A nested polymerase chain reaction (Ln-PCR) for diagnosing and monitoring Leishmania infantum infection in patients co-infected with human immunodeficiency virus

Abstract

We investigated a Leishmania-specific nested polymerase chain reaction (Ln-PCR) for the diagnosis and treatment monitoring of L. infantum infections in patients co-infected with human immunodeficiency virus (HIV). Peripheral blood and bone marrow samples from 89 HIV patients in Spain suspected of having leishmaniasis were examined by different diagnostic techniques (Ln-PCR, microscopy, NNN culture and indirect fluorescent antibody test). The sensitivity of Ln-PCR compared with microscopy and culture of bone marrow was 95.45% using blood and 100% when using bone marrow. 38 of these patients with confirmed leishmaniasis were entered in a chemotherapy trial (reported elsewhere), and samples from them were collected before treatment, one month after treatment ended and during follow-up (1-20 months), and examined similarly. Ln-PCR was shown to be a good method for testing efficacy of treatment and for predicting relapses after treatment (relapses were predicted on average 5 months earlier than when using classical diagnostic techniques). We suggest that Ln-PCR (especially using peripheral blood) should be the technique of choice for diagnosis, monitoring the success of treatment, and predicting relapses in patients with HIV and suspected or confirmed L. infantum infection.

Reference Values for Leishmania Infantum Parasitemia in Different Clinical Presentations: Quantitative Polymerase Chain Reaction for Therapeutic Monitoring and Patient Follow-Up

“…[21][22][23] These assays gave negative results after treatment and were unable to analyze the decrease in parasitic load during treatment. In typical cases of VL, the quantitative PCR showed that parasitemia decreases to undetectable levels between the third and the fourth administration of liposomal amphotericin B independently of the initial parasitemia.…”

Evaluation of Two rK39 Dipstick Tests, Direct Agglutination Test, and Indirect Fluorescent Antibody Test for Diagnosis of Visceral Leishmaniasis in a New Epidemic Site in Highland Ethiopia

“…One hundred microliters of peripheral blood and 100 μL of spleen aspirate dilution were subjected to a classical phenol-chloroform DNA extraction and ethanol precipitation, and 10 μL of the extracted DNA were analyzed by PCR according to the method of Cruz and others . 25,26 Standard quality control procedures were followed. In all assays, negative controls without DNA and controls with healthy human DNA were used.…”

“…DNA from an estimated 10 promastigotes was used as the positive control in each experiment. 25,26 Statistical analysis. Data were entered into an Excel (Microsoft, Redmond, WA) spreadsheet and statistical analysis was performed by using Epidat version 3.1 software ( http:// www.epidata.dk/ ).…”

Nuevos desarrollos en el diagnóstico de parásitos

“…Estos métodos para patologías parasitarias como leishmaniasis, enfermedad del sueño, toxoplasmosis o criptosporidiasis han pasado del laboratorio de investigación a los hospitales y laboratorios de referencia basados generalmente en métodos de PCR anillada (nested PCR) para aumentar la sensibilidad del diagnóstico a partir de muestras clínicas (Cruz, et al, 2002). También se han incorporado o desarrollado métodos para la detección de marcadores de resistencia, virulencia y genotipificación molecular (Fuentes, et al, 2001).…”