key: cord-296197-ohfhnpma
authors: Deborggraeve, Stijn; Laurent, Thierry; Espinosa, Diego; Van der Auwera, Gert; Mbuchi, Margaret; Wasunna, Monique; El-Safi, Sayda; Al-Basheer, Ahmed Almustafa; Arévalo, Jorge; Miranda-Verástegui, César; Leclipteux, Thierry; Mertens, Pascal; Dujardin, Jean-Claude; Herdewijn, Piet; Büscher, Philippe
title: A Simplified and Standardized Polymerase Chain Reaction Format for the Diagnosis of Leishmaniasis
date: 2008-11-15
journal: J Infect Dis
DOI: 10.1086/592509
sha: 
doc_id: 296197
cord_uid: ohfhnpma

Background. Definite diagnosis of Leishmania infections is based on demonstration of the parasite by microscopic analysis of tissue biopsy specimens or aspirate samples. However, microscopy generally shows low sensitivity and requires invasive sampling. Methods. We describe here the development of a simple and rapid test for the detection of polymerase chain reaction-amplified Leishmania DNA. A phase 1 evaluation of the text was conducted in clinical samples from 60 nonendemic and 45 endemic control subjects and from 44 patients with confirmed cutaneous leishmaniasis (CL), 12 with mucocutaneous leishmaniasis (MCL), and 43 with visceral leishmaniasis (VL) from Peru, Kenya, and Sudan. Results. The lower detection limits of the assay are 10 fg of Leishmania DNA and 1 parasite in 180 µL of blood. The specificity was 98.3% (95% confidence interval [CI], 91.1%–99.7%) and 95.6% (95% CI, 85.2%–98.8%) for nonendemic and endemic control samples, respectively, and the sensitivity was 93.2% (95% CI, 81.8%–97.7%), 91.7% (95% CI, 64.6%–98.5%), and 86% (95% CI, 72.7%–93.4%) for lesions from patients with CL or MCL and blood from patients with VL, respectively. Conclusions. The Leishmania OligoC-TesT showed high specificity and sensitivity in clinical samples and was able to detect the parasite in samples obtained by less invasive means, such as blood, lymph, and lesion scrapings. The assay is a promising new tool for simplified and standardized molecular detection of Leishmania parasites.

Leishmaniasis is a vectorborne disease caused by protozoa of the genus Leishmania and is endemic in many areas of the tropics, subtropics, and Mediterranean basin [1] . Clinical presentations, depending on the infecting species and the immune status of the patient, range from self-healing skin sores to devastating cutaneous and mucocutaneous ulcers and the lethal visceral form [2] . The disease is re-sponsible for important public health and economic problems in affected regions [3] .

Definite diagnosis of Leishmania infections is currently based on microscopic demonstration of the parasites in skin biopsy specimens or mucosal aspirate samples for cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) and in spleen or bone marrow aspirate samples for visceral leishmaniasis (VL) [1] . However, this conventional method is hampered by its low and variable sensitivity and the need for invasive sampling techniques. Sensitivity may be increased by prior in vitro cultivation of the parasite, but this technique is cumbersome and time consuming. Serologic tests, such as the direct agglutination test (DAT) [4] , support the clinical diagnosis of VL, but antibodies remain detectable for years after successful treatment [5, 6] . Furthermore, serologic tests are less useful in patients with VL coinfected with HIV [7] and in those with CL or MCL [8] .

Amplification of the parasite DNA by the polymerase chain reaction (PCR) has evolved into one of the most specific and sensitive methods for Leishmania detection [9, 10] . Despite its promising features, PCR is restricted to wellequipped laboratory settings, partly due to technical complexity, from DNA extraction to PCR and product detection. There is a need for simplification, and a first step concerns the visualization of amplified DNA, which is usually identified using electrophoresis followed by ultraviolet transillumination in the presence of the carcinogenic ethidium bromide. Safer nucleic acid stains based on fluorescent dyes are much more expensive and not routinely used in most laboratories. Alternative methods for PCR product detection, such as real-time PCR [11] , PCR-ELISA [12, 13] , and mass spectrometry [14] , have been developed but remain complex, expensive, and highly dependent on specific equipment. Along with the need for simplification of the PCR assay, there is a demand for standardization and optimization [10] . At present, standardization of PCR for Leishmania detection is largely neglected, and the abundance of in-house PCR assays may lead to diagnostic inconsistencies.

