Skip to main content
Download PDF
  • Research note
  • Open access
  • Published:

Ability of real-time PCR for differential diagnosis of various forms of cutaneous leishmaniasis: a comparative study with histopathology

BMC Research Notes volume 12, Article number: 615 (2019) Cite this article

Abstract

Objective

Histopathological studies suggest that parasite load is different between acute and chronic forms of cutaneous leishmaniasis (CL). However, highly sensitive detection methods are still needed to distinguish different forms of leishmaniasis. In the present study, we developed a quantitative real-time polymerase chain reaction (PCR) to detect and quantify Leishmania tropica parasites in paraffin-embedded tissue samples.

Results

The ability of real-time PCR for leishmania detection was higher than histopathological evaluation. The quantitative real-time PCR (qPCR) quantified parasite loads were highly correlated with microscopic results (r = 0.598; P < 0.001). Among patients, the parasite load was inversely correlated with disease duration (acute CL lesions had very higher parasite load than chronic CL lesions), but there was no difference in the parasite load according to the patients’ age and sex as well as location of the lesions. In contrast to Ridley scoring system (P < 0.001), there were no statistically significant differences in the relative number of parasites among the lupoid and non-lupoid forms of chronic lesions in real-time PCR (P = 0.549), which indicates the superiority of histopathological evaluation for chronic forms differentiation.

Introduction

Dry cutaneous leishmaniasis (CL) caused by Leishmania tropica is a significant parasitic disease in Iran [1]. The clinical phenotype, histopathology, and the number of organisms are diverse among acute, chronic lupoid, and chronic non-lupoid forms of this infectious disease [2]. In histopathology of acute CL, plasma cells, histiocytes, epithelioid cells, and occasionally eosinophils and giant cells, and dense dermal infiltrate of lymphocytes are seen. Also, numerous intracytoplasmic Leishman bodies parasitized macrophages and sometimes neutrophils are seen throughout the reticular dermis. A small number of infected macrophages and multifocal small tuberculoid granulomas composed of epithelioid cells, histiocytes, and occasional giant cells are seen more in chronic form. In addition, mild to moderate mononuclear infiltrates (lymphocytes and plasma cells) adjacent to the granuloma along with fibrosis and telangiectasia are present. Low numbers of organisms, erythematous papules at the periphery of a scar of a healed acute lesion, and granulomas consisting of tubercles surrounded by lymphocytes, histiocytes, and giant cells are the most pathological findings in the lupoid forms of the disease; although, because of scant organisms in cutaneous lesions specifically in chronic leishmaniasis, microscopic studies has less sensitivity [2,3,4,5,6,7,8,9].

Laboratory diagnosis of CL relies on either the microscopic detection of Leishman bodies in cutaneous tissue or the culture and isolation of parasites from lesions biopsy samples [10, 11]. Apart from high specificity, inadequate sensitivity, difficulty, and time consuming nature are among disadvantages of these methods [12]. Nowadays, PCR-based testing of skin lesion biopsies is known as a sensitive and specific test for diagnosis and quantification of leishmaniasis [13,14,15,16]. The analysis of the load of leishmania parasites within the skin lesions would be important not only for diagnostic purposes, but also for an eventual follow-up of a patient’s response to treatment [17]. Accordingly, in the present study we applied a standardized qPCR assay to detect Leishmania tropica load in paraffin blocks of various CL forms. The differentiation ability of this quantitative method was compared with semi-quantitative pathological scoring system.

Main text

Materials and methods

Patients and sampling

Forty patients presenting with acute (n = 10), chronic lupoid (n = 16), and chronic non-lupoid (n = 14) forms of CL who were referred to the Dermatopathology Department of Afzalipour Hospital (2010–2013) were selected to participate in our study. Patient selection was performed after evaluation of inclusion/exclusion criteria. The patients were considered to be included in the study if they have confirmed and long-term CL (≥ 3 years), had received at least 3 times glucantime treatment, and were able to give contact information for the follow-up. We excluded patients with other skin diseases or with small biopsy samples.

