title: Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis.
abstract: BACKGROUND
  Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite's DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite's DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis.
  
  
  RESULTS
  Two primer pairs were designed to detect the plasmid pUC18 and a triplex PCR assay targeting the Leishmania braziliensis kinetoplast DNA, the external control and the internal control was standardized. The triplex PCR assay was assessed for its ability to detect the three target DNA fragments simultaneously. PCR products from pUC18 DNA resulted in bands of 368 (P1) and 316 (P2) base pairs (bp). The triplex PCR optimized with the chosen external control system (P1) allowed the simultaneous detection of the internal control (G3PD - 567 bp) as well as of small quantities (10 pg) of the target parasite's DNA, detected by amplification of a 138 bp product.
  
  
  CONCLUSIONS
  The new tool standardized herein enables a more reliable interpretation of PCR results, mainly by contributing to quality assurance of leishmaniasis diagnosis. Furthermore, after simple standardization steps, this protocol could be applied to the diagnosis of other infectious diseases in reference laboratories. This triplex PCR enables the assessment of small losses during the DNA extraction process, problems concerning DNA degradation (sample quality) and the detection of L. braziliensis kDNA.
authors.affiliation: Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães (CPqAM), Av. Prof. Moraes Rego, s/n, Recife, Pernambuco CEP 50670-420, Brasil.
authors.forename: Suênia da Cunha
authors.initials: Sda C
authors.lastname: Gonçalves-de-Albuquerque
authors.affiliation: Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães (CPqAM), Av. Prof. Moraes Rego, s/n, Recife, Pernambuco CEP 50670-420, Brasil.
authors.forename: Rômulo
authors.initials: R
authors.lastname: Pessoa E Silva
authors.affiliation: Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães (CPqAM), Av. Prof. Moraes Rego, s/n, Recife, Pernambuco CEP 50670-420, Brasil.
authors.forename: Rayana Carla Silva
authors.initials: RC
authors.lastname: de Morais
authors.affiliation: Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães (CPqAM), Av. Prof. Moraes Rego, s/n, Recife, Pernambuco CEP 50670-420, Brasil.
authors.forename: Lays Adrianne Mendonça
authors.initials: LA
authors.lastname: Trajano-Silva
authors.affiliation: Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães (CPqAM), Av. Prof. Moraes Rego, s/n, Recife, Pernambuco CEP 50670-420, Brasil.
authors.forename: Carlos Gustavo
authors.initials: CG
authors.lastname: Régis-da-Silva
authors.affiliation: Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães (CPqAM), Av. Prof. Moraes Rego, s/n, Recife, Pernambuco CEP 50670-420, Brasil.
authors.forename: Sinval Pinto
authors.initials: SP
authors.lastname: Brandão-Filho
authors.affiliation: Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães (CPqAM), Av. Prof. Moraes Rego, s/n, Recife, Pernambuco CEP 50670-420, Brasil.
authors.forename: Milena
authors.initials: M
authors.lastname: de Paiva-Cavalcanti
keywords: Diagnosis; Extraction control; False-negative result; Leishmaniasis; Multiplex PCR; pUC18