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Neurocysticercosis diagnosis is based on a combination of clinical, epidemiological, radiological, and immunological findings. We describe a real-time PCR assay for the confirmation of neurocysticercosis diagnosis in cerebrospinal fluid. The assay, tested on samples from nine patients living in France and diagnosed with neurocysticercosis, had a detection rate of 83.3% and 100% specificity.

Trypanosoma brucei gambiense group 1 is the major causative agent of the Gambian human African trypanosomiasis (HAT). Accurate diagnosis of Gambian HAT is still challenged by lack of precise diagnostic methods, low and fluctuating parasitemia, and generally poor services in the areas of endemicity. In this study, we designed a rapid loop-mediated isothermal amplification (LAMP) test for T. b. gambiense based on the 3' end of the T. b. gambiense-specific glycoprotein (TgsGP) gene. The test is specific and amplifies DNA from T. b. gambiense isolates and clinical samples at 62°C within 40 min using a normal water bath. The analytical sensitivity of the TgsGP LAMP was equivalent to 10 trypanosomes/ml using purified DNA and ∼1 trypanosome/ml using supernatant prepared from boiled blood, while those of classical PCR tests ranged from 10 to 10(3) trypanosomes/ml. There was 100% agreement in the detection of the LAMP product by real-time gel electrophoresis and the DNA-intercalating dye SYBR green I. The LAMP amplicons were unequivocally confirmed through sequencing and analysis of melting curves. The assay was able to amplify parasite DNA from native cerebrospinal fluid (CSF) and double-centrifuged supernatant prepared from boiled buffy coat and bone marrow aspirate. The robustness, superior sensitivity, and ability to inspect results visually through color change indicate the potential of TgsGP LAMP as a future point-of-care test.

Neurocysticercosis (NC), caused by the larval stage of Taenia solium, is one of the most common parasitic diseases of the central nervous system. The diagnosis of NC is mostly based on costly brain neuroimaging (computed tomography and/or nuclear magnetic resonance), which is rarely accessible in most affected areas. The most sensitive and specific tools for NC diagnosis are imagery techniques. The identification of specific antibodies and antigens is currently used only to support NC diagnosis due to their limited specificity and sensitivity. This study was performed to compare immunodiagnostic assays (antibody detection by enzyme-linked immunosorbent assay [ELISA] and enzyme-linked immunoelectrotransfer blotting [EITB] and HP10 antigen detection by ELISA) with the detection of parasite DNA by PCR amplification of a repetitive element of the parasite genome in the cerebrospinal fluid (CSF) of 121 radiologically and clinically characterized NC patients. Patients were divided into six groups according to the stage of the parasites and their localization. The CSF cellularity of each patient was also recorded. When all patients were considered, PCR exhibited the highest sensitivity (95.9%) and variable specificity (80% or 100%) depending on the controls used. The sensitivities of antibody detection by ELISA and EITB were not significantly different, and ELISA identified HP10 antigen mostly when vesicular cysticerci were located in the subarachnoideal basal cisterns. These results can help in the selection of different individual assays or combinations of assays to be used in NC diagnosis according to different requirements.

Molecular tools, such as real-time nucleic acid sequence-based amplification (NASBA) and PCR, have been developed to detect Trypanosoma brucei parasites in blood for the diagnosis of human African trypanosomiasis (HAT). Despite good sensitivity, these techniques are not implemented in HAT control programs due to the high cost of the equipment, which is unaffordable for laboratories in developing countries where HAT is endemic. In this study, a simplified technique, oligochromatography (OC), was developed for the detection of amplification products of T. brucei 18S rRNA by NASBA. The T. brucei NASBA-OC test has analytical sensitivities of 1 to 10 parasites/ml on nucleic acids extracted from parasite culture and 10 parasites/ml on spiked blood. The test showed no reaction with nontarget pathogens or with blood from healthy controls. Compared to the composite standard applied in the present study, i.e., parasitological confirmation of a HAT case by direct microscopy or by microscopy after concentration of parasites using either a microhematocrit centrifugation technique or a mini-anion-exchange centrifugation technique, NASBA-OC on blood samples had a sensitivity of 73.0% (95% confidence interval, 60 to 83%), while standard expert microscopy had a sensitivity of 57.1% (95% confidence interval, 44 to 69%). On cerebrospinal fluid samples, NASBA-OC had a sensitivity of 88.2% (95% confidence interval, 75 to 95%) and standard microscopy had a sensitivity of 86.2% (95% confidence interval, 64 to 88%). The T. brucei NASBA-OC test developed in this study can be employed in field laboratories, because it does not require a thermocycler; a simple heat block or a water bath maintained at two different temperatures is sufficient for amplification.

