• View in gallery

    Kaplan-Meier probability estimates for relapse in patients co-infected with Leishmania and human immunodeficiency virus during post-treatment monitoring based on blood culture results.

  • View in gallery

    Kaplan-Meier probability estimates for relapse in patients co-infected with Leishmania and human immunodeficiency virus during post-treatment monitoring based on nested polymerase chain reaction (PCR) results.

  • 1

    Alvar J, Cañavate C, Gutierrez-Solar B, Jimenez M, Laguna F, Lopez-Velez R, Molina R, Moreno J, 1997. Leishmania and human immunodeficiency virus coinfection: the first 10 years. Clin Microbiol Rev 10 :298–319.

    • Search Google Scholar
    • Export Citation
  • 2

    Ribera E, Ocana I, de Otero J, Cortes E, Gasser I, Pahissa A, 1996. Prophylaxis of visceral leishmaniasis in human immunodeficiency virus-infected patients. Am J Med 100 :496–501.

    • Search Google Scholar
    • Export Citation
  • 3

    Lopez-Velez R, Perez-Molina JA, Guerrero A, Baquero F, Villarrubia J, Escribano L, Bellas C, Perez-Corral F, Alvar J, 1998. Clinicoepidemiologic characteristics, prognostic factors, and survival analysis of patients coinfected with human immunodeficiency virus and Leishmania in an area of Madrid, Spain. Am J Trop Med Hyg 58 :436–443.

    • Search Google Scholar
    • Export Citation
  • 4

    Pintado V, Lopez-Velez R, 2001. HIV-associated visceral leishmaniasis. Clin Microbiol Infect 7 :291–300.

  • 5

    Casado JL, Lopez-Velez R, Pintado V, Quereda C, Antela A, Moreno S, 2001. Relapsing visceral leishmaniasis in HIV-infected patients undergoing successful protease inhibitor therapy. Eur J Clin Microbiol Infect Dis 20 :202–205.

    • Search Google Scholar
    • Export Citation
  • 6

    Fisa R, Riera C, Gallego M, Manubens J, Portus M, 2001. Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates. Vet Parasitol 99 :105–111.

    • Search Google Scholar
    • Export Citation
  • 7

    López Vélez R, Laguna F, Alvar J, Pérez-Molina A, Molina R, Martínez P, Villarrrubia J, 1995. Parasitic culture of buffy coat for diagnosis of visceral leishmaniasis in human immunodeficiency virus-infected patients. J Clin Microbiol 33 :937–939.

    • Search Google Scholar
    • Export Citation
  • 8

    Dereure J, Pratlong F, Reynes J, Basset D, Bastient P, Dedet JP, 1998. Haemoculture as a tool for diagnosing visceral leishmaniasis in HIV-negative and HIV-positive patients: interest for parasitic identification. Bull World Health Organ 76 :203–206.

    • Search Google Scholar
    • Export Citation
  • 9

    Lachaud L, Dereure J, Chabbert E, Reynes J, Mauboussin JM, Oziol E, Dedet JP, Bastien P, 2000. Optimized PCR using patient blood samples for diagnosis and follow-up of visceral leishmaniasis, with special reference to AIDS patients. J Clin Microbiol 38 :236–240.

    • Search Google Scholar
    • Export Citation
  • 10

    World Health Organization (WHO), 2000. Leishmania/HIV Coinfection: South-Western Europe 1990–1998 (WHO/LEISH/2000). Geneva: World Health Organization.

  • 11

    Piarroux R, Gambarelli F, Toga B, Dumon H, Fontes M, Dunan S, Quilici M, 1996. Interest and reliability of a polymerase chain reaction on bone-marrow samples in the diagnosis of visceral leishmaniasis in AIDS. AIDS 10 :452–453.

    • Search Google Scholar
    • Export Citation
  • 12

    Mathis A, Deplazes P, 1995. PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs. J Clin Microbiol 5 :1145–1149.

    • Search Google Scholar
    • Export Citation
  • 13

    Costa JM, Durand R, Deniau M, Rivollet D, Izri M, Houin R, Vidaud M, Bretagne S, 1996. PCR enzyme-linked immunosorbent assay for diagnosis of leishmaniasis in human immunodeficiency virus-infected patients. J Clin Microbiol 34 :1831–1833.

