TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (2002) 96, SUPPLEMENT 1 S1/185-S11189 Diagnosis A nested polymerase chain reaction (Ln-PCR) for diagnosing and monitoring Leishmania infanturn infection in patients co-infected with human immuno- deficiency virus I. Cruz’, C. Caiiavate’, J. M. Rubio’, M. A. Morales I, C. Chicharro’, F. Laguna’, M. Jimknez-Mejias3, G. Sirera4, S. Videla4, J. Alvar’ and the Spanish HIV-Leishmania Study Group* ‘WHO Collaborating Centre for Leishmaniasis, Sewicio de Pavasitologia, Centro National de Microbiologia, Instituto de Salud Carlos III, Madrid, Spain; ‘Servicio de Enfermedades Infecciosas, Hospital Carlos IIL Institute de Salud Carlos III, Madrid, Spain; 3Hospital Universitario Virgen de1 Rocio, Sevilla, Spain; 4Laboratorios Dv Esteve, S. A., Investigacidn Clinica, Barcelona, Spain Abstract We investigated a Leishmania-specific nested polymerase chain reaction (Ln-PCR) for the diagnosis and treatment monitoring of L. infanturn infections in patients co-infected with human immunodeficiency virus (HIV’). Perinheral blood and bone marrow samules from 89 HIV uatients in Suain susuected 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 (l-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. infanturn infection. Keywords: leishmaniasis, Leishmaniu infantum, human immunodeticiency virus, polymerase chain reaction, diag- nosis, follow-up, Spain Introduction Visceral leishmaniasis (VL) is a frequent oppor- tunistic disease in patients infected with human immunodeficiency virus (HIV) in countries of the Mediterranean basin. To date, L&hnzaniu/HIV co- infection has been reported in 33 countries (WHO, 2000), with most of the cases being in southern Europe, where 2570% of adult patients with VL are co-infected with HIV, and it is estimated that 1.5- 9% of patients with acquired immune deficiency syndrome (AIDS) will develop leishmaniasis (WHO, 1995). Several studies have demonstrated that HIV/ AIDS patients living in Lekhmania-endemic areas are at greater risk of developing leishmaniasis (LOPEZ- SLEZ et al., 1998) and that dual infection accelerates the clinical course of HIV disease (BEFWIER et al., 1995; WOLDAY et al., 1999). In addition, VL seems to be emerging as an important infection in persons infected with HIV in areas where this disease is not endemic, such as northern Europe (ALBRECHT et al., 1996). Another cause for concern is the increase in the number of cases of co-infection in eastern Africa, Brazil and the Indian subcontinent, owing to the simultaneous spread and geographical overlap of both diseases as well as the periodic occurrence of epi- demics of VL (WHO, 2000). *In addition to the authors, the members of the Spanish HIV- Leishmania Study Group include Miguel Gorgolas (Fundacion Jimenez Diaz, Madrid), Ana Salas (Hospital Son Dureta, Palma, Mallorca), Federico Pulido (Hospital 12 de Octubre, Madrid), Julian de la Terre (Hospital Reina Sofia, Cordoba), Vicente Boix (Hospital General Universitario de Alicante), J. Rambn Arribas (Hospital La Paz, Madrid), Este- ban Ribera (Hospital Universitario Vall d’Hebron, Barcelona) and Manuel Marquez (Hospital Virgen de la Victoria, Mala- gal. Address for correspondence: Dr Jorge Alvar, WHO Collabor- ating Centre for Leishmaniasis, Servicio de Parasitologia, Centro National de Microbiologia, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain; phone +34 91 5097901, fax +34 91 5097034, e-mail jalvar@isciii.es Examination of Giemsa-stained slides of parasitized tissue is still the technique of choice for diagnosis, but its efficacy depends on the skill of microscopists, who often have an extremely difficult time due to the scar- city of parasites. Alternative diagnostic methods, for example NNN culture of bone marrow aspirates, can be performed only in advanced laboratories and are time-consuming. On the other hand, specific immuno- globulin G (IgG) antibodies are easily detected in immunocompetent patients with VL, due to the hu- moral response which they develop; the indirect immu- nofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and direct agglutina- tion test, are all highly sensitive and specific (&AR, 1995). However, 40% of VL patients co-infected with HIV do not develop a humoral immune response to the parasite (ALVAR et al., 1997; MEDRANO et al., 1998). The detection of the parasite by the polymerase chain reaction (PCR) is a useful alternative technique. PCR has been used.for the detection of different species of Leishmania (SMYTH et al.. 1992: OSMAN et al.. 1997; LACHAUD et al., 2000) and it> has the potential oi increasing the sensitivity of diagnosis and detecting very low numbers of parasites in biological samples, even in the presence of foreign deoxyribonucleic acid (DNA). Another advantage of PCR is the possibility of circum- venting the need for invasive procedures, owing to the high sensitivity of diagnosis in samples such as periph- eral blood (ADHYA et al., 1995; LE FICHOLJX et al., 1999). Previously it has been shown that PCR, using the gene SSUrRNA (small subunit ribosomal ribonucleic acid) as the target, is highly specific and sensitive for detecting Leishmaniu (VAN EYS et al., 1992). We have developed a Leishmuniu nested PCR (Ln-PCR), based on previously designed primers, aimed at the SSU- rRNA region, and evaluated its use for diagnosis with blood and bone marrow samples obtained from Leish- mania/HIV co-infected uatients before and after treat- ment for leishmaniasii, in comparison with other diagnostic techniques. An advantage of this Ln-PCR is