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An ante-mortem diagnosis of equine protozoal myeloencephalitis (EPM) is presently based on clinical presentation, immunodiagnostics performed on serum and cerebrospinal fluid (CSF), and ruling out other neurological disorders. Molecular techniques introduce a novel and promising approach for the detection of protozoal agents in CSF. Hypothesizing that real-time PCR (rtPCR) can be a useful complement to EPM diagnostics, 210 CSF samples from horses suspected of neurological disease with EPM included as a differential diagnosis were tested using rtPCR to detect Sarcocystis neurona DNA and immunodiagnostics targeting antibodies against the same pathogen, performed on serum and CSF samples. Molecular and immunological results were compared with respect to origin of the horse, time of the year, signalment, clinical signs and treatment history. Twenty-five horses tested positive in CSF for S. neurona by rtPCR only, while 30 horses had intrathecally-derived antibodies to S. neurona only (serum to CSF ratio ≤ 64 by indirect fluorescent antibody test - IFAT), and 13 horses tested rtPCR-positive in CSF with evidence of intrathecally-derived antibodies to S. neurona. Previous treatment for EPM was the only variable presenting statistical difference between the two testing modalities, highlighting that animals with history of anti-protozoal treatment were more likely to test positive solely in IFAT, while horses without treatment were more likely to test positive by rtPCR only. The results support the use of molecular diagnosis for EPM caused by S. neurona as a complement to immunodiagnostics. The use of rtPCR in CSF for the detection of S. neurona may improve the diagnostic work-up of neurologic disease suspected horses, especially in animals without previous anti-protozoal treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Congenital toxoplasmosis (CT) is a parasitic disease that causes serious fetal and neonatal harm or death. In countries that do not have antenatal screening programs, the initiation of CT treatment relies on a postnatal diagnosis. Until recently, diagnosis was based on clinical signs and immunoglobulin seropositivity, which is fraught with difficulty. In these cases, diagnosis was often delayed or treatment, which carries risk, started empirically. We highlight the use of polymerase chain reaction to diagnose a case of congenital toxoplasmosis, allowing early treatment and justifying the treatment burden., (Copyright © 2018 American Association for Pediatric Ophthalmology and Strabismus. Published by Elsevier Inc. All rights reserved.)

Congenital toxoplasmosis (CT) results from transplacental passage of Toxoplasma gondii to the fetus during acute maternal infection. Our study aims to report clinical and biological patterns of 35 cases of CT diagnosed at the department of the Parasitology of the Pasteur Institute of Tunis and to access the performance of prenatal and early postnatal diagnosis techniques. Serological screening of maternal infection was performed by Immunoglobulin (Ig) M and IgG detection and IgG avidity determination. Prenatal diagnosis was based on both Toxoplasma DNA detection in the amniotic fluid and monthly ultrasound examinations. polymerase chain reaction analysis on amniotic fluid, performed only in 15 cases, detected Toxoplasma 's DNA in five cases (33.3%). Ultrasound examination did not reveal any morphological abnormalities. Thirty newborns had serological criteria of Toxoplasma infection. Congenital toxoplasmosis diagnosis was confirmed in 23 cases (76.6%) by immunoblot. Among the 35 born-infants, five (14.3%) were symptomatic: three had chorioretinitis at the first clinical ocular examination, one had neurological symptoms (seizures) with positive parasite DNA in cerebral spinal fluid, and one had both ophthalmological and neurological damages- chorioretinitis and intracranial calcifications in the computed tomography scan. Thirty-four of 35 infected children were treated with pyrimethamine-sulfadiazine combination. Four (11.7%) of the treated infants showed abnormal hematological values because of the treatment side effect. Serological rebound was observed in seven infants. A screening program and a diagnostic algorithm in pregnant women should be implemented in Tunisia to improve the follow-up of seronegative ones and to prevent CT cases.

Human cryptosporidiosis is an intestinal infection caused by different species belonging to the genus Cryptosporidium in both immunocompetent and immunocompromised individuals. The life cycle of Cryptosporidium sp. when affecting the digestive system is well known but the infection of other organs is less studied. Molecular methods are necessary for species and subtypes identification. The goal of this work is to propose a new approach that contributes to the diagnosis of the extra-intestinal dissemination process of Cryptosporidium infection. Cryptosporidium sp. was detected in stool and biopsy samples of two HIV-infected patients. DNA was extracted from feces, biopsy specimens, blood, and cerebrospinal fluid (CSF). All samples were analyzed by nested PCR-RFLP of the 18S rDNA, real-time PCR, and gp60 subtyping. Cryptosporidium DNA was detected in stool and tissue samples and it was also present in blood and CSF samples. Both cases were characterized as Cryptosporidium hominis subtype IeA11G3T3. This is the first report that demonstrates the presence of Cryptosporidium DNA in blood and CSF of HIV-infected patients.

This report describes a case of congenital toxoplasmosis in a newborn in Southern Italy. A pregnant mother had been admitted at the 20th week of her pregnancy on account of pharyngodynia and laterocervical lymphadenopathy. Although serological testing of the mother's serum documented a seroconversion with positive IgG and IgM anti-Toxoplasma antibodies during II trimester, the woman refused to perform prenatal diagnosis for congenital toxoplasmosis. Fetal ultrasound scan already showed mild asymmetrical triventricular hydrocephaly and cerebral calcifications. After birth, real-time PCR on cerebrospinal fluid and blood samples of the newborn showed a positive result for 529bp-repeat element DNA of T. gondii, In addition brain magnetic resonance imaging and computed tomography showed a characteristic diffuse brain tissue loss associated with hydrocephalus. For the first time molecular characterization of T. gondii isolate was performed directly from the newborn's CSF samples by using nested-PCR-RFLP of sag-2 and pk1 genes. The PCR-RLFP analysis revealed that the isolate belongs to the clonal type II, the predominant lineage causing human toxoplasmosis, as confirmed by DNA sequencing.