Oligochromatography provides a simple and rapid dipstick format for detection of PCR products (Coris BioConcept; patent WO 2004/099438A1) [15] . PCR products are visualized on the dipstick by hybridization with a gold-conjugated probe, allowing sequence-specific PCR product detection. This detection format takes only 5-10 min and requires no equipment other than a water bath and a pipette. Internal controls for amplification and chromatographic migration are incorporated in the assay. The technique has already been successfully applied for the diagnosis of human African trypanosomiasis [16] , toxoplasmosis [17] , and severe acute respiratory syndrome [18] , and high sensitivity and specificity were reported.

We present here the development and evaluation of a Leishmania-specific PCR-oligochromatographic test (Leishmania OligoC-TesT), targeting a short sequence within the Leishmania 18S rRNA gene. After providing proof of concept in experimental samples, we evaluated (phase 1) the Leishmania OligoC-TesT in a larger series of clinical samples from nonendemic and endemic control subjects and patients with CL, MCL, or VL.

DNA from Leishmania species (L. donovani, L. amazonensis, L. infantum, L. braziliensis, L. peruviana, L. guyanensis, L. panamensis, L. mexicana, L. major, L. lainsoni, L. tropica, and L. aethiopica) and from Trypanosoma brucei gambiense and Trypanosoma cruzi was obtained from the DNA reference bank at the Institute of Tropical Medicine Antwerp (ITMA). DNA from other pathogens (Sporothrix schenckii, Plasmodium falciparum, Mycobacterium tuberculosis, and Schistosoma mansoni) was obtained from other research groups.

Informed consent was obtained from patients or guardians and from persons without disease. The human and animal experimentation guidelines of the ITMA were followed. Ethical clearance for the study was obtained from the institutional review boards in Belgium, Kenya, Sudan, and Peru.

Spiked blood. L. donovani promastigotes were grown in glucose-lactalbumin-serum-hemoglobin medium [19] with 10% fetal calf serum at 26°C. A 10-fold dilution series of parasites, ranging from 10,000 parasites to 1 parasite per 180 L of blood, was made in fresh blood obtained on EDTA from a healthy volunteer. Nonspiked blood was used as a negative control.

Nonendemic controls. DNA extracts from the blood of 20 persons with confirmed T. brucei gambiense infection from the Democratic Republic of the Congo (DRC), 20 with confirmed P. falciparum infection from Zambia, and 20 with confirmed T. cruzi infection from Chile were obtained from other research groups. T. brucei gambiense and P. falciparum infections were confirmed by direct parasite detection with microscopy, and T. cruzi infections were confirmed by kinetoplast DNA PCR followed by Southern blot hybridization [20] .

Patients with CL or MCL and healthy endemic controls (Peru). Lesion biopsy specimens from 36 patients with CL, mucosal aspirate samples from 12 patients with MCL, and lesion scrapings from 8 patients with CL were collected in 2006 and 2007 at the Instituto de Medicina Tropical Alexander von Humboldt, Lima, Peru. CL and MCL were confirmed by means of microscopy and/or cultivation in 3 mL of Novy-MacNeal-Nicolle medium (Difco Laboratories) containing 15% defibrinated rabbit blood. Forty-one of the 56 CL and MCL lesion samples had a positive direct microscopy result, and the remaining 15 became positive after culture. All biopsy specimens were obtained from the active edge of the lesion with a sterile 4-mmdiameter biopsy puncher, and simple sterile lancets were used for the lesion scrapings. Dental biopsy specimens from 8 healthy endemic control subjects were collected in 2007 in a dental clinic in Lima, Peru. Biopsy specimens and lesion scrapings were stored in absolute ethanol at Ϫ20°C until DNA was extracted.

Patients with VL and healthy endemic controls (Kenya and Sudan). Sample collections in Kenya and Sudan were performed by the Kenya Medical Research Institute and Khartoum University, respectively. Blood samples from patients with confirmed VL and from healthy endemic control subjects were collected in 2007 in the Baringo district in Kenya (25 VL and 18 control samples) and Gedarif state in Sudan (18 VL and 19 control samples). A patient was classified as having confirmed VL if parasites were observed during microscopic analysis of lymph or bone marrow aspirate samples for Sudan and of spleen aspirate samples for Kenya. A person was classified as a healthy endemic control if there was no clinical suspicion for VL and if the DAT titer was below 1:3200 for Sudan and 1:12,000 for Kenya (threshold values used routinely at the 2 institutions). Along with the blood samples, bone marrow from 12 and lymph from the remaining 6 of the 18 Sudanese patients with confirmed VL were collected for subsequent analysis with the Leishmania OligoC-TesT; 200 L of blood, bone marrow, or lymph was used for DNA extraction.

