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MULTILOCUS ENZYME ELECTROPHORESIS AND CYTOCHROME B GENE SEQUENCING–BASED IDENTIFICATION OF LEISHMANIA ISOLATES FROM DIFFERENT FOCI OF CUTANEOUS LEISHMANIASIS IN PAKISTAN

ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Seventeen Leishmania stocks isolated from cutaneous lesions of Pakistani patients were studied by multilocus enzyme electrophoresis and by polymerase chain reaction amplification and sequencing of the cytochrome b (Cyt b) gene. Eleven stocks that expressed nine zymodemes were assigned to L. (Leishmania) major. All of them were isolated from patients in the lowlands of Larkana district and Sibi city in Sindh and Balochistan provinces, respectively. The remaining six, distributed in two zymodemes (five and one), isolated from the highland of Quetta city, Balochistan, were identified as L. (L.) tropica. The same result at species level was obtained by the Cyt b sequencing for all the stocks examined. No clear-cut association between the clinical features (wet or dry type lesions) and the Leishmania species involved was found. Leishmania (L.) major was highly polymorphic compared with L. (L.) tropica. This difference may be explained by the fact that humans may act as a sole reservoir of L. (L.) tropica in anthroponotic cycles; however, many wild mammals can be reservoirs of L. (L.) major in zoonotic cycles.

INTRODUCTION

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The leishmaniases are a group of diseases that threaten 350 million people in 88 countries with an incidence estimated at between 1.5 and 2 million new cases per year. Nevertheless, this estimated global burden of disease might be inaccurate, because of passive case detection data from many endemic areas.¹ The diseases are caused by parasitic protozoan flagellates of the genus Leishmania, which includes ~30 different taxa. The intrinsic diversity and the immune response of the host are responsible for the variability of clinical manifestation of the diseases,² which can be pleomorphic, and one species can be responsible for more than one clinical form. Therefore, the differentiation of these parasites and their accurate identification, which are relevant to eco-epidemiology, clinical diagnosis, and management of patients, must be based on molecular approaches, because parasitologic, clinical, and epidemiologic features are, by themselves, insufficient for this task.³ Among the molecular techniques that have been proposed for the characterization of Leishmania, multi-locus enzyme electrophoresis (MLEE) is the gold standard method, whereas DNA-based techniques are being used increasingly.² One of these techniques, the polymerase chain reaction (PCR) amplification and sequencing of cytochrome b (Cyt b) gene was recently established,⁴ has been very promising for species identification over wild isolates.⁵

Pakistan, a tropical and subtropical country located in the northwest of South Asia, is highly endemic for the leishmaniases. The Old World visceral leishmaniasis (VL), which is considered deadly if it is not treated, mainly occurs in the northern region of the country, in areas such as Baltistan, Chilas, and Azad Jammu and Kashmir (AJK).⁶ It has also been reported sporadically from the vicinity of the Northwest Frontier Province (NWFP), Balochistan, and Punjab provinces, although no cases of VL have yet been documented from Sindh province.⁷ In the Himalayan region, L. (L.) infantum has been incriminated as the causal agent of VL using MLEE, with dogs as a reservoir.^6,8

Two types of cutaneous leishmaniasis (CL), Old World anthroponotic (ACL) and zoonotic (ZCL) forms, are also prevalent in Pakistan. Locally, the lesions are often called tropical or oriental sores, Baghdad or Delhi boils, and Kandahar or Lahore sores.⁹

The ACL that usually affects urban or city dwellers and is clinically characterized as "dry-type lesions" has been reported from Multan city located in the southern part of Punjab province, Quetta, and other cities of Balochistan province, and also from AJK and Timargara Afghan refugee camps in the NWFP. In addition, some districts in Sindh province could be considered endemic for ACL.^10–13 Recently, it has been suggested that ACL transmission is autochthonous in a given endemic area of Pakistan, characterized by household clustering of the cases and a higher risk among children.^13,14 In most of the studies done in Pakistan to date, the etiologic Leishmania identification was made, mainly based on clinical features, epidemiology, and vector sand fly fauna. Only in limited leishmaniasis-endemic areas has L. (L.) tropica been incriminated as the causal agent of ACL by using molecular methods. The species has been isolated from patients and characterized by MLEE in Multan city,¹⁵ AJK, Rawalpindi, Besham (NWFP), and Afghan refugee camps of Islamabad.¹⁶ Leishmania (L.) tropica was also identified by a nested PCR-based schizodeme method in clinical samples from Timargara refugee camps in the NWFP.^10,17

