1887

Journal of Medical Microbiology logo

Volume 59, Issue 6

Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients Free

Abstract

Cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients in Brazil, and results in high mortality and morbidity, despite free access to HAART (highly active antiretroviral treatment). Molecular diagnosis based on conventional PCR (cnPCR) or real-time quantitative PCR (qrtPCR) has been indispensable for definitive diagnosis. We report here the evaluation of qrtPCR with blood and cerebrospinal fluid (CSF) samples from AIDS patients in Brazil. This prospective study was conducted for 2 years, analysing DNA samples extracted from 149 AIDS patients (98 blood and 51 CSF samples) with confirmed clinical and radiological diagnosis. The laboratory diagnosis included cnPCR (with the B22/B23 primer set) and indirect immunofluorescence (IF). For qrtPCR, two primer sets were simultaneously designed based on described genes and using a 6-carboxyfluorescein dye-labelled TaqMan MGB (minor groove binder) probe. One was B1Tg, which amplified a sequence from the B1 gene. The other was the RETg, which amplified a PCR product of the 529 bp sequence. The overall cnPCR and qrtPCR results were: positive results were observed in 33.6 % (50) patients. The sensitivities were 98 % for cnPCR (B22/B23), and 86 and 98 % for qrtPCR (B1Tg and RETg, respectively). Negative reactions were observed in 66.4 % patients. The specificities were 97 % for cnPCR and qrtPCR (B1Tg), and 88.8 % for RETg. These data show that RETg PCR is highly sensitive as it amplifies a repeat region with many copies; however, its specificity is lower than the other markers. However, B1Tg PCR had good specificity, but lower sensitivity. Among the patients, 20 had blood and CSF collected simultaneously. Thus, their results permitted us to analyse and compare molecular, serological and clinical diagnosis for a better understanding of the different scenarios of laboratorial and clinical diagnosis. For nine patients with confirmed cerebral toxoplasmosis diagnosis, four scenarios were observed: (i) and (ii) negative molecular diagnosis for CSF and positive for blood with variable IF titres for the sera and CSF (negative or positive); (iii) positive molecular diagnosis with CSF and negative with blood; and (iv) positive molecular diagnosis in both samples. In the latter two situations, normally the IF titres in sera and CSF are variable. Other opportunistic infections were shown in 11 patients. Despite the IF titres in sera and CSF being variable, all of them had negative molecular diagnosis for both samples. qrtPCR allows for a rapid identification of DNA in patient samples; in a minority of cases discrepancies occur with the cnPCR.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): cnPCR, conventional PCR , CSF, cerebrospinal fluid , FAM, 6-carboxyfluorescein , HAART, highly active antiretroviral treatment , HIV, human immunodeficiency virus , IF, indirect immunofluorescence , NPV, negative predictive value , PPV, positive predictive value and qrtPCR, real-time quantitative PCR
SGM
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.016261-0
2010-06-01
2024-04-25
Loading full text...

Full text loading...

/deliver/fulltext/jmm/59/6/641.html?itemId=/content/journal/jmm/10.1099/jmm.0.016261-0&mimeType=html&fmt=ahah