Histopathology

Skin biopsies were fixed in formalin, routinely processed, and after embedding in paraffin, sections were stained with hematoxylin and eosin, based on general approach. After grouping of cutaneous lesions according to Azadeh [18] classification (Anergic macrophage reaction, Focalized histiocytic reaction, Diffuse necrotizing reaction, Diffuse lympho-histiocytic reaction, and Lupoid granulomatous reaction), the Ridley scoring system [19] was applied for determination of parasite load, as follows from 0 to + 4:

  • 0: None amastigote

  • + 1: One or more amastigotes

  • + 2: 10 or more amastigotes

  • + 3: 100 or more amastigotes

  • + 4: 1000 or more amastigotes.

In should be noted that for uniform inflammatory cell-counting in all samples, it was decided to count the cells in inflammatory centers near the parasite and around granulomas. In addition, histopathologic alterations including necrosis, unorganized or organized granuloma, cellular (polymorphonuclears, eosinophils, giant cells, lymphocytes, plasma cells, and macrophages) infiltration and parasite index were estimated through an arbitrary semiquantitative procedure.

DNA extraction

For DNA extraction, 5 μm sections from paraffin-embedded blocks were cut using disposable blades and deparaffinized by hot xylene and then, were hydrated (descending grades of alcohol) and incubated in proteinase K (20 μg/μL, at 60 °C). After digestion completed (3 days), the DNA was isolated using a QIAamp® DNA Mini Kit (QIAGEN, 51304), according to the manufacturer′s protocol.

Real-time PCR assay

We applied a probe-based assay targeting rRNAITS region to detect and quantify parasites in the samples (Table 1). PCR amplification reaction was fulfilled using ABI StepOne system (Applied Biosystems, USA) and in a 25 μL of reaction mixture, containing 12.5 μL of master mix, 2 μL of forward and reverse primers for beta-actin and rRNAITS regions, 1.5 μL probe, 2 μL of H2O, and 5 μL of extracted DNA. Thermal cycling conditions started at 95 °C for 2 min followed by 95 °C for 20 s (denaturation), and 60 °C for 30 s (annealing and extension), which were programmed for 45 cycles. A cycle threshold (Ct) for each sample was determined based on the required cycles for the fluorescent signal to cross the background level.

Table 1 Primers used in our study
Full size table

Quantification of parasite DNA load

For absolute quantification, the standard strain (MHOM/Sudan/58/OD) of L. tropica was cultured in RPMI1640 medium and serial dilutions (10 to 107) were prepared. Subsequently, a standard curve was set by plotting the Ct values against different standards with known concentration of the parasite’s DNA.

Statistical analysis

The differences between experimental groups were analyzed using the ANOVA (Tukey test). The Spearman’s rank correlation coefficient was used for evaluation of the relationship between real-time PCR and histopathological results. The SPSS software (version 22) was applied in this study.

Results

Histopathology and real-time PCR results in studied patients with different forms of CL are summarized in Table 2 and 3. Forty patients with confirmed CL were enrolled: 25 (62.5%) men and 15 (37.5%) women, with mean age of 32 years (range 6–73 years). To evaluate the correlation between the qPCR assay and histopathological evaluation, collected samples were analyzed in parallel by both methods. The linearity of qPCR results was approved (diagram slope of − 3.23 and correlation coefficient (r2) ≥ 0.997) [20]. This assay allowed the quantification of the parasite load in all samples, while the microscopic evaluation allowed this in 32 samples (80%, 8 negative samples corresponded to lupoid patients), which is indicating that the former method is more sensitive than the latter.

