with low IgG avidity indices. Of these nine women, three had congenital toxoplasmosis. None of women with high or border line IgG avidity indices had a positive PCR result in the amniotic fluid or congenital toxoplasmosis. No congenital toxoplasmosis was detected in women whose amniotic fluids yielded negative PCR results. Ingestion of raw or undercooked meat was found to be the main risk factor for acute T. gondii infection. Congenital toxoplasmosis screening with a combination of IgG avidity in the maternal blood and multiplex nested PCR in the amniotic fluid was useful for detecting a high risk pregnancy and diagnosing congenital toxoplasmosis.     

Toxoplasma gondii virulence markers identified by random amplified polymorphic DNA polymerase chain reaction

Genomic DNAs from 35 Toxoplasma gondii strains were amplified by random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) using 18 arbitrary 10-mer primers. At least four primers were found to generate DNA fragments that discriminate the 35 T. gondii strains into a genotype of virulent strains and a genotype of avirulent strains. Primer B12 was found to generate a virulence-specific fragment and primers B5, C8, and C20 were found to generate avirulence-specific fragments, which in all cases clearly identified either the virulence phenotype or the avirulence phenotype, respectively. In addition, the DNA polymorphic bands detected were analyzed by parsimony and distance analysis. A similar genetic relationship among the T. gondii strains was determined by the two phylogenetic methods, which use completely different assumptions. Consistent with the division of the 35 strains into a genotype of virulent strains and a genotype of avirulent strains, both analyses revealed 2 clonal lineages directly correlated with murine virulence. These results strongly support the hypothesis that the genus Toxoplasma may actually contain two clonal lineages correlated with virulence, which have evolved independently following their initial separation. Received: 1 October 1996 / Accepted: 15 October 1996     

Use of GRA6-derived synthetic polymorphic peptides in an immunoenzymatic assay to serotype Toxoplasma gondii in human serum samples collected from three continents

Serotyping is a simple typing method that consists of an immunoenzymatic assay (enzyme-linked immunosorbent assay [ELISA]) using synthetic polymorphic peptides derived from Toxoplasma gondii antigens. We developed a new ELISA based on GRA6 C-terminal polymorphic peptides. Serum samples from 41 human infections due to 23 archetypal (type I, II, or III) and 18 nonarchetypal strains were selected in order to validate this approach. For 20 out of the 23 archetypal infections, there was a clear correlation between microsatellite genotype and GRA6 serotyping. All infections due to nonarchetypal strains were misclassified as archetypal strain infections. The GRA6 C-terminal peptides from these strains were analyzed to explain this misclassification. A second group of 455 patients with acute and chronic toxoplasmosis due to unknown genotypes from different European, African, and Latin American countries were included in this study, and the strain type predicted by this method. The results suggest that serotyping is a promising method for typing strains, although limitations exist for African and South American strains as a consequence of higher peptide polymorphism. Other peptides from different markers must be studied in order to discriminate archetypal from nonarchetypal strains.     

Microsatellite in the beta-tubulin gene of Toxoplasma gondii as a new genetic marker for use in direct screening of amniotic fluids.

To examine the correlation between Toxoplasma gondii genotype and congenital human toxoplasmosis, the polymorphism of the microsatellite consisting of a dinucleotide (TG) repeat in the intron of the beta-tubulin gene was investigated by PCR. Thirty-four reference strains were studied, including 7 strains virulent in mice and 27 strains avirulent in mice. The seven virulent strains had a (TG)8 microsatellite, and the avirulent strains had a (TG)7 microsatellite. This confirms the dichotomy already observed for virulent and avirulent strains. Additionally, 37 samples of amniotic fluid from infected fetuses were tested. All of them had the (TG)7 microsatellite marker. This result confirms that most of the human cases of congenital toxoplasmosis are due to strains avirulent in mice. Nevertheless, their virulence in human fetuses was obvious, as numerous abnormalities were observed on ultrasonic examination. The new genetic marker is the first one directly used for typing T. gondii isolates without any bias due to cultivation of the parasite. This microsatellite marker is not sufficient to type the strains which are avirulent in mice; however, seeking more polymorphic microsatellites should be worthwhile to obtain new genetic markers for direct screening of biological samples.     

