Prospective Value of PCR Amplification and Sequencing for Diagnosis ...

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Sep 23, 2002 - Osman, O. F., L. Oskam, E. E. Zijlstra, N. C. M. Kroon, G. J. Schoone, ... Robert-Gangneux, F., M. T. Baixench, R. Piarroux, F. Pratlong, and C.
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2003, p. 1419–1422 0095-1137/03/$08.00⫹0 DOI: 10.1128/JCM.41.4.1419–1422.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 41, No. 4

Prospective Value of PCR Amplification and Sequencing for Diagnosis and Typing of Old World Leishmania Infections in an Area of Nonendemicity Jean-Pierre Gangneux,1,2* Jean Menotti,1 Fre´de´ric Lorenzo,1 Claudine Sarfati,1 He´le`ne Blanche,3 Hung Bui,3 Francine Pratlong,4 Yves-Jean-Franc¸ois Garin,1 and Francis Derouin1 Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire Lariboisie`re-Saint-Louis, 75010 Paris,1 Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire de Rennes, 35000 Rennes,2 Centre d’Etude du Polymorphisme Humain, Ho ˆpital Saint-Louis, 75475 Paris,3 and Laboratoire d’Ecologie Me´dicale et Pathologie Parasitaire, 34090 Montpellier,4 France Received 23 September 2002/Returned for modification 5 November 2002/Accepted 7 January 2003

We assessed the prospective value of PCR amplification of a repetitive sequence from Leishmania nuclear DNA and sequencing for the diagnosis and typing of Old World Leishmania infection in an area of nonendemicity. During this 42-month study, 29 of 168 consecutive samples were examined and classified as positive for Leishmania by direct examination and/or in vitro culture. This molecular approach showed excellent sensitivity (97%) and specificity (100%) compared to direct examination (86 and 100%, respectively) and in vitro culture (72 and 100%, respectively). Isoenzymatic and molecular typing allowed similar identification for 12 samples. Besides, PCR and subsequent sequencing of DNA products permitted the species identification of 14 samples for which parasite culture remained negative or did not allow isoenzymatic characterization, indicating the complementarity of parasitological and molecular tools. The biological diagnosis of Leishmania infections usually relies on direct examination of smears after Giemsa staining and in vitro culture of clinical samples, i.e., bone marrow or blood in visceral leishmaniasis (VL) and dermal and mucous scrapings, aspirates, or biopsy material in cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis. These tools confirm the diagnosis when (i) amastigotes are detected in tissues or (ii) promastigotes are obtained in culture, thus allowing species identification by enzymatic characterization. However, these methods are tedious and time-consuming and require culture facilities and individual expertise, particularly in areas of nonendemicity where the number of cases detected is small. Few of the 50 to 100 samples that are collected in our laboratory each year for the diagnosis of Old World VL and CL are positive. This underlines the need for a trained microscopist, especially as in vitro culture is sometimes negative or contaminated by fungi. In case of positive culture, zymodeme analysis, which is performed only in reference laboratories (22), gives a definitive species identification within 2 or 3 months. More recently, PCR amplification was also reported to be a useful method for the diagnosis of Leishmania infection. Several research groups designed and evaluated various PCR amplification targets (nuclear or minicircle kinetoplastic DNA, small-subunit rRNA, or miniexon RNA sequences) with primers defined according to their own endemic species (3, 4, 6, 7, 10–16, 18, 20, 21, 24). Typing methods for Leishmania species include multiplex PCR, randomly amplified polymorphic DNA, single-strand conformation polymorphism, restriction