Background: Laboratory diagnosis of neurosyphilis is complicated especially when it is asymptomatic, no single laboratory test result being appropriate to diagnose central nervous system infectivity caused by Treponema pallidum. Our objective was to evaluate two polymerase chain reaction (PCR) techniques for the detection of T. pallidum DNA in the cerebrospinal fluid (CSF) of patients with syphilis., Methods: One hundred twenty-four CSF samples from patients with reactive blood tests for syphilis were obtained. Two PCR techniques (47-PCR, polA-PCR) were used to detect T. pallidum DNA. The laboratory criteria used for the diagnosis of neurosyphilis to which the PCR techniques were compared were those recommended by the IUSTI: 2008 European guidelines on the management of syphilis., Results: Treponema pallidum DNA was detected amplified in 37 of 124 (29.8%) and 30 of 124 (24.2%) samples with the 47-PCR and polA-PCR, respectively. Sensitivities were 75.8% and 69.7% and specificities 86.8% and 92.3%, respectively, for 47-PCR and polA-PCR techniques, respectively. The three CSF samples of patients with primary syphilis did not fulfill the criteria of neurosyphilis and DNA was only detected in one by the 47-PCR. In samples from secondary syphilis and neurosyphilis, three of nine and nine of nine respectively, results were coincident for the two PCR techniques and neurosyphilis criteria. Major discrepancies between the two PCR techniques and neurosyphilis diagnostic criteria were observed in latent syphilis., Conclusion: Beyond some limitations of the study, which are discussed here, both PCR techniques seem to be useful for the diagnosis of neurosyphilis, although 47-PCR presents a higher sensitivity and polA-PCR a higher specificity., (© 2016 Wiley Periodicals, Inc.)

Genotyping of Toxoplasma gondii is traditionally performed using DNA obtained from tachyzoites after isolation by bioassay in mice. In this study, genotyping of T. gondii was performed by multiplex nested polymerase chain reaction restriction fragment length polymorphism (Mn-PCR-RFLP) in DNA obtained from the lungs of experimentally infected mice, the hearts of naturally infected free-range chickens, and human blood samples of newborns with congenital toxoplasmosis. The efficiency of Mn-PCR varied according to the marker. We obtained complete genotypes of all of the mice lung samples. In chickens, total or partial genotyping was performed on all of the 15 samples. Two complete genotypes were obtained, including one identified for the first time, and another previously described in different hosts including dogs, cats, and humans. In blood from infants, partial genotypes were obtained in 8 of the 12 samples. Mouse bioassay is the most efficient method to obtain DNA from T. gondii , but direct tissue genotyping enhances the likelihood of obtaining molecular information on T. gondii and is an effective tool as a complement to isolation in mice. In this study, we genotyped Toxoplasma gondii directly from human (blood samples of newborns with congenital toxoplasmosis) and free-range chickens (hearts) by Mn-PCR-RFLP. We present partial and complete genotypes and provide technical and scientific information about T. gondii genotyping methods.

A two-year-old male, neutered, basset hound-beagle mix with progressive neurological impairment was examined postmortem. Grossly, the dog had multiple raised masses on the spinal cord between nerve roots. Microscopically, the dog had protozoal myeloencephalitis. Toxoplasma gondii and Sarcocystis neurona were detected in the CNS by immunohistochemistry and polymerase chain reaction (PCR). Sarcocysts in formalin-fixed muscle were negative for Sarcocystis by PCR. Banked serum was negative for T. gondii using the modified agglutination test, suggesting an acute case of T. gondii infection or immunosuppression; however, no predisposing immunosuppressive diseases, including canine distemper, were found. To the authors' knowledge, this is the first report of dual T. gondii and S. neurona infection in a dog., (Published by Elsevier B.V.)

Background: Knowledge of central nervous system (CNS) opportunistic infections (OIs) among people living with human immunodeficiency virus (HIV) in sub-Saharan Africa is limited., Methods: We analyzed 1 cerebrospinal fluid (CSF) sample from each of 331 HIV-infected adults with symptoms suggestive of CNS OI at a tertiary care center in Zambia. We used pathogen-specific primers to detect DNA from JC virus (JCV), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV) types 1 and 2, Mycobacterium tuberculosis, and Toxoplasma gondii via real-time polymerase chain reaction (PCR)., Results: The patients' median CD4(+) T-cell count was 89 cells/µL (interquartile range, 38-191 cells/µL). Of 331 CSF samples, 189 (57.1%) had at least 1 pathogen. PCR detected DNA from EBV in 91 (27.5%) patients, M. tuberculosis in 48 (14.5%), JCV in 20 (6.0%), CMV in 20 (6.0%), VZV in 13 (3.9%), HSV-1 in 5 (1.5%), and HSV-2 and T. gondii in none. Fungal and bacteriological studies showed Cryptococcus in 64 (19.5%) patients, pneumococcus in 8 (2.4%), and meningococcus in 2 (0.6%). Multiple pathogens were found in 68 of 189 (36.0%) samples. One hundred seventeen of 331 (35.3%) inpatients died during their hospitalization. Men were older than women (median, 37 vs 34 years; P = .01), more recently diagnosed with HIV (median, 30 vs 63 days; P = .03), and tended to have a higher mortality rate (40.2% vs 30.2%; P = .07)., Conclusions: CNS OIs are frequent, potentially treatable complications of AIDS in Zambia. Multiple pathogens often coexist in CSF. EBV is the most prevalent CNS organism in isolation and in coinfection. Whether it is associated with CNS disease or a marker of inflammation requires further investigation. More comprehensive testing for CNS pathogens could improve treatment and patient outcomes in Zambia., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Congenital toxoplasmosis can result in visual impairment, hearing loss, serious neurologic sequelae and death in the infant. We studied the potential of the polymerase chain reaction (PCR) in cerebrospinal fluid (CSF) for diagnosis of congenital toxoplasmosis., Methods: For this purpose, we studied both congenitally infected (diagnosed clinically and serologically) and noninfected infants born to untreated mothers., Results: The infants ranged in age from 0 to 180 days. CSF PCR was positive in 27 of the 58 (46.5%) congenitally infected infants and was negative in each of the 103 infants without congenital toxoplasmosis. The frequency of positive CSF PCR varied according to whether infants had major clinical signs of the disease; PCR was positive in 70.9%, 53.3% and 50.9% of those with hydrocephalus, cerebral calcifications and/or eye disease, respectively. Of 6 infants who were negative for both IgM and IgA antibodies, 3 had a positive PCR in their CSF as the confirmatory test for diagnosis of congenital toxoplasmosis. IgM and IgA antibodies and CSF PCR, when combined, yielded a higher sensitivity for diagnosis of congenital toxoplasmosis when compared with the performance of each test alone., Conclusions: Our findings reveal that in infants with clinical and serologic findings suggestive of congenital toxoplasmosis and born to untreated mothers, CSF PCR has the potential to increase the frequency of cases in which the diagnosis is confirmed.

Case Description: A 9-year-old male Miniature Poodle was evaluated because of progressive severe right hemiparesis, right forelimb lameness, and signs of cervical pain., Clinical Findings: A low body condition score (2/9) and popliteal lymphadenopathy were detected. Results of a CBC, serum biochemical analyses, urinalysis, cytologic examination of bone marrow and popliteal lymph node aspirates, and serum ELISA were consistent with systemic leishmaniasis. Magnetic resonance imaging of the cervical spinal cord revealed an intramedullary mass extending from the caudal aspect of the C5 vertebral body to the C5-6 intervertebral disk space with a contrast medium-enhanced pattern that had 3 zones (central contrast medium-enhanced core, intermediate isointense zone, and peripheral contrast medium-enhanced ring). Surgical biopsy of the mass was performed by means of a right C5-6 dorsal hemilaminectomy. Results of PCR assays for detection of Leishmania DNA in CSF and tissue biopsy samples were positive., Treatment and Outcome: Treatment for systemic leishmaniasis was initiated. Two months later, body condition, neurologic signs, and gait of the dog had substantially improved; the dog had mild right forelimb paresis at that time. Results of follow-up MRI indicated resolution of the cervical spinal cord lesion. Four months after diagnosis, the dog's neurologic condition was stable., Clinical Relevance: To the authors' knowledge, this report is the first in which clinical findings, clinicopathologic data, and MRI characteristics of an intramedullary inflammatory spinal cord lesion presumptively attributable to leishmaniasis in a dog have been reported, and the first report of CNS leishmaniasis in a dog with MRI resolution and a successful clinical response to treatment.

Introduction: Neurotoxoplasmosis (NT) sometimes manifests unusual characteristics., Methods: We analyzed 85 patients with NT and AIDS according to clinical, cerebrospinal fluid, cranial magnetic resonance, and polymerase chain reaction (PCR) characteristics., Results: In 8.5%, focal neurological deficits were absent and 16.4% had single cerebral lesions. Increased sensitivity of PCR for Toxoplasma gondii DNA in the central nervous system was associated with pleocytosis and presence of >4 encephalic lesions., Conclusions: Patients with NT may present without focal neurological deficit and NT may occur with presence of a single cerebral lesion. Greater numbers of lesions and greater cellularity in cerebrospinal fluid improve the sensitivity of PCR to T gondii.

The aim of this study was to assess the efficacy of nested polymerase chain reaction (PCR) and the loop-mediated isothermal amplification (LAMP) assay, which were developed to detect and identify toxoplasma parasites in human cerebrospinal fluid (CSF). Nested PCR was performed using primers generated by Dr. L.D. Sibley to target the 18S rDNA instead of the conventionally used primers which target the B1 gene. We also designed Toxoplasma gondii-specific LAMP primers targeting both genes. In vitro detection sensitivity was evaluated using 10-fold serially diluted genomic DNA purified from RH tachyzoites, and clinical sensitivity and specificity were evaluated using clinical CSF samples from 16 patients with toxoplasmic encephalitis (TE) and from 12 patients with other diseases. The 18S rDNA nested PCR showed the highest detection sensitivity limit with a minimum of 1.0 × 10(-8) ng/μL. However, sensitivity and specificity of nested PCR with clinical specimens were 50% and 100%, respectively. The sensitivity of molecular diagnosis of TE is not sufficient; therefore, patients clinically suspected of having TE should be treated promptly. Our molecular diagnostic tool would restrictively facilitate a definitive diagnosis of TE at an early stage in approximately 50% of patients., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Polymerase chain reaction (PCR) has made a significant improvement in the diagnosis of toxoplasmic encephalitis (TE). Nevertheless, a wide variety of targets and primers has been used in different assays, and few comparative studies had been carried out. The aim of the present study was to compare the efficiency of 3 conventional PCR methods by using 3 sets of primers targeting the repetitive B1 gene in the diagnosis of TE. Diagnostic sensitivity and specificity of PCR and nested-PCR protocols were assessed for 207 (nested-PCR/T1-T4), 200 (nested-PCR/S1-AS1), and 206 (PCR/B22-B23) cerebrospinal fluid (CSF) samples, including AIDS and HIV-negative patients. The diagnostic sensitivity of PCR and nested-PCR assays was 50.85%, 68.97%, and 72.41% for T1-T4, S1-AS1, and B22-B23, respectively. The diagnostic specificity was high for all the assays showing values between 95% and 97%. In general, the best results were obtained for the B22-B23 set of primers, suggesting their usefulness compared with 2 nested-PCR protocols and showing that this simple and rapid strategy may be the preferred one for the diagnosis of TE in AIDS patients., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Very limited data exists on the genetic diversity of Toxoplasma gondii from Eastern Europe. We present the first Romanian case of symptomatic congenital toxoplasmosis in which the T. gondii strain was isolated after inoculation in mice of a cerebrospinal fluid sample from a living neonate. The T. gondii strain was genotyped with 15 microsatellite markers distributed on 10 of the 14 chromosomes of T. gondii. The strain had a type II genotype., (© C.A. Costache et al., published by EDP Sciences, 2013.)