    • Search Google Scholar
    • Export Citation
  • 14

    Osman OF, Oskam L, Zijlstra EE, Kroon NCM, Schoone GJ, Khalil ETAG, El-Hassan AM, Kager PA, 1997. Evaluation of PCR for diagnosis of visceral leishmaniasis. J Clin Microbiol 35 :2454–2457.

    • Search Google Scholar
    • Export Citation
  • 15

    Fisa R, Riera C, Ribera E, Gállego M, Portús M, 2002. A nested polymerase chain reaction for diagnosis and follow-up of human visceral leishmaniasis patients using blood samples. Trans R Soc Trop Med Hyg 96 (Suppl 1):S191–S194.

    • Search Google Scholar
    • Export Citation
  • 16

    Cruz I, Canavate C, Rubio JM, Morales MA, Chicharro C, Laguna F, Jimenez-Mejias M, Sirera G, Videla S, Alvar J, Spanish HIV-Leishmania Study Group, 2002. A nested polymerase chain reaction (Ln-PCR) for diagnosing and monitoring Leishmania infantum infection in patients co-infected with human immunodeficiency virus. Trans R Soc Trop Med Hyg 96 (Suppl 1):S185–S189.

    • Search Google Scholar
    • Export Citation
  • 17

    Lemesre JL, Rizvi F, Santoro F, Loyens M, Sadigursky M, Capron A, 1988. Autorrégulation de la croissance in vitro des Trypanosomatidae. C R Acad Sci III 307 :283–288.

    • Search Google Scholar
    • Export Citation
  • 18

    Le Fichoux Y, Quaranta JF, Aufeuvre JP, Lelievre A, Marty P, Suffia I, Rousseau D, Kubar J, 1999. Occurrence of Leishmania infantum parasitemia in asymptomatic blood donors living in an area of endemicity in southern France. J Clin Microbiol 37 :1953–1957.

    • Search Google Scholar
    • Export Citation
  • 19

    Riera C, Fisa R, Udina M, Gállego M, Portús M, 2004. Detection of Leishmania infantum cryptic infection in asymptomatic blood donors living in an endemic area (Eivissa, Balearic Islands, Spain) by different diagnostic methods. Trans R Soc Trop Med Hyg 98 :102–110.

    • Search Google Scholar
    • Export Citation
  • 20

    Pizzuto M, Piazza M, Senese D, Scalamogna C, Calattini S, Corsico L, Persico T, Adriani B, Magni C, Guaraldi G, Gaiera G, Ludovisi A, Gramiccia M, Galli M, Moroni M, Corbellino M, Antinori S, 2001. Role of PCR in diagnosis and prognosis of visceral leishmaniasis in patients coinfected with human immunodeficiency virus type 1. J Clin Microbiol 39 :357–361.

    • Search Google Scholar
    • Export Citation
  • 21

    Bossolasco S, Gaiera G, Olchini D, Gulletta M, Martello L, Bestetti A, Bossi L, Germagnoli L, Lazzarin A, Uberti-Foppa C, Cinque P, 2003. Real-time PCR assay for clinical management of human immunodeficiency virus-infected patients with visceral leishmaniasis. J Clin Microbiol 41 :5080–5084.

    • Search Google Scholar
    • Export Citation

 

 

 

 

VALUE OF CULTURE AND NESTED POLYMERASE CHAIN REACTION OF BLOOD IN THE PREDICTION OF RELAPSES IN PATIENTS CO-INFECTED WITH LEISHMANIA AND HUMAN IMMUNODEFICIENCY VIRUS

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  • 1 Laboratori de Parasitologia, Facultat de Farmàcia, Universitat de Barcelona, Barcelona, Spain; Servicio de Enfermedades Infecciosas, Hospital General Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain; Servicio de Medicina Interna, Hospital Residencia Sant Camil, Sant Pere de Ribes, Barcelona, Spain

