Prospective Value of PCR Amplification and Sequencing for Diagnosis and Typing of Old World Leishmania Infections in an Area of Nonendemicity

We assessed the prospective value of PCR amplification of arepetitive sequence from Leishmania nuclear DNA and sequencingfor the diagnosis and typing of Old World Leishmania infectionin an area of nonendemicity. During this 42-month study, 29of 168 consecutive samples were examined and classified as positivefor Leishmania by direct examination and/or in vitro culture.This molecular approach showed excellent sensitivity (97%) andspecificity (100%) compared to direct examination (86 and 100%,respectively) and in vitro culture (72 and 100%, respectively).Isoenzymatic and molecular typing allowed similar identificationfor 12 samples. Besides, PCR and subsequent sequencing of DNAproducts permitted the species identification of 14 samplesfor which parasite culture remained negative or did not allowisoenzymatic characterization, indicating the complementarityof parasitological and molecular tools.

INTRODUCTION

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Introduction

The biological diagnosis of Leishmania infections usually relieson direct examination of smears after Giemsa staining and invitro culture of clinical samples, i.e., bone marrow or bloodin visceral leishmaniasis (VL) and dermal and mucous scrapings,aspirates, or biopsy material in cutaneous leishmaniasis (CL)and mucocutaneous leishmaniasis. These tools confirm the diagnosiswhen (i) amastigotes are detected in tissues or (ii) promastigotesare obtained in culture, thus allowing species identificationby enzymatic characterization. However, these methods are tediousand time-consuming and require culture facilities and individualexpertise, particularly in areas of nonendemicity where thenumber of cases detected is small. Few of the 50 to 100 samplesthat are collected in our laboratory each year for the diagnosisof Old World VL and CL are positive. This underlines the needfor a trained microscopist, especially as in vitro culture issometimes negative or contaminated by fungi. In case of positiveculture, zymodeme analysis, which is performed only in referencelaboratories (22), gives a definitive species identificationwithin 2 or 3 months.

More recently, PCR amplification was also reported to be a usefulmethod for the diagnosis of Leishmania infection. Several researchgroups designed and evaluated various PCR amplification targets(nuclear or minicircle kinetoplastic DNA, small-subunit rRNA,or miniexon RNA sequences) with primers defined according totheir 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 conformationpolymorphism, restriction fragment length polymorphism, andsequence analysis (3, 4, 8, 9, 13, 17, 19, 23-25).

We prospectively evaluated the value of a PCR method for rapiddiagnosis and typing of Old World VL or CL. We used PCR amplificationof a repetitive sequence from Leishmania nuclear DNA as describedby Minodier et al. (13), with further molecular typing by sequenceanalysis, for the diagnosis of Mediterranean VL. The sensitivityand specificity of PCR were compared with direct examinationand parasite culture. Species identification by subsequent sequencingof PCR products was compared with zymodeme analysis of culturedpromastigotes.

MATERIALS AND METHODS

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Materials and Methods

One hundred sixty-eight consecutive samples obtained from patientsoriginating from Europe or Africa were examined for Leishmaniabetween 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 scrapingsand 19 biopsy specimens). Both conventional parasitologicalexamination and molecular diagnosis were applied to each sample.

Parasitological diagnosis consisted of microscopic examination(magnification, x1,000) of smears for amastigotes after Giemsastaining and in vitro culture in RPMI 1640 medium (Gibco, CergyPontoise, France) containing penicillin and streptomycin (BioMérieux,Marcy l'Etoile, France) and supplemented with 15% fetal calfserum (Gibco). When promastigotes were obtained in culture,isoenzymatic typing was performed by the French National ReferenceCenter for Leishmaniasis (Montpellier, France), as previouslydescribed (22).

For molecular diagnosis, nucleic acid extraction was performedwith QIAamp DNA mini kits (Qiagen, Courtaboeuf, France), accordingto the manufacturer's recommendations. PCR amplification wascarried out with the modified primers T2 (5'-CGGCTTCGCACCATGCGGTG-3')and B4 (5'-ACATCCCTGCCCACATACGC-3') as previously described(13, 19). The amplification reaction mixture (50 µl) contained10 µl of DNA extracted from a patient sample (either pureor diluted 1/10), 1x Applied Biosystems gold buffer, 2 mM MgCl₂,200 µM dATP, 200 µM dCTP, 200 µM dGTP, 400µM dUTP, 0.5 µM B4 primer, 0.5 µM T2 primer,0.5 U of AmpErase uracil DNA glycosylase, and 1.25 U of AmpliTaqgold DNA polymerase. All reagents were purchased from AppliedBiosystems except for the primers, which were obtained fromGenset Oligos (Evry, France). Amplification was performed (pureand diluted 1/10 for each sample) on a Perkin Elmer GeneAmpPCR System 2400 thermocycler (Applied Biosystems) with a profileof 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 annealingat 56°C, and 30 s of extension at 72°C; and 1 cycleof 5 min of terminal extension at 72°C. The presence ofamplification products was confirmed with 1% agarose gel electrophoresisanalysis. For sequencing analysis, PCR products were purifiedon a Microcon 100 column (Amicon, Millipore, Molsheim, France).The two strands of amplified DNA were sequenced with the PCRprimers and the BigDye Terminator kit (Applied Biosystems) onan automated sequencer (Applied Biosystems 377XL).