In Pakistan, ZCL mainly occurs in rural and semi-urban areas of Balochistan and neighboring Punjab and Sindh provinces; clinically, the disease form has been associated with "moist or wet-type lesions,"^12,13,18 and unusual clinical forms have been reported.¹⁹ Only L. (L.) major, a causal agent of ZCL, was reported from Quetta, Balochistan, by using monoclonal antibodies, without indicating the detailed data of isolates tested, such as origins, altitudes of localities, lesion types, etc.¹⁹

This paper reports for the first time on the application of numerical zymotaxonomy and the Cyt b gene sequencing, in a double blind assay, in the characterization and identification of Leishmania isolated from CL patients who were actively recruited in endemic areas of Balochistan and Sindh provinces, Pakistan, and gives a brief discussion on the implications of the findings on the clinical and epidemiologic aspects of this disease.

MATERIALS AND METHODS

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Parasites and study area. Seventeen stocks of Leishmania were isolated from lesions of patients with CL and analyzed enzymatically in this study. They were collected in two campaigns during January and December of 2003 in several villages in highlands (1,600–1,800 m, above sea level [a.s.l.]) and lowlands (~100 m, a.s.l.) of Balochistan and Sindh provinces of Pakistan (Table 1; Figure 1).

View larger version (37K): [in this window] [in a new window]       FIGURE 1. Map of Pakistan showing the geographical origin of the Leishmania zymodemes in Balochistan (highland CL focus, 1,600–1,800 m, a.s.l.: Quetta city and Mari Abad) and Sindh provinces (lowland CL focus, ~100 m, a.s.l.: Sono Khan, Tharri Hajra, Warah, Lalu Raunk, Shadadkot, and Gabi Dero; Sibi city, Balochistan, also belongs to this lowland focus). NWFP, Northwest Frontier Province; CL, Old World cutaneous leishmaniasis.

Six World Health Organization (WHO) reference strains of Leishmania were included in the analysis: L. (L.) major, MHOM/SU/73/5ASKH and MHOM/IL/80/Friedlin; L (L.) aethiopica, MHOM/ET/72/L100; L (L.) infantum, MHOM/TN/80/IPT1; L. (L.) donovani, MHOM/IN/80/DD8; and L. (L.) tropica, MHOM/SU/74/K27.

Isolation, mass cultivation, soluble enzyme extract, and sample for DNA analysis preparation. Seventy-three aspirated materials were taken from lesion edges of suspected CL patients using a syringe with 0.5 mL of sterile proline balanced salts solution (PBSS) containing 100 U/mL penicillin and 50 µg/mL streptomycin (PS). The materials obtained were inoculated into "Difco" Blood agar (USMARU) medium containing 20% defibrinated rabbit blood.^20,21 Approximately 1 mL of PBSS was added after 4 days of cultivation. The cultures were maintained at ~25°C during field transportation. In the laboratory, the liquid phases of cultures were centrifuged at 2,500 rpm for 10 minutes, and the used USMARU mediums were replaced with fresh ones after washing the pellets twice with 5 mL of PBSS. The cultures were maintained at 23°C and examined regularly for 1 month.

All Leishmania stocks isolated were mass cultured using Schneider Drosophila medium supplemented with PS and 20% heat inactivated fetal bovine serum, after a maximum of two subcultures in USMARU. The soluble enzyme extracts were prepared from the promastigotes pellets obtained following the protocols previously described.²¹ For the DNA analysis, a small amount of the promastigotes pellets was re-suspended in 2 mL of TE buffer (10 mmol/L Tris-HCl, pH = 8, 1 mmol/L EDTA). These samples were aliquoted and stored at –20°C until use.