Cerebral toxoplasmosis is one of the leading causes of neurologic diseases with high mortality rates in patients with HIV infection. Invasion was difficult to diagnose for a number of objective reasons. The objective of the investigation was to determine the clinical sensitivity of different laboratory techniques as both a single study and their various combinations to verify the diagnosis of cerebral toxoplasmosis in HIV-infected patients. Blood and cerebrospinal fluid were tested in 51 patients with Stage 4B HIV infection (AIDS) with the verified diagnosis of cerebral toxoplasmosis. Separate determination of specific antibodies of IgG, IgM, IgA and toxoplasma DNA in the blood and cerebrospinal fluid was shown to have an insufficient clinical sensitivity (37.3-68.6%). The benefits of various combinations of immunological and molecular biological assays enhancing the diagnostic efficiency up to 76.5-96.1% are demonstrated.

A one-month male Greyhound dog presented with a swinging gait of the hindlimbs, and later developed muscular atrophy of the femoral region and hyperextension of hindlimbs. The dog had positive serum IFAT titers to Neospora caninum, but a negative titer in the cerebrospinal fluid (CSF). N. caninum-specific DNA was amplified from the CSF using a semi-nested polymerase chain reaction assay. Clusters of protozoa in biopsied muscle fibers were subsequently confirmed as N. caninum tachyzoites by immunohistochemical examination. Early recognition and treatment are necessary for effective recovery of clinical canine neosporosis, but antemortem diagnosis is difficult. We suggest that the detection of parasite deoxyribonucleic acid in the CSF is a useful antemortem diagnostic method in facilitating treatment of this disease.

Molecular methods are used increasingly for the detection of Toxoplasma gondii infection. This study developed a rapid, sensitive, and specific conventional triplex PCR for the detection of the B1 gene and ITS1 region of T. gondii using newly designed primers and an internal control based on the Vibrio cholerae HemM gene. The annealing temperature and concentrations of the primers, MgCl(2), and dNTPs were optimized. Two sets of primers (set 1 and 2) were tested, which contained different segments of the T. gondii B1 gene, 529 repeat region and ITS1 region. A series of sensitivity tests were performed using parasite DNA, whole parasites, and spiked human body fluids. Specificity tests were performed using DNA from common protozoa and bacteria. The newly developed assay based on set 2 primers was found to be specific and sensitive. The test was capable of detecting as little as 10 pg T. gondii DNA, 10(4) tachyzoites in spiked body fluids, and T. gondii DNA in the organ tissues of experimentally infected mice. The assay developed in this study will be useful for the laboratory detection of T. gondii infection., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Human African trypanosomosis, caused by Trypanosoma brucei gambiense, is a chronic disease, although various clinical patterns have been observed, from asymptomatic to acute forms. Since 2001 in Angola, 80% of patients have been found to be in the meningoencephalitic stage of the disease. The existence of an acute form of the disease caused by virulent strains of trypanosomes was suspected. To test this hypothesis, four sensitive and polymorphic microsatellite markers were used to characterize the trypanosome DNA extracted from the blood and cerebrospinal fluid of 100 patients in the meningoencephalitic stage. Twenty-three patients were found with mixed T. b. gambiense genotypes in the blood and/or cerebrospinal fluid. The absence of association between the number of infecting genotypes, the presence of neurological signs and white blood cell counts in the cerebrospinal fluid, seems to indicate, at least in the context of the present study, the absence of virulent strains. However, out of five patients who died from encephalopathy syndrome during treatment with eflornithine, three harbored multiple infections., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This study investigated the genetic characteristics of Toxoplasma gondii samples collected from 62 patients with toxoplasmosis in Sao Paulo State, Brazil. DNA samples were isolated from blood, cerebrospinal fluid and amniotic fluids of 25 patients with cerebral toxoplasmosis and AIDS, two patients with acute toxoplasmosis, 12 patients with ocular toxoplasmosis, six newborns with congenital toxoplasmosis and 17 pregnant women with acute infection. Diagnosis of toxoplasmosis was based in clinical, radiological and laboratory features. Genotyping was performed using multilocus PCR-RFLP genetic markers including SAG1, SAG2, 5'- and 3'-SAG2, alt.SAG2, SAG3, BTUB, GRA6, C22-8, c29-2, L358, PK1 and Apico. Among the 62 clinical samples, 20 (32%) were successfully genotyped at eight or more genetic loci and were grouped to three distinct genotypes. Eighteen samples belonged to ToxoDB Genotype #65 and the other two samples were identified as ToxoDB Genotypes #6 and #71, respectively (http://toxodb.org/toxo/). Patients presenting Genotypes #6 and #71 had severe and atypical cerebral toxoplasmosis, characterized by diffuse encephalitis without extensive brain lesions. These results indicate that T. gondii Genotype #65 may have a high frequency in causing human toxoplasmosis in Sao Paulo State, Brazil. This unusual finding highlights the need to investigate the possible association of parasite genotypes with human toxoplasmosis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: The loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 10(3) per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay., Methodology/principal Findings: For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 10(3) parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards., Conclusions/significance: This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world.

Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence. Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri., (Copyright 2009 Elsevier Inc. All rights reserved.)

We report the first case of primary amoebic meningoencephalitis in a 9-year-old boy in Guadeloupe. The outcome was rapidly fatal in 7 days. The patient presumably acquired the infection by swimming and diving in a basin supplied by natural thermal water 1 week before onset of the disease. The possibility of a free-living amoeba infection was suspected both on the negativity of all bacterial and viral initial tests and on the observation of peculiar cells in stained cerebrospinal fluid samples. Although the amoeba was not isolated, Naegleria fowleri could be identified by polymerase chain reaction with specific primers on DNA extracted from frozen cerebrospinal fluid samples. Furthermore, as the internal transcribed spacer (ITS1) region of DNA is variable in length between the different strains of N. fowleri, sequencing of the amplified ITS1 demonstrated that the responsible N. fowleri strain belongs to a common genotype present in the American and European continent.