Table 2 Summary of patients informations
Full size table
Table 3 Detailed analysis of each patient
Full size table

As presented in Tables 2 and 3, acute form has higher parasite load than chronic ones (P < 0.001) by real-time PCR. The mean parasite load in chronic lesions (n = 30) was 0.08 × 103 parasites, compared with 13.064 × 103 in acute lesions (n = 10, P < 0.001). Interestingly, there was no significant difference in parasite load among lupoid and non-lupoid lesions by real-time PCR (P = 0.549). According to histopathological analysis, there were statistically significant differences in the relative number of parasites among the acute and chronic (P< 0.01) and chronic-lupoid and non-lupoid forms (P < 0.001). These results indicate the superiority of histopathological evaluation (Ridley scoring system) for differentiation of various forms of CL.

Discussion

In order to accurately and confidently quantify parasites in paraffin-embedded biopsy samples, we evaluated the parasitic load in acute and chronic forms using real-time PCR and histopathological scoring system. The focus of the present study was to compare the diagnostic ability of two common methods in a relatively large number of patients with CL. The power of the used qPCR assay [21] has allowed the quantification of a broad range of parasite load levels in tissue lesions. In terms of diagnostic sensitivity, our results confirmed that the sensitivity of real-time PCR is indeed higher than histopathological scoring system. Our findings are also consistent with the findings of previous studies that focused on different abundance of parasite in various forms of CL, pointing to inversely correlation of parasite load with the disease duration. Namely, in both methods of this study, acute form has higher parasite load than chronic ones. Interestingly, in contrast to Ridley scoring system (P < 0.001), there were no statistically significant differences in the relative number of parasites among the lupoid and non-lupoid forms of chronic lesions in real-time PCR (P = 0.549), which indicates the superiority of histopathological evaluation in differentiation of chronic forms. It should be noted that the analysis performed here revealed no significant differences in parasite load with regard to the age, sex, and location of skin lesions. These findings were consistent with other studies [22,23,24,25]. For example, Mashayekhi et al. in a study on 11 male and 9 female patients with a mean age of 17.5 years showed that PCR was positive in 60% of the samples and no correlation was found between the results of PCR and age, sex, duration, and location of the lesions [26]. Venkataram et al. indicated that 65% of acute, subacute, and chronic lesions manifested leishmania parasites in tissues. But they could not find the relationship between the duration of lesions and PCR results [25]. Weigle and others showed that PCR sensitivity was higher than the conventional assays for the diagnosis of acute lesions while for chronic samples, the sensitivity of PCR was much higher than the conventional assays [27]. In this regard, Verma et al. conducted real-time assay to estimate parasite burden in clinical samples of visceral leishmaniasis and patients with post kala-azar dermal leishmaniasis. Concurrent diagnostic and prognostic ability of this assay, provide a simple molecular instrument to detect parasite and show the efficacy of anti-leishmanial drugs or vaccines [28]. In line with this, Dabiri et al. compared the effect of different treatments on DNA load of leishmania using real-time PCR method [29]. Jara et al. improved a quantitative real-time PCR (qPCR) method targeting mini-circle kinetoplast DNA (kDNA) to find and quantify Leishmania (Viannia) parasites. According to the parasite species, the patients’ age, and number or area of lesions, there was no difference in parasite load [17]. Sirian et al. conducted a comparison between conventional, molecular, and immunohistochemical methods for CL detection and reported that immunohistochemical and molecular techniques were more sensitive [30,31,32].

Our observations support the validity of using real-time PCR to simultaneously detect and quantify the leishmania load in human lesions, particularly in chronic lesions. This highly sensitive quantitative technique [10, 20, 21] can be employed also for monitoring the parasite load during treatment and follow-up as a way to assess the outcome of treatment.

Limitations

Accurately and confidently quantify parasites in biopsy samples help to evaluated the parasitic load in acute and chronic forms using real-time PCR in scoring CL. Using the paraffin-embedded biopsy samples make our samples collection time short. But it’s make the DNA extraction laboratories and effect on the quality of extracted DNA.

Availability of data and materials

Please contact corresponding author (S.D) for data requests.

