Summary: The apicomplexan parasite Toxoplasma gondii was discovered a little over 100 years ago, but knowledge of its biological life cycle and its medical importance has grown in the last 40 years. This obligate intracellular parasite was identified early as a pathogen responsible for congenital infection, but its clinical expression and the importance of reactivations of infections in immunocompromised patients were recognized later, in the era of organ transplantation and HIV infection. Recent knowledge of host cell-parasite interactions and of parasite virulence has brought new insights into the comprehension of the pathophysiology of infection. In this review, we focus on epidemiological and diagnostic aspects, putting them in perspective with current knowledge of parasite genotypes. In particular, we provide critical information on diagnostic methods according to the patient's background and discuss the implementation of screening tools for congenital toxoplasmosis according to health policies.

https://hal.archives-ouvertes.fr/hal-00696903/document

Epidemiology of and Diagnostic Strategies for Toxoplasmosis

Outbreaks of infectious disease often result from exposure to a common source of the etiologic agent. Generally, the etiologic agent causing an outbreak of infection is derived from a single cell whose progeny are genetically identical or closely related to the source organism. In epidemiological terms, the organisms involved in the outbreak are clonally related; that is, they have a common origin. Clonally related organisms are members of the same species that share virulence factors, biochemical traits, and genomic characteristics. However, there is sufficient diversity at the species level that organisms isolated from different sources at different times and in different geographical regions may be differentiated or classified into subtypes or strains.

Principles and Applications of Methods for DNA-Based Typing of Microbial Organisms

Toxoplasmosis: a history of clinical observations

DNA fingerprinting methods have evolved as major tools in fungal epidemiology. However, no single method has emerged as the method of choice, and some methods perform better than others at different levels of resolution. In this review, requirements for an effective DNA fingerprinting method are proposed and procedures are described for testing the efficacy of a method. In light of the proposed requirements, the most common methods now being used to DNA fingerprint the infectious fungi are described and assessed. These methods include restriction fragment length polymorphisms (RFLP), RFLP with hybridization probes, randomly amplified polymorphic DNA and other PCR-based methods, electrophoretic karyotyping, and sequencing-based methods. Procedures for computing similarity coefficients, generating phylogenetic trees, and testing the stability of clusters are then described. To facilitate the analysis of DNA fingerprinting data, computer-assisted methods are described. Finally, the problems inherent in the collection of test and control isolates are considered, and DNA fingerprinting studies of strain maintenance during persistent or recurrent infections, microevolution in infecting strains, and the origin of nosocomial infections are assessed in light of the preceding discussion of the ins and outs of DNA fingerprinting. The intent of this review is to generate an awareness of the need to verify the efficacy of each DNA fingerprinting method for the level of genetic relatedness necessary to answer the epidemiological question posed, to use quantitative methods to analyze DNA fingerprint data, to use computer-assisted DNA fingerprint analysis systems to analyze data, and to file data in a form that can be used in the future for retrospective and comparative studies.

The Ins and Outs of DNA Fingerprinting the Infectious Fungi

Toxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damnage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid. Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity. Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii. We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis.

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Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes

A case of congenital toxoplasmic chorioretinitis was diagnosed (specific-immunoglobulin G [IgG] and -IgM comparative Western blot analysis) in a baby whose mother was immune during pregnancy. Maternal sera showed an increase in specific IgG and emergence of both IgM and IgA during pregnancy. The mother was probably reinfected through contact with kittens.

Congenital toxoplasmosis due to maternal reinfection during pregnancy.

Burn patients are particularly exposed to deep-seated nosocomial infections caused by Candida species. Superficial carriage of C. albicans is a potential source of infection and dissemination, and typing methods could be useful to trace the different isolates. We report the use of random amplified polymorphic DNA to type isolates of C. albicans in the Hopital Cochin burn unit. This molecular typing method, which is based on PCR with arbitrary short primers, was evaluated on a panel of 32 C. albicans strains isolated from various anatomical sites of unrelated patients, and the strains showed 22 different patterns. Random amplified polymorphic DNA was then used in the epidemiological surveillance of the patients in the burn unit over a 9-month period. Seven patterns were identified among 84 isolates from 18 patients. One pattern (pattern A) corresponding to isolates from 7 of the 18 patients (68% of isolates) predominated throughout the 9-month study, while some strains with other profiles were isolated only once. Some profiles appeared to show a particular geographic pattern within the unit, suggesting transmission from room to room. These results underline the importance of fungal surveillance in such patients and the need to inform nursing staff of measures to prevent the spread of Candida spp. from patient to patient.

Use of random amplified polymorphic DNA as a typing method for Candida albicans in epidemiological surveillance of a burn unit.