Cerebral toxoplasmosis among individuals with AIDS may be difficult to diagnose and needs to be differentiated from other neurological diseases. A validation study was performed on real-time PCR for detecting the B1 gene of Toxoplasma gondii in the blood and cerebrospinal fluid (CSF) of AIDS patients with cerebral toxoplasmosis. The study included 135 AIDS patients divided into two groups: Group I comprised 85 patients with neurotoxoplasmosis; and Group II comprised 50 patients with non-toxoplasmic neurological diseases. Real-time PCR on blood showed a sensitivity of 1.5%, specificity of 100.0%, positive predictive value (PPV) of 100.0% and negative predictive value (NPV) of 36.5%. CSF testing produced better results, with a sensitivity of 35.3%, specificity of 100.0%, PPV of 100.0% and NPV of 44.7%. The group presenting with pleocytosis and four or more encephalic lesions was associated with greater CSF positivity on PCR. In conclusion, real-time PCR on blood was not useful for diagnosis. CSF testing showed low sensitivity but high specificity. Greater numbers of lesions and greater CSF cellularity may improve the sensitivity of the method.

Highly active antiretroviral therapy (HAART) has decreased the incidence of opportunistic infections in the central nervous system (CNS) in AIDS patients. However, toxoplasmic encephalitis (TE) still represents the most common cerebral mass lesion in patients infected with human immunodeficiency virus (HIV). The aim of this study was to evaluate nested PCR-B1 using cerebrospinal fluid (CSF) to detect Toxoplasma gondii DNA for the diagnosis of TE. A total of 114 samples were evaluated, and 33/44 samples from patients with TE were positive by PCR (sensitivity 75%), demonstrating the diagnostic usefulness of PCR technique. PCR-B1 products were analyzed by restriction fragment length polymorphism (RFLP) in 30 samples. Only type I allele at B1 was identified in these samples according banding patterns. This is the first report of evaluation of S1-AS1/S2-AS2 set of primers in more than 100 clinical samples as well as the first genotyping study of T. gondii in Cuba.

Background: We present data from 9 years (1999-2008) of tests for Balamuthia mandrillaris, an agent of amebic encephalitis that were conducted as part of the California Encephalitis Project., Methods: Specimens obtained from patients with encephalitis were sent to the California Encephalitis Project for diagnostic testing; a subset of these specimens were tested for Balamuthia species. Tests included indirect immunofluorescent staining of sections for amebae, fluorescent antibody staining and enzyme-linked immunosorbent assay for serum titers, and polymerase chain reaction for Balamuthia 16S mitochondrial DNA. Cerebrospinal fluid (CSF) samples obtained from patients with diverse types of encephalitis were also tested for a broad range of cytokines., Results: Of >3500 cases referred to the California Encephalitis Project, 10 were found to be amebic encephalitis on the basis of serologic and CSF tests and examination of stained tissue sections. Most of these cases would have been described as "encephalitis of unknown origin" if it were not for the California Encephalitis Project. Nine of the 10 patients were male; ages ranged from 1.5 to 72 years. All patients had abnormal neuroimaging findings and abnormal CSF composition. The more common symptoms at presentation included headache, seizures, cranial nerve palsies, and lethargy. CSF specimens from patients with Balamuthia infection had significant elevations in the levels of cytokines IL-6 and IL-8, compared with specimens obtained from persons with viral or noninfectious encephalitides., Conclusions: Balamuthiasis is difficult to diagnose, and it is likely that cases go unrecognized because clinicians and laboratorians are unfamiliar with the disease. Alerting the medical community to this disease may lead to earlier diagnosis and improve the chances of survival.

Encephalitis caused by Toxoplasma gondii is the most common cause of central nervous system damage in patients with acquired immunodeficiency syndrome (AIDS). Toxoplasma may infect any of the brain cells, thus leading to non-specific neurotoxoplasmosis clinical manifestations including focused or non-focused signs and symptoms of central nervous system malfunction. Clinical development ranges from insidious display during weeks to experiencing acute general confusion or ultimately fatal onset. Cerebral toxoplasmosis occurs in advanced stages of immunodeficiency, and the absence of anti-toxoplasmosis antibodies by the immunofluorescence method does not allow us to rule out its diagnosis. As specific therapy begins, diagnosis confirmation is sought through clinical and radiological response. There are few accurate diagnosis methods to confirm such cases. We present a method for T. gondii DNA detection by real time PCR-Multiplex. Fifty-one patients were evaluated; 16 patients had AIDS and a presumptive diagnosis for toxoplasmosis, 23 patients were HIV-positive with further morbidities except neurotoxoplasmosis, and 12 subjects were HIV-negative control patients. Real time PCR-Multiplex was applied to these patients' cephalorachidian liquid with a specific T. gondii genome sequence from the 529bp fragment. This test is usually carried out within four hours. Test sensitivity, specificity, positive predictive value, and negative predictive value were calculated according to applicable tables. Toxoplasma gondii assay by real time Multiplex of cephalorachidian fluid was positive for 11 out of 16 patients with AIDS and a presumptive diagnosis for cerebral toxoplasmosis, while none of the 35 control patients displayed such a result. Therefore, this method allowed us to achieve 68.8% sensitivity, 100% specificity, 100% positive predictive value, and 87.8% negative predictive value. Real time PCR on CSF allowed high specificity and good sensitivity among patients who presumably had cerebral toxoplasmosis. Since this is a low invasive method, it could be included in the diagnosis algorithm of patients with AIDS and central nervous system damage.

Background: Toxoplasmosis is a serious and often life-threatening disease in immunodeficient patients. Polymerase chain reaction (PCR) assays allow a rapid diagnosis of Toxoplasma infection by direct detection of the parasite's DNA. To perform a sensitive, specific, and reliable PCR-based diagnostic test, the availability of pure DNA lacking PCR inhibitors as well as a rapid and easy-to-perform DNA extraction protocol are essential. The aim of the present study was to compare four DNA extraction methods for the detection of T. gondii on cerebrospinal fluid (CSF) using the PCR technology., Material/methods: Four DNA extraction methods (boiling, lysis + centrifugation, the miniMAG commercial system, and phenol-chloroform) were compared with respect to the time of completion, the manual labor involved, and PCR analytical sensitivity for the detection of T. gondii in CSF. The optimal DNA extraction method for the detection of the parasite was evaluated in CSF from 43 AIDS patients using the nest-PCR B1 assay., Results: According to the time required for completion, labor, and PCR analytical sensitivity, the lysis + centrifugation protocol proved to be a simple, efficient, and economical in-house procedure to recover the T. gondii DNA present in the CSF. The diagnostic sensitivity of nest-PCR, according to Centers for Disease Control and Prevention (CDC) criteria, was 86.3% and the diagnostic specificity was 100%., Conclusions: We report a simple, rapid, reproducible, and economical in-house method for T. gondii DNA extraction from CSF. This method is recommended for diagnostic PCR of Toxoplasmic encephalitis (TE) in places with economical shortage.