The use of culture and a nested polymerase chain reaction (PCR) of blood in predicting the probability of relapse was evaluated in 20 patients co-infected with Leishmania and human immunodeficiency virus (HIV). Fourteen of 20 patients relapsed, with 24 clinical relapses diagnosed. During clinical relapse, the parasite was detected by culture in 21 of 24 blood samples and by nested PCR in 23 of 24 blood samples. After treatment and during asymptomatic periods, the parasite was detected by culture in 18 (19.1%) of 94 blood samples and by nested PCR in 58 (61.7%) of 94 blood samples. For positive blood cultures, the Kaplan-Meier probability estimates for relapse at 6, 12, 18, and 24 months were 44%, 68%, 76%, and 76%, respectively, while for positive nested PCRs, the estimates were 20%, 33%, 45%, and 50%, respectively. For negative blood cultures, relapse probabilities for the same time points were 7%, 12%, 12%, and 12%, while for negative nested PCRs, these probabilities were 8%, 14%, 21%, and 26%. Nested PCR-positive results in asymptomatic periods indicated presence of the parasite, but not necessarily relapse. However, the presence of viable parasites during post-treatment follow-up increased the probability of relapse and showed that culture positivity could be a good relapse marker.

INTRODUCTION

Visceral leishmaniasis (VL), which is caused by Leishmania infantum, is endemic throughout the Mediterranean basin. It is a frequent opportunistic disease in patients infected with human immunodeficiency virus (HIV) in southern Europe, where 25–70% of adult patients with VL are co-infected with HIV, and it is estimated that 1.5–9% of patients with acquired immunodeficiency syndrome (AIDS) will develop leishmaniasis.1

Relapse of VL is an early event, occurring in most HIV-Leishmania co-infected patients during the first year after treatment of VL.2,3 It does not depend on the clinical features of the leishmanial infection, the degree of immunosuppression, or the presence of AIDS. Despite the use of highly active anti-retroviral therapy, high VL relapse rates (26–70%) have been observed in co-infected patients.4,5 Clinical monitoring does not provide sufficient information to predict relapse because most patients are asymptomatic during the post-treatment period and develop symptoms only weeks before relapse.

The aim of the present study was to evaluate two diagnostic methods, culture and nested-polymerase chain reaction (PCR), in blood samples of co-infected patients to establish the value of these techniques as potential relapse markers.

MATERIALS AND METHODS

Patients.

A prospective study was conducted in Barcelona, Spain from June 1998 to June 2001. Included in this study were 20 HIV-infected patients with VL at two different hospitals. All patients gave signed, informed consent for participation in this study. The study was reviewed and approved by the ethics committees of each center. Diagnosis of VL was based in each case on an evaluation of the symptoms and signs associated with VL, including intermittent fever, hepatoesplenomegaly, pancytopenia and anemia, and identification of Leishmania amastigotes by direct examination or/and by culture in bone marrow. The patients received one of the following treatments at standard doses: meglumine antimoniate, 20 mg of pentavalent antimony/kg/day for 28 days, or amphotericin B lipidic complex, 3 mg/kg/day for 10 days. After clinical recovery, all patients were monitored. The treatment was considered successful when patients showed clinical recovery and no parasites were detected in bone marrow one month after completion of therapy. The diagnosis of clinical relapse or therapeutic failure was confirmed by direct examination or/and culture of bone marrow.

Samples and methods.

Nine milliliters of peripheral blood obtained by sterile venipuncture and collected into tubes containing EDTA for in vitro cultivation and nested PCR were collected at monthly intervals during post-treatment follow-up and, in patients who relapsed, upon diagnosis of relapse. Peripheral blood mononuclear cells (PBMCs) were isolated from blood by the Ficoll-Paque™ Plus procedure (Amersham Pharmacia Biotech, Piscataway, NJ). Preparations were divided into two aliquots, one used immediately for culture and the other stored at −40°C for nested PCR.

The PBMC samples were seeded into Schneider’s insect culture medium (Sigma, St. Louis, MO) spplemented with 20% heat-inactivated fetal calf serum, 1% sterile human urine, and gentamicin (25 μg/mL) (Sigma). Cultures were maintained at 24–26°C, examined by inverted microscopy twice a week, and sub-cultured every 2 weeks for 6 months before being pronounced negative.