Abbreviations

CL:

cutaneous leishmaniasis

PCR:

polymerase chain reaction

Ct:

cycle threshold

CNL:

chronic non-lupoid form

qPCR:

quantitative real-time PCR

References

  1. Murray HW, Berman JD, Davies CR, Saravia NG. Advances in leishmaniasis. Lancet. 2005;366(9496):1561–77.

    Article  CAS  Google Scholar 

  2. Meymandi S, Dabiri S, Dabiri D, Crawford RI, Kharazmi A. A quantitative study of epidermal Langerhans cells in cutaneous leishmaniasis caused by Leishmania tropica. Int J Dermatol. 2004;43(11):819–23.

    Article  Google Scholar 

  3. Choi CM, Lerner EA. Leishmaniasis as an emerging infection. Journal of investigative dermatology symposium proceedings. New York: Elsevier; 2001.

    Google Scholar 

  4. Lallas A, Apalla Z, Argenziano G, Longo C, Moscarella E, Specchio F, et al. The dermatoscopic universe of basal cell carcinoma. Dermatol Pract Conceptual. 2014;4(3):11.

    Google Scholar 

  5. Salman SM, Rubeiz NG, Kibbi A-G. Cutaneous leishmaniasis: clinical features and diagnosis. Clin Dermatol. 1999;17(3):291–6.

    Article  CAS  Google Scholar 

  6. Zvulunov A, Cagnano E, Frankenburg S, Barenholz Y, Vardy D. Topical treatment of persistent cutaneous leishmaniasis with ethanolic lipid amphotericin B. Pediatr Infect Dis J. 2003;22(6):567–9.

    PubMed  Google Scholar 

  7. Oliveira-Neto MP, Mattos M, da Silva C, de Souza F, Fernandes O, Pirmez C. Leishmaniasis recidiva cutis in New World cutaneous leishmaniasis. Int J Dermatol. 1998;37(11):846–9.

    Article  CAS  Google Scholar 

  8. Gurel MS, Ulukanligil M, Ozbilge H. Cutaneous leishmaniasis in Sanliurfa: epidemiologic and clinical features of the last four years (1997–2000). Int J Dermatol. 2002;41(1):32–7.

    Article  Google Scholar 

  9. Ardehali S, Sodeiphy M, Haghighi P, Rezai H, Vollum D. Studies on chronic (lupoid) leishmaniasis. Ann Trop Med Parasitol. 1980;74(4):439–45.

    Article  CAS  Google Scholar 

  10. Sundar S, Rai M. Laboratory diagnosis of visceral leishmaniasis. Clin Diagn Lab Immunol. 2002;9(5):951–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Ramírez JR, Agudelo S, Muskus C, Alzate JF, Berberich C, Barker D, et al. Diagnosis of cutaneous leishmaniasis in Colombia: the sampling site within lesions influences the sensitivity of parasitologic diagnosis. J Clin Microbiol. 2000;38(10):3768–73.

    PubMed  PubMed Central  Google Scholar 

  12. Beheshti N, Ghafarifar F, Dalimiasl A, Eslamirad Z, Sharifi Z, Farivar SM. Detection of cutaneous leishmanioasis isolated from Iranian patients by using ITS1 gene and apol enzyme via PCR-RFLP molecular method. Sci J Ilam Univ Med Sci. 2013;20(4):71–8.

    Google Scholar 

  13. Moradabadi AL, Farsinejad A, Fekri SM. Fast method for diagnosis of leishmania by PCR and FLASH PCR. J Arak Univ Med Sci. 2017;19(11):79–86.

    Google Scholar 

  14. Al-Jawabreh A, Schnur L, Nasereddin A, Schwenkenbecher J, Abdeen Z, Barghuthy F, et al. The recent emergence of Leishmania tropica in Jericho (A’riha) and its environs, a classical focus of L. major. Trop Med Int Health. 2004;9(7):812–6.