The genome and EMBL databases were consulted to obtain specificnucleotide information and, in particular, known polymorphismsof the available sequences. The nucleotide homologies of thesequenced products were studied with the BLASTN program (1)without the low-complexity filter (27).

The determination of consensus sequences and informative motifswas performed by using FASTA formatted sequences aligned withthe MULTALIGN program (5) or the XALIGN program (26).

Nucleotide sequence accession number. The repeat region sequence of Leishmania archibaldi has beensubmitted to GenBank under accession number AF344178 to enlargethe databases on this rare species. Original sequences of onestrain of Leishmania major and one strain of Leishmania infantumobtained 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

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Results and Discussion

Leishmania infection was considered definite when the clinicaldiagnosis was confirmed by positive direct examination and/orin vitro culture. The sensitivity, specificity, and positiveand negative predictive values were calculated for each diagnostictest. One hundred thirty-nine samples (83%) were negative byboth parasitological and molecular tests.

Twenty-nine (17%) of the 168 samples were positive by directexamination, culture, or PCR, corresponding to 25 cases of CLand 4 cases of VL (Table 1). Clinical and biological findingsallowed us to retrospectively exclude Leishmania infection forthe 139 negative samples. No amplification was obtained witha few samples of New World Leishmania infections (data not shown).

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TABLE 1. Patient data and results^a

Taken alone, direct examination had a sensitivity of 86% (25of 29), whereas parasite culture had a sensitivity of 72% (21of 29) (Table 2). PCR amplification yielded a specific productfor 28 of the 29 samples from patients with proven leishmaniasis.Regarding patient 5, the false negativity of molecular amplification(pure and diluted 1/10) was attributed to PCR inhibitors sinceretrospective analysis of the sample allowed a positive resultwhen diluted 1/50. Finally, this molecular method showed highsensitivity (97%) and excellent specificity (100%) (Table 2).Similar results have been obtained by some other research groupswith various populations of patients (3, 4, 7, 8, 10-16, 18,20, 21), particularly human immunodeficiency virus-infectedpatients, for whom tests with excellent sensitivity and specificityare necessary (2). Our results illustrate the complementarityof the parasitological and molecular methods for increasingthe rapidity and sensitivity of diagnosis in areas of nonendemicity,where the small yearly number of positive samples can resultin inefficient microscopic diagnosis.

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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

Regarding species identification by sequence analysis, the targetsequence of PCR amplification is localized at the 5' end ofthe previously described repetitive DNA sequence (L42476) ofthe sequenced clone LA6 from PstI-digested L. infantum MCAN/FR/73/LPMA/56DNA (17). We have performed the alignments of the 31 repetitiveLeishmania sequences available in the databases. The consensussequence (Fig. 1) highlighted 3 polymorphisms (positions 65,94, and 95) when the analyzed sequence was aligned with theeasy-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 speciesgroups, L. infantum plus Leishmania donovani plus L. archibaldi(A group) and Leishmania tropica plus Leishmania killicki (Cgroup) (Table 3). The analysis of other polymorphisms did notallow the differentiation of Leishmania species between theA and C groups. In addition to this typing method, we have determined15 types of the polymorphic poly(A) regions (nucleotides 90to 115 of the consensus sequence) (Table 4). However, analysisof this region did not allow us to discriminate one L. tropicastrain from L. killicki (type 9) or one L. archibaldi strainfrom L. donovani and L infantum (types 1 to 2). Epidemiologicaldata (clinical presentation and area of endemicity, etc.) aretherefore of prime interest to interpret the molecular diagnosis.As a whole, sequence analysis of PCR products identified thespecies or the species group for 26 (90%) of the 29 patients.By comparison, in vitro culture of promastigotes was positivefor 21 (72%) of the 29 samples. However, as isoenzymatic characterizationfailed for 9 strains, this method only yielded strain identificationfor 13 (45%) of the 29 patients. Most isoenzymatic characterizationfailures can be explained by insufficient growth of promastigotesin culture for isoenzymatic typing or by fungal contaminationof the cultures. Results of the two typing methods were complementary,as shown in Table 1. In particular, PCR and sequence analysisallowed parasite identification in 14 samples (at the specieslevel for 11 samples and at the species group level for 3 samples)that could not be cultured or identified by enzymatic methodsand yielded the result within a few days, compared to severalweeks for culture and subsequent isoenzymatic electrophoresis.Among the 12 patients for whom both isoenzymatic and moleculartyping could be realized, 100% correlation between the two typingmethods was observed. The reference method of identificationby isoenzymatic characterization allows for these cases a precisespecies identification with a zymodeme determination that correspondsto a useful epidemiological tool.

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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).

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

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

These data make PCR amplification and DNA sequencing sensitiveand efficient tools for rapid diagnosis and epidemiologicalstudies of Old World leishmaniasis. However, remaining limitationsincite us to use complementary parasitological and moleculartools to optimize the diagnosis.

ACKNOWLEDGMENTS

We are indebted to C. Giudicelli and I. Le Gall, from the Centred'Etude du Polymorphisme Humain (CEPH), Hôpital Saint-Louis,Paris, France, for sequencing the PCR amplification products.We thank J.-P. Dedet and P. Bastien for critical review of themanuscript and retrospective analysis of the PCR false-negativesample. We thank D. Young for reviewing the manuscript.

FOOTNOTES

* 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.gangneux{at}univ-rennes1.fr.

REFERENCES

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