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Comparative Study
. 2015 Jan-Feb;19(1):62-7.
doi: 10.1016/j.bjid.2014.09.004. Epub 2014 Dec 16.

Molecular diagnosis of cryptococcal meningitis in cerebrospinal fluid: comparison of primer sets for Cryptococcus neoformans and Cryptococcus gattii species complex

Marilena dos Anjos Martins  1 , Kate Bastos Santos Brighente  2 , Terezinha Aparecida de Matos  3 , Jose Ernesto Vidal  4 , Daise Damaris Carnietto de Hipólito  5 , Vera Lucia Pereira-Chioccola  6
Affiliations
Comparative Study

Molecular diagnosis of cryptococcal meningitis in cerebrospinal fluid: comparison of primer sets for Cryptococcus neoformans and Cryptococcus gattii species complex

Marilena dos Anjos Martins et al. Braz J Infect Dis. 2015 Jan-Feb.

Abstract

Aim: This study evaluated the use of polymerase chain reaction for cryptococcal meningitis diagnosis in clinical samples.

Materials and methods: The sensitivity and specificity of the methodology were evaluated using eight Cryptococcus neoformans/C. gattii species complex reference strains and 165 cerebrospinal fluid samples from patients with neurological diseases divided into two groups: 96 patients with cryptococcal meningitis and AIDS; and 69 patients with other neurological opportunistic diseases (CRL/AIDS). Two primer sets were tested (CN4-CN5 and the multiplex CNa70S-CNa70A/CNb49S-CNb-49A that amplify a specific product for C. neoformans and another for C. gattii).

Results: CN4-CN5 primer set was positive in all Cryptococcus standard strains and in 94.8% in DNA samples from cryptococcal meningitis and AIDS group. With the multiplex, no 448-bp product of C. gattii was observed in the clinical samples of either group. The 695bp products of C. neoformans were observed only in 64.6% of the cryptococcal meningitis and AIDS group. This primer set was negative for two standard strains. The specificity based on the negative samples from the CTL/AIDS group was 98.5% in both primer sets.

Conclusions: These data suggest that the CN4/CN5 primer set was highly sensitive for the identification of C. neoformans/C. gattii species complex in cerebrospinal fluid samples from patients with clinical suspicion of cryptococcal meningitis.

Keywords: Acquired immunodeficiency syndrome; Cerebrospinal fluid; Cryptococcal meningitis; Polymerase chain reaction.

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Figures

Fig. 1
Fig. 1
Amplified PCR products of two different target regions of Cryptococcus neoformans/C. gattii species complex. CNa70S–CNa70A/CNb49S–CNb-49A multiplex amplified a 695-bp product of C. neoformans (line 1) and 448-bp of C. gattii (line 2). CN4–CN5 primer set, amplified a 136-bp product (lines 3 and 4). The DNA fragments were resolved in 2% agarose gels stained with ethidium bromide. Lane MM, 100-bp ladder.
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References

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