    Article  CAS  Google Scholar 

  15. Khosravi S, Hejazi H, Hashemzadeh-Chaleshtori M, Eslami G, Yousofi Darani H. Molecular diagnosis of Old World leishmaniasis: real-time PCR based on tryparedoxin peroxidase gene for the detection and identification of Leishmania spp. J Vector Borne Dis. 2012;49(1):15–8.

    CAS  PubMed  Google Scholar 

  16. Nasreen SA, Hossain MA, Paul SK, Mahmud MC, Ahmed S, Ghosh S, et al. PCR-based detection of Leishmania DNA in skin samples of post kala-azar dermal leishmaniasis patients from an endemic area of Bangladesh. Jap J Infect Dis. 2012;65(4):315–7.

    Article  Google Scholar 

  17. Suárez M, Valencia BM, Jara M, Alba M, Boggild AK, Dujardin J-C, et al. Quantification of Leishmania (Viannia) kinetoplast DNA in ulcers of cutaneous leishmaniasis reveals inter-site and inter-sampling variability in parasite load. PLoS Negl Trop Dis. 2015;9(7):e0003936.

    Article  Google Scholar 

  18. Azadeh B, Samad A, Ardehali S. Histological spectrum of cutaneous leishmaniasis due to Leishmania tropica. Trans R Soc Trop Med Hyg. 1985;79(5):631–6.

    Article  CAS  Google Scholar 

  19. Hassan AE, Kadaru A, Khalil E, Fadl A, Hassan ME. The pathology of cutaneous leishmaniasis in the Sudan: a comparison with that in other geographical areas. Ann Trop Med Parasitol. 1996;90(5):485–90.

    Article  Google Scholar 

  20. Fekri SM, Dabiri S, Fotouhi AR, Fani ML, Amirpoor RS, Ziasistani M, et al. Design and validation of real-time PCR: quantitative diagnosis of common leishmania species in Iran. J Clin Microbiol. 2016;19:496–501.

    Google Scholar 

  21. Jara M, Adaui V, Valencia BM, Martinez D, Alba M, Castrillon C, et al. A real-time PCR assay for detection and quantification of Leishmania (Viannia) in skin and mucosal lesions: an exploratory study of parasite load and clinical parameters. J Clin Microbiol. 2013;51:1826–33.

    Article  Google Scholar 

  22. Noazin S, Khamesipour A, Moulton LH, Tanner M, Nasseri K, Modabber F, et al. Efficacy of killed whole-parasite vaccines in the prevention of leishmaniasis—a meta-analysis. Vaccine. 2009;27(35):4747–53.

    Article  CAS  Google Scholar 

  23. Momeni AZ, Yotsumoto S, Mehregan DR, Mehregan AH, Mehregan DA, Aminjavaheri M, et al. Chronic lupoid leishmaniasis: evaluation by polymerase chain reaction. Arch Dermatol. 1996;132(2):198–202.

    Article  CAS  Google Scholar 

  24. El-On J, Weinrauch L, Livshin R, Even-Paz Z, Jacobs G. Topical treatment of recurrent cutaneous leishmaniasis with ointment containing paromomycin and methylbenzethonium chloride. Br Med J (Clinical Research ed). 1985;291(6497):704.

    Article  CAS  Google Scholar 

  25. Venkataram M, Moosa M, Devi L. Histopathological spectrum in cutaneous leishmaniasis: a study in Oman. Indian J Dermatol Venereol Leprol. 2001;67(6):294.

    CAS  PubMed  Google Scholar 

  26. Mashayekhi V, Mahmoudi M, Rastin M, Tayebi N, Taheri AR, Tavakoli M. Detection of Leishmania DNA in paraffin embedded specimens of chronic lupoid leishmaniasis using polymerase chain reaction. J Infect Public Health. 2012;9:557–63.