Experimental approaches to understanding virulence in toxoplasmosis

Toxoplasma gondii is a widespread protozoan parasite that causes severe disease only in immunocompromised individuals. Equipped with excellent animal models and relatively advanced systems for genetics, T. gondii provides an excellent system for understanding pathogenesis. Resistance to toxoplasmosis is governed by rapid innate and adaptive immunity that is characterized by a Th1 type profile of cytokines. Despite this effective response, acute infections can cause considerable damage and the parasite effectively establishes a long-term chronic infection that predisposes the host to reactivation and provides a means of eventual transmission. This complex interaction is brought about by the differentiation of the parasite from a rapidly replicating, lytic form (known as the tachyzoite) to a slow-growing form (known as the bradyzoite) that gives rise to chronic infection. The population structure of T. gondii is remarkably clonal, consisting of just three predominant lineages that are geographically widespread and found in a variety of hosts including humans. Acute virulence is strongly associated with the type I genotype which exhibits an enhanced replication rate in vitro and higher tissue burdens in vivo relative to non-virulent lineages. The pathology associated with acute infection appears to be due to excessive production of acute inflammatory mediators, suggesting that disease is partly due to over-response of the host immune system. A combination of refined animal models and newly developed genetic tools for establishing the relative contribution of genes to pathogenesis will enable a comprehensive analysis of the molecular basis of virulence in toxoplasmosis.     

Association of ocular toxoplasmosis with type I Toxoplasma gondii strains: direct genotyping from peripheral blood samples

Toxoplasma gondii strains were genotyped directly from blood samples of patients with ocular toxoplasmosis. Analysis of nontranscribed spacer 2 revealed that all detected strains belonged to type I, suggesting an association of ocular toxoplasmosis with this type. The method shows the usefulness of blood samples for genotyping in ocular toxoplasmosis.     

Molecular and virulence characterization of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> strains isolated from humans in Portugal

Toxoplasma gondii is an apicomplexan parasite responsible for toxoplasmosis which infects all warm-blooded vertebrates, including mammals and birds. The majority of studies conducted in Europe have revealed that more than 80 % of strains isolated from human infections belong to genotype II, whereas genotypes I and III are responsible for a small number of cases. Atypical and recombinant strains are generally associated with more severe infections. In Portugal, there is a lack of data concerning genetic diversity as the classical typing studies in humans have never been performed. We aimed to determine the Sag2 and microsatellite-based (TUB2, TgM-A, W35, B17, B18) genotypes of T. gondii isolated from humans in Portugal, as well as to study their virulence in mice. We analyzed 48 strains from congenital and acquired toxoplasmosis collected during the last two decades. Sag2-based genotyping of T. gondii was achieved in all 48 strains where 35 (73 %) were classified as type II and 13 (27 %) were type I. The multilocus PCR of five microsatellites allowed the classification of 10 strains (21 %) as recombinant strains that had been previously identified as type II or I by Sag2 typing. Seven out of the 48 strains, including three type I, three recombinant, and one type I, were virulent in mice. This study constitutes the first evidence of recombinant strains circulating in Portugal in humans from congenital infection, highlighting the need for a better evaluation of these strains as their phenotype is still barely understood.     

Acute toxoplasmoses in immunocompetent patients hospitalized in an intensive care unit in French Guiana

Atypical Toxoplasma gondii strains, unrelated to archetypal clonal lineages (I, II, III), have been reported more frequently over the last decade in areas other than Europe and North America. A newly described form of toxoplasmosis, 'Amazonian toxoplasmosis' (AT), has been reported since 2002 in French Guiana. It is characterized by severe cases and atypical strains linked to a neotropical forest-based cycle. We report on the cases of AT that required intensive care management. We performed a prospective observational study on hospitalized adults in the Intensive Care Unit (ICU) from 2002 to 2008. Clinical and laboratory data, microbiological findings and outcomes were recorded. Data, including the ICU simplified acute physiology score and the pneumonia severity index, were calculated. Epidemiological risk factors for AT were assessed through questionnaires. Eleven non-immunodeficient patients were admitted to the ICU in Cayenne for life-threatening pneumonia associated with disseminated toxoplasmosis. Mechanical ventilation was necessary in seven patients, four of whom required immediate orotracheal intubation. Cardiac and ophthalmological abnormalities were found in five and four patients, respectively. One patient died from multiple organ failure. The genetic characterization of Toxoplasma DNA using six microsatellite markers revealed unique and atypical genotypes in eight patients. All patients presented epidemiological risk factors for AT. In French Guiana, significant T. gondii-related infectious syndrome associated with the lungs, a high level of LDH activity and the reported risk factors for AT was strongly suggestive of disseminated toxoplasmosis with a possible trend toward life-threatening pneumonia.     

Listeria monocytogenes serotype 4b strains belonging to lineages I and III possess distinct molecular features

A collection of Listeria monocytogenes serotype 4b strains belonging to lineages I and III were examined by PCR and Southern blot analysis using species-, virulence-, and serotype-specific primers and probes. Whereas four serotype 4b lineage I strains reacted in PCR with the serotype 4b-, 4d-, and 4e-specific ORF2110 and virulence-specific lmo1134 and lmo2821 primers, all nine serotype 4b lineage III strains were negative by ORF2110 and lmo1134 primers. In addition, the nine serotype 4b lineage III strains formed two separate groups through their reactions in PCR with virulence-specific lmo2821 primers. Southern blot analysis using species-specific lmo0733 and virulence-specific lmo2821 gene probes largely confirmed the PCR results. These findings indicate that L. monocytogenes serotype 4b strains belonging to lineages I and III possess distinct molecular features.     