Nested PCR amplification was performed on PBMCs according to a standard technique.6 DNA was extracted from 0.2 mL of PBMCs adjusted to a concentration of 1 × 106 PBMCs/mL with the High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, Mannheim, Germany), according to the manufacturer’s instruction. External primers Ext (5′-AAT TCG ACG ATC ACG AGG TC-3′) and E2b (5′-CGA CTC GGT TGG CAC ACT GC-3′) and internal primers P-1 (5′-ACG AGG TCA GCT CCA CTC C-3′) and P-2 (5′-CTG CAA CGC CTG TGT CTA CG-3′) were used. The PCR mixture contained 5 μL of DNA, 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 0.01% gelatin), 1.5 mM MgCl2, 0.1 mM of each deoxynucleotide tryphosphate, 0.2 μM of each primer to include Ext/E2b (Amersham Pharmacia Biotech), and 0.5 units of RedTaq polymerase (Sigma) in a final volume of 20 μL. Reactions were cycled in a PTC-200 thermocycler (MJ Research, Waltham, MA) using the following conditions: 96°C for 5 minutes; 30 cycles at 94°C for 30 seconds, 59°C for 30 seconds, and 72°C for 30 seconds; and 72°C for 5 minutes. The second-round amplification contained 2 μL of the first-round amplification product as the template and primers for P-1 and P-2. Reactions were visualized by electrophoresis on a 3% agarose gel. Samples were positive when a product of 100 basepairs was detected.

Statistical analysis.

The Kaplan-Meier method was used in survival analysis, and curves for the two groups (positive versus negative by culture, and positive versus negative by nested PCR) were compared by the Mantel-Cox log-rank and Breslow tests (SPSS Inc., Chicago, IL).

RESULTS

During the study period, the efficacy of specific drug treatment was monitored by blood samples over a period of one month to three years. Fourteen of the 20 patients exhibited clinical relapse, with 24 clinical relapses diagnosed. Nine patients had a single relapse, two had two, one had three, and two had four. The remaining six patients remained free of new episodes throughout the study period.

A total of 118 peripheral blood samples were collected and analyzed simultaneously by culture and nested PCR. These two methods gave concordant results in 72 cases, 37 of which were positive and 35 negative. In two instances, culture was positive and nested PCR negative; one of these cases was a relapse. In 43 instances, PBMC culture was negative and nested PCR positive; three of the cases were relapses. At time of clinical relapse, the parasite was detected in blood by culture in 21 of 24 samples and by nested PCR in 23 of 24 samples. During the post-treatment monitoring, and in absence of clinical symptoms, the parasite was detected by culture in 18 (19.1%) of 94 samples and by nested PCR in 58 (61.7%) of 94 samples, with corresponding negative predictive values of 80.8% (76 of 94) and 38.2% (36 of 94), respectively (Table 1).

Kaplan-Meier estimates of the probability of relapse at 6, 12, 18, and 24 months were 8% (95% confidence interval [CI] = 0–14%), 14% (95% CI = 5–22%), 21% (95% CI = 10–33%), and 26% (95% CI = 12–39%), respectively, in those patients with a negative blood culture. However, in those patients with a positive culture, relapse probabilities were 44% (95% CI = 22–67%), 68% (95% CI = 46–89%), 76% (95% CI = 54–97%), and 76% (95% CI = 54–97%), respectively (Figure 1). There were significant differences in the probability of relapse between patients with negative and positive blood cultures (P < 0.001).

Kaplan-Meier estimates of the probability of relapse at 6, 12, 18, and 24 months were 7% (95% CI = 0–16%), 12% (95% CI = 0–24%), 12% (95% CI = 0–24%), and 12% (95% CI = 0–24%), respectively, in patients with negative nested PCR results, and 20% (95% CI = 10–31%), 33% (95% CI = 20–46%), 45% (95% CI = 30–60%), and 50% (95% CI = 33–67%), respectively, in those patients with positive nested PCR results (Figure 2). Differences in the probability of relapse between patients with negative nested PCR results and patients with positive nested PCR results were significant (P < 0.001).

DISCUSSION

The high percentage of relapses in co-infected patients following treatment13 highlights the need not only for post-treatment parasite control, but also for alternative diagnostic techniques that use non-invasive samples. Peripheral blood has the advantage over such conventional samples as bone marrow or lymph aspirate in ease of collection and being a relatively simple and non-invasive procedure. Blood culture and/or PCR in VL diagnosis has been used extensively and shows a range of sensitivities depending on the study: 55–88% for culture710 and 82–98% for PCR.9,1116 Our results show that culture and the nested PCR showed high sensitivity during clinical episodes, when parasitemia is generally higher (87.5% and 95.8%, respectively).