Enzyme electrophoresis. The electrophoresis, on cellulose acetate support (Sebiagel; Moulineux, France), staining, and photographic procedures were performed following the methods previously described.²¹ Each enzyme extract was analyzed by 11 enzyme systems for 12 putative enzymatic loci: alanine aminotransferase (E.C.2.6.1.2; ALAT), aspartate aminotransferase (E.C.2.6.1.1; ASAT), glucose-6-phosphate dehydrogenase (E.C.1.1.1.49; G6PDH), 6-phosphogluconate dehydrogenase (E.C.1.1.1.44; 6GPDH), glucose-phosphate isomerase (E.C.5.3.1.9; GPI), malate dehydrogenase (E.C.1.1.1.37; MDH), malic enzyme (E.C.1.1.1.40; ME), mannose-phosphate isomerase (E.C.5.3.1.8; MPI), nucleoside hydrolase (inosine) (E.C.2.4.2; NHi1 and NHi2), phosphoglucomutase (E.C.2.7.5.1; PGM), and pyruvate kinase (E.C.2.7.1.40; PK).

Electrophoretic data analysis. The distance that each reproducible enzyme band migrated from the origin (anode) was measured. The bands were numerated from the fastest (the one nearest to the cathode) to the slowest for each enzyme gel or zymogram. The obtained set of bands defined an electrophoretic profile for each extract. Stocks with the same profile were grouped in the same zymodeme; the species attribution was made comparing the profiles with the WHO reference Leishmania strains. To examine the relationship between the zymodemes, Jaccard distances were obtained for all pairwise comparisons, and a distance matrix was built using software designed on practical extraction and report language (Perl) for this work and transformed into a phenogram by the UPGMA method using the MEGA version 2.1 software.^21,22

Detection of cytochrome b gene using PCR. To extract the genomic DNA from each of the samples prepared, a genomic extraction kit (i.e., Genomic Prep^TM Cell and Tissue DNA Isolation Kit; Amersham Biosciences, Piscataway, NJ) were used, following the protocol and methods described by the company. PCR was performed with Ex-Taq polymerase (Takara Bio Inc., Takara, Japan) under the following conditions: initial denaturation at 94°C for 3 minutes followed by 35 cycles of 94°C for one minute, 50°C for one minute, and 72°C for one minute. Two hundred nanograms of parasite DNA as a template, fragments of LCBF1 forward primer (5'-GGTGTAGGTTTTAGTYTAGG-3'), and LCBR2 reverse primer (5'-CTACAATAAACAAATCATAATATR-CAATT-3') were used for Cyt b gene amplification. The PCR products were visualized on a 1% agarose gel.

Cytochrome b gene direct sequencing analysis. The Cyt b gene sequences for each stock were determined following the procedure described previously.^4,5 Briefly, the amplified fragments were electrophoresed on 1% Seakam GTG agarose gel (FMC, Germany), the band was excised, and the DNA was purified by using the QIA quick Gel Extraction kit (Qiagen, Valencia, CA). The PCR products were examined by direct sequencing with the BigDye Terminator Cycle Sequencing kit (PE Biosystems, Foster City, CA) and ABI PRISMTM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Sequencing primers used were LCBF1, LCBR2, LCYT B F4L (5'-TGTTATTGAATATGAGGTAGTG-3'), and LCYT B R4 (5'-GAACTCATAAAATAATGTAAACAAAA-3'). The obtained sequences, assembled and edited by Genetyx Mac 11.0.0 (Software Development Co. Ltd., Japan) were compared with the reference strains, available from DDBJ/EMBL/GenBank nucleotide sequence databases with accession numbers from AB095957 to AB095970.

Statistical analysis. The Fisher exact test was used for analyzing the relation between clinical features (wet or dry type lesions) and Leishmania species involved.

RESULTS

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Multi-Locus enzyme electrophoresis analysis. Seventeen Leishmania stocks isolated from patients infected in Pakistani CL-endemic areas were analyzed studying 12 putative enzymatic loci. The denomination, species assignation of the isolates, the clinical data of the patients and their geographic origin are shown in Table 1 and Figure 1. Based on phenotypic comparison, 11 stocks that expressed nine zymodemes were assigned to L. (L.) major. All were isolated from the lowlands (~100 m, a.s.l.) of the Larkana district (Sono Khan, Tharri Hajra, Warah, Lalu Raunk, Shadadkot, and Gaibi Dero), Sindh, and Sibi city, Balochistan.

