Leishmania aethiopica: Development of specific and sensitive PCR diagnostic test

Experimental Parasitology 128 (2011) 391–395

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Leishmania aethiopica: Development of specific and sensitive PCR diagnostic test Teklu Kuru a,⇑, Nick Janusz a, Endalamaw Gadisa b, Lashitew Gedamu a, Abraham Aseffa b a b

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4 Armauer Hansen Research Institute, P.O. Box 1005, Jimma Road, ALERT Campus, Addis Ababa, Ethiopia

a r t i c l e

i n f o

Article history: Received 1 February 2011 Received in revised form 28 April 2011 Accepted 10 May 2011 Available online 15 May 2011 Keywords: Leishmania aethiopica Cpb Cutaneous leishmaniasis

a b s t r a c t PCR has proved useful for rapid diagnosis and typing of Leishmania. Lack of specificity to discriminate between species and/or sensitivity to detect from clinical samples has always been an issue. Previously developed primers either require PCR–RFLP analysis for Leishmania aethiopica discrimination or lack sensitivity to detect L. aethiopica from clinical samples. Here we report the development and validation of L. aethiopica specific PCR primers (V5F/V10R) based on cysteine protease B (cpb), a multicopy and polymorphic gene of Leishmania. V5F/V10R primers differentiate L. aethiopica from Leishmania tropica, Leishmania major, Leishmania donovani and Leishmania infantum by direct PCR. In addition, they are sensitive enough to detect L. aethiopica from biopsy samples. The primers can be very useful for epidemiological studies, species typing and diagnosis of L. aethiopica directly from clinical samples. Implementation of these primers in routine L. aethiopica diagnosis can improve detection rate, save time, money and labor required for culturing Leishmania. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Leishmania is responsible for 1.5–2 million leishmaniasis cases every year and 350 million people are at risk of infection worldwide (Desjeux, 2004). Both visceral and cutaneous forms of leishmaniasis are prevalent in Ethiopia. While Leishmania donovani is responsible for the visceral form, Leishmania aethiopica is the main causative agent of cutaneous leishmaniasis (CL) in Ethiopia with occasional cases due to Leishmania major and Leishmania tropica (Gadisa et al., 2007; Negera et al., 2008). Ethiopian cutaneous leishmaniasis manifests itself in three different forms: localized, mucocutaneous and diffused. The number of CL cases in Ethiopia are on the rise and a new epidemic site has recently been reported (Negera et al., 2008). CL patients referred to the central Leishmania clinical laboratory from several regions in Ethiopia also suggest the possible spread of the disease to non-endemic sites. The prevalence of CL due to L. aethiopica and the contribution of L. major and L. tropica to CL cases in Ethiopia is unknown. A rapid, specific and sensitive L. aethiopica detection method is of importance for epidemiological studies. The importance of species typing to guide treatment is also justifiable by the fact that L. aethiopica is less responsive to conventional dosage of antimonial drugs (Akuffo et al., 1990). Advances in molecular identification of Leishmania have resulted in the development and use of PCR primers for the diagnosis of leishmaniasis. PCR primers based on rDNA, kDNA and ⇑ Corresponding author. E-mail address: [email protected] (T. Kuru). 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.05.006

miniexon DNA sequences have been developed for identification of Leishmania (Abdalla et al., 2003; Schonian et al., 2003; Seridi et al., 2008; Oshaghi et al., 2009; Abdalla, 2010). Permissively primed intergenic polymorphic primers of unknown target have been used to discriminate species of Leishmania based on pattern of bands (Eisenberger and Jaffe, 1999). PCR primers based on GP63 (Mauricio et al., 2001; Seridi et al., 2008) and cysteine protease B (cpb) genes (Hide and Banuls, 2006; Seridi et al., 2008; Laurent et al., 2009; Oshaghi et al., 2009) have also been developed. However, most of the primers were not validated for L. aethiopica or only a subset of species are discriminated from one another or require additional RFLP analysis for discrimination of L. aethiopica from L. tropica (Hide and Banuls, 2006; Schonian et al., 2003; Gadisa et al., 2007, 2010; Laurent et al., 2009). The primers developed in Laurent et al. (2009) do not require RFLP analysis for species discrimination. However, since the specific primers target each copy of the cpb genes, they lack sufficient specificity for detection from clinical samples (Laurent et al., 2009). Previously, we have identified cpb genes from various Leishmania species including L. aethiopica (Mundodi et al., 2002; Kuru et al., 2007). The polymorphic and multi-copy nature of the cpb genes presents an excellent opportunity for the development of species specific and sensitive primers. In this study, we report the development of L. aethiopica specific primers using cpb genes as a target. The primers discriminate L. aethiopica from other old world Leishmania species and are sensitive enough for detection from biopsy samples.

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2. Methods

2.5. Sequencing and primer designing

2.1. Ethics statement

Open reading frames of cpb sequences from eight L. aethiopica culture isolates were amplified using cpbATG/cpbTAG primers. The primer sequences, product size and PCR conditions are given in Table 1. The PCR products were purified using PCR purification kit (Qiagen) and sequenced at the University of Calgary UCDNA sequencing facility (Calgary, Canada). The sequences were submitted to GenBank and their accession numbers (HM178934– HM17894) are shown in Table 2. The cpb sequence of one of the isolates (DQ071678) was reported previously from our group (Kuru et al., 2007). Previously published full coding cpb sequences of Leishmania species were obtained from GenBank; L. tropica (GenBank accession No. DQ286773), L. mexicana (Z49962, Z14061), L. major (U43706), L. donovani (AF309626), L. infantum (AJ420286, XM_001463395, AJ628942), L. pifanoi (M97695). The cpb sequences of L. aethiopica isolates reported in this study and the above sequences obtained from GenBank were compared to

The study was approved by the institutional (Armauer Hansen Research Institute (AHRI)/All Africa Leprosy, Tuberculosis and Rehabilitation Training Centre (ALERT), Ethiopia) and national (National Ethics Review Committee, NERC, Ethiopia) ethics review committees. Written informed consent was obtained before sample collection. 2.2. Sample collection Clinical samples were obtained from suspected cutaneous leishmaniasis (CL) patients attending the ALERT Leishmania diagnostics clinic in Addis Ababa and field sites in Silti Woreda and Ocholo Kebele, Ethiopia. Skin lesion scrapings and biopsy samples were collected for culturing and molecular typing of Leishmania, respectively. 2.3. Leishmania culture and strains Leishmania clinical isolates were prepared by culturing skin lesion scrapings in Novy-MacNeal-Nicolle (NNN) solid media with an overlay Locke’s solution at 26 °C. The reference strains: L. donovani, MHOM/SD/00/IS2D; Leishmania infantum, MHOM/FR/LEM75; L. aethiopica, MHOM/ET/72/L100; L. major, MHOM/SU/73/ 5ASKH; L. tropica, MHOM/SU/74/K27, were obtained from WHO collaborating center for leishmaniasis, Health Institute of Carlos III, Madrid, Spain.

Table 2 Leishmania aethiopica culture isolates and biopsy samples. A total of 15 culture isolates were used and cpb was sequenced from nine of the isolates. Accession numbers for the cpb sequences are given. In addition to 15 culture isolates, seven biopsy samples were used for validation. Samples were typed with Isoenzyme (I) or PCR–RFLP (P) or both Isoenzyme and PCR–RFLP (IP). LCL, localized cutaneous leishmaniasis. DCL, diffuse cutaneous leishmaniasis. Biopsy sample 1275 is negative for Leishmania. NS, Not sequenced. NA, Not applicable. Sample codes

Sample

Clinical form

Accession

Typing, reference

1018-04

Culture isolate Culture isolate Culture isolate Culture isolate Culture isolate Culture isolate Culture isolate Culture isolate Culture isolate Culture isolate Culture isolate Culture isolate Culture isolate Culture isolate Culture isolate Biopsy sample Biopsy sample Biopsy sample Biopsy sample Biopsy sample Biopsy sample Biopsy sample