Post-treatment monitoring of parasitemia during asymptomatic periods showed markedly different sensitivities between the two diagnostic methods used in this study. Although Leishmania DNA was detected in 58 (61.7%) of 94 blood samples, only 17 were associated with the presence of parasites in blood culture. No association between positive results and clinical relapse during asymptomatic periods was observed. These differences were observed by Lachaud and others,9 as well as in a preliminary study done for the work presented here.15 Low parasitic loads, which are likely present after treatment and during asymptomatic periods, could be the principle cause of low culture sensitivity, and may explain the high sensitivity of the nested PCR in detecting infections, even when levels of circulating parasites are low. However, inoculum size affects the growth of Leishmania; cultures initiated with low inoculums failed to promote cell growth.17 Recent studies on blood donors in disease-endemic areas as southern France and Spain reported low blood culture sensitivity, versus PCR, when parasitemia was low.18,19

Multivariate proportional hazard analyses showed that positive result in the nested PCR was not always associated with clinical disease and that at 24 months post-treatment the probability of relapse was 50%. These data differ from those of another group who reported positive PCR results predictive of clinical relapse.20 Most likely, the use of a nested PCR procedure, compared with a conventional PCR, resulted in an increase in overall sensitivity, but with a decrease in the negative predictive value with respect to the culture (38.2% versus 80.8%). However, when the culture was positive, risk of relapse increased to 76%. These results suggest that positive PBMC cultures, in relation to greater parasitic loads, are associated with higher risks for a new clinical episode, thereby corroborating the data of Bossolasco and others,21 who reported that clinical relapse was preceded by a substantial increase in blood parasite levels, as quantified by real-time PCR. However, during follow-up, the risk of relapse in patients with a negative nested PCR result was low (12%), whereas patients with a negative blood culture had a two-fold risk of relapse (26%).

In conclusion, nested PCR is an appropriate technique for monitoring long-term efficacy of treatment. A negative result is strongly indicative of successful control of the infection and efficacy of treatment, whereas a positive nested PCR result during an asymptomatic period is indicative of the presence of the parasite, but not necessarily of relapse. In contrast, the probability of relapse when a blood culture is positive is significantly higher than when a nested PCR result is positive. The presence of viable parasites during post-treatment follow-up increases the likelihood of relapse, demonstrating that a positive culture could be a good relapse marker.

Table 1

Results of blood cultures and nested-polymerase chain reactions (PCRs) obtained during post-treatment in 20 patients with human immunodeficiency virus–Leishmania co-infection during both clinical relapse and asymptomatic periods

Number of blood samples
At time of clinical relapsesPost-treatment follow-up during asymptomatic period*
Culture+Culture-TotalCulture+Culture-TotalTotal
* Negative predictive value of culture = 80.8% (76 of 94); negative predictive value of nested PCR = 38.2% (36 of 94).
PCR+2032317415881
PCR-1011353637
Total21324187694118
Figure 1.
Figure 1.

Kaplan-Meier probability estimates for relapse in patients co-infected with Leishmania and human immunodeficiency virus during post-treatment monitoring based on blood culture results.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 6; 10.4269/ajtmh.2005.73.1012

Figure 2.
Figure 2.

Kaplan-Meier probability estimates for relapse in patients co-infected with Leishmania and human immunodeficiency virus during post-treatment monitoring based on nested polymerase chain reaction (PCR) results.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 6; 10.4269/ajtmh.2005.73.1012

*

Address correspondence to Cristina Riera, Laboratori de Parasitologia, Facultat de Farmàcia, Universitat de Barcelona, Avenida Joan XXIII s.n., E-08028 Barcelona, Spain. E-mail: mcriera@ub.edu

Authors’ addresses: Cristina Riera, Roser Fisa, Jaume Carrió, Montserrat Gállego, and Montserrat Portús, Laboratori de Parasitologia, Facultat de Farmàcia, Universitat de Barcelona, Avenida Joan XXIII s.n., E-08028 Barcelona, Spain, Telephone: 34-93-402-4500, Fax: 34-93-402-4504, E-mails: mcriera@ub.edu, rfisa@ub.edu, jcarrio@ub.edu, mgallego@ub.edu, and mportus@ub.edu. Esteban Ribera, Vicenç Falcó, and Israel Molina, Servicio de Enfermedades Infecciosas, Hospital General Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Paseo Vall d’Hebron 119-129, 08035, Barcelona, Spain, Telephone: 34-93-274-6090, Fax: 34-93-274-6057, E-mail: eribera@vhebron.net, vfalco@vhebron.net, and imolro@yahoo.es. Lluis Moner, Servicio de Medicina Interna, Hospital Residencia Sant Camil, Sant Pere de Ribes, Carreter Puigmoltó Km 0.8, Sant Pere de Ribes 08810, Barcelona, Spain, Telephone: 34-93-896-0025, Fax: 34-93-896-1287, E-mail: llmoner@terra.es.