The remaining six, distributed in two zymodemes (five and one for each) isolated from the highland (1,600–1,800 m, a.s.l.) of Quetta (Quetta city and Mari Abad), Balochistan, were identified as L. (L.) tropica. None of the Pakistani isolates showed the same enzymatic profile as the WHO reference strains used (Table 2; Figure 2). The ALAT, PK, and MPI were the loci that had clearly differentiated between L. (L.) major and L. (L.) tropica (Figure 3A). The GPI, PGM, and G6PDH loci showed the phenotypic diversity among the L. (L.) major stocks (Figure 3B), whereas GPI and MDH loci were polymorphous for L. (L.) tropica (Figure 3C). The enzymatic profiles are shown in Table 2.

View larger version (18K): [in this window] [in a new window]       FIGURE 2. Phenogram built by the UPGMA method showing the species assignation based on the phylogenetic relationship among the zymodemes expressed by Pakistani and WHO reference strains of Leishmania. The denomination and characteristics of the stocks and zymodemes are shown in Tables 1 and 2.

View larger version (35K): [in this window] [in a new window]       FIGURE 3. (A) Mannose-phosphate isomerase (MPI) zymogram showing the Leishmania species assignation of Pakistani stocks. Line 1, band expressed indifferently by L. (L.) aethiopica, L. (L.) infantum, and L. (L.) donovani reference strains; lines 2–5, Pakistani and reference strains of L. (L.) tropica; line 6, Pakistani and reference strains of L. (L.) major. (B) Glucose-6-phosphate dehydrogenase (G6PDH) zymogram. The intraspecific polymorphism of L. (L.) major is shown. Lines 1–5 and 7, zymodemes KMS 16, 18, 11, 15, 10 (local), and reference strain MHOM/SU/73/5ASKH of L. (L.) major, respectively; line 6, L. (L.) tropica reference strain; line 8, L. (L.) donovani reference strain. (C) Malate dehydrogenase (MDH) zymogram. Intraspecific polymorphism of L. (L.) tropica. Lines 1 and 5, zymodemes KMS 13 and reference strain of L. (L.) major; lines 2 and 3, local zymodeme KMS 26 of L. (L.) tropica; line 4, reference strain of L. (L.) tropica.

There was complete agreement on the Leishmania species identification between MLEE and Cyt b gene sequencing (Table 1).

No association was found between Leishmania species and the induction of clinical features (wet or dry type lesions; P > 0.05).

DISCUSSION

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Accurate identification of etiologic agents for Leishmania spp. by molecular approaches in the transmission foci of the disease is highly relevant not only for clinical aspects but also for eco-epidemiologic features, because of the diversity of putative vectors and reservoirs associated with Leishmania transmission.³

In this study, two Leishmania spp., L. (L.) tropica and L. (L.) major, were found at different altitudes in Pakistan; the former from highlands and the latter from lowlands. This is the first report of the incrimination by molecular techniques of L. (L.) major as a causal agent of CL in Sindh province, Pakistan. The species was also found in Sibi, a lowland city of Balochistan province. L. (L.) major has been reported from the highlands (Quetta city) of Balochistan,¹⁹ without indicating, however, the clinico-epidemiologic data of the parasite sources including locality origins of the patients and the basic features of the technique used. In contrast, we only found a single species, L. (L.) tropica, in the highland areas (1,600–1,800 m, a.s.l.) of Quetta city, Balochistan.

Although L. (L.) major was the only species found in the lowland CL-endemic foci, and L. (L.) tropica the only one in the highland foci of these study sites, the clinical features of CL described as wet and dry type lesions were detected in both foci. Because no statistical association between these clinical presentations and the Leishmania species involved were found, the lesion types such as dry and wet clinical forms might be affected by the secondary infections, environmental factors, and/or host-related factors. Therefore, this lack of association suggests that the identification or estimation of the etiological Leishmania species from the clinical features may not be reliable.^11,12,18,23 This criterion might be supported by the work of Aljeboori and Evans²⁴ in which L. (L.) major was isolated from dry type lesions (usually associated with L. (L.) tropica) in Iraq.