DNA from spiked blood samples and from biopsy specimens and lesion scrapings was extracted using the QIAamp DNA Blood Mini Kit and the QIAamp DNA Mini Kit (Qiagen), respectively. DNA from blood, bone marrow, and lymph node samples from patients with VL and healthy endemic controls was extracted according to the method of Boom et al. [21] . DNA was eluted in 50 L of pure water or Tris-EDTA (TE) buffer and stored at Ϫ20°C, except for the Sudanese clinical samples, which were eluted in 100 L of TE buffer.

Primers, probes, and IC DNA were synthesized by Biomers.net (figure 1). Sequences of the 18S rRNA gene of the trypanosomatid parasites L. donovani (GenBank accession number X07773), T. brucei gambiense (GenBank accession number AJ009141), and T. cruzi (GenBank accession number AF303660) were aligned and Leishmania-specific primers were designed using DNAMAN software, version 5.0 (Lynnon). The sense primer 18S-L-F and the antisense primer 18S-L-R amplify a 115-bp sequence within the 18S rRNA gene of Leishmania.

The IC DNA has the same length as the Leishmania target sequence. Both sequences are identical except for a 17-bp central part.

The detection probe was designed, synthesized, and conjugated with gold particles using the procedure described in patent WO 2004/099438A1 [15] . The Leishmania capture probe and the IC capture probe, which hybridize to the central part of the Leishmania amplicon and the IC DNA amplicon, respectively, were synthesized and biotinylated at the 5' end. The 5'-acg and 5'-a linkers were added to avoid steric hindrance by the biotinavidin binding during hybridization. The migration control se-quence is the reverse complement of the detection probe sequence.

The 50-L PCR mixture was prepared by adding 5 L of sample DNA and 1 U of HotStar Taq polymerase (Qiagen) to 44.8 L of Leishmania Ampli-Mix (Coris BioConcept). This premade PCR mix contains all components to allow PCR amplification, the primers 18S-L-F and 18S-L-R and the IC DNA at a concentration of 3.2 ϫ 10 Ϫ18 mol/L. The commonly used dTTP was replaced by dUTP to allow elimination of carryover contamination with uracil-DNA N-glycosylase (UNG). An initial denaturation step at 94°C for 15 min to activate the HotStar Taq polymerase was followed by 40 cycles of 94°C for 30 s, 60°C for 1 min, 72°C for 1 min, and a single final extension at 72°C for 5 min. Amplification was done in 200-L thin-wall PCR tubes (Abgene) in a T3 thermocycler 48 (Biometra).

Preparation. The Leishmania Oligo-Strip was constructed as described by Deborggraeve et al. [16] but with the following modifications. The lower absorbent pad on the test side was impregnated with the detection probe and the Leishmania capture probe. The lower absorbent pad on the control side was impregnated with the detection probe and the IC capture probe. On both sides of the nitrocellulose membrane, 2 lines were coated, a line with Neutralite avidin (Belovo SA) and a line with the migration control probe.

Principle. A schematic overview of the Leishmania OligoC-TesT principle is presented in figure 2 . The assay was repeated when the control lines indicated migration or PCR failure.

Assay procedure. After PCR amplification, the PCR product was denaturated at 94°C for 30 s and transferred immediately to ice. Then, 40 L was mixed with an equal volume of migration buffer preheated at 55°C, and the Leishmania Oligo-Strip was dipped into the solution. Test results were read after 10 min. (1) is performed using a PCR mix containing single-stranded internal control (IC) template (2) . This IC template is amplified with the same primers as the Leishmania DNA target but contains a specific internal sequence (dotted line). When the PCR and subsequent denaturation is completed, the PCR product solution contains single-stranded Leishmania and IC DNA (3) and is mixed with an equal volume of migration buffer preheated at 55°C. The Leishmania Oligo-Strip is dipped into the solution, and test results are read after a 10-min migration at 55°C (4). During migration, the solution takes up the impregnated probes: the gold-labeled detection probe (5) hybridizes with both types of amplicons, while the biotinylated Leishmania capture probe on the test side (6) and the biotinylated IC capture probe on the control side (7) hybridize with their respective amplicons. Gold and biotin labeling is indicated by white circles and black circles, respectively. The biotinylated capture probes accumulate the hybridized complex on the Neutralite avidin lines on both sides of the strip, resulting in visible red lines (8) . For a negative sample, only the IC amplicon is present and is captured on the control side (9) . The excess detection probes migrate further and hybridize on the complementary probes coated on both sides of the dipstick as a control for migration (10) . The IC line and the migration control lines determine whether the test is valid or invalid. An invisible migration control line indicates an invalid detection step, while an invalid PCR is indicated by a negative IC line in combination with a negative test line. The latter is possibly due to inhibitory factors in the extracted DNA. When a sample contains high concentrations of Leishmania DNA, competition between the Leishmania DNA and the IC template DNA during PCR can result in an invisible IC control line combined with a strongly visible Leishmania test line. In this case, the test is considered valid. 