References

  1. Al-Soud W. A., Rådström P. 1998; Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples. Appl Environ Microbiol 6:3748–3753
     [Google Scholar]
  2. Antinori A., Larussa D., Cingolani A., Lorenzini P., Bossolasco S., Finazzi M. G., Bongiovanni M., Guaraldi G., Grisetti S. other authors 2004; Prevalence, associated factors, and prognostic determinants of AIDS-related toxoplasmic encephalitis in the era of advanced highly active antiretroviral therapy. Clin Infect Dis 39:1681–1691 [CrossRef]
     [Google Scholar]
  3. Bastien P. 2002; Molecular diagnosis of toxoplasmosis. Trans R Soc Trop Med Hyg 96:S205–S215 [CrossRef]
     [Google Scholar]
  4. Bastien P., Procop G. W., Reischl U. 2008; Quantitative real-time PCR is not more sensitive than conventional PCR. J Clin Microbiol 46:1897–1900 [CrossRef]
     [Google Scholar]
  5. Brenier-Pinchart M. P., Morand-Bui V., Fricker-Hidalgo H., Equy V., Marlu R., Pelloux H. 2007; Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control. Parasite 14:149–154 [CrossRef]
     [Google Scholar]
  6. Bretagne S. 2003; Molecular diagnostics in clinical parasitology and mycology: limits of the current polymerase chain reaction (PCR) assays and interest of the real-time PCR assays. Clin Microbiol Infect 9:505–511 [CrossRef]
     [Google Scholar]
  7. Buchbinder S., Blatz R., Rodloff A. C. 2003; Comparison of real-time PCR detection methods for B1 and P30 genes of Toxoplasma gondii . Diagn Microbiol Infect Dis 45:269–271 [CrossRef]
     [Google Scholar]
  8. Burg J. L., Grover C. M., Pouletty P., Boothroyd J. C. 1989; Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii , by polymerase chain reaction. J Clin Microbiol 27:1787–1792
     [Google Scholar]
  9. Calderaro A., Piccolo G., Gorrini C., Peruzzi S., Zerbini L., Bommezzadri S., Dettori G., Chezzi C. 2006; Comparison between two real-time PCR assays and a nested-PCR for the detection of Toxoplasma gondii . Acta Biomed 77:75–80
     [Google Scholar]
  10. Cassaing S., Bessières M. H., Berry A., Berrebi A., Fabre R., Magnaval J. F. 2006; Comparison between two amplification sets for molecular diagnosis of toxoplasmosis by real-time PCR. J Clin Microbiol 44:720–724 [CrossRef]
     [Google Scholar]
  11. Chabbert E., Lachaud L., Crobu L., Bastien P. 2004; Comparison of two widely used PCR primer systems for detection of Toxoplasma in amniotic fluid, blood and tissues. J Clin Microbiol 42:1719–1722 [CrossRef]
     [Google Scholar]
  12. Cingolani A., De Luca A., Ammassari A., Murri R., Linzalone A., Grillo R., Antinori A. 1996; PCR detection of Toxoplasma gondii DNA in CSF for the differential diagnosis of AIDS-related focal brain lesions. J Med Microbiol 45:472–476 [CrossRef]
     [Google Scholar]
  13. Cohen B. A. 1999; Neurological manifestations of toxoplasmosis in AIDS. Semin Neurol 19:201–211 [CrossRef]
     [Google Scholar]
  14. Collazos J. 2003; Opportunistic infections of the CNS in patients with AIDS. CNS Drugs 17:869–887 [CrossRef]
     [Google Scholar]
  15. Colombo F. A., Vidal J. E., Penalva de Oliveira A. C., Hernández A. V., Bonasser-Filho F., Nogueira R. S., Focaccia R., Pereira-Chioccola V. L. 2005; Diagnosis of cerebral toxoplasmosis in AIDS patients in Brazil: importance of molecular and immunological methods using peripheral blood samples. J Clin Microbiol 43:5044–5047 [CrossRef]
     [Google Scholar]
  16. Contini C., Seraceni S., Cultrera R., Incorvaia C., Sebastiani A., Picot S. 2005; Evaluation of a real-time PCR-based assay using the lightcycler system for detection of Toxoplasma gondii bradyzoite genes in blood specimens from patients with toxoplasmic retinochoroiditis. Int J Parasitol 35:275–283 [CrossRef]
     [Google Scholar]
  17. Correia C. C., Melo H. R. L., Costa V. M. A. 2010; Influence of neurotoxoplasmosis characteristics on real-time PCR sensitivity among AIDS patients in Brazil. Trans R Soc Trop Med Hyg 104:24–28 [CrossRef]
     [Google Scholar]
  18. Costa J. M., Pautas C., Ernault P., Foulet F., Cordonnier C., Bretagne S. 2000; 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 38:2929–2932
     [Google Scholar]
  19. Dubey J. P. 1996; Toxoplasma gondii . In Medical Microbiology , 5th edn. Edited by Baron S., Peake R. C., James D. A., Susman M., Kennedy C. A., Singleton M. J. D., Schuenke S. Galveston, TX: University of Texas Medical Branch;
     [Google Scholar]
  20. Dubey J. P. 1998; Advances in the life cycle of Toxoplasma gondii . Int J Parasitol 28:1019–1024 [CrossRef]
     [Google Scholar]
  21. Edvinsson B., Lappalainen M., Evengård B. on behalf of the ESCMID Study Group for Toxoplasmosis; 2006; Real-time PCR targeting a 529-bp repeat element for diagnosis of toxoplasmosis. Clin Microbiol Infect 12:131–136 [CrossRef]
     [Google Scholar]