fragment length polymorphism, and sequence analysis (3, 4, 8, 9, 13, 17, 19, 23–25). We prospectively evaluated the value of a PCR method for rapid diagnosis and typing of Old World VL or CL. We used PCR amplification of a repetitive sequence from Leishmania nuclear DNA as described by Minodier et al. (13), with further molecular typing by sequence analysis, for the diagnosis of Mediterranean VL. The sensitivity and specificity of PCR were compared with direct examination and parasite culture. Species identification by subsequent sequencing of PCR products was compared with zymodeme analysis of cultured promastigotes. MATERIALS AND METHODS One hundred sixty-eight consecutive samples obtained from patients originating from Europe or Africa were examined for Leishmania between June 1998 and December 2001. Twenty-eight samples (17%) were bone marrow aspirates, 46 samples (27%) were blood samples, and 94 samples (56%) were cutaneous samples (75 dermal scrapings and 19 biopsy specimens). Both conventional parasitological examination and molecular diagnosis were applied to each sample. Parasitological diagnosis consisted of microscopic examination (magnification, ⫻1,000) of smears for amastigotes after Giemsa staining and in vitro culture in RPMI 1640 medium (Gibco, Cergy Pontoise, France) containing penicillin and streptomycin (BioMe´rieux, Marcy l’Etoile, France) and supplemented with 15% fetal calf serum (Gibco). When promastigotes were obtained in culture, isoenzymatic typing was performed by the French National Reference Center for Leishmaniasis (Montpellier, France), as previously described (22). For molecular diagnosis, nucleic acid extraction was performed with QIAamp DNA mini kits (Qiagen, Courtaboeuf, France), according to the manufacturer’s recommendations. PCR amplification was carried out with the modified primers T2 (5⬘-CGGCTTCGCACCATGCGGTG-3⬘) and B4 (5⬘-ACATCCCTGCCCA CATACGC-3⬘) as previously described (13, 19). The amplification reaction mixture (50 ␮l) contained 10 ␮l of DNA extracted from a patient sample (either pure or diluted 1/10), 1⫻ Applied Biosystems gold buffer, 2 mM MgCl2, 200 ␮M dATP, 200 ␮M dCTP, 200 ␮M dGTP, 400 ␮M dUTP, 0.5 ␮M B4 primer, 0.5 ␮M T2 primer, 0.5 U of AmpErase uracil DNA glycosylase, and 1.25 U of AmpliTaq

* Corresponding author. Mailing address: Laboratoire de Parasitologie-Mycologie, Rue Henri le Guilloux, 35000 Rennes, France. Phone: 33 2 23 23 44 90. Fax: 33 2 23 23 46 29. E-mail: jean-pierre [email protected]. 1419

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J. CLIN. MICROBIOL. TABLE 1. Patient data and resultsa

Patient no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30–168 a

Sample origina

DS CB BM CB DS CB DS B DS DS CB DS DS B DS DS DS DS DS DS DS DS DS DS DS B DS DS DS Various

Country(ies) visited

Senegal Algeria France Mali Algeria Mauritania Mali France Algeria Algeria Mali Algeria Turkey Sudan Senegal Senegal Tunisia Algeria Turkey Mali Algeria Sicilia, Baleares Senegal Algeria Mauritania France Mauritania Senegal Algeria Various

Result by: Direct examination

In vitro culture

⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺

⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺

Isoenzymatic identification

PCR result

L. major MON-26 ND L. infantum MON-24 ND L. major MON-25 ND ND L. infantum MON-1 Failed L. major MON-25 ND L. major MON-25 L. tropica L. archibaldi L. major MON-74 Failed Failed ND Failed Failed ND L. infantum MON-34 L. major MON-74 Failed Failed L. infantum MON-11 Failed ND L. major MON-25 ND

⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺

Molecular identification

L. major Failed L. infantum, L. major ND L. major L. major L. infantum, L. tropica L. major Failed L. major L. tropica L. infantum, L. major L. major L. infantum, L. major L. infantum, L. major L. major L. infantum, L. major L. infantum, L. major L. infantum, L. major L. major L. major ND

L. donovani, L. archibaldi

L. donovani, L. archibaldi

L. donovani, L. archibaldi L. donovani, L. archibaldi L. donovani, L. archibaldi L. donovani, L. archibaldi L. donovani, L. archibaldi L. donovani, L. archibaldi

DS, dermal scraping; CB, cutaneous biopsy; BM, bone marrow; B, blood; ND, not determined; ⫹, positive; ⫺, negative.