Objectifs : La toxoplasmose cerebrale (TC) est l'une des manifestations opportunistes les plus repandues en France chez les patients sideens. Nous rapportons les resultats d'une etude retrospective d'un an, ayant pour but d'evaluer la valeur de la PCR (reaction de polymerisation en chaine) dans le diagnostic de toxoplasmose en routine hospitaliere, chez les patients infectes par le VIH. Methodes : De janvier 1993 a fevrier 1994, 83 patients VIH +, avec un nombre de CD4 inferieur a 200/mm 3 , ont beneficie d'une recherche de toxoplasme par amplification par PCR de deux cibles geniques (gene B1 et sequence TGR1E), dans divers prelevements (93 echantillons sanguins, 33 liquides cephalorachidiens (LCR), 1 humeur aqueuse), a l'occasion de 93 episodes cliniques differents (29 lesions cerebrales focales, 21 encephalites, 38 fievres avec ou sans pneumonie, 5 retinites). Les dossiers ont ete etudies a posteriori, et l'etiologie toxoplasmique a ete retenue sur les criteres d'evolution clinique et radiologique sous traitement anti-toxoplasmique specifique. Resultats : Dix-huit des 29 patients avec des lesions cerebrales focales a l'imagerie par resonance magnetique et/ou au scanner avaient une TC ; 17 prelevements de sang et 6 LCR etaient disponibles. Un seul LCR etait positif. Quatre des 17 prelevements de sang etaient positifs a la fois pour B1 et TGRIE (sensibilite 23,5 p. 100 et specificite 100 p. 100), et 10/17 etaient positifs pour l'une ou l'autre des cibles analysees (sensibilite 58,9 p. 100 et specificite 72,7 p. 100). Par ailleurs, la PCR etait positive chez 3/11 patients (uniquement en TGRIE) sans TC. Dans le groupe des 21 patients avec encephalite diffuse, 5 avaient une PCR positive dans le sang et/ou le LCR, sans qu'aucune amelioration clinique sous traitement anti-toxoplasmique ne soit decelee. Deux des 38 patients avec fievre et/ou pneumopathie avaient une toxoplasmose disseminee prouvee, avec une PCR positive pour les deux cibles analysees. Sept autres patients avaient une PCR positive dans le sang et/ou le LCR, parmi lesquels 3 se sont ameliores sous traitement specifique, et 2 sont decedes rapidement. Sur les 5 cas de retinite, un patient avait une retinite toxoplasmique et etait positif dans le sang et l'humeur aqueuse. Conclusion : La technique PCR ne presente pas un interet diagnostique majeur dans le cadre des TC isolees, mais son utilite pourrait resider dans le diagnostic des TC associees a un site extracerebral ou des toxoplasmoses disseminees.

Retrospective evaluation of the detection of Toxoplasma gondii by polymerase chain reaction in AIDS patients

Au cours des dix dernieres annees, l'incidence des candidoses profondes a considerablement augmente en raison des facteurs d'immunodepression, des traitements (corticotherapie, antibiotherapie), des catheters et de la chirurgie... On note, par ailleurs, la progression de levures du genre Candida autres que Candida albicans, en tant qu'opportunistes. Nous rapportons le cas d'une septicemie a Candida glabrata, diagnostiquee a l'occasion d'une recherche de Plasmodium sur sang peripherique, chez un patient cancereux. Si des cas d'histoplasmose, de cryptococcose ou de penicilliose disseminees ont deja ete diagnotiquees a partir du sang peripherique de patients atteints du SIDA, c'est la premiere fois, a notre connaissance, qu'une septicemie a C. glabrata est decouverte par la mise en evidence de levures intra-leucocytaires

Septicémie à Candida glabrata diagnostiquée sur frottis sanguin à l'occasion d'une recherche de Plasmodium

Resume Les techniques traditionnelles de diagnostic de la toxoplasmose (serologie ou mise en evidence du parasite par examen microscopique, culture cellulaire ou inoculation a la souris) ont ete perfectionnees au fil des annees. Toutefois, dans certaines situations, ces techniques peuvent etre mises en defaut, soit par leur manque de sensibilite, soit par leur delai de reponse trop important. L'amplification genique par polymerase chain reaction (PCR), mise au point sur plusieurs genes et sequences toxoplasmiques, tels que les genes B1 et P30, l'ADNr et la sequence TGR1E, est theoriquement capable de detecter la presence d'un seul parasite. Cette technique a ete evaluee depuis plusieurs annees dans un certain nombre d'etudes prospectives et retrospectives. II en ressort que la PCR est a l'heure actuelle un outil de choix pour le diagnostic antenatal de la toxoplasmose congenitale par simple examen du liquide amniotique. Dans le diagnostic de toxoplasmose cerebrale chez l'immunodeprime, la PCR sur le sang ou le LCR, bien que plus performante que les techniques habituelles, manque de sensibilite, probablement parce que la dissemination hematogene du parasite est inconstante et/ou transitoire. Toutefois, il semble qu'elle soit particulierement interessante dans le diagnostic de toxoplasmose disseminee, y compris chez les patients recevant une prophylaxie.

F Robert

Papers

Congenital toxoplasmosis due to maternal reinfection during pregnancy.

Use of random amplified polymorphic DNA as a typing method for Candida albicans in epidemiological surveillance of a burn unit.

Retrospective evaluation of the detection of Toxoplasma gondii by polymerase chain reaction in AIDS patients

Septicémie à Candida glabrata diagnostiquée sur frottis sanguin à l'occasion d'une recherche de Plasmodium

Intérêts et limites de la polymerase chain reaction dans le diagnostic de la toxoplasmose