    Google Scholar 

  27. Weigle KA, Labrada LA, Lozano C, Santrich C, Barker DC. PCR-based diagnosis of acute and chronic cutaneous leishmaniasis caused by Leishmania (Viannia). J Clin Microbiol. 2002;40(2):601–6.

    Article  CAS  Google Scholar 

  28. Verma S, Bhandari V, Avishek K, Ramesh V, Salotra P. Reliable diagnosis of post-kala-azar dermal leishmaniasis (PKDL) using slit aspirate specimen to avoid invasive sampling procedures. Trop Med Int Health. 2013;18(3):268–75.

    PubMed  Google Scholar 

  29. Dabiri S, Manafi Anari H, Shamsi Meymandi S, Fotouhi Ardakani R, Amirpour Rostami S, Meymandi MS, et al. DNA load analysis using real time PCR in comparison with immunohistochemical findings of dry type cutaneous leishmaniasis; before and after treatment by imiquimode, glucantime and combination of both drugs. Iran J Pathol. 2013;8(4):247–54.

    Google Scholar 

  30. Shirian S, Oryan A, Hatam G-R, Panahi S, Daneshbod Y. Comparison of conventional, molecular, and immunohistochemical methods in diagnosis of typical and atypical cutaneous leishmaniasis. Arch Pathol Lab Med. 2014;138(2):235–40.

    Article  CAS  Google Scholar 

  31. Shirian S, Oryan A, Hatam GR, Daneshbod Y. Three Leishmania/L. species–L. infantum, L. major, L. tropica—as causative agents of mucosal leishmaniasis in Iran. Pathog Glob Health. 2013;107(5):267–72.

    Article  Google Scholar 

  32. Daneshbod Y, Oryan A, Davarmanesh M, Shirian S, Negahban S, Aledavood A, et al. Clinical, histopathologic, and cytologic diagnosis of mucosal leishmaniasis and literature review. Arch Pathol Lab Med. 2011;135(4):478–82.

    PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank all friends and colleagues of Pathology and Stem Cell Research Center of Kerman University of Medical Sciences for their relentless efforts.

Funding

No funding sources used in this study.

Author information

Authors and Affiliations

  1. Pathology and Stem Cell Research Center, Department of Pathology, Afzalipour Medical School, Kerman University of Medical Sciences, 22 Bahman Blvd, 7616913555, Kerman, Iran

    Maryam Fekri-SoofiAbadi, Alireza moradabadi, Reza Vahidi, Donya Dabiri & Shahriar Dabiri

  2. Dermatology Department, Afzalipour Hospital, Kerman University of Medical Sciences, Kerman, Iran

    Simin Shamsi-Meymandi

  3. Department of Medicine, Montefiore New Rochelle Hospital, Albert Einstein College of Medicine, New York, USA

    Meisam Fekri

Authors
  1. Maryam Fekri-SoofiAbadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Meisam Fekri
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alireza moradabadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Reza Vahidi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Simin Shamsi-Meymandi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Donya Dabiri
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shahriar Dabiri
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SD and SM proposed the original concept and designed the experiment and supervised all aspects of the work. MAF, MEF, AM, RV, and DD equally participated in the data acquisition and analysis. All authors contributed to writing the manuscript. SD and SM provided critical reviews in order to promote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Shahriar Dabiri.

Ethics declarations

Ethics approval and consent to participate

The study approved in Ethical Committee of Kerman University of Medical Sciences and the ethic approval code is IR.KMU.REC.1397.813. Informed consent was obtained from all the participants prior to enrolment.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fekri-SoofiAbadi, M., Fekri, M., moradabadi, A. et al. Ability of real-time PCR for differential diagnosis of various forms of cutaneous leishmaniasis: a comparative study with histopathology. BMC Res Notes 12, 615 (2019). https://doi.org/10.1186/s13104-019-4666-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13104-019-4666-5

Keywords

BMC Research Notes

ISSN: 1756-0500

Contact us