Accurate detection of the parasite is of utmost importance in the diagnosis of Leishmania infections and in disease control. We here report on the development, proof-of-concept analysis, and phase 1 evaluation of a simplified molecular test for detecting Leishmania organisms in clinical samples. The Leishmania OligoC-TesT is based on PCR amplification of Leishmania DNA followed by simple and rapid detection of the PCR product in dipstick format. The lower detection limits of the assay were evaluated in L. donovani promastigotes, and 10 fg of purified DNA and 1 parasite in 180 L of human blood could be detected.

Fluctuations in the analytical sensitivity between different isolates might be expected. In 1998, Inga et al. [23] reported a lower copy number of the rRNA genes in L. (Viannia) peruviana from southern Peru compared with L. (V.) peruviana from northern Peru. In our study, however, we did not observe a significantly lower sensitivity of the Leishmania OligoC-TesT in 13 patients originating from L. (V.) peruviana-endemic regions in southern Peru, because only one of these patients had a negative test result. No cross-reaction with nontarget human pathogens was observed, and all Leishmania species tested had a positive test result. Considering the high similarity of the 18S rRNA gene sequence in Leishmania and Endotrypanum, Crithidia, Wallaceina, and Leptomonas organisms [24] , our assay might have positive results with these nonpathogenic lower trypanosomatids. Although the chances of finding such protozoa in immunocompetent patients are negligible, they may show up as opportunistic infections in immunocompromised patients [25] . If needed, confirmation could be provided by direct sequencing of the PCR product, because Leishmania-specific point mutations are present (authors' unpublished data).

The Leishmania OligoC-TesT showed a high diagnostic specificity when tested in 60 nonendemic control blood samples from persons infected with T. brucei gambiense, P. falciparum, or T. cruzi. One T. brucei gambiense-infected person from the DRC had a positive Leishmania OligoC-TesT result. We cannot exclude the possibility that this could be due to a Leishmania infection, because VL has been occasionally reported in the DRC [26, 27] .

When evaluated in 56 clinical samples from patients with confirmed CL or MCL (definition based on microscopy and/or culture) collected in Peru, the Leishmania OligoC-TesT showed an overall diagnostic sensitivity of 92.9%, whereas all 8 dental biopsy specimens from healthy control subjects were negative. This finding is similar to the observed sensitivities of other PCR assays reviewed by Vega-López [8] . Direct microscopy of the lesion showed a lower sensitivity (78.8%). The lack of a positive PCR result in some of the confirmed CL and MCL lesion samples might be explained by (1) heterogeneity in parasite distribution in the lesion, (2) delay between sample collection and testing, or (3) false-positive microscopic results caused by staining artifacts. This is supported by the fact that 3 of the 4 Leishmania OligoC-TesT negative samples were also negative in a second PCR analysis performed in Peru targeting the Leishmania kinetoplast DNA [28] (authors' unpublished data). Leishmania DNA could be detected in all lesion scrapings, which is encouraging, because this offers a less invasive sampling procedure compared with biopsy punctures. The higher sensitivity for lesion scrapings can be explained by the lower amount of PCR-inhibiting factors. This is confirmed by the observation of Garcia et al. [29] , who reported a higher sensitivity of PCR for lesion scrapings than for biopsy specimens in 44 Bolivian patients with CL or MCL. The similar sensitivities of our method for CL and MCL samples is encouraging, considering the generally low parasite load in the latter. The sensitivity of 86% in blood from patients with confirmed VL is promising and suggests that the OligoC-TesT can contribute significantly to less invasive VL diagnosis. Patients with clinically suspected infection who have positive serologic results and positive Leishmania OligoC-TesT results in blood might not need to undergo the invasive bone marrow or spleen sampling. Previous PCR studies found the same range of sensitivity in blood [9, 30] . The lower sensitivity of the test in blood samples from Sudan (77.8%) versus those from Kenya (92%) could be due to bias in parasite load or experimental differences during DNA extraction. Further evaluations with different types of extraction methods might be recommended. Despite the low number of available lymph node samples, the observation that all of them showed positive Leishmania OligoC-TesT results is encouraging. Indeed, if molecular diagnosis in lymph nodes proves to have high sensitivity, this would be a major step forward in the search for a noninvasive method for diagnosing VL. Previous studies showed similar findings in a larger number of lymph node samples [31, 32] . Further studies of molecular diagnosis in the lymph would be interesting, especially for East African VL, for which the majority of patients have enlarged lymph nodes [33] .