Acknowledgments: We thank Dr. Adil M. Allahverdiyev (Tropical Diseases Research Center, School of Medicine, Cukurova University, Adana, Turkey) and Dr. R. Ortiz de Lejarazu (Servicio de Microbiología, Hospital Universitario de Valladolid, Spain) for useful suggestions and comments. We also thank Dr. I. Gasser (Laboratorio de Microbiología, Hospital Universitari Vall d’Hebron, Barcelona, Spain) and R. Angrill and F. Corcoll (Laboratorio de Microbiología, Hospital Residencia Sant Camil, St. Pere de Ribes, Barcelona, Spain) for providing biological samples, R. Rycroft for correcting the manuscript, and S. Tebar for excellent technical assistance.

Financial support: This work was supported by the Comissionat per Universitats i Recerca, Generalitat de Catalunya (exp. 1997 SGR 00341; exp. 2001 SGR 00136).

REFERENCES

  • 1

    Alvar J, Cañavate C, Gutierrez-Solar B, Jimenez M, Laguna F, Lopez-Velez R, Molina R, Moreno J, 1997. Leishmania and human immunodeficiency virus coinfection: the first 10 years. Clin Microbiol Rev 10 :298–319.

    • Search Google Scholar
    • Export Citation
  • 2

    Ribera E, Ocana I, de Otero J, Cortes E, Gasser I, Pahissa A, 1996. Prophylaxis of visceral leishmaniasis in human immunodeficiency virus-infected patients. Am J Med 100 :496–501.

    • Search Google Scholar
    • Export Citation
  • 3

    Lopez-Velez R, Perez-Molina JA, Guerrero A, Baquero F, Villarrubia J, Escribano L, Bellas C, Perez-Corral F, Alvar J, 1998. Clinicoepidemiologic characteristics, prognostic factors, and survival analysis of patients coinfected with human immunodeficiency virus and Leishmania in an area of Madrid, Spain. Am J Trop Med Hyg 58 :436–443.

    • Search Google Scholar
    • Export Citation
  • 4

    Pintado V, Lopez-Velez R, 2001. HIV-associated visceral leishmaniasis. Clin Microbiol Infect 7 :291–300.

  • 5

    Casado JL, Lopez-Velez R, Pintado V, Quereda C, Antela A, Moreno S, 2001. Relapsing visceral leishmaniasis in HIV-infected patients undergoing successful protease inhibitor therapy. Eur J Clin Microbiol Infect Dis 20 :202–205.

    • Search Google Scholar
    • Export Citation
  • 6

    Fisa R, Riera C, Gallego M, Manubens J, Portus M, 2001. Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates. Vet Parasitol 99 :105–111.

    • Search Google Scholar
    • Export Citation
  • 7

    López Vélez R, Laguna F, Alvar J, Pérez-Molina A, Molina R, Martínez P, Villarrrubia J, 1995. Parasitic culture of buffy coat for diagnosis of visceral leishmaniasis in human immunodeficiency virus-infected patients. J Clin Microbiol 33 :937–939.

    • Search Google Scholar
    • Export Citation
  • 8

    Dereure J, Pratlong F, Reynes J, Basset D, Bastient P, Dedet JP, 1998. Haemoculture as a tool for diagnosing visceral leishmaniasis in HIV-negative and HIV-positive patients: interest for parasitic identification. Bull World Health Organ 76 :203–206.

    • Search Google Scholar
    • Export Citation
  • 9

    Lachaud L, Dereure J, Chabbert E, Reynes J, Mauboussin JM, Oziol E, Dedet JP, Bastien P, 2000. Optimized PCR using patient blood samples for diagnosis and follow-up of visceral leishmaniasis, with special reference to AIDS patients. J Clin Microbiol 38 :236–240.

    • Search Google Scholar
    • Export Citation
  • 10

    World Health Organization (WHO), 2000. Leishmania/HIV Coinfection: South-Western Europe 1990–1998 (WHO/LEISH/2000). Geneva: World Health Organization.

  • 11

    Piarroux R, Gambarelli F, Toga B, Dumon H, Fontes M, Dunan S, Quilici M, 1996. Interest and reliability of a polymerase chain reaction on bone-marrow samples in the diagnosis of visceral leishmaniasis in AIDS. AIDS 10 :452–453.

    • Search Google Scholar
    • Export Citation
  • 12

    Mathis A, Deplazes P, 1995. PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs. J Clin Microbiol 5 :1145–1149.

    • Search Google Scholar
    • Export Citation
  • 13

    Costa JM, Durand R, Deniau M, Rivollet D, Izri M, Houin R, Vidaud M, Bretagne S, 1996. PCR enzyme-linked immunosorbent assay for diagnosis of leishmaniasis in human immunodeficiency virus-infected patients. J Clin Microbiol 34 :1831–1833.

    • Search Google Scholar
    • Export Citation
  • 14

    Osman OF, Oskam L, Zijlstra EE, Kroon NCM, Schoone GJ, Khalil ETAG, El-Hassan AM, Kager PA, 1997. Evaluation of PCR for diagnosis of visceral leishmaniasis. J Clin Microbiol 35 :2454–2457.

    • Search Google Scholar
    • Export Citation
  • 15

    Fisa R, Riera C, Ribera E, Gállego M, Portús M, 2002. A nested polymerase chain reaction for diagnosis and follow-up of human visceral leishmaniasis patients using blood samples. Trans R Soc Trop Med Hyg 96 (Suppl 1):S191–S194.

    • Search Google Scholar
    • Export Citation
  • 16

    Cruz I, Canavate C, Rubio JM, Morales MA, Chicharro C, Laguna F, Jimenez-Mejias M, Sirera G, Videla S, Alvar J, Spanish HIV-Leishmania Study Group, 2002. A nested polymerase chain reaction (Ln-PCR) for diagnosing and monitoring Leishmania infantum infection in patients co-infected with human immunodeficiency virus. Trans R Soc Trop Med Hyg 96 (Suppl 1):S185–S189.

    • Search Google Scholar
    • Export Citation
  • 17

    Lemesre JL, Rizvi F, Santoro F, Loyens M, Sadigursky M, Capron A, 1988. Autorrégulation de la croissance in vitro des Trypanosomatidae. C R Acad Sci III 307 :283–288.

    • Search Google Scholar
    • Export Citation
  • 18

    Le Fichoux Y, Quaranta JF, Aufeuvre JP, Lelievre A, Marty P, Suffia I, Rousseau D, Kubar J, 1999. Occurrence of Leishmania infantum parasitemia in asymptomatic blood donors living in an area of endemicity in southern France. J Clin Microbiol 37 :1953–1957.

    • Search Google Scholar
    • Export Citation
  • 19

    Riera C, Fisa R, Udina M, Gállego M, Portús M, 2004. Detection of Leishmania infantum cryptic infection in asymptomatic blood donors living in an endemic area (Eivissa, Balearic Islands, Spain) by different diagnostic methods. Trans R Soc Trop Med Hyg 98 :102–110.

    • Search Google Scholar
    • Export Citation
  • 20

    Pizzuto M, Piazza M, Senese D, Scalamogna C, Calattini S, Corsico L, Persico T, Adriani B, Magni C, Guaraldi G, Gaiera G, Ludovisi A, Gramiccia M, Galli M, Moroni M, Corbellino M, Antinori S, 2001. Role of PCR in diagnosis and prognosis of visceral leishmaniasis in patients coinfected with human immunodeficiency virus type 1. J Clin Microbiol 39 :357–361.

    • Search Google Scholar
    • Export Citation
  • 21

    Bossolasco S, Gaiera G, Olchini D, Gulletta M, Martello L, Bestetti A, Bossi L, Germagnoli L, Lazzarin A, Uberti-Foppa C, Cinque P, 2003. Real-time PCR assay for clinical management of human immunodeficiency virus-infected patients with visceral leishmaniasis. J Clin Microbiol 41 :5080–5084.

    • Search Google Scholar
    • Export Citation
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