LCL

HM178941

LCL

DQ071678

LCL

HM178934

LCL

HM178938

LCL

HM178940

LCL

HM178939

LCL

HM178935

DCL

HM178936

DCL

HM178937

LCL

NS

LCL

NS

LCL

NS

LCL

NS

LCL

NS

LCL

NS

LCL

NS

LCL

NS

LCL

NS

LCL

NS

LCL

NS

LCL

NS

Negative

NA

IP, Negera et al. (2008) I, Genetu et al. (2006) IP, Gadisa et al. (2007) I, Genetu et al. (2006) IP, Gadisa et al. (2007) IP, Negera et al. (2008) IP, Gadisa et al. (2007) IP, Gadisa et al. (2007) P, Gadisa et al. (2007) IP, Gadisa et al. (2007) IP, Gadisa et al. (2007) IP, Gadisa et al. (2007) IP, Gadisa et al. (2007) IP, Negera et al. (2008) IP, Gadisa et al. (2007) P, Negera et al. (2008) P, Negera et al. (2008) P, Negera et al. (2008) P, Negera et al. (2008) P, Negera et al. (2008) P, Negera et al. (2008) NA

1093-02

2.4. DNA extraction 1114-03

DNA was extracted from culture and biopsy samples using phenol chloroform extraction method as described previously (Paramchuk et al., 1997). Briefly, samples were incubated in 10 mM Tris– HCl pH 8.3, 50 mM EDTA, 1% SDS and 100 lg ml1 of RNaseA (Boehringer Mannheim, Germany) at 37 °C for 1 h. Proteinase K (Boehringer Mannheim, Germany) was added at a final concentration of 100 lg ml1 and incubated overnight at 42 °C. The DNA was further purified by phenol–chloroform extraction and ethanol precipitation. The extracted DNA was quantified using spectrophotometer.

Table 1 Primers and PCR conditions used in the study. V5F/V10R primers are L. aethiopica specific primers amplifying 564 bp product from cpb genes. CpbF/CpbR primers amplify 735 bp product from all Leishmania species. b-actinF/b-actinR primers amplify 800 bp product from human DNA sample. CpbATG/cpbTAG was used in the study for amplification of cpb open reading frames (1300 bp) from Leishmania species for sequencing. Genes from Initial denaturation at 95 °C for 5 min; 30 cycles of denaturation (95 °C, 1 min), annealing (à65 or  60 °C, 2 min), extension (72 °C, 2 min); final extension at 72 °C for 10 min. Primers

Sequence

Product (bp)

Condition

V5F V10R

50 -GGTGATGTGCCCGAGTGCA-30 50 -CGTGCACATCAGCACATGGG-30

564

à

CpbF CpbR

50 -GTGCGTGCGGGTCGTGC-30 50 -AAAGCCCCGGACCAAAGCA-30

735

à

800

à

1300

 

b-actin F b-actin R cpbATG cpbTAG

0

5 -ATCTGGCACCACACCTTCTACAAT GAGCTGCG-30 50 -CGTCATACTCCTGCTTGCTGATC CACATCTGC-30 0

5 -GGCGCCGGATCCATGGCGAC GTCGAGG-30 50 -GCTCCAGGATCCCGTGTACTG GCAGGTGTT-30

1185-02 1540-03 1630-04 985-03 260-04 Och01 292-04 112-04 257-04 1630-03 390-03 1502-03 173 1004 1276 1090 1321 1091 1275