A great intraspecific polymorphism was found by MLEE among the Pakistani L. (L.) major isolates, whereas the L. (L.) tropica stocks have been shown to be a homogeneous group. Although genetic diversity can be an intrinsic characteristic for each species, it may be correlated with the number of vectors and/or animal reservoirs involved in the transmission cycles.³ Thus, although many wild mammals can be the reservoirs of L. (L.) major in a zoonotic cycle, humans may act as a sole reservoir of L. (L.) tropica in a restricted anthroponotic cycle.^13,14 This also has implications for the occurrence of CL in Balochistan and Sindh provinces. Whereas the dispersion for L. (L.) tropica is connected with humans and their activities, for L. (L.) major, it may be more related with the movement or ecological aspects of the animal primary reservoirs than the human activities, because this species conforms to zoonotic cycles.²⁵

The little intra-specific variations found on the Cyt b gene among Pakistani zymodemes for both of the Leishmania species analyzed have indicated that the former is highly conserved sequences inside these species. On the other hand, there are clear inter-specific differences in the Cyt b gene sequences.⁴ Thus, these data validate and highlight the usefulness of Cyt b gene sequencing on Leishmania species identification among wild Pakistani stocks.

In conclusion, the accurate identification by the gold standard and a novel molecular technique of L. (L.) tropica and L. (L.) major in two different foci of Balochistan and Sindh provinces, CL-endemic areas in Pakistan, reported here contributes not only to the clinical aspect but also to the eco-epidemiology of the diseases. Such data represent a first step for future incrimination of vectors and/or reservoirs, disclosing the eco-epidemiologic picture of these diseases.

Received October 20, 2005. Accepted for publication March 9, 2006.

Acknowledgments: The authors thank all the people and health care providers for cooperation in the study; Leandro A. Llanza for the design of the software used to calculate the Jaccard distances and the distances matrix; Patricio Diosque, Manuel Calvopiña, and Daniel Ribble for contributions and valuable comments; and Debbie Nolder for providing the Leishmania reference strains. We also thank M. Iqbal Tareen, A. Malik Tareen, Irfanullah K. Tareen, and Hashim for providing facilities and support for the examination of patients at Quetta city and Mari Abad, Quetta, Balochistan, Pakistan.

Financial support: This study was supported by Ministry of Education, Science, Culture and Sports of Japan Grants 14256002 and 18256004.

^* Address correspondence to Jorge D. Marco, Department of Parasitology, Kochi Medical School, Kochi University, Kohasu, Oko, Nankoku, Kochi 783-8505, Japan. E-mail: marcojd{at}med.kochi-ms.ac.jp

Authors’ addresses: Jorge D. Marco, Paola A. Barroso, Masataka Korenaga, and Yoshihisa Hashiguchi, Department of Parasitology, Kochi Medical School, Kochi University, Kohasu, Oko, Nankoku, Kochi 783-8505, Japan, E-mails: marcojd{at}med.kochi-ms.ac.jp, paola{at}med.kochi-u.ac.jp, korenaga{at}med.kochi-u.ac.jp, and hasiguti{at}med.kochi-u.ac.jp. Abdul M. Bhutto, Doctors’ colony, Bunglow no. 14, VIP Road, Larkana, Sindh, Pakistan, E-mail: bhutto_manan{at}hotmail.com. Farooq R. Soomro and Javed H. Baloch, Incharge Leprosy Unit, CMC Hospital, Larkana, Sindh, Pakistan, E-mails: farooq_soomro5{at}hotmail.com and javedhussainb{at}yahoo.com. Hirotomo Kato, Department of Veterinary Hygiene, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan, E-mail: katoh{at}yamaguchi-u.ac.jp. Hiroshi Uezato and Shigeo Nonaka, Department of Dermatology, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0125, Japan, E-mails: huezato{at}med.u-ryukyu.ac.jp and shigeo{at}med.u-ryukyu.ac.jp. Ken Katakura, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Kita, Sapporo 060-0818, Japan, E-mail: kenkata{at}vetmed.hokudai.ac.jp.

Reprint requests: Jorge D. Marco, Department of Parasitology, Kochi Medical School, Kochi University, Kohasu, Oko, Nankoku, Kochi 783-8505, Japan. E-mail: marcojd{at}med.kochi-ms.ac.jp.

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