Two of 37 DAT-negative blood samples from Kenyan and Sudanese endemic control subjects were positive with the Leishmania OligoC-TesT. Although PCR contamination can never be 100% excluded and low sensitivity of the DAT in Sudan has been reported [34] , these positive results in the healthy control group are probably due to asymptomatic infections. In VL-endemic regions, there is a high prevalence of asymptomatic infections, which are generally not treated because the available drugs are very toxic. Therefore, the interpretation of PCR results should always take into account possible asymptomatic infections [35] .

Multiple attempts to simplify and speed up the detection of PCR products have been reported, such as PCR-ELISA [12, 13] and fluorescent self-probing amplicons [36] , but none of them merges simplicity and speed with "low-tech" approaches. The Leishmania OligoC-TesT combines the sensitivity and specificity of PCR with the simplicity and speed of membrane oligochromatography. Detection of PCR products is performed in 10 min and does not require any equipment except for a water bath and a pipette. The cost of the Leishmania Ampli-Mix components is similar to that of conventional PCR mix, and the cost of the Oligo-Strip is much lower than that of commercial agarose for the same number of samples. The global cost of diagnosis is reduced with the OligoC-TesT, because the internal amplification control eliminates the need for additional PCRs to check inhibition.

A common drawback of PCR in the diagnostic process is carryover contamination leading to false-positive results. The implementation of the dUTP/UNG decontamination system and the single-step detection format of the Leishmania OligoC-TesT facilitate contamination control. Presentation of the Leishmania OligoC-TesT as a self-containing kit with a ready-to-use PCR mix and dipsticks will allow quality control and standardization. However, sampling and DNA extraction are also important steps for accurate molecular diagnosis and need to be standardized. The described tool may help in the rapid and simple molecular diagnosis of leishmaniasis and in epidemiological studies for which a sensitive marker of infection is required, although the current format is not directly designed for high-throughput applications.

There is a need for further phase 2 and 3 studies to estimate the diagnostic accuracy of the Leishmania OligoC-TesT in patients with suspected infection and with positive serologic test results, healthy endemic controls, and patients with other pathologic conditions. Furthermore, a multicentric study in which different PCR methods are evaluated on the same samples would be highly valuable. In addition, a standardized oligochromatographic format for identification of Leishmania species would be welcome, given the high importance of accurate species identification in patient management, disease control, and epidemiological studies. Recently, a first prototype to discriminate between L. infantum and L. donovani by means of the oligochromatographic approach has been developed (T. Laurent, G.V.d.A., M. Hide, et al., unpublished data). The combination of the dipstick with novel isothermal nucleic acid amplification techniques, such as nucleic acid sequence-based amplification [37, 38] and loop-mediated isothermal amplification [39] , may circumvent the need for thermocycling reactions and therefore further simplify molecular diagnosis.

Clinical spectrum of leishmaniasis

Leishmaniasis and poverty

Operational validation of the direct agglutination test for diagnosis of visceral leishmaniasis

Immunologic tests in patients after clinical cure of visceral leishmaniasis

Pre-and post-treatment antibody levels in visceral leishmaniasis

Contribution of serological tests to the diagnosis of visceral leishmaniasis in patients infected with the human immunodeficiency virus

Diagnosis of cutaneous leishmaniasis

Clinical use of polymerase chain reaction performed on peripheral blood and bone marrow samples for the diagnosis and monitoring of visceral leishmaniasis in HIV-infected and HIV-uