Neurotoxoplasmosis diagnosis for HIV-1 patients by real-time PCR of cerebrospinal fluid

Federal University of São Paulo, Brazil.
The Brazilian journal of infectious diseases: an official publication of the Brazilian Society of Infectious Diseases (Impact Factor: 1.3). 03/2009; 13(1):18-23. DOI: 10.1590/S1413-86702009000100006
Source: PubMed


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.

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    • "Due to the important role of contamination of soil in the transmission dynamics of T. gondii, previous studies developed several methods such as loop-mediated isothermal amplification (LAMP) and QT-PCR to investigate its oocysts in soils [17-20]. However, no information about the T. gondii oocysts prevalence in soils could be available in northwestern China, especially in the pasturing areas. "
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    ABSTRACT: Background Toxoplasma gondii is a zoonotic pathogen that can infect a range of animals and humans. Ingestion of T. gondii oocysts in soil is a significant transmission route for humans and animals acquiring toxoplasmosis. In the present study, we developed a new semi-nested PCR method to determine T. gondii oocysts distribution in soils in northwestern China.ResultsThe one tube semi-nested PCR assay was developed to detect the oocysts of T. gondii in soil, targeting the repetitive 529 bp fragment of T. gondii genomic DNA. Then a total of 268 soil samples, including 148 samples from Gansu Province and 120 samples from Qinghai Province, northwestern China, were examined by the semi-nested PCR method. One third of the positive samples were sequenced. The sensitivity of the semi-nested PCR assay was 102¿ T. gondii oocysts in 5 g soil sample. Investigation of soil samples from northwestern China showed that 34 out of 268 soil samples (12.69%) were T. gondii positive. Sequences of the partial 529 bp fragments varied from 0¿1.2% among the sequenced samples. The prevalence of T. gondii oocysts in soil from cities (24/163) was slightly higher than that in soils from pasturing areas (10/105) (P¿=¿0.21). Among the different regions in cities, the prevalence of T. gondii oocysts in soils from parks was 14.15%, whereas that in soils from schools was 19.05%.Conclusions The present study firstly reported the prevalence of T. gondii oocysts in soils in northwest China using a novel semi-nested PCR assay, which provided baseline data for the effective prevention and control of toxoplasmosis in this region.
    BMC Veterinary Research 09/2014; 10(1):238. DOI:10.1186/s12917-014-0238-z · 1.78 Impact Factor
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    • "An accurate diagnosis of these neurological complications is crucial for HIV infected patients, since most such complications are likely to be treated, and a prompt effective intervention may eventually yield to longer survival or better quality of living. Several studies have demonstrated the usefulness of PCR on cerebrospinal fluid (CSF) samples for the diagnosis of cerebral toxoplasmosis [1] [15] [19]. However, a lumbar puncture could be contraindicated in a subgroup of patients with expansive cerebral lesions [6]. "
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    ABSTRACT: We determine if peripheral blood sample could be used for diagnosis of cerebral toxoplasmosis by a real-time PCR assay and we analyzed the clinical and laboratory findings in 22 patients with confirmed cerebral toxoplasmosis compared to 27 patients with other neuro-opportunistic infections. We compared two gene targets (B1 and RE) in the Taqman-PCR real-time assay. Efficiency values were also calculated. We found that 18.8% (4/22) of cases with cerebral toxoplasmosis and 7.4% (2/27) of patients with other neuroinfections had positive results with the Taqman PCR assay. The mean number of parasites was 67.7 (SD 69.0) tachyzoites/mL in patients with cerebral toxoplasmosis and of 31.8 (SD 6.3) tachyzoites/mL in other neuroinfections. Clinical symptoms as headache were significantly less frequent and focal neurological symptoms were significantly more frequent in cerebral toxoplasmosis than in other neuroinfections. In our conditions, real-time PCR on peripheral blood samples was not useful for diagnosis of cerebral toxoplasmosis.
    Journal of neuroparasitology 01/2011; 2. DOI:10.4303/jnp/N110402
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    ABSTRACT: There are many neglected nonenteric protozoa able to cause serious morbidity and mortality in humans, particularly in the developing world. Diseases caused by certain protozoa are often more severe in the presence of HIV. While information regarding neglected tropical diseases caused by trypanosomatids and Plasmodium is abundant, these protozoa are often not a first consideration in Western countries where they are not endemic. As such, diagnostics may not be available in these regions. Due to global travel and immigration, this has become an increasing problem. Inversely, in certain parts of the world (particularly sub-Saharan Africa), the HIV problem is so severe that diseases like microsporidiosis and toxoplasmosis are common. In Western countries, due to the availability of highly active antiretroviral therapy (HAART), these diseases are infrequently encountered. While free-living amoebae are rarely encountered in a clinical setting, when infections do occur, they are often fatal. Rapid diagnosis and treatment are essential to the survival of patients infected with these organisms. This paper reviews information on the diagnosis and treatment of nonenteric protozoal diseases in immunocompromised people, with a focus on patients infected with HIV. The nonenteric microsporidia, some trypanosomatids, Toxoplasma spp., Neospora spp., some free-living amoebae, Plasmodium spp., and Babesia spp. are discussed.
    Clinical microbiology reviews 10/2010; 23(4):795-836. DOI:10.1128/CMR.00001-10 · 17.41 Impact Factor
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