gold DNA polymerase. All reagents were purchased from Applied Biosystems except for the primers, which were obtained from Genset Oligos (Evry, France). Amplification was performed (pure and diluted 1/10 for each sample) on a Perkin Elmer GeneAmp PCR System 2400 thermocycler (Applied Biosystems) with a profile of 1 cycle of 5 min at 50°C; 1 cycle of 9 min at 95°C; 40 cycles of 30 s of denaturation at 95°C, 30 s of annealing at 56°C, and 30 s of extension at 72°C; and 1 cycle of 5 min of terminal extension at 72°C. The presence of amplification products was confirmed with 1% agarose gel electrophoresis analysis. For sequencing analysis, PCR products were purified on a Microcon 100 column (Amicon, Millipore, Molsheim, France). The two strands of amplified DNA were sequenced with the PCR primers and the BigDye Terminator kit (Applied Biosystems) on an automated sequencer (Applied Biosystems 377XL). The genome and EMBL databases were consulted to obtain specific nucleotide information and, in particular, known polymorphisms of the available sequences. The nucleotide homologies of the sequenced products were studied with the BLASTN program (1) without the low-complexity filter (27). The determination of consensus sequences and informative motifs was performed by using FASTA formatted sequences aligned with the MULTALIGN program (5) or the XALIGN program (26). Nucleotide sequence accession number. The repeat region sequence of Leishmania archibaldi has been submitted to GenBank under accession number AF344178 to enlarge the databases on this rare species. Original sequences of one strain of Leishmania major and one strain of Leishmania infantum obtained in this study have also been submitted to GenBank, under accession numbers AF421497 and AF421498, respectively, in order to improve the efficiency of sequence alignment.

RESULTS AND DISCUSSION Leishmania infection was considered definite when the clinical diagnosis was confirmed by positive direct examination and/or in vitro culture. The sensitivity, specificity, and positive

and negative predictive values were calculated for each diagnostic test. One hundred thirty-nine samples (83%) were negative by both parasitological and molecular tests. Twenty-nine (17%) of the 168 samples were positive by direct examination, culture, or PCR, corresponding to 25 cases of CL and 4 cases of VL (Table 1). Clinical and biological findings allowed us to retrospectively exclude Leishmania infection for the 139 negative samples. No amplification was obtained with a few samples of New World Leishmania infections (data not shown). Taken alone, direct examination had a sensitivity of 86% (25 of 29), whereas parasite culture had a sensitivity of 72% (21 of 29) (Table 2). PCR amplification yielded a specific product for 28 of the 29 samples from patients with proven leishmaniasis.

TABLE 2. Sensitivity, specificity, and positive and negative predictive values of direct examination, in vitro culture, and PCR for the diagnosis of Old World CL and VL Result (% [no. positive/no. total]) for: Diagnostic method

Sensitivity

Specificity

Positive predictive value

Negative predictive value

Direct 86 (25/29) 100 (139/139) 100 (25/25) 96 (139/144) examination In vitro culture 72 (21/29) 100 (139/139) 100 (21/21) 95 (139/147) PCR 97 (28/29) 100 (139/139) 100 (28/28) 99 (139/140)

TOOLS FOR DIAGNOSIS AND TYPING OF OLD WORLD LEISHMANIA

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TABLE 3. Genetic diagnostic polymorphisms at nucleotides 65, 94, and 95 Organism

L. L. L. L. L. L. L.

archibaldi donovani infantum aethiopica killicki major tropica

Polymorphism(s) at nucleotide position(s)a: 65*

94*95*

G G G C C ⌬ C

⌬⌬ ⌬⌬ ⌬⌬ ⌬⌬ CC ⌬⌬ CC

Group

A A A B C D C

a *, unambiguous polymorphisms selected for the genetic diagnostic; ⌬ and ⌬⌬, simple and double deletions, respectively.

Regarding patient 5, the false negativity of molecular amplification (pure and diluted 1/10) was attributed to PCR inhibitors since retrospective analysis of the sample allowed a positive result when diluted 1/50. Finally, this molecular method showed high sensitivity (97%) and excellent specificity (100%) (Table 2). Similar results have been obtained by some other research groups with various populations of patients (3, 4, 7, 8, 10–16, 18, 20, 21), particularly human immunodeficiency virusinfected patients, for whom tests with excellent sensitivity and specificity are necessary (2). Our results illustrate the complementarity of the parasitological and molecular methods for increasing the rapidity and sensitivity of diagnosis in areas of nonendemicity, where the small yearly number of positive samples can result in inefficient microscopic diagnosis. Regarding species identification by sequence analysis, the target sequence of PCR amplification is localized at the 5⬘ end of the previously described repetitive DNA sequence (L42476) of the sequenced clone LA6 from PstI-digested L. infantum MCAN/FR/73/LPMA/56 DNA (17). We have performed the alignments of the 31 repetitive Leishmania sequences available in the databases. The consensus sequence (Fig. 1) highlighted

FIG. 1. Human pathogenic Old World Leishmania consensus sequence. The consensus sequence (bottom line) was aligned with the reference sequence of L. infantum L42476 (top line). *, unambiguous polymorphisms selected for the genetic diagnostic; —, ambiguous polymorphisms not selected. The G5 region is underlined (position 38).

3 polymorphisms (positions 65, 94, and 95) when the analyzed sequence was aligned with the easy-to-spot G5 (5 consecutive guanines at positions 38 to 42). These polymorphisms easily categorize two species, L. major (D group) and Leishmania aethiopica (B group), and two species groups, L. infantum plus Leishmania donovani plus L. archibaldi (A group) and Leishmania tropica plus Leishmania killicki (C group) (Table 3). The analysis of other polymorphisms did not allow the differentiation of Leishmania species between the A and C groups. In addition to this typing method, we have determined 15 types of the polymorphic poly(A) regions (nucleotides 90 to 115 of the consensus sequence) (Table 4). However, analysis of this region did not allow us to discriminate one L. tropica strain from L. killicki (type 9) or one L. archibaldi strain from L. donovani and L infantum (types 1 to 2). Epidemiological data (clinical presentation and area of endemicity, etc.) are therefore of prime interest to interpret the molecular diagnosis. As a whole, sequence analysis of PCR products identified the species or the species group for 26 (90%) of the 29 patients. By comparison, in vitro culture of promastigotes was positive for 21 (72%) of the 29 samples. However, as isoenzymatic characterization failed for 9 strains, this method only yielded strain identification for 13 (45%) of the 29 patients. Most isoenzymatic characterization failures can be explained by insufficient growth of

TABLE 4. Genetic diagnostic polymorphisms of the polymorphic poly(A) region (nucleotides 90 to 115) Type

Sequence (nucleotides 90 to 115)a

Reference strain(s)

0 1

—GA———A——AA———AA———AGA—A—— GGAA——A—GAAA—AAAAGCAGA—ACA

2

GGAA——A—GAAA—AAAGGCAGA—AGA

3 4

GGAG——A—GAAA—AAAAG—AGAGAGA GGAA——A—GAAA—AAAAG—AGA—AGA

5 6 7 8 9 10

CGAA——A—GAAA—AAAAG—AGA—AGA GGAA——A—GAAA—AAAAGCAGA—ACT —GAA——ATGAAA—AAAAGCAGA—AGA —GAA——ACGAAA—AAAAGCAGA—AGA GGAACCA——AAA—AAAAGCAGA—AGA GGAA——A—GAAC—GAAAGTAGA—AGA

11 12 13 14

GGAA——A—GAAA—GAAAGCAGA—AGA GGAA——A—GAACGAAAAGTAGA—AGA GGAACCA——AA——AAAAGCAGA—AGA GGAACCA—AAAA—AAAA—CAGA—AGA

15

GGAACCA——AAA—AAAA—CAGA—AGA

Consensus L. archibaldi AF344178, L. infantum L42481, L. infantum AF421498 L. archibaldi L42499, L. donovani L42475, L. donovani L42478 L. infantum L42476 L. infantum L42477, L. infantum L42479, L. infantum L42483, L. infantum L42486 L. infantum L42482 L. infantum L42480 L. aethiopica L42484 L. aethiopica L42485 L. killicki L42500, L. tropica L42495 L. major L42501, L. major L42502, L. major L42503, L. major L42504 L. major L42505 L. major AF421497 L. tropica L42487, L. tropica L42492 L. tropica L42488, L. tropica L42490, L. tropica L42491, L. tropica L42493, L. tropica L42494 L. tropica L42489

a

—, ambiguous polymorphisms not selected.

Diagnosis

L. archibaldi or L. infantum L. archibaldi or L. donovani L. infantum L. infantum L. L. L. L. L. L.

infantum infantum aethiopica aethiopica killicki or L. tropica major

L. L. L. L.

major major tropica tropica

L. tropica

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promastigotes in culture for isoenzymatic typing or by fungal contamination of the cultures. Results of the two typing methods were complementary, as shown in Table 1. In particular, PCR and sequence analysis allowed parasite identification in 14 samples (at the species level for 11 samples and at the species group level for 3 samples) that could not be cultured or identified by enzymatic methods and yielded the result within a few days, compared to several weeks for culture and subsequent isoenzymatic electrophoresis. Among the 12 patients for whom both isoenzymatic and molecular typing could be realized, 100% correlation between the two typing methods was observed. The reference method of identification by isoenzymatic characterization allows for these cases a precise species identification with a zymodeme determination that corresponds to a useful epidemiological tool. These data make PCR amplification and DNA sequencing sensitive and efficient tools for rapid diagnosis and epidemiological studies of Old World leishmaniasis. However, remaining limitations incite us to use complementary parasitological and molecular tools to optimize the diagnosis.