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identify L. aethiopica specific sites. Based on the specific sites, L. aethiopica specific primers (V5F/V10R) were designed. V5F/V10R primers amplify a 564 bp specific product from L. aethiopica. CpbF/CpbR primers were designed to amplify 735 bp product from all Leishmania species. b-actinF/b-actinR primers amplify 800 bp product from human DNA sample. The sequences, product size and PCR conditions of the primers are presented in Table 1. Alignments for sequence comparison were generated by using Clustal_X program (Thompson et al., 1997). 2.6. PCR amplification PCR amplification was done using100 ng template DNA in a 50 ll reaction mix containing 1 U Taq Polymerase (Amersham), 1 PCR buffer (Amersham; 10 mM Tris–HCl, pH 9.0 1.5 mM MgCl2, 50 mM KCl), 0.25 mM final concentration of dNTPs, 10 pmol of each forward and reverse primers, and sterile distilled H2O adjusted to final volume. The PCR products were run on 1.5% Agarose gel and Ethidium Bromide stained for detection. One kb plus ladder (Invitrogen) was used for product size determination. PCR conditions and sequence of primers used in this study are presented in Table 1. 3. Results In order to design a specific primer for the diagnosis of L. aethiopica from clinical samples we have sequenced cpb coding sequence from nine L. aethiopica clinical isolates (Table 1), aligned the sequences and identified intra-species polymorphic sites for exclusion from specific primers (data not shown). The sequences were then aligned with cpb sequences of L. major, L. tropica, L. donovani and L. infantum to identify L. aethiopica specific polymorphic sites. We found several L. aethiopica specific polymorphic sites and specific primers (V5F/V10R) were designed with polymorphic nucleotides on the 30 end (Supplementary data). V5F/V10R primers specifically amplify from L. aethiopica (Fig. 1A). The genus level primers (cpbF/cpbR) amplify from all the Leishmania species (Fig. 1B). To check for the existence of intra-species variability, we validated V5F/V10R primers on L. aethiopica isolate DNA extracted from parasite cultures. A total of 13 isolates previously typed as L. aethiopica (Table 2) were used for the validation test. Nine of these isolates were initially used in the bioinformatics analysis of cpb sequences for the identification of the L. aethiopica specific polymorphic sites. As shown in Fig. 2, the primers amplify from all the 13 L. aethiopica isolates. We then tested the primers on biopsy samples to determine their ability to detect L. aethiopica from clinical samples. As shown in Fig. 3A, the primers amplify from all the tested biopsy samples except one (sample 1275). Human beta-actin primers amplified from all the biopsy samples (Fig. 3B). Biopsy sample 1275 was confirmed to be negative for Leishmania using genus level primers (Fig. 3C). To quantitate the sensitivity of the primers, we tested them on serially diluted L. aethiopica DNA. Ten fold serial dilutions starting from 10 ng DNA was used and amplification was detectable from as low as 10 pg DNA (Fig. 4). Taken together, the above data indicate that V5F/V10R primers are not only specific but also sensitive for detection of L. aethiopica from biopsy samples. 4. Discussion L. aethiopica is one of the most neglected species of Leishmania endemic in East Africa, Ethiopia. L. aethiopica causes all the three different forms of cutaneous leishmaniasis (localized, diffused and mucocutaneous). The outcome of the disease ranges from benign and self healing to severe and persistent disfiguring lesions. L.

Fig. 1. Development of L. aethiopica specific primers. V5F/V10R primers amplify 564 bp product from L. aethiopica but not from other Leishmania species (A). The quality of culture DNA was checked by amplification using genus level primer (cpbF/cpbR) that can amplify 735 bp product (indicated by arrow) from cpb of Leishmania (B). L. aethiopica, L. donovani, L. infantum, L. major and L. tropica are WHO reference strains described in Section 2. Marker, 1 kb plus DNA ladder. Negative, no template control. The size of DNA marker bands running before and after the amplified product are shown.

aethiopica responds poorly to conventional drugs. Recent spread of L. aethiopica to new localities and the overlap with HIV has posed serious public health concern. Currently, studies are underway to understand the extent and nature of L. aethiopica dissemination. Rapid, sensitive and specific diagnostics for L. aethiopica is required to facilitate large scale epidemiological studies. Routine diagnosis of leishmaniasis using Microscopy is prone to false negativity and is not species specific. Isoenzyme electrophoresis, gold standard for Leishmania typing, is labor intensive and relies on parasite culture making it impractical for routine diagnosis. In contrast, PCR based molecular diagnostics has proved very useful in the diagnostics and typing of Leishmania (Abdalla, 2011). Development of diagnostics specific to discriminate between Leishmania species, sensitive for detection from clinical samples and easy for application in resource poor settings is of importance. In this study, we exploit the multicopy and polymorphic nature of cysteine protease (cpb) genes of Leishmania to develop L. aethiopica specific and sensitive PCR primers (V5F/V10R). ITS1 primers that has been routinely used for typing Leishmania requires PCR–RFLP analysis for discrimination of L. aethiopica from L. tropica (Schonian et al., 2003; Gadisa et al., 2007; Negera et al., 2008). V5F/V10R primers developed in this study discriminate L. aethiopica with direct PCR eliminating the requirement of additional RFLP analysis. Specific primers capable of discriminating L. aethiopica by direct PCR has been recently reported by Laurent et al. (2009). These primers were designed to amplify a single copy cpb gene and thus lack sufficient

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Fig. 2. Validation of L. aethiopica specific V5F/V10R primers on L. aethiopica clinical isolates. DNA extracted from parasite culture was used for amplification. Numbers indicate, the isolate codes. All isolates were previously typed to be L. aethiopica (Table 2). Marker, 1 kb plus DNA ladder. Negative, no template control. V5F/V10R primers amplify the L. aethiopica specific 564 bp product. The size of DNA marker bands running before and after the amplified product are shown.

