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Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients
JNS-13402; No of Pages 4 Journal of the Neurological Sciences xxx (2014) xxx–xxx
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Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients Lívia Maria Pala Anselmo a,⁎, Fernando Crivelenti Vilar b, José Eduardo Lima c, Aparecida Yule Yamamoto d, Valdes Roberto Bollela e, Osvaldo Massaiti Takayanagui a a
School of Medicine of Ribeirão Preto, University of São Paulo, Department of Neurology, Brazil Clinics Hospital, School of Medicine of Ribeirão Preto, University of São Paulo, Department of Internal Medicine — Infectious Diseases, Brazil Clinics Hospital, School of Medicine of Ribeirão Preto, University of São Paulo, Department of Neurology, Brazil d School of Medicine of Ribeirão Preto, University of São Paulo, Department of Pediatrics—Molecular and Infectious Diseases, Brazil e School of Medicine of Ribeirão Preto, University of São Paulo, Department of Internal Medicine — Infectious Diseases, Brazil b c
a r t i c l e
i n f o
Article history: Received 11 June 2014 Received in revised form 29 July 2014 Accepted 24 August 2014 Available online xxxx Keywords: Toxoplasmosis HIV Acquired immunodeficiency syndrome Cerebrospinal fluid Central nervous system
a b s t r a c t The objective of the present study was to assess the performance and the best indication of the polymerase chain reaction (PCR) for the detection of Toxoplasmosis gondii DNA in cerebrospinal fluid (CSF) from patients with suspected neurotoxoplasmosis. CSF samples were collected from 79 patients for amplification of the T. gondii genome (gene B1) by two PCR techniques (nested and real time). Twenty-seven of the 79 patients were classified as probable cases of neurotoxoplasmosis on the basis of clinical criteria, neuroimaging and therapeutic response. PCR showed high sensitivity (86.6%) when performed in CSF samples which were collected up to the seventh day of specific toxoplasmosis treatment. There was no positive test after 1 week of treatment. These results suggest the usefulness of PCR for the diagnosis of cerebral toxoplasmosis, and support the first week as the window for the best performance of toxoplasmosis PCR in CSF. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Among AIDS patients, toxoplasmosis usually occurs in individuals with CD4 + T lymphocyte counts b100/μl by reactivation of chronic infection [20]. The clinical signs and symptoms of neurotoxoplasmosis in immunosuppressed patients can be acute or subacute and a definite diagnosis of toxoplasmosis requires the identification of Toxoplasma gondii in tissue biopsies and eventually in the cerebrospinal fluid (CSF) [19]. In practice, the diagnosis of neurotoxoplasmosis is usually presumptive and is based on a set of clinical and neuroimaging data, in addition to the response to specific treatment against the agent [2]. The differential diagnosis of neurotoxoplasmosis includes primary lymphoma of the central nervous system (CNS), cytomegalovirus encephalitis, abscesses and other granulomatous diseases such as tuberculosis, syphilis, Cryptococcus infection, histoplasmosis, cysticercosis, and Chagas disease [17,25]. Imaging exams, i.e., computed tomography (CT) and magnetic resonance (MRI), typically demonstrate multiple
⁎ Corresponding author at: Avenida Bandeirantes, 3900 - Monte Alegre - CEP: 14049900 Ribeirão Preto; São Paulo-Brazil. Tel.: +55 16 3602-2747; fax: +55 16 3633 66 95. E-mail addresses: [email protected] (L.M.P. Anselmo), [email protected] (F.C. Vilar), [email protected] (J.E. Lima), [email protected] (A.Y. Yamamoto), [email protected] (V.R. Bollela), [email protected] (O.M. Takayanagui).
granulomatous lesions but do not differentiate toxoplasmosis from other lesions such as abscesses and lymphoma [13,25]. The methods for the detection of IgG and IgM antibodies are of little use, although the presence of high IgG titers is common in patients with a diagnosis of neurotoxoplasmosis. However, the absence of antibodies does not rule out the diagnosis in a definitive manner [19,23,27]. In this respect, molecular biology and direct detection methods such as the polymerase chain reaction (PCR) represent a good alternative for the etiologic diagnosis of the disease or for decision making in cases with an unfavorable course despite specific treatment [1,11,17]. The objective of the present study was to assess the performance and the best indication of PCR for T. gondii detection in patients with suspected CNS neurotoxoplasmosis. 2. Materials and methods 2.1. Patients Inclusion criteria: immunosuppressed patients older than 18 years suspected of having neurotoxoplasmosis based on history, physical examination and CNS images, who were seen at a reference hospital in the Northeast of São Paulo State, Brazil. Patients included were from January 2010 to February 2012. Exclusion criteria were: patients with severe intracranial hypertension, coagulopathy or any condition that would contraindicate lumbar or occipital puncture. All selected
http://dx.doi.org/10.1016/j.jns.2014.08.034 0022-510X/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Anselmo LMP, et al, Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.08.034
2
L.M.P. Anselmo et al. / Journal of the Neurological Sciences xxx (2014) xxx–xxx
patients gave written informed consent to participate. A 1 ml CSF sample was then obtained from each subject and submitted to PCR. Amplification was performed according to two protocols, i.e., nested PCR and real time PCR, with all samples being tested simultaneously. The target of both reactions was amplification of the B1 gene of T. gondii, which is the region of the most conserved DNA of the parasite [5,14]. The criteria were those established by the Centers for Disease Control and Prevention [6] as follows: (i) the onset of a focal neurologic abnormality consistent with intracranial disease or reduced level of consciousness, (ii) a lesion having a mass effect demonstrated by neuroimaging (CT or MRI) or a lesion with radiographic appearance enhanced by injection of contrast medium, and (iii) a positive test for anti-T. gondii in serum or a response to specific treatment for toxoplasmosis. The study was conducted at the Clinical Hospital of Ribeirão Preto and approved by the Ethics Committee.
2.6. Neuroimaging exams All patients with suspected neurotoxoplasmosis were submitted to CT and/or MRI exams performed by specialists. The imaging exams were decisive for the diagnostic suspicion and/or case definition, in addition to clinical signs and symptoms and response to specific treatment. 2.7. Statistical analysis
DNA was extracted from the CSF samples using a commercial kit (Qiamp Blood kit, Qiagen Inc., Valencia, CA, USA) according to manufacturer specifications.
The PCR results were analyzed for the 79 CSF samples (total group) and compared to the definition of toxoplasmosis in the CNS of immunosuppressed patients. In cases suspected of neurotoxoplasmosis, the PCR results were analyzed considering the duration of specific treatment at the time of CSF collection. Data were analyzed in 2 × 2 tables for the calculation of PCR sensitivity and specificity for the 27 patients with cerebral toxoplasmosis and also for a subgroup with a time of treatment of seven days or less. The Chi-square test was used to analyze the clinical manifestations and the presence or absence of the disease. The Fisher test was used for the analysis of serology and ELISA exams and the Mann–Whitney test was used to compare the numerical means between CD4+ T cell count and CSF protein.
2.3. Nested PCR
3. Results
In the first reaction step, the following external primers described by Burg et al. [5] were used: 5-GGAACTGCATCCGTTCATGAG-3 and 5-TCTTTAAAGCGTTCGTGGTC-3 (Invitrogen — USA), which correspond to positions 757 to 776 and 887 to 868 bp, respectively, in the T. gondii DNA genome. A 1 μl aliquot of each product amplified in the first reaction was submitted to a second amplification step using the internal primers 5-TGCATAGGTTGCAGTCACTG-3 and 5-GGCGACCAATCTGCGAATACACC3, which correspond to positions 694 to 714 and 853 to 831 bp, respectively, in the T. gondii DNA genome [14]. DNA extracted from a T. gondii suspension in mouse ascitic fluid was used as the positive control and deionized water was used as a negative control. The samples containing T. gondii DNA with a final amplified product of 97 bp were visualized on 0.2% agarose gel under ultraviolet light. The lower detection limit of this assay was 1 parasite/ml.
Seventy-eight of the 79 patients studied were HIV infected. The noninfected patient was being treated for an autoimmune disease with high corticoid doses and had developed clinical and tomographic signs and symptoms compatible with neurotoxoplasmosis, which responded to the specific treatment instituted for the disease. The CSF of this patient was analyzed and found to be positive for toxoplasmosis. Of the 79 patients, 24 (30%) were females and 55 (70%) were males, with a mean age of 43 years (range: 16–75 years). Table 1 compares the results for patients with cerebral toxoplasmosis and patients for whom this diagnosis was excluded. Twenty-seven of the 79 patients were classified as probable cases of neurotoxoplasmosis on the basis of clinical criteria, neuroimaging and therapeutic response. In two of these cases, the diagnosis was confirmed after death by anatomopathological exams which revealed T. gondii cysts in brain tissue. Further confirmation was obtained by immunohistochemistry using specific monoclonal anti-toxoplasma antibodies. Of the 27 probable patients with neurotoxoplasmosis, 13 showed positive PCR for the detection of T. gondii DNA and one of them was not HIV infected. In all cases of positive PCR, CSF had been collected up to the seventh day after the beginning of specific treatment for the disease, as shown in Table 2. Clinical, laboratory and radiological parameters (CT and MRI) were used to monitor the clinical and therapeutic outcomes of these patients. Most of the 27 patients with probable cerebral toxoplasmosis improved
2.2. DNA extraction
2.4. Real time PCR — Sybr Green dye Real time PCR was based on the fluorescent dye Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen — Carlsbad, CA, USA) and was carried out using the Rotor-Gene 3000 instrument (Corbett, Australia) and the Rotor-Gene software version 6.0. The primers used were 5-GGAACTGCATCCGTTCATGAG-3 and 5-TCTTTAAAGCGTTCGTGGTC-3, which amplify the same 97 bp region of the B1 gene. The reaction was carried out for 2 min at 50 °C and for 2 min at 95 °C for the initial activation of Taq polymerase, followed by 40 denaturation cycles at 95 °C for 25 s, annealing at 55 °C for 30 s, and denaturation at 72 °C for 30 s [5]. At the end of amplification, the temperature was reduced to 60 °C and then gradually increased to 95 °C (0.2 °C/s). Samples with a dissociation temperature of 84.5 °C to 84.8 °C were defined as positive and the detection limit of this assay was 1 parasite/ml. 2.5. Serologic diagnosis The exams for the serologic diagnosis of toxoplasmosis and for the investigation of infection with acquired human immunodeficiency virus (HIV) in cases suspected of toxoplasmosis in the CNS were carried out according to the routine of the serology laboratory of the hospital. Serum and CSF samples were submitted to an automated qualitative test that permits the detection of anti-toxoplasma IgM/IgG by Enzyme Linked Fluorescent Assay (ELFA) according to manufacturer specifications (VIDAS TOXO IgM/IgG — Biomérieux).
Table 1 Comparison of the clinical manifestations and complementary exams of patients with neurotoxoplasmosis and of patients with other diagnoses. Clinical signs and symptoms
Toxoplasmosis (27)
Other diagnoses (52)
X2test p b 0,05
Fever Headache Vomiting Paresis Seizures
63% (17/27) 52% (14/27) 44% (12/27) 37% (10/27) 30% (8/27)
48% (25/52) 46% (24/52) 27% (14/52) 25% (13/52) 30% (16/52)
1.58 0.63 0.12 0.57 0.66
Exams
Toxoplasmosis (27)
Other diagnoses (52)
p Value
Blood Toxo.-reagent IgG CD4+ T lymphocytes CSF protein CSF cellularity/mm3
100 (27/27) 28 (25/27) 82 (27/27) 9.65 (22/27)
36,6 (32/52) 147 (39/52) 47 (47/52) 3,66 (27/52)
p p p p
b 0.001 b 0.001 = 0.04 = 0.03
Please cite this article as: Anselmo LMP, et al, Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.08.034
L.M.P. Anselmo et al. / Journal of the Neurological Sciences xxx (2014) xxx–xxx
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Table 2 Clinical and laboratory data for the 15 patients with a defined diagnosis of neurotoxoplasmosis and the detection of T. gondii genome by PCR. No
Sex
Age
CD4+ T lymphocytes (/μl)
IgG antibodies (blood) N300
Neuro-imaging
PCR
Days of treatment pre-CSF analysis
Clinical improvement
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
M F F F M M M F F M M M M M F
50 40 58 38 40 40 44 19 75 45 40 45 32 23 30
92 4 13 9 11 11 20 Non-HIV 28 117 230 37 15 98 49
+ + + + + + + + + + + + + + +
Typical Typical Typical Typical Typical Typical Typical Typical Typical Typical Typical Typical Typical Typical Typical
+ + + + + + + + + + + + + – –
1 1 4 7 5 3 5 1 4 6 4 1 3 2 6
Good evolution Good evolution Good evolution Death Good evolution Good evolution Good evolution Good evolution Good evolution Death Good evolution Good evolution Good evolution Good evolution Good evolution
after 14 days of treatment with specific sulfadiazine (4 g/day) and pyrimethamine (50 mg/day) or clindamycin (2.7 g/day) and pyrimethamine (50 mg/day). Most patients improved after 14 days of treatment, and it happened after 1 to 4 weeks. All of these patients underwent imaging control. Regarding the serologic exam for toxoplasmosis, all 27 patients had positive IgG serology, 24 of them with titers higher than 300 IU/ml. Of the 52 patients without a diagnosis of neurotoxoplasmosis and with negative PCR, 19 showed positive serology for toxoplasmosis and the remaining ones were negative or did not perform the exam. The 52 patients for whom neurotoxoplasmosis had been excluded, had a definite diagnosis of other infectious diseases such as fungal, bacterial and viral meningitis, and associated syndromes (Cryptococcus infection, histoplasmosis, polyoma, CMV, TB, type I and II herpes, PML, Vogt–Koyanagi–Harada (VKH) syndrome, polyneuropathy, Guillain– Barré syndrome, stroke). The median CD4+ T lymphocyte count was lower among patients with neurotoxoplasmosis (28 cells/mm3) than among patients without the disease (147 cells/mm3), and CSF protein levels were higher among patients with neurotoxoplasmosis (82 versus 47 mg/dL). In 15 of the 27 patients, CSF was collected within the first 7 days of treatment, with PCR being positive in 13 of them (86.6%). In 8 cases, CSF was collected between the 8th and 21st day after the beginning of treatment. In these patients, all PCR tests were negative. In 4 cases in which CSF was collected after 21 days of treatment, PCR of CSF did not show positivity (Table 3). All CSF PCR tests were negative in the 52 patients with no confirmation of neurotoxoplasmosis (Table 2). In cases in which there was amplification for CSF samples collected up to seven days after the beginning of treatment, both PCR techniques (nested and real time) showed the same performance, with 86.6% sensitivity and 100% specificity.
Table 3 Nested PCR and Real Time PCR results for the 27 CSF samples from patients with a diagnosis of neurotoxoplasmosis according to time between CSF collection and the beginning of specific treatment. PCR — toxo
Time between CSF collection and the beginning of treatment
PCR + toxo
1 to 7 days
13
2
8 to 21 days N21 days Total
0 0 13
8 4 14
PCR sensitivity 86.6% (13/15) 0 0 48.1% (13/27)
4. Discussion The presumptive diagnosis of cerebral toxoplasmosis in AIDS patients is based on the presence of clinical manifestations of intracranial hypertension syndrome with localizing signals, a CD4+ T lymphocyte count lower than 200/μl, the detection of anti-T. gondii IgG antibodies in serum and in the CSF, granulomatous lesions revealed by neuroimaging exams, and a positive response to specific treatment [6]. For any immunosuppressed patient with suspected cerebral toxoplasmosis who is submitted to empirical treatment without clinical or imaging exam improvement, alternative diagnoses should be considered. Despite the decline observed in the prevalence of opportunistic diseases after the introduction of highly active antiretroviral therapy among AIDS patients, neurotoxoplasmosis still is the most important opportunistic disease affecting the nervous system of HIV-infected patients [21,22]. However, there are other infectious or neoplastic diseases that mimic the clinical and radiologic signs and symptoms of neurotoxoplasmosis, posing a challenge for a definite diagnosis before two weeks of empirical treatment of the disease. This time may represent a significant delay for the diagnosis of other causes of disease, such as primary CNS lymphoma, neurotuberculosis or reactivated Chagas disease in the CNS. In addition, there are atypical forms of cerebral toxoplasmosis that have been described after the advent of highly active antiretroviral therapy [24]. The search of anti-T. gondii antibodies in serum or CSF, although sensitive, is of low specificity for the characterization of toxoplasmosis activity in the CNS. The ideal exam, a brain biopsy, in addition to being of difficult access, is highly invasive [27]. In the present study, the role of PCR was assessed as a diagnostic method for CSF samples from patients with suspected neurotoxoplasmosis, and the best conditions for its indication in clinical practice were identified in order to obtain the best possible yield of the test and to determine its limitations. Previous studies have demonstrated the applicability of this molecular test to the diagnosis of CNS toxoplasmosis, although its sensitivity showed wide variability among different studies [3,4,9,10,12]. In the present study, the option was to amplify the region of the B1 gene of T. gondii because this is the best known, conserved and highly repetitive DNA region of the parasite, using a pair of primers that amplify a 97 bp sequence. Two amplification protocols were established and, when tested, they demonstrated a similar performance with high sensitivity for samples from patients who had started treatment less than seven days before, and excellent specificity regardless of the time of treatment or other factors. Nested PCR and real time PCR carried out in CSF samples from patients with suspected neurotoxoplasmosis were fully comparable in terms of sensitivity and specificity. This information favors real time PCR as the method to be implanted in the
Please cite this article as: Anselmo LMP, et al, Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.08.034
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diagnostic routine, since this is a faster and safer procedure that minimizes the risk of contamination and reduces observer-dependent errors. Among the 27 cases of CNS toxoplasmosis, PCR carried out by both techniques was positive only in those from whom the CSF sample was collected up to seven days after the beginning of specific treatment, with 86.6% sensitivity and 100% specificity. Vidal et al. [26] detected a PCR sensitivity close to 100% in CSF samples collected from adults with AIDS and from neonates up to the third day of specific treatment for toxoplasmosis Nogui et al. [18] and Mesquita et al. [15,16] reported 68% PCR sensitivity in CSF samples collected from 12 patients between the first and ninth days of treatment. There is no doubt about the role of PCR in the confirmatory diagnosis of CNS toxoplasmosis, especially if the CSF samples are collected as early as possible and the test is performed within the first three days between sample collection and the beginning of specific treatment. In practice, however, the presence of an expansive lesion in the CNS may contraindicate an early CSF collection for these patients, with collection being possible only after the beginning of specific treatment and the eventual use of drugs that reduce edema and intracranial hypertension. Within this context, it is essential to define the time interval during which it is still possible to amplify the T. gondii genome even after the beginning of treatment of toxoplasmosis. This variation of PCR sensitivity in CSF samples has been investigated and can be explained by factors such as optimization of PCR by different laboratories, sample processing within less than 48 to 72 h, increased CSF cellularity and protein, the number of lesions observed in neuroimaging exams, and CD4+ T lymphocyte count. Mesquita et al. [15,16] and Correia et al. [7,8] observed that patients with four or more lesions in the CNS and with CSF cellularity N4 cells/mm3 appear to show increased positivity and sensitivity to the test. The present patients had CD4 + T lymphocyte counts of b100 cells/mm 3 , increased CSF protein and mean cellularity of 43 cells/mm 3; however, the decisive factor for PCR positivity was the time of treatment when CSF was collected. In a sample of 79 patients, 27 had a diagnosis of toxoplasmosis at different times during treatment, and these results suggest the usefulness of PCR for the diagnosis of cerebral toxoplasmosis, and reinforce the first week as the window for the best performance of toxoplasmosis PCR in CSF. This information is important for the optimization of PCR sensitivity when the procedure is performed under ideal conditions, with the added advantage of cost reduction by avoiding sample collection under conditions that would practically render unviable PCR positivity in these patients. The conventional PCR method showed similar accuracy, with the advantage of being of lower cost. Conflict of interest The authors have no conflict of interest. Acknowledgments The authors are grateful to the National Council for Scientific and Technological Development (151965/2010-2) (CNPq) for granting a research fellowship to Osvaldo Massaiti Takayanagui and a Master's fellowship to Lívia Maria Pala Anselmo.
[2] Borges AS, Figueiredo JF. Evaluation of intrathecal synthesis of specific IgG antibodies against Toxoplasma gondii in the diagnosis assessment of presumptive toxoplasma encephalitis in AIDS patients. Rev Soc Bras Med Trop 2004;37:480–4. [3] Brenier-Pinchart MP, Morand-Bui V, Fricker-Hidalgo H, Equy V, Marlu R, Pelloux H. Adapting a conventional PCR assay for Toxoplasma gondii detection to realtime quantitative PCR including a competitive internal control. Parasite 2007; 14:149–54. [4] Buchbinder S, Blatz R, Rodloff AC. Comparison of real-time PCR detection methods for B1 and P30 genes of Toxoplasma gondii. Diagn Microbiol Infect Dis 2003;45: 269–71. [5] Burg JL, Grover CM, Pouletty P, Boothroyd JC. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma Gondii, by polymerase chain reaction. J Clin Microbiol 1989;27:1787–92. [6] Centers for Disease Control, Prevention (CDC). 1993 revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. JAMA 1993;269:729–30. [7] Correia CC, Melo HRL, Costa VMA. Influence of neurotoxoplasmosis characteristics on real-time PCR sensitivity among AIDS patients in Brazil. Trans R Soc Trop Med Hyg 2010;104:24–8. [8] Correia CC, Melo HRL, Costa VMA, Brainer AM. Features to validate cerebral toxoplasmosis. Rev Soc Bras Med Trop 2013;46(3):373–6. [9] Costa JM, Pautas C, Ernault P, Foulet F, Cordonnier C, Bretagne S. Real-time PCR for diagnosis and follow-up of Toxoplasma reactivation after allogeneic stem cell transplantation using fluorescence resonance energy transfer hybridization probes. J Clin Microbiol 2000;38:2929–32. [10] Edvinsson B, Lappalainen M, Evengard B. ESCMID Study Group for Toxoplasmosis. Real-time PCR targeting a 529-bp repeat element for diagnosis of toxoplasmosis. Clin Microbiol Infect 2006;12:131–6. [11] Ferreira IM, Vidal JE, Costa-Silva TA, Meira CS, Hiramoto RM, Oliveira PAC, et al. Toxoplasma gondii: genotyping of strains from Brazilian AIDS patients with cerebral toxoplasmosis by multilocus PCR-RFLP markers. Exp Parasitol 2008;118:221–7. [12] Hierl T, Reischl U, Lang P, Hebart H, Stark M, Kyme P, et al. Preliminary evaluation of one conventional nested and two real-time PCR assays for the detection of Toxoplasma gondii in immunocompromised patients. J Med Microbiol 2004;53:629–32. [13] Kastrup O, Wanke I, Maschke M. Neuroimaging of infections of the central nervous system. Semin Neurol 2008;28:511–22. [14] Khalifa Kel-S, Roth A, Roth B, Arasteh KN, Janitschke K. Value of PCR for evaluating occurrence of parasitemia in immunocompromised patients with cerebral and extracerebral toxoplasmosis. J Clin Microbiol 1994;32(11):2813–9. [15] Mesquita RT, Vidal JE, Pereira-Chioccola VL. Molecular diagnosis of cerebral toxoplasmosis: comparing markers that determine Toxoplasma gondii by PCR in peripheral blood from HIV-infected patients. Braz J Infect Dis 2010;14:346–50. [16] Mesquita RT, Ziegler AP, Hiramoto RM, Vidal JE, Pereira-Chioccola VL. Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients. J Med Microbiol 2010;59(Pt 6):641–7. [17] Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet 2008;363:1965–76. [18] Nogui FL, Mattas S, Turcato Júnior G, Lewi DS. Neurotoxoplasmosis diagnosis for HIV-1 patients by real-time PCR of cerebrospinal fluid. Braz J Infect Dis 2009;13: 18–23. [19] Palm C, Tumani H, Pietzcker T, Bengel D. Diagnosis of cerebral toxoplasmosis by detection of Toxoplasma gondii tachyzoites in cerebrospinal fluid. J Neurol 2008; 255:939–41. [20] Porter SB, Sande MA. Toxoplasmosis of the central nervous system in the acquired immunodeficiency syndrome. N Engl J Med 1992;327:1643–8. [21] Sacktor N, Mcarthur J. Prospects for therapy of HIV-associated neurologic disease. J Neurovirol 1997;3:89–101. [22] Sacktor N. The epidemiology of human immunodeficiency virus-associated neurological disease in the era of highly active antiretroviral therapy. J Neurovirol 2002;8:115–21. [23] Sukthana Y. Toxoplasmosis: beyond animals to humans. Trends Parasitol 2006;22: 137–42. [24] Vallat-Decouvelaere AV, Chrétien F, Grandmaison LG, Carlier R, Force G, Gray F. The neuropathology of HIV infection in the era of highly active antiretroviral therapy. Ann Pathol 2003;23:408–23. [25] Verma A, Berger JR. Infections of the nervous system: neurological manifestations of human immunodeficiency virus infection. In: Daroff RB, Fenichel GM, Jankovic J, Mazziotta JC, editors. Bradley's neurology in clinical practice. 6th ed. Philadelphia Pa: Elsevier Saunders; 2012. p. 1211–30. [26] Vidal JE, Colombo FA, de Oliveira ACP, Focaccia R, Pereira-Chioccola VL. 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Please cite this article as: Anselmo LMP, et al, Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.08.034
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Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients Lívia Maria Pala Anselmo a,⁎, Fernando Crivelenti Vilar b, José Eduardo Lima c, Aparecida Yule Yamamoto d, Valdes Roberto Bollela e, Osvaldo Massaiti Takayanagui a a
School of Medicine of Ribeirão Preto, University of São Paulo, Department of Neurology, Brazil Clinics Hospital, School of Medicine of Ribeirão Preto, University of São Paulo, Department of Internal Medicine — Infectious Diseases, Brazil Clinics Hospital, School of Medicine of Ribeirão Preto, University of São Paulo, Department of Neurology, Brazil d School of Medicine of Ribeirão Preto, University of São Paulo, Department of Pediatrics—Molecular and Infectious Diseases, Brazil e School of Medicine of Ribeirão Preto, University of São Paulo, Department of Internal Medicine — Infectious Diseases, Brazil b c
a r t i c l e
i n f o
Article history: Received 11 June 2014 Received in revised form 29 July 2014 Accepted 24 August 2014 Available online xxxx Keywords: Toxoplasmosis HIV Acquired immunodeficiency syndrome Cerebrospinal fluid Central nervous system
a b s t r a c t The objective of the present study was to assess the performance and the best indication of the polymerase chain reaction (PCR) for the detection of Toxoplasmosis gondii DNA in cerebrospinal fluid (CSF) from patients with suspected neurotoxoplasmosis. CSF samples were collected from 79 patients for amplification of the T. gondii genome (gene B1) by two PCR techniques (nested and real time). Twenty-seven of the 79 patients were classified as probable cases of neurotoxoplasmosis on the basis of clinical criteria, neuroimaging and therapeutic response. PCR showed high sensitivity (86.6%) when performed in CSF samples which were collected up to the seventh day of specific toxoplasmosis treatment. There was no positive test after 1 week of treatment. These results suggest the usefulness of PCR for the diagnosis of cerebral toxoplasmosis, and support the first week as the window for the best performance of toxoplasmosis PCR in CSF. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Among AIDS patients, toxoplasmosis usually occurs in individuals with CD4 + T lymphocyte counts b100/μl by reactivation of chronic infection [20]. The clinical signs and symptoms of neurotoxoplasmosis in immunosuppressed patients can be acute or subacute and a definite diagnosis of toxoplasmosis requires the identification of Toxoplasma gondii in tissue biopsies and eventually in the cerebrospinal fluid (CSF) [19]. In practice, the diagnosis of neurotoxoplasmosis is usually presumptive and is based on a set of clinical and neuroimaging data, in addition to the response to specific treatment against the agent [2]. The differential diagnosis of neurotoxoplasmosis includes primary lymphoma of the central nervous system (CNS), cytomegalovirus encephalitis, abscesses and other granulomatous diseases such as tuberculosis, syphilis, Cryptococcus infection, histoplasmosis, cysticercosis, and Chagas disease [17,25]. Imaging exams, i.e., computed tomography (CT) and magnetic resonance (MRI), typically demonstrate multiple
⁎ Corresponding author at: Avenida Bandeirantes, 3900 - Monte Alegre - CEP: 14049900 Ribeirão Preto; São Paulo-Brazil. Tel.: +55 16 3602-2747; fax: +55 16 3633 66 95. E-mail addresses: [email protected] (L.M.P. Anselmo), [email protected] (F.C. Vilar), [email protected] (J.E. Lima), [email protected] (A.Y. Yamamoto), [email protected] (V.R. Bollela), [email protected] (O.M. Takayanagui).
granulomatous lesions but do not differentiate toxoplasmosis from other lesions such as abscesses and lymphoma [13,25]. The methods for the detection of IgG and IgM antibodies are of little use, although the presence of high IgG titers is common in patients with a diagnosis of neurotoxoplasmosis. However, the absence of antibodies does not rule out the diagnosis in a definitive manner [19,23,27]. In this respect, molecular biology and direct detection methods such as the polymerase chain reaction (PCR) represent a good alternative for the etiologic diagnosis of the disease or for decision making in cases with an unfavorable course despite specific treatment [1,11,17]. The objective of the present study was to assess the performance and the best indication of PCR for T. gondii detection in patients with suspected CNS neurotoxoplasmosis. 2. Materials and methods 2.1. Patients Inclusion criteria: immunosuppressed patients older than 18 years suspected of having neurotoxoplasmosis based on history, physical examination and CNS images, who were seen at a reference hospital in the Northeast of São Paulo State, Brazil. Patients included were from January 2010 to February 2012. Exclusion criteria were: patients with severe intracranial hypertension, coagulopathy or any condition that would contraindicate lumbar or occipital puncture. All selected
http://dx.doi.org/10.1016/j.jns.2014.08.034 0022-510X/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Anselmo LMP, et al, Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.08.034
2
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patients gave written informed consent to participate. A 1 ml CSF sample was then obtained from each subject and submitted to PCR. Amplification was performed according to two protocols, i.e., nested PCR and real time PCR, with all samples being tested simultaneously. The target of both reactions was amplification of the B1 gene of T. gondii, which is the region of the most conserved DNA of the parasite [5,14]. The criteria were those established by the Centers for Disease Control and Prevention [6] as follows: (i) the onset of a focal neurologic abnormality consistent with intracranial disease or reduced level of consciousness, (ii) a lesion having a mass effect demonstrated by neuroimaging (CT or MRI) or a lesion with radiographic appearance enhanced by injection of contrast medium, and (iii) a positive test for anti-T. gondii in serum or a response to specific treatment for toxoplasmosis. The study was conducted at the Clinical Hospital of Ribeirão Preto and approved by the Ethics Committee.
2.6. Neuroimaging exams All patients with suspected neurotoxoplasmosis were submitted to CT and/or MRI exams performed by specialists. The imaging exams were decisive for the diagnostic suspicion and/or case definition, in addition to clinical signs and symptoms and response to specific treatment. 2.7. Statistical analysis
DNA was extracted from the CSF samples using a commercial kit (Qiamp Blood kit, Qiagen Inc., Valencia, CA, USA) according to manufacturer specifications.
The PCR results were analyzed for the 79 CSF samples (total group) and compared to the definition of toxoplasmosis in the CNS of immunosuppressed patients. In cases suspected of neurotoxoplasmosis, the PCR results were analyzed considering the duration of specific treatment at the time of CSF collection. Data were analyzed in 2 × 2 tables for the calculation of PCR sensitivity and specificity for the 27 patients with cerebral toxoplasmosis and also for a subgroup with a time of treatment of seven days or less. The Chi-square test was used to analyze the clinical manifestations and the presence or absence of the disease. The Fisher test was used for the analysis of serology and ELISA exams and the Mann–Whitney test was used to compare the numerical means between CD4+ T cell count and CSF protein.
2.3. Nested PCR
3. Results
In the first reaction step, the following external primers described by Burg et al. [5] were used: 5-GGAACTGCATCCGTTCATGAG-3 and 5-TCTTTAAAGCGTTCGTGGTC-3 (Invitrogen — USA), which correspond to positions 757 to 776 and 887 to 868 bp, respectively, in the T. gondii DNA genome. A 1 μl aliquot of each product amplified in the first reaction was submitted to a second amplification step using the internal primers 5-TGCATAGGTTGCAGTCACTG-3 and 5-GGCGACCAATCTGCGAATACACC3, which correspond to positions 694 to 714 and 853 to 831 bp, respectively, in the T. gondii DNA genome [14]. DNA extracted from a T. gondii suspension in mouse ascitic fluid was used as the positive control and deionized water was used as a negative control. The samples containing T. gondii DNA with a final amplified product of 97 bp were visualized on 0.2% agarose gel under ultraviolet light. The lower detection limit of this assay was 1 parasite/ml.
Seventy-eight of the 79 patients studied were HIV infected. The noninfected patient was being treated for an autoimmune disease with high corticoid doses and had developed clinical and tomographic signs and symptoms compatible with neurotoxoplasmosis, which responded to the specific treatment instituted for the disease. The CSF of this patient was analyzed and found to be positive for toxoplasmosis. Of the 79 patients, 24 (30%) were females and 55 (70%) were males, with a mean age of 43 years (range: 16–75 years). Table 1 compares the results for patients with cerebral toxoplasmosis and patients for whom this diagnosis was excluded. Twenty-seven of the 79 patients were classified as probable cases of neurotoxoplasmosis on the basis of clinical criteria, neuroimaging and therapeutic response. In two of these cases, the diagnosis was confirmed after death by anatomopathological exams which revealed T. gondii cysts in brain tissue. Further confirmation was obtained by immunohistochemistry using specific monoclonal anti-toxoplasma antibodies. Of the 27 probable patients with neurotoxoplasmosis, 13 showed positive PCR for the detection of T. gondii DNA and one of them was not HIV infected. In all cases of positive PCR, CSF had been collected up to the seventh day after the beginning of specific treatment for the disease, as shown in Table 2. Clinical, laboratory and radiological parameters (CT and MRI) were used to monitor the clinical and therapeutic outcomes of these patients. Most of the 27 patients with probable cerebral toxoplasmosis improved
2.2. DNA extraction
2.4. Real time PCR — Sybr Green dye Real time PCR was based on the fluorescent dye Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen — Carlsbad, CA, USA) and was carried out using the Rotor-Gene 3000 instrument (Corbett, Australia) and the Rotor-Gene software version 6.0. The primers used were 5-GGAACTGCATCCGTTCATGAG-3 and 5-TCTTTAAAGCGTTCGTGGTC-3, which amplify the same 97 bp region of the B1 gene. The reaction was carried out for 2 min at 50 °C and for 2 min at 95 °C for the initial activation of Taq polymerase, followed by 40 denaturation cycles at 95 °C for 25 s, annealing at 55 °C for 30 s, and denaturation at 72 °C for 30 s [5]. At the end of amplification, the temperature was reduced to 60 °C and then gradually increased to 95 °C (0.2 °C/s). Samples with a dissociation temperature of 84.5 °C to 84.8 °C were defined as positive and the detection limit of this assay was 1 parasite/ml. 2.5. Serologic diagnosis The exams for the serologic diagnosis of toxoplasmosis and for the investigation of infection with acquired human immunodeficiency virus (HIV) in cases suspected of toxoplasmosis in the CNS were carried out according to the routine of the serology laboratory of the hospital. Serum and CSF samples were submitted to an automated qualitative test that permits the detection of anti-toxoplasma IgM/IgG by Enzyme Linked Fluorescent Assay (ELFA) according to manufacturer specifications (VIDAS TOXO IgM/IgG — Biomérieux).
Table 1 Comparison of the clinical manifestations and complementary exams of patients with neurotoxoplasmosis and of patients with other diagnoses. Clinical signs and symptoms
Toxoplasmosis (27)
Other diagnoses (52)
X2test p b 0,05
Fever Headache Vomiting Paresis Seizures
63% (17/27) 52% (14/27) 44% (12/27) 37% (10/27) 30% (8/27)
48% (25/52) 46% (24/52) 27% (14/52) 25% (13/52) 30% (16/52)
1.58 0.63 0.12 0.57 0.66
Exams
Toxoplasmosis (27)
Other diagnoses (52)
p Value
Blood Toxo.-reagent IgG CD4+ T lymphocytes CSF protein CSF cellularity/mm3
100 (27/27) 28 (25/27) 82 (27/27) 9.65 (22/27)
36,6 (32/52) 147 (39/52) 47 (47/52) 3,66 (27/52)
p p p p
b 0.001 b 0.001 = 0.04 = 0.03
Please cite this article as: Anselmo LMP, et al, Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.08.034
L.M.P. Anselmo et al. / Journal of the Neurological Sciences xxx (2014) xxx–xxx
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Table 2 Clinical and laboratory data for the 15 patients with a defined diagnosis of neurotoxoplasmosis and the detection of T. gondii genome by PCR. No
Sex
Age
CD4+ T lymphocytes (/μl)
IgG antibodies (blood) N300
Neuro-imaging
PCR
Days of treatment pre-CSF analysis
Clinical improvement
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
M F F F M M M F F M M M M M F
50 40 58 38 40 40 44 19 75 45 40 45 32 23 30
92 4 13 9 11 11 20 Non-HIV 28 117 230 37 15 98 49
+ + + + + + + + + + + + + + +
Typical Typical Typical Typical Typical Typical Typical Typical Typical Typical Typical Typical Typical Typical Typical
+ + + + + + + + + + + + + – –
1 1 4 7 5 3 5 1 4 6 4 1 3 2 6
Good evolution Good evolution Good evolution Death Good evolution Good evolution Good evolution Good evolution Good evolution Death Good evolution Good evolution Good evolution Good evolution Good evolution
after 14 days of treatment with specific sulfadiazine (4 g/day) and pyrimethamine (50 mg/day) or clindamycin (2.7 g/day) and pyrimethamine (50 mg/day). Most patients improved after 14 days of treatment, and it happened after 1 to 4 weeks. All of these patients underwent imaging control. Regarding the serologic exam for toxoplasmosis, all 27 patients had positive IgG serology, 24 of them with titers higher than 300 IU/ml. Of the 52 patients without a diagnosis of neurotoxoplasmosis and with negative PCR, 19 showed positive serology for toxoplasmosis and the remaining ones were negative or did not perform the exam. The 52 patients for whom neurotoxoplasmosis had been excluded, had a definite diagnosis of other infectious diseases such as fungal, bacterial and viral meningitis, and associated syndromes (Cryptococcus infection, histoplasmosis, polyoma, CMV, TB, type I and II herpes, PML, Vogt–Koyanagi–Harada (VKH) syndrome, polyneuropathy, Guillain– Barré syndrome, stroke). The median CD4+ T lymphocyte count was lower among patients with neurotoxoplasmosis (28 cells/mm3) than among patients without the disease (147 cells/mm3), and CSF protein levels were higher among patients with neurotoxoplasmosis (82 versus 47 mg/dL). In 15 of the 27 patients, CSF was collected within the first 7 days of treatment, with PCR being positive in 13 of them (86.6%). In 8 cases, CSF was collected between the 8th and 21st day after the beginning of treatment. In these patients, all PCR tests were negative. In 4 cases in which CSF was collected after 21 days of treatment, PCR of CSF did not show positivity (Table 3). All CSF PCR tests were negative in the 52 patients with no confirmation of neurotoxoplasmosis (Table 2). In cases in which there was amplification for CSF samples collected up to seven days after the beginning of treatment, both PCR techniques (nested and real time) showed the same performance, with 86.6% sensitivity and 100% specificity.
Table 3 Nested PCR and Real Time PCR results for the 27 CSF samples from patients with a diagnosis of neurotoxoplasmosis according to time between CSF collection and the beginning of specific treatment. PCR — toxo
Time between CSF collection and the beginning of treatment
PCR + toxo
1 to 7 days
13
2
8 to 21 days N21 days Total
0 0 13
8 4 14
PCR sensitivity 86.6% (13/15) 0 0 48.1% (13/27)
4. Discussion The presumptive diagnosis of cerebral toxoplasmosis in AIDS patients is based on the presence of clinical manifestations of intracranial hypertension syndrome with localizing signals, a CD4+ T lymphocyte count lower than 200/μl, the detection of anti-T. gondii IgG antibodies in serum and in the CSF, granulomatous lesions revealed by neuroimaging exams, and a positive response to specific treatment [6]. For any immunosuppressed patient with suspected cerebral toxoplasmosis who is submitted to empirical treatment without clinical or imaging exam improvement, alternative diagnoses should be considered. Despite the decline observed in the prevalence of opportunistic diseases after the introduction of highly active antiretroviral therapy among AIDS patients, neurotoxoplasmosis still is the most important opportunistic disease affecting the nervous system of HIV-infected patients [21,22]. However, there are other infectious or neoplastic diseases that mimic the clinical and radiologic signs and symptoms of neurotoxoplasmosis, posing a challenge for a definite diagnosis before two weeks of empirical treatment of the disease. This time may represent a significant delay for the diagnosis of other causes of disease, such as primary CNS lymphoma, neurotuberculosis or reactivated Chagas disease in the CNS. In addition, there are atypical forms of cerebral toxoplasmosis that have been described after the advent of highly active antiretroviral therapy [24]. The search of anti-T. gondii antibodies in serum or CSF, although sensitive, is of low specificity for the characterization of toxoplasmosis activity in the CNS. The ideal exam, a brain biopsy, in addition to being of difficult access, is highly invasive [27]. In the present study, the role of PCR was assessed as a diagnostic method for CSF samples from patients with suspected neurotoxoplasmosis, and the best conditions for its indication in clinical practice were identified in order to obtain the best possible yield of the test and to determine its limitations. Previous studies have demonstrated the applicability of this molecular test to the diagnosis of CNS toxoplasmosis, although its sensitivity showed wide variability among different studies [3,4,9,10,12]. In the present study, the option was to amplify the region of the B1 gene of T. gondii because this is the best known, conserved and highly repetitive DNA region of the parasite, using a pair of primers that amplify a 97 bp sequence. Two amplification protocols were established and, when tested, they demonstrated a similar performance with high sensitivity for samples from patients who had started treatment less than seven days before, and excellent specificity regardless of the time of treatment or other factors. Nested PCR and real time PCR carried out in CSF samples from patients with suspected neurotoxoplasmosis were fully comparable in terms of sensitivity and specificity. This information favors real time PCR as the method to be implanted in the
Please cite this article as: Anselmo LMP, et al, Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.08.034
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diagnostic routine, since this is a faster and safer procedure that minimizes the risk of contamination and reduces observer-dependent errors. Among the 27 cases of CNS toxoplasmosis, PCR carried out by both techniques was positive only in those from whom the CSF sample was collected up to seven days after the beginning of specific treatment, with 86.6% sensitivity and 100% specificity. Vidal et al. [26] detected a PCR sensitivity close to 100% in CSF samples collected from adults with AIDS and from neonates up to the third day of specific treatment for toxoplasmosis Nogui et al. [18] and Mesquita et al. [15,16] reported 68% PCR sensitivity in CSF samples collected from 12 patients between the first and ninth days of treatment. There is no doubt about the role of PCR in the confirmatory diagnosis of CNS toxoplasmosis, especially if the CSF samples are collected as early as possible and the test is performed within the first three days between sample collection and the beginning of specific treatment. In practice, however, the presence of an expansive lesion in the CNS may contraindicate an early CSF collection for these patients, with collection being possible only after the beginning of specific treatment and the eventual use of drugs that reduce edema and intracranial hypertension. Within this context, it is essential to define the time interval during which it is still possible to amplify the T. gondii genome even after the beginning of treatment of toxoplasmosis. This variation of PCR sensitivity in CSF samples has been investigated and can be explained by factors such as optimization of PCR by different laboratories, sample processing within less than 48 to 72 h, increased CSF cellularity and protein, the number of lesions observed in neuroimaging exams, and CD4+ T lymphocyte count. Mesquita et al. [15,16] and Correia et al. [7,8] observed that patients with four or more lesions in the CNS and with CSF cellularity N4 cells/mm3 appear to show increased positivity and sensitivity to the test. The present patients had CD4 + T lymphocyte counts of b100 cells/mm 3 , increased CSF protein and mean cellularity of 43 cells/mm 3; however, the decisive factor for PCR positivity was the time of treatment when CSF was collected. In a sample of 79 patients, 27 had a diagnosis of toxoplasmosis at different times during treatment, and these results suggest the usefulness of PCR for the diagnosis of cerebral toxoplasmosis, and reinforce the first week as the window for the best performance of toxoplasmosis PCR in CSF. This information is important for the optimization of PCR sensitivity when the procedure is performed under ideal conditions, with the added advantage of cost reduction by avoiding sample collection under conditions that would practically render unviable PCR positivity in these patients. The conventional PCR method showed similar accuracy, with the advantage of being of lower cost. Conflict of interest The authors have no conflict of interest. Acknowledgments The authors are grateful to the National Council for Scientific and Technological Development (151965/2010-2) (CNPq) for granting a research fellowship to Osvaldo Massaiti Takayanagui and a Master's fellowship to Lívia Maria Pala Anselmo.
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Please cite this article as: Anselmo LMP, et al, Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.08.034