J. CLIN. MICROBIOL.

10.

11.

12. 13. 14.

15. 16. 17.

ACKNOWLEDGMENTS We are indebted to C. Giudicelli and I. Le Gall, from the Centre d’Etude du Polymorphisme Humain (CEPH), Ho ˆpital Saint-Louis, Paris, France, for sequencing the PCR amplification products. We thank J.-P. Dedet and P. Bastien for critical review of the manuscript and retrospective analysis of the PCR false-negative sample. We thank D. Young for reviewing the manuscript.

18.

19.

REFERENCES 1. Altschul, S. F., T. L. Madden, A. A. Scha ¨ffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a next generation of protein database search programs. Nucleic Acids Res. 25:3389–3402. 2. Alvar, J., C. Canavate, B. Gutie´rrez-Solar, M. Jime´nez, F. Laguna, R. LopezVe´lez, R. Molina, and J. Moreno. 1997. Leishmania and human immunodeficiency virus coinfection: the first 10 years. Clin. Microbiol. Rev. 10:298–319. 3. Aviles, H., A. Belli, R. Armijos, F. P. Monroy, and E. Harris. 1999. PCR detection and identification of Leishmania parasites in clinical specimens in Ecuador: a comparison with classical diagnostic methods. J. Parasitol. 85: 181–187. 4. Belli, A., B. Rodriguez, H. Aviles, and E. Harris. 1998. Simplified polymerase chain reaction detection of new world Leishmania in clinical specimens of cutaneous leishmaniasis. Am. J. Trop. Med. Hyg. 58:102–109. 5. Brodskii, L. I., V. V. Ivanov, I. L. Kalaidzidis, A. M. Leontovich, V. K. Nikolaev, S. I. Feranchuk, and V. A. Drachev. 1995. GeneBee-NET: an internet-based server for biopolymer structure analysis. Biokhimiya 60:1221– 1230. 6. Costa, J. M., R. Durand, M. Deniau, D. Rivollet, M. Izri, R. Houin, M. Vidaud, and S. Bretagne. 1996. PCR enzyme-linked immunosorbent assay for diagnosis of leishmaniasis in human immunodeficiency virus-infected patients. J. Clin. Microbiol. 34:1831–1833. 7. Delgado, J., J. A. Pineda, J. M. C. Regordan, J. A. Gallardo, M. Leal, A. Sanchez-Quijano, and E. Lissen. 1998. Low sensitivity of peripheral blood smear for diagnosis of subclinical visceral leishmaniasis in human immunodeficiency virus type 1-infected patients. J. Clin. Microbiol. 36:315–316. 8. Eisenberg, C. L., and C. L. Jaffe. 1999. Leishmania: identification of old world species using a permissively prime intergenic polymorphic-polymerase chain reaction. Exp. Parasitol. 91:70–77. 9. Harris, E., G. Kropp, A. Belli, B. Rodriguez, and N. Agabian. 1998. Single-

20.

21.

22. 23.

24.

25.

26. 27.