This study investigated the genetic characteristics of the Toxoplasma gondii strains isolated from 87 patients with cerebral toxoplasmosis and AIDS, treated in Sao Paulo State, Brazil. The laboratorial diagnosis of cerebral toxoplasmosis was based on positive serological exams and PCR of blood and/or cerebrospinal fluid. Four markers (5'-SAG2, 3'-SAG2, SAG3 and GRA6) were chosen to analyze the samples. Each having clear resolution to distinguish the three clonal lineages after PCR amplified targets were treated with restriction enzyme digestion (PCR-RFLP). The genotyping provided the following results: 40 patients (46%) were infected with strains classified as type I; 4 (4%), as type III; 13 (15%) were infected with polymorphic strains (unusual genotype); 6 patients with type I or II alleles; and 15 (17%) patients had strains not classified for any marker. PCR-RFLP, also classified 9 (11%) clinical isolates as type II, which is uncommon in South America. However, the sequencing of the nested-PCR products (of SAG3 marker) of type II and polymorphic isolates (of 5'-SAG2, SAG3 and GRA6 markers) showed a nucleotide polymorphism compared with the archetypal clonal genotypes (types I, II and III) and these isolates were considered as polymorphic strains. The markers used here were inappropriate to distinguish the most isolates considered as polymorphic strains. These data confirm other studies showing the high rate of genetic polymorphism in T. gondii strains isolated in Brazil.

Trypanosoma cruzi lineages, microsatellite allelic polymorphism, and mithocondrial gene haplotypes were directly typified from peripheral blood and cerebrospinal fluid specimens of a Bolivian patient with Chagas disease with accompanying AIDS and central nervous system severe involvement. Of note, the patient's blood was infected by a mixture of T. cruzi I and T. cruzi IId/e polyclonal populations while the cerebrospinal fluid showed only a monoclonal T. cruzi I population. Our findings do not corroborate the original assumption of innocuity for T. cruzi I in the southern cone of the Americas and highlight lineage I tropism for central nervous system causing lethal Chagas reactivation.

Toxoplasmosis is a rare but rapidly fatal complication that can occur following hematopoietic stem cell transplantation (HSCT). Over a 17-yr period at our institutions, a definite diagnosis of toxoplasmosis was made in only two of 925 allogeneic HSCT recipients (0.22%) and none of 641 autologous HSCT recipients. These two patients received a conventional conditioning regimen followed by transplantation from an HLA-matched donor; however, they developed severe graft-vs.-host disease, which required intensive immunosuppressive therapy. Despite prophylactic treatment with trimethoprim/sulfamethoxazole, their immunosuppressive state, as indicated by a low CD4(+) cell count, might have resulted in toxoplasmosis encephalitis. Rapid and non-invasive methods such as a polymerase chain reaction (PCR) test of their cerebrospinal fluid for Toxoplasma gondii and magnetic resonance imaging of the brain were useful for providing a definitive diagnosis and prompt therapy in these patients: one patient stabilized and survived after responding to treatment with pyrimethamine/sulfodiazine whereas the other died of bacterial infection. In addition, retrospective PCR analyses of the frozen stored peripheral blood samples disclosed that detection of T. gondii preceded the onset of disease, indicating routine PCR testing of peripheral blood specimens may be an early diagnostic tool. It should be noted that when patients receiving HSCT have an unexplained fever and/or neurological complications, PCR tests should be considered to avoid cerebral lesions and improve the outcome of the patients.

Extra-intestinal amebiasis is secondary to invasive intestinal infections and usually results in an amebic liver abscess. Other organs, including lungs, brain, skin, spleen and kidneys, may be involved. Diagnosis of the cerebral infection is of the utmost importance and is most often made by detection of the organism at the time of brain biopsy or at autopsy. We report the first case of Entamoeba histolytica encephalitis diagnosed by PCR of the cerebrospinal fluid. The patient was treated successfully with metronidazole. PCR is an increasingly useful tool for the diagnosis of central nervous system infection and can provide rapid diagnosis.

Infections caused by Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris occur throughout the world and pose many diagnostic challenges. To date, at least 440 cases of severe central nervous system infections caused by these amebas have been documented worldwide. Rapid and specific identification of these free-living amebas in clinical samples is of crucial importance for efficient case management. We have developed a triplex real-time TaqMan PCR assay that can simultaneously identify Acanthamoeba spp., B. mandrillaris, and N. fowleri in the same PCR vessel. The assay was validated with 22 well-characterized amebic strains harvested from cultures and nine clinical specimens that were previously characterized by in vitro culture and/or immunofluorescence assay. The triplex assay demonstrated high specificity and a rapid test completion time of less than 5 h from the reception of the specimen in the laboratory. This assay was able to detect one single ameba per sample analyzed, as determined with cerebrospinal fluid spiked with diluted cultured amebas. This assay could become useful for fast laboratory diagnostic assessment of amebic infections (caused by free-living amebas) in laboratories with adequate infrastructure to perform real-time PCR testing.

PCR is now commonly applied to the diagnosis of toxoplasmosis. Although several methods are available, comparative studies are few, making it difficult to compare the performance of each technique. We compared the sensitivities of two real-time PCR assays through a prospective study on fetuses, neonates, and immunocompromised patients and on the ocular diagnosis of toxoplasmosis. The first system targeted the widely used B1 gene (GenBank accession number AF179871) while the second (RE) targeted a more recently described sequence repeated roughly 200 to 300 times (GenBank accession number AF146527). We demonstrated that molecular diagnosis requires the duplication of PCR assays, especially with the B1 system, as only one PCR was positive in 33.3% of cases. Our study showed that the RE target was more sensitive for all biological samples (amniotic fluid, placenta, aqueous humor, whole blood, and cerebrospinal and bronchoalveolar fluids) and significantly improved the performance of the diagnosis of toxoplasmosis. Taking into consideration all clinical samples, the mean gain in the crossing point value was 4.2 +/- 1.7 cycles and was even more significant for amniotic fluid (5.8 +/- 1.7 cycles).