Study of genotypes and virB4 secretion gene of Bartonella henselae strains from patients with clinically defined cat scratch disease

Bartonella henselae is the causative agent of cat scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy. Occasionally, the bacteria will spread and be responsible for tissue and visceral involvement. Two B. henselae genotypes (genotypes I and II) have been described to be responsible for uncomplicated CSD on the basis of 16S rRNA sequence analysis. A type IV secretion system (T4SS) similar to the virulence-associated VirB system of Agrobacterium tumefaciens was recently identified in the B. henselae Houston-1 genotype I strain. We studied the correlations of the B. henselae genotypes with the clinical presentations and with the presence of T4SS. Isolates originated from CSD patients whose lymph nodes were prospectively analyzed. B. henselae genotype I was identified in 13 of 42 patients (30%). Among these, two teenage twins presented with hepatosplenic CSD and one immunocompetent adult presented with osteomyelitis. Genotype II was detected in 28 of 42 patients (67%), all of whom presented with uncomplicated CSD. The last patient was infected with both genotypes. T4SS was studied by PCR amplification of the virB4 gene. Amplification of virB4 codons 146 to 256, 273 to 357, and 480 to 537 enabled us to detect 66, 90, and 100% of the B. henselae isolates, respectively. Sequence analysis revealed sequence variations that correlated with genotype distribution. Our studies suggest that B. henselae genotype I strains harbor virB4 genes that are different from those harbored by genotype II strains and that genotype I strains might be more pathogenic.     

Development of touch down-multiplex PCR for the diagnosis of toxoplasmosis

Purpose: The diagnosis of toxoplasmosis is challenging since conventional methods like culture and immunofluorescence are not universally available. Serology, which is used regularly might be negative during early phase of infection and in immunosuppressed patients or may remain positive for a long time. Several molecular tests have been used for the diagnosis of toxoplasmosis, but none of them have an internal control which would inform us regarding the presence of polymerase chain reaction (PCR) inhibitors thus, undermining the confidence of a laboratory physician. Materials and Methods: We designed a multiplex PCR containing primers targeting human beta globin gene which would act as internal control and two primers against the B1 gene and 5s gene which aid in sensitive detection of T. gondii. Results: Multiplex PCR had a sensitivity of 83.3% and specificity of 100%. Conclusion: Multiplex PCR may provide a sensitive and specific tool for diagnosis of human toxoplasmosis.     

Urine sample used for congenital toxoplasmosis diagnosis by PCR.

The diagnosis of toxoplasmosis in congenitally infected infants can be difficult; serology is unreliable, and diagnosis must be based on the combination of symptomatology and direct demonstration of the parasite. Four infants suspected of having Toxoplasma gondii infection were studied by serological analysis, tissue culture, and PCR determination. T. gondii was isolated from the urine of one patient. The parasite was detected by PCR in the blood and cerebrospinal fluid of three infants and in the urine in all patients. Because nested PCR proved to be a sensitive, relatively rapid, and specific method and because it can be applied to a variety of different clinical samples, PCR can be a valuable technique for the identification of T. gondii infections in children. The present study indicates that PCR examination of urine, a fluid never before used for diagnosis in this age group, may be valuable in diagnosing cases of congenital toxoplasmosis.     

Brazilian P[8],G1, P[8],G5, P[8],G9, and P[4],G2 rotavirus strains: nucleotide sequence and phylogenetic analysis

Rotavirus epidemiological surveys with molecular analysis of strains are required for gastroenteritis control and prevention. Twenty-nine human rotavirus strains detected in Rio de Janeiro, Brazil, from 1986 to 2004 were characterized as P[8],G1, P[8],G5, P[8],G9, and P[4],G2 genotypes. The VP7 genes were sequenced and phylogenetic analysis was performed. Strains of genotype G1 revealed two distinct lineages, G1-3 and G1-4; strains of genotype G2 grouped in lineage G2-1; G5 strains clustered with other Brazilians G5 strains and G9 strains were closely related to each other in lineage G9-3, distinct from the original G9 strains detected in 1980s. The VP4 genes were analyzed and P[8] strains fell into two major genetic lineages, P[8]-2 and P[8]-3. Our findings document an intragenotype diversity represented by lineages and sublineages within rotavirus circulating in Rio de Janeiro from 1986 to 2004, before application of a vaccine (Rotarix) in Brazil. This report emphasizes the importance of continuing monitor genotypes to verify if uncommon strains or newly strains are emerging to be specifically addressed in future vaccine trials.