step multiplex PCR assay for characterization of new world Leishmania complexes. J. Clin. Microbiol. 36:1989–1995. Katakura, K., S. I. Kawazu, T. Naya, K. Nagakura, M. Ito, M. Aikawa, J. Q. Qu, L. R. Guan, X. P. Zuo, J. J. Chai, K. P. Chang, and Y. Matsumoto. 1998. Diagnosis of kala azar by nested PCR based on amplification of the Leishmania mini-exon gene. J. Clin. Microbiol. 36:2173–2177. Lachaud, L., J. Dereure, E. Chabbert, J. Reynes, J. M. Mauboussin, E. Oziol, J. P. Dedet, and P. Bastien. 2000. Optimized PCR using patient blood sample for diagnosis and follow-up of visceral leishmaniasis, with special reference to AIDS patients. J. Clin. Microbiol. 38:236–240. Mathis, A., and P. Deplazes. 1995. PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs. J. Clin. Microbiol. 33:1145–1149. Minodier, P., R. Piarroux, F. Gambarelli, C. Joblet, and H. Dumon. 1997. Rapid identification of causative species in patients with Old World leishmaniasis. J. Clin. Microbiol. 35:2551–2555. Noyes, H. A., H. Reyburn, J. W. Bailey, and D. Smith. 1998. A nested-PCRbased schizodeme method for identifying Leishmania kinetoplast minicircle classes from clinical sample and its application to the study of the epidemiology of Leishmania tropica in Pakistan. J. Clin. Microbiol. 36:2877–2881. Nuzum, E., F. White III, C. Thakur, R. Dietze, J. Wages, M. Grogl, and J. Berman. 1995. Diagnosis of symptomatic visceral leishmaniasis by use of the polymerase chain reaction on patient blood. J. Infect. Dis. 171:751–754. Osman, O. F., L. Oskam, E. E. Zijlstra, N. C. M. Kroon, G. J. Schoone, E. A. G. Khalil, A. M. El-Hassan, and P. A. Kager. 1997. Evaluation of PCR for diagnosis of visceral leishmaniasis. J. Clin. Microbiol. 35:2454–2457. Piarroux, R., R. Azaiez, A. M. Lossi, P. Reynier, F. Muscatelli, F. Gambarelli, M. Fontes, H. Dumon, and M. Quilici. 1993. Isolation and characterization of a repetitive DNA sequence from Leishmania infantum: development of a visceral leishmaniasis polymerase chain reaction. Am. J. Trop. Hyg. 49:364–369. Piarroux, R., F. Gambarelli, H. Dumon, M. Fontes, S. Dunan, C. Mary, B Toga, and M. Quilici. 1994. Comparison of PCR with direct examination of bone marrow aspiration, myeloculture, and serology for diagnosis of visceral leishmaniasis in immunocompromised patients. J. Clin. Microbiol. 32:746– 749. Piarroux, R., M. Fontes, R. Perasso, F. Gambarelli, C. Joblet, H. Dumon, and M. Quilici. 1995. Phylogenic relationships between Old World Leishmania strains revealed by analysis of a repetitive DNA sequence. Mol. Biochem. Parasitol. 73:249–252. Pirmez, C., V. Da-Silva-Trajano, M. P. Oliveira-Neto, A. M. Da-Cruz, S. C. Goncalves-da-Costa, M. Catanho, W. Degrave, and O. Fernandes. 1999. Use of PCR in diagnosis of human American tegumentary leishmaniasis in Rio de Janeiro, Brazil. J. Clin. Microbiol. 37:1819–1823. Pizzuto, M., M. Piazza, D. Senese, C. Scallamogna, S. Calattini, L. Corsico, T. Persico, B. Adriani, C. Magni, G. Guaraldi, G. Gaiera, A. Ludovisi, M. Gramiccia, M. Gali, M. Moroni, M. Corbellino, and S. Antinori. 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. Rioux, J. A., G. Lanotte, E. Serres, F. Pratlong, P. Bastien, and J. Perie`res. 1990. Taxonomy of Leishmania. Use of isoenzymes. Suggestions for a new classification. Ann. Parasitol. Hum. Comp. 65:111–125. Robert-Gangneux, F., M. T. Baixench, R. Piarroux, F. Pratlong, and C. Tourte-Schaefer. 1999. Use of molecular tools for the diagnosis and typing of a Leishmania major strain isolated from an HIV-infected patient in Burkina Faso. Trans. R. Soc. Trop. Med. Hyg. 93:396–397. Schonian, G., C. Schweynoch, K. Zlateva, L. Oskam, N. Kroon, Y. Graser, and W. Presber. 1996. Identification and determination of the relationships of species and strains within the genus Leishmania using single primers in the polymerase chain reaction. Mol. Biochem. Parasitol. 77:19–29. van Eys, G. J. J. M., G. J. Schoone, N. C. M. Kroon, and S. B. Ebeling. 1992. Sequence analysis of small subunit ribosome RNA genes and its use for detection and identification of Leishmania parasites. Mol. Biochem. Parasitol. 51:133–142. Wishart, D. S., R. F. Boyko, and B. D. Sykes. 1994. Constrained multiple sequence alignment using XALIGN. Comput. Appl. Biosci. 10:687–688. Wootton, J. C., and S. Federhen. 1996. Analysis of compositionally biased regions in sequence databases. Methods Enzymol. 266:554–571.