We aimed to determine the effectiveness of using the polymerase chain reaction (PCR) to detect Toxoplasma gondii in cerebrospinal fluid (CSF) specimens from Japanese patients infected with human immunodeficiency virus (HIV)-1. Twenty-six HIV-positive individuals presenting with focal neurological signs and a possible diagnosis of T. gondii encephalitis (TE) were enrolled in the study between April 1997 and March 2003. Eight patients were diagnosed as having TE using the accepted diagnostic criteria; PCR amplified the T. gondii B1 gene in CSF samples from five of these eight patients. CSF samples from the 18 patients without TE were negative for T. gondii DNA. The sensitivity, specificity and positive and negative predictive values for detecting T. gondii in CSF using PCR were 62.5%, 100%, 100% and 85.7%, respectively. These results suggest that PCR might be a clinically useful technique for detecting T. gondii DNA in patients infected with HIV showing focal neurological signs. Improvements in sensitivity are needed, however.

In human African trypanosomiasis (HAT), two disease stages are defined: the first, or haemo-lymphatic stage, and the second, or meningo-encephalitic stage. Stage determination forms the basis of therapeutic decision and is of prime importance, as the drug used to cure second-stage patients has considerable side-effects. However, the tests currently used for stage determination have low sensitivity or specificity. Two new tests for stage determination in the cerebrospinal fluid (CSF) were evaluated on 73 patients diagnosed with HAT in Côte d'Ivoire. The polymerase chain reaction (PCR) detecting trypanosome DNA (PCR/CSF) is an indirect test for trypanosome detection whereas the latex agglutination test detecting immunoglobulin M (LATEX/IgM) is an indicator for neuro-inflammation. Both tests were compared with classically used tests, double centrifugation and white blood cell count of the CSF. PCR/CSF appeared to be the most sensitive test (96%), and may be of use to improve stage determination. However, its value for therapeutic decision appears limited, as patients whose CSF was positive with PCR were successfully treated with pentamidine. This result confirms those of previous works that showed that some patients with trypanosomes in the CSF could be treated successfully with pentamidine. LATEX/IgM, which depending on the cut-off, showed lower sensitivity of 76% and 88%, but higher specificity of 83% and 71% for LATEX/IgM 16 and LATEX/IgM 8 respectively, appears more appropriate for therapeutic decision making.

Object: Toxoplasmic encephalitis (TE), primary central nervous system lymphoma (PCNSL) and progressive multifocal leukoencephalopathy (PML) are major central nervous system (CNS) diseases in patients with acquired immunodeficiency syndrome (AIDS). We assessed the diagnostic value of polymerase chain reaction (PCR) in the detection of DNAs of Toxoplasma gondii (T. gondii), Epstein-Barr virus (EBV) and JC virus (JCV) in the cerebrospinal fluid (CSF)., Methods: We compared the PCR results with those of pathological findings at autopsy., Patients or Materials: The present study included 23 autopsies representing those in whom CSF samples were obtained before death while the patient was hospitalized or at autopsy., Results: The threshold levels for PCR detection were 4 tachyzoites of T. gondii, 5-15 genomes of EBV and 10 genomes of JCV. We identified T. gondii DNA in 4 out of 5 autopsy-defined cases of TE, EBV DNA in 5 out of 5 cases with PCNSL, and JCV DNA in 2 out of 2 cases with PML. The specificity of PCR was 100% in TE, 78% in PCNSL, and 100% in PML., Conclusion: Although the number of cases was relatively small in this study, PCR correctly identified T. gondii DNA in those cases in which PML or PCNSL was the sole clinical diagnosis. Our results indicate that PCR examination of CSF is a clinically useful tool for the diagnosis of focal brain lesions in patients with AIDS.

This study evaluated the detection of antibodies, circulating antigens and parasite DNA by polymerase chain reaction (PCR) in the diagnosis of toxoplasma encephalitis. The detection of antibody classes and IgG avidity were not useful diagnostically. The detection of circulating antigens by the ELISA system described was not sufficiently sensitive. The detection of DNA by PCR was the most useful test especially in untreated patients, with a sensitivity of 62% overall, 81% in untreated patients and only 20% in treated patients. The use of non-isotopic probes makes the use of this technique feasible in routine diagnostic parasitology laboratories.

Objective: To identify disease patterns in AIDS-related focal brain lesions (FBL) and to design a decision-making strategy for differential diagnosis., Design: Prospective study. Probabilities of CNS disorders were calculated using Bayes' theorem according to clinical variables (mass effect at CT or MRI, Toxoplasma serology, anti-Toxoplasma prophylaxis) and to the results of polymerase chain reaction (PCR) assays., Patients: 136 consecutive HIV-infected patients with a definitive diagnosis of FBL-causing disorder observed from 1991 to 1995 in a single clinical setting., Interventions: Patients underwent empiric anti-Toxoplasma therapy. After 3 weeks, patients with progressive/stable disease underwent brain biopsy. In 66 patients Epstein-Barr virus (EBV)-DNA, JC virus (JCV)-DNA, and T gondii-DNA amplification was performed by PCR in CSF. Diagnostic criteria were histopathologic examination of bioptic or autoptic tissue specimens for all disorders and complete/partial resolution of FBL after empiric therapy for toxoplasmic encephalitis (TE)., Results: Neuroradiologic characteristics did not discriminate between TE and primary CNS lymphoma (PCNSL). Probability of TE was 0.87 in Toxoplasma-seropositive patients with mass effect who were not receiving anti-Toxoplasma prophylaxis, but only 0.59 if prophylaxis was performed. In seronegative patients with mass effect, the likelihood of PCNSL was 0.74. If EBV-DNA or T gondii-DNA tests were positive, the probability of PCNSL or TE increased to more than 0.96. The absence of T gondii-DNA did not exclude the possibility of a TE diagnosis. Among FBL without mass effect, the probability of progressive multifocal leukoencephalopathy (PML) was 0.81; this increased to 0.99 if JCV-DNA testing was positive. Sensitivity of brain biopsy was 93%, with a perioperative morbidity of 12% and a mortality of 2%., Conclusions: Due to the low diagnostic capability of clinical variables, PCR amplifications in CSF, especially for EBV-DNA and for JCV-DNA, represent, in most cases, an essential step in the differential diagnosis of AIDS-related FBL. This is particularly true in patients with FBL without mass effect or with mass effect and who are either seronegative or undergoing anti-Toxoplasma prophylaxis. Brain biopsy remains a necessary procedure in EBV-DNA-positive cases and in seronegative patients with FBL displaying a mass effect. Positive JCV-DNA testing may obviate the need for brain biopsy in patients with FBL without mass effect. An advanced diagnostic strategy based on combined clinical criteria and PCR tests may allow rapid and accurate identification of patients for prompt brain biopsy or specific therapy.

