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.

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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

For the past 40 years, the Toxoplasma Serology Laboratory at the Palo Alto Medical Foundation Research Institute (TSL-PAMFRI) has been dedicated to the laboratory diagnosis of Toxoplasma gondii infection and toxoplasmosis. TSL-PAMFRI is the "brain child" of Jack S. Remington. Jack's ceaseless devotion to objectivity and uncompromising excellence has made TSL-PAMFRI the Toxoplasma reference laboratory for the Centers for Disease Control and Prevention, the US Food and Drug Administration, and health care providers and clinical laboratories in the United States and other countries. Jack's leadership and vision created, defined, and significantly contributed to the development of laboratory methods for the diagnosis of the infection and diseases caused by T. gondii. A summary of the laboratory tests currently available at TSL-PAMFRI for the diagnosis of infection and disease caused by the parasite is presented here.

Laboratory Diagnosis of Toxoplasma gondii Infection and Toxoplasmosis

Monitoring of verotoxigenic Escherichia coli (VTEC) and identification of human pathogenic VTEC types 1 Scientific Opinion of the Panel on Biological Hazards

We reviewed the files of 110 women with Toxoplasma seroconversion during pregnancy. Prenatal diagnosis was attempted for 94 women by amniotic fluid sampling. Toxoplasma gondii was detected by PCR, with or without tissue culture and mouse inoculation. The early neonatal diagnostic procedure included placental testing by PCR and/or mouse inoculation, cord blood serological testing, and comparison of maternal and newborn antibodies by Western blotting (WB). Serological follow-up of the infants was conducted during the first year of life or until the diagnosis of congenital toxoplasmosis (CT) could be ruled out. Congenital infection was diagnosed in 27 individuals (20 live births) in the prenatal and/or neonatal period. The sensitivity and specificity of prenatal diagnosis were 81 and 100%, respectively. Placental examination was positive for 66.7% of individuals with CT and was always negative for neonates without CT. Cord blood serology detected immunoglobulin M (IgM) and/or IgA in 80% of infected newborns, with respective specificities of 91.2 and 87.7%. By WB we detected bands on IgG and IgM blots recognized by the newborn serum but not by the maternal serum (neosynthesized IgG and/or IgM) for 88.2% of infected infants within the first 2 months of life with a specificity of 100%. Early postnatal diagnosis was negative for 2 of the 20 neonates with CT. Both of these newborns had a negative prenatal diagnosis and were asymptomatic, suggesting a very low parasite load. In conclusion, despite the use of advanced methods, some cases of congenital toxoplasmosis cannot be detected early, which underlines the importance of careful follow-up of newborns who are at risk.

Value of Prenatal Diagnosis and Early Postnatal Diagnosis of Congenital Toxoplasmosis: Retrospective Study of 110 Cases

Detection of Toxoplasma gondii in blood by means of the polymerase chain reaction (PCR) may facilitate the diagnosis and follow-up of cerebral toxoplasmosis in patients with AIDS. We evaluated this approach with seven patients with tissue culture-proven parasitemia, 14 patients with presumptive cerebral toxoplasmosis, and 17 healthy human immunodeficiency virus-positive controls. Each sample of blood was assayed on three different occasions by a PCR assay based on detection of the gene encoding the P30 surface protein. A positive PCR diagnosis required positivity in at least two of the three PCR tests. None of the controls had a positive PCR diagnosis, but six of the seven patients with parasitemia did. Cerebral toxoplasmosis was confirmed in 13 of the 14 patients with a presumptive diagnosis; diagnosis by PCR was positive before treatment for 9 of these 13 patients, whereas tissue culture was positive for only 1 patient. During treatment, blood samples were taken from 14 patients at regular intervals until day 12. PCR diagnosis became negative on ethidium-stained gels, but persistent signals were observed after hybridization, in some cases, for up to 12 days after initiation of therapy. PCR on venous blood could thus be a sensitive and noninvasive method for the diagnosis of cerebral and disseminated toxoplasmosis in AIDS patients and could be a potential tool for monitoring the effects of treatment.

Detection of Toxoplasma gondii in venous blood from AIDS patients by polymerase chain reaction.

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.

C Tourte-Schaefer

Papers

Value of Prenatal Diagnosis and Early Postnatal Diagnosis of Congenital Toxoplasmosis: Retrospective Study of 110 Cases

Detection of Toxoplasma gondii in venous blood from AIDS patients by polymerase chain reaction.

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