Fig. 3. Validation of L. aethiopica specific V5F/V10R primers on biopsy DNA samples. (A) V5F/V10R primers amplify the L. aethiopica specific 564 bp product (indicated by arrow) from biopsy samples (1091, 173, 1004, 1276, 1090 and 1321). (B) Human b-actin primers amplify 800 bp product (indicated by arrow) from all biopsy samples. (C) Biopsy sample 1275 is Leishmania negative. Genus level primers (cpbF/cpbR) were used for amplification of 735 bp product. 985/03 and U937 are L. aethiopica culture isolate and human cell line DNA samples, respectively. The size of DNA marker bands running before and after the amplified product are shown.

sensitivity for amplification from clinical samples (Laurent et al., 2009). V5F/V10R primers were designed to amplify from multiple copies of cpb genes and validated for sensitive detection of L. aethiopica from biopsy samples (Fig. 3). The sensitivity of V5F/V10R primers for application on clinical samples can save time, money and labor required for culturing Leishmania. Moreover, it can improve the detection rate of L. aethiopica by avoiding false negative results due to culture contamination. Therefore, V5F/V10R primers can be exploited for large scale epidemiological studies and clinical diagnostics. This study presents proof of principle that protein coding DNA targets with multi copy and interspecies polymorphism can provide next generation of molecular markers for diagnosis and typing of Leishmania capable of detecting clinical infection with high sensitivity

and specificity. V5F/V10R primers can be adapted for quantitative and faster detection by labeling the primers for real time PCR and developing them for dipstick based system, respectively. Real time PCR (Mortarino et al., 2004; de Paiva Cavalcanti et al., 2009; Talmi-Frank et al., 2009) and dipstick based detection has been applied for Leishmania (Espinosa et al., 2009; Laurent et al., 2009). Although we validated V5F/V10R primers on recent culture isolates and biopsy samples of L. aethiopica and reference strains of old world Leishmania, we have not yet tested them on biopsy samples of non L. aethiopica old world Leishmania species. Therefore, a validation study on L. tropica, L. major, L. donovani and L. infantum clinical samples and more L. aethiopica isolates would further strengthen the validity of V5F/V10R primers for routine diagnosis.

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Fig. 4. Sensitivity analysis of V5F/V10R primers. L. aethiopica DNA sample was diluted 10-fold starting from 10 ng and tested for amplification. The L. aethiopica specific V5F/V10R primers amplify the 564 bp product (indicated by arrow) from at least as low as 10 pg DNA. Marker, 1 kb plus DNA ladder. Negative, no template control. The size of DNA marker bands running before and after the amplified product are shown.

Acknowledgments This work was supported by the Armauer Hansen Research Institute (AHRI) core budget (funded by NORAD, SIDA and Government of Ethiopia) and a grant provided to LG (University of Calgary, Canada) by the Natural Sciences and Engineering Council of Canada (NSERC; Grant No. 12034) and Canadian Institute of Health Research (CIHR; Grant No. 64318). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.exppara.2011.05.006. References Abdalla, N.M., 2010. Evaluation of gene targeted PCR and molecular hybridization used in diagnosis of human Leishmania isolates. Biotechnology 9, 212–217. Abdalla, N.M., 2011. Comparative study of immune-diagnostic tools with polymerase chain reaction in sub-clinical leishmaniasis isolates. Journal of Medicine 12, 34–39. Abdalla, N.M., ELdosh, A.A., Abdulgani, A.M., Yusif, B.E., Magzoub, M.M., 2003. Typing and characterization of Leishmania sub-clinical isolates from Nuba Mountain, west of Sudan. Infection, Genetic and Evolution 2, 277. Akuffo, H., Dietz, M., Teklemariam, S., Tadesse, T., Amare, G., Berhan, T.Y., 1990. The use of itraconazole in the treatment of leishmaniasis caused by Leishmania aethiopica. Transactions of the Royal Society of Tropical Medicine and Hygiene 84, 532–534. de Paiva Cavalcanti, M., Felinto de Brito, M.E., de Souza, W.V., de Miranda Gomes, Y., Abath, F.G., 2009. The development of a real-time PCR assay for the quantification of Leishmania infantum DNA in canine blood. Veterinary Journal 182, 356–358.

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