To evaluate whether the detection of Toxoplasma gondii DNA in CSF could contribute to the differential diagnosis of AIDS-related focal brain lesions, CSF samples from 88 HIV-infected patients (56 with focal brain lesions and 32 without) were tested prospectively by a nested PCR for the B1 gene of T. gondii. The assay had a detection limit of 10 trophozoite equivalents. Six of 18 patients with toxoplasmic encephalitis, but none of the 70 patients with other disorders, were PCR-positive (33.3% sensitivity and 100% specificity). Considering only those patients with cerebral toxoplasmosis from whom CSF was collected before or during the first week of antitoxoplasmic therapy, sensitivity rose to 50%. This was higher than the sensitivity in patients whose CSF was collected after the first week of treatment (odds ratio (OR) of 7.0; 95% CI: 0.46-218.2). The administration of antitoxoplasmic prophylaxis did not affect the PCR results. Patients with a poor response to therapy had a higher probability of detectable T. gondii DNA in their CSF (OR of 5.0; 95% CI: 0.37-86.6). All patients with other central nervous system disorders were PCR-negative. Despite the moderate sensitivity, the high specificity and positive predictive value (100%) make this assay a useful tool in the differential diagnosis of AIDS-related focal brain lesions as part of a series of CSF and neuroradiological examinations.

Objective: To develop a polymerase chain reaction (PCR) technique to identify Toxoplasma gondii DNA in biological samples from cats and dogs., Design: To artificially create samples that would mimic those acquired in a clinical setting from animals with naturally acquired toxoplasmosis. Using these samples, a PCR test to identify T gondii DNA was developed., Sample Population: Feline and canine aqueous humor, CSF, serum, and blood samples., Procedure: Tachyzoites of several strains of T gondii grown in cell culture were added to feline and canine aqueous humor, CSF, serum, and blood samples. Protocols for identifying T gondii DNA by use of the PCR were developed., Results: The DNA from as few as 10 tachyzoites of T gondii could be identified in feline and canine aqueous humor, CSF, and serum samples. One hundred tachyzoites could be identified in blood samples., Conclusions: Toxoplasma gondii can be identified in feline and canine biological samples by use of the PCR., Clinical Relevance: Correlation of clinical disease to T gondii serum antibodies provides only a presumptive diagnosis of toxoplasmosis. Use of PCR to detect T gondii DNA in biological samples from cats and dogs may provide a sensitive tool for the antemortem diagnosis of toxoplasmosis and may be most beneficial when used in conjunction with serum antibody titers.

Objective: To study the accuracy of polymerase chain reaction (PCR) for Toxoplasma gondii DNA in the cerebrospinal fluid (CSF) of AIDS patients for the diagnosis of T. gondii encephalitis., Patients: Eighty-two AIDS patients with brain lesions. At autopsy, 19 patients (group A) had toxoplasmic encephalitis and 33 (group B) primary brain lymphoma or other infections. Brain histology was not available for 30 patients; cerebral lesions improved after anti-Toxoplasma therapy in 16 (group C), but there was no improvement in 14 patients (group D)., Methods: T. gondii RH strain was serially diluted in microplate wells. After heat denaturation, nested PCR was performed on diluted tachyzoites and on 10 microliters CSF with primers flanking the B1 repetitive region of T. gondii genome., Results: DNA from one to five tachyzoites was detected in each experiment. PCR was positive in eight (42.1%) out of 19 group A samples, none of the group B samples, 10 (62.5%) out of 16 group C samples and none of the group D samples. Among group A and C patients, PCR was positive in all 11, and in seven out of 24 (29.1%; P < 0.04) patients who had received anti-Toxoplasma therapy for less or more than 1 week at the time of rachicentesis, respectively., Conclusions: Nested PCR for T. gondii in CSF may improve early differential diagnosis of AIDS-associated focal brain lesions. Higher diagnostic accuracy was achieved when lumbar puncture was performed in the first week of anti-Toxoplasma therapy.

Central nervous system toxoplasmosis is a life-threatening infection with a mortality rate of higher than 60%. An early and rapid diagnosis is important for effective treatment of the disease. A new approach for detection of cerebral toxoplasmosis is described here. DNAs extracted from cells in cerebrospinal fluid samples (0.3 to 0.8 ml) of patients suspected of having cerebral toxoplasmosis were analyzed by a dot blot hybridization technique. A highly repetitive DNA sequence of Toxoplasma gondii (ABGTg4) was nonisotopically labelled with digoxigenin-dUTP and used as a specific DNA probe. Four of six patients analyzed gave positive signals in our hybridization assay. Two of them recovered with pyrimethamine-sulfadiazine, a drug recommended for treatment of toxoplasmosis. The other two patients with positive signals died soon after diagnosis. Patients with negative signals were found to suffer from mycobacterial infection (patient 1) or varicella-zoster virus infection (patient 6).

The polymerase chain reaction (PCR) was used to detect Toxoplasma gondii DNA in cerebrospinal fluid (CSF) from 14 patients with AIDS by amplification of the repetitive B1 gene. Positive PCRs were obtained in CSF from four of nine patients with toxoplasmic encephalitis. CSF samples from five control patients were negative for T. gondii DNA by PCR.