Early identification of Acanthamoeba in cerebrospinal fluid is mandatory to prevent fatal granulomatous amebic encephalitis. In the case presented here amebic trophozoites were detected in a routine cerebrospinal fluid sample. The antibiotic treatment and the apparently low virulence of this isolate were responsible for the benign progression of the infection.

Toxoplasma gondii is an important food- and waterborne opportunistic pathogen that causes severe disease in immunocompromised patients. T. gondii has an unusual clonal population structure consisting of three widespread lineages known as I, II, and III. To establish the genotypes of strains of T. gondii associated with human toxoplasmosis, we have developed a set of four highly sensitive and polymorphic nested PCR markers. Multiplex nested PCR analysis was used to genotype parasites in cerebral spinal fluid samples from 8 of 10 human immunodeficiency virus-positive patients. Remarkably, a majority of these patients had infections with type I strains or strains containing type I alleles, despite the fact that this lineage is normally uncommon in humans and animals. Multiplex analysis of these four unlinked makers was able to distinguish all three common genotypes and also detected two strains with mixed genotypes. Further analysis based on sequencing of a polymorphic intron revealed that one of these recombinant strains was an exotic lineage distinct from the archetypal clonal lineages. The multiplex nested PCR analysis described here will be useful for analyzing the contribution of parasite genotype to toxoplasmosis.

Human African trypanosomiasis (HAT) or sleeping sickness is a disease characterized by a hemolymphatic stage 1 followed by a meningoencephalitic stage 2 which is fatal without specific treatment. Furthermore, due to the toxicity of drugs used to treat stage 2 (mainly melarsoprol) accurate staging is required. Actual criteria employed during field surveys are not sensitive enough for precise staging. Antineurofilament (anti-NF) and antigalactocerebrosides (anti-GalC) antibodies have been identified in cerebrospinal fluid (CSF) as potential markers of central nervous system (CNS) involvement. We describe a dot enzyme-linked immunosorbent assay (dot-ELISA) to detect anti-GalC and anti-NF antibodies and its value in staging. NF- and GalC-dotted nitrocellulose strips were first developed in our laboratory. They were then evaluated in Angola and Central African Republic on 140 CSF samples. Compared to our staging criteria (i.e., CSF cell count > or = 20 cells/microl, CSF immunoglobulin M concentration > or = 100 mg/liter, and/or the presence of trypanosomes in the CSF), combined detection of both CSF anti-NF and CSF anti-GalC by dot-ELISA showed 83.2% sensitivity and 100.0% specificity. Dot-ELISA could be a useful test to diagnose CNS involvement in HAT in the less-equipped laboratories or in the field situation and to improve patient treatment.

Balamuthia mandrillaris is a free-living ameba that causes granulomatous amebic encephalitis in both immunocompromised and immunocompetent individuals. Because of a lack of pathognomonic symptoms and the difficulty in recognizing amebas in biopsied tissues, most cases are not diagnosed or effectively treated, leading to a >95% mortality. We report here on five cases of balamuthiasis that were diagnosed by indirect immunofluorescence (IIF) staining of serum for anti-Balamuthia antibodies (titer > or = 1:128) and confirmed by IIF of unstained brain tissue sections and/or detection of amebas in hematoxylin-eosin-stained slides. Additionally, we have used the PCR for the detection of mitochondrial 16S rRNA gene DNA from the ameba in clinical specimens such as brain tissue and cerebrospinal fluid (CSF) from individuals with Balamuthia encephalitis. Balamuthia DNA was successfully detected by the PCR in clinical samples from all five individuals. It was detected in brain tissue from three cases, in CSF from three cases, and in one of two samples of lung tissue from two individuals, but not in two samples of kidney tissue tested. One sample of unfixed brain tissue was culture positive for Balamuthia. In order to test the sensitivity of the PCR for detection of Balamuthia DNA, CSF specimens from two individuals negative for amebic infection were spiked with Balamuthia amebas. We found that it was possible to detect Balamuthia DNA in the PCR mixtures containing mitochondrial DNA from 1 to as little as 0.2 ameba per reaction mixture. A single Balamuthia ameba contains multiple mitochondrial targets; thus, 0.2 ameba represents multiple targets for amplification and is not equivalent to 0.2 of an ameba as a target.

Highly active antiretroviral therapy has decreased the incidence of opportunistic infections in the central nervous system in AIDS patients. However, neurological abnormalities still remain important causes of mortality and morbidity in developing countries. In Brazil, cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients. For these reasons, early, inexpensive, and sensitive diagnostic tests must be evaluated. The aim of this study was to evaluate PCR, using cerebrospinal fluid (CSF) samples to detect Toxoplasma gondii DNA, and to determine if the association of PCR with immunological assays can contribute to a timely diagnosis. We studied two sample groups. First, we analyzed stored CSF samples from 29 newborns and from 39 adults with AIDS without a definitive diagnosis of toxoplasmosis. The goal of this step was to standardize the methodology with a simple and economical procedure to recover the T. gondii DNA. Next, we prospectively evaluated CSF samples from 12 AIDS patients with a first episode of cerebral toxoplasmosis and 18 AIDS patients with other neurological opportunistic diseases and without previous cerebral toxoplasmosis. In all PCR samples, an indirect immunofluorescent assay and an enzyme-linked immunosorbent assay were performed. Samples from all patients with cerebral toxoplasmosis presented positive PCR results (sensitivity, 100%), and a sample from one of the 18 AIDS patients with other neurological diseases also presented positive PCR results (specificity, 94.4%). These findings suggest the clinical utility of PCR in the diagnosis of cerebral toxoplasmosis in developing countries.

Toxoplasma gondii is a common life-threatening opportunistic infection. We used experimental murine T. gondii infection to optimize the PCR for diagnostic use, define its sensitivity, and characterize the time course and tissue distribution of experimental toxoplasmosis. PCR conditions were adjusted until the assay reliably detected quantities of DNA derived from less than a single parasite. Forty-two mice were inoculated intraperitoneally with T. gondii tachyzoites and sacrificed from 6 to 72 h later. Examination of tissues with PCR and histology revealed progression of infection from blood to lung, heart, liver, and brain, with PCR consistently detecting parasites earlier than microscopy and with no false-positive results. We then evaluated the diagnostic value of this PCR assay in human patients. We studied cerebrospinal fluid and serum samples from 12 patients with AIDS and confirmed toxoplasmic encephalitis (defined as positive mouse inoculation and/or all of the Centers for Disease Control clinical diagnostic criteria), 12 human immunodeficiency virus-infected patients with suspected cerebral toxoplasmosis who had neither CDC diagnostic criteria nor positive mouse inoculation, 26 human immunodeficiency virus-infected patients with other opportunistic infections and no signs of cerebral toxoplasmosis, and 18 immunocompetent patients with neurocysticercosis. Eleven of the 12 patients with confirmed toxoplasmosis had positive PCR results in either blood or cerebrospinal fluid samples (6 of 9 blood samples and 8 of 12 cerebrospinal fluid samples). All samples from control patients were negative. This study demonstrates the high sensitivity, specificity, and clinical utility of PCR in the diagnosis of toxoplasmic encephalitis in a resource-poor setting.

A fatal case of primary amebic meningoencephalitis (PAM) in a 5-month-old infant is described. The disease may have been contracted during bathing. The source of water was from an artificial well. The clinical presentation, the isolation of the ameba from the cerebrospinal fluid, the poor response to amphotericin B, and the ultimate fatal outcome are all consistent with the diagnosis of PAM. On the basis of its ability to grow at temperatures above 30 degrees C, the morphology of the trophozoite, and the presence of flagellate forms, the ameba was identified as Naegleria fowleri. Pathogenic N. fowleri amebae were recovered from samples of water from the well. To our knowledge this case represents the second case of PAM in an infant in the absence of the history of swimming.

Antigens were detected in cerebrospinal fluid (CSF) samples from patients with neurocysticercosis (NC) by enzyme-linked immunosorbent assay (ELISA) using polyclonal sera of rabbit anti-Taenia solium cysticerci (anti-Tso) and anti- Taenia crassiceps cysticerci vesicular fluid (anti-Tcra or anti-Tcra <30 kDa). A group of NC patients (n = 174) were studied (NC), including 40 patients in different phases of the disease. ELISAs carried out with the anti-Tso, anti-Tcra, and anti-Tcra <30 kDa showed sensitivities of 81.2, 90, and 95.8% and specificities of 82, 98, and 100%, respectively. The 14- and 18-kDa low-molecular-weight peptides were only detected in CSF samples from patients with NC by immunoblotting with anti-Tso and anti-Tcra sera. Because of the importance of the diagnosis and prognosis of cysticercosis, the detection of antigens may contribute as an additional marker to the study and clarification of the parasite-host relationship.

In this study we have devised a simple and robust PCR strategy to detect a wide range of viruses, bacteria, and parasites, all of which are capable of causing aseptic meningitis and encephalitis. The techniques developed have been used in a routine diagnostic virology laboratory to test prospectively 2,233 cerebrospinal fluid specimens. A virus was detected in 147 specimens of cerebrospinal fluid from 143 patients. Four sets of primers were sufficient to detect the virus in 135 (94%) of the PCR-positive patients. We conclude that with appropriate primers, PCR can be systematically and economically applied to test for a range of organisms in a routine diagnostic laboratory. In our opinion, PCR will soon become the "gold standard" test for viral infections of the central nervous system.

Viable Angiostrongylus cantonensis was recovered from the cerebrospinal fluid of a 17-month-old boy with eosinophilic meningitis. Neurological findings were minimal, and the child had an uneventful recovery.