  22. Espy M. J., Uhl J. R., Sloan L. M., Buckwalter S. P., Jones M. F., Vetter E. A., Yao J. D., Wengenack N. L., Rosenblatt J. E. other authors 2006; Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 19:165–256 [CrossRef]
     [Google Scholar]
  23. Gianotti N., Cinque P., Castagna A., Novati R., Moro M., Lazzarin A. 1997; Diagnosis of toxoplasmic encephalitis in HIV-infected patients. AIDS 11:1529–1530
     [Google Scholar]
  24. Hierl T., Reischl U., Lang P., Hebart H., Stark M., Kyme P., Autenrieth I. B. 2004; Preliminary evaluation of one conventional nested and two real-time PCR assays for the detection of Toxoplasma gondii in immunocompromised patients. J Med Microbiol 53:629–632 [CrossRef]
     [Google Scholar]
  25. Hill D. E., Chirukandoth S., Dubey J. P. 2005; Biology and epidemiology of Toxoplasma gondii in man and animals. Anim Health Res Rev 6:41–61 [CrossRef]
     [Google Scholar]
  26. Hoffmann C. 2005; Opportunistic infections. In HIV Medicine Edited by Hoffmann C., Rockstroh J. K., Kamps B. S. Paris: Flying Publisher; (in Spanish) www.HIVMedicine.com
     [Google Scholar]
  27. Homan W. L., Vercammen M., De Braekeleer J., Verschueren H. 2000; Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii , and its use for diagnostic and quantitative PCR. Int J Parasitol 30:69–75 [CrossRef]
     [Google Scholar]
  28. Lee C. N., Cavanagh H. M., Lo S. T., Ng C. S. 2001; Human papillomavirus infection in non-neoplastic uterine cervical disease in Hong Kong. Br J Biomed Sci 58:85–91
     [Google Scholar]
  29. Luft B. J., Remington J. S. 1992; Toxoplasmic encephalitis. Clin Infect Dis 15:211–222 [CrossRef]
     [Google Scholar]
  30. Meira C. S., Costa-Silva T. A., Vidal J. E., Ferreira I. M. R., Hiramoto R. M., Pereira-Chioccola V. L. 2008; Use of the serum reactivity against Toxoplasma gondii excreted–secreted antigen in cerebral toxoplasmosis diagnosis in HIV-infected patients. J Med Microbiol 57:845–850 [CrossRef]
     [Google Scholar]
  31. Montoya J. G., Liesenfeld O. 2004; Toxoplasmosis. Lancet 363:1965–1976 [CrossRef]
     [Google Scholar]
  32. Nagy B., Ban Z., Beke A., Nagy G. R., Lazar L., Papp C., Toth-Pal E., Papp Z. 2006; Detection of Toxoplasma gondii from amniotic fluid, a comparison of four different molecular biological methods. Clin Chim Acta 368:131–137 [CrossRef]
     [Google Scholar]
  33. Okay T. S., Yamamoto L., Oliveira L. C., Manuli E. R., Andrade H. F. Jr, Del Negro G. M. B. 2009; Significant performance variation among PCR systems in diagnosing congenital toxoplasmosis in São Paulo, Brazil: analysis of 467 amniotic fluid samples. Clinics 64:171–176
     [Google Scholar]
  34. Pereira-Chioccola V. L., Vidal J. E., Su C. 2009; Cerebral toxoplasmosis in HIV-infected patients. Future Microbiol 4:1363–1379 [CrossRef]
     [Google Scholar]
  35. Portegies P., Solod L., Cinque P., Chaudhuri A., Begovac J., Everall I., Weber T., Bojar M., Martinez-Martin P., Kennedy G. E. 2004; Guidelines for the diagnosis and management of neurological complications of HIV infection. Eur J Neurol 11:297–304 [CrossRef]
     [Google Scholar]
  36. Raffi F., Aboulker J. P., Michelet C., Reliquet V., Pelloux H., Huart A., Poizot-Martin I., Morlat P., Dupas B. other authors 1997; A prospective study of criteria for the diagnosis of toxoplasmic encephalitis in 186 AIDS patients. AIDS 11:177–184 [CrossRef]
     [Google Scholar]
  37. Reischl U., Bretagne S., Kruger D., Ernault P., Costa J. M. 2003; Comparison of two DNA targets for the diagnosis of toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes. BMC Infect Dis 3: 7 [CrossRef]
     [Google Scholar]
  38. Vidal J. E., Colombo F. A., Penalva de Oliveira A. C., Focaccia R., Pereira-Chioccola V. L. 2004; PCR assay using cerebrospinal fluid for diagnosis of cerebral toxoplasmosis in Brazilian AIDS patients. J Clin Microbiol 42:4765–4768 [CrossRef]
     [Google Scholar]
  39. Vidal J. E., Hernandez A. V., Penalva de Oliveira A. C., Dauar R. F., Barboza S. P., Focaccia R. 2005; Cerebral toxoplasmosis in HIV-positive patients in Brazil: clinical features and predictors of treatment response in the HAART era. AIDS Patient Care STDS 19:626–634 [CrossRef]
     [Google Scholar]
  40. Wahab T., Edvinsson B., Palm D., Lindh J. 2010; Comparison of the AF146527 and B1 repeated elements, two real-time PCR targets used for detection of Toxoplasma gondii . J Clin Microbiol 48:591–592 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.016261-0
Loading
Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients
J. Med. Microbiol. 59, 641 (2010); https://doi.org/10.1099/jmm.0.016261-0
/content/journal/jmm/10.1099/jmm.0.016261-0
/content/journal/jmm/10.1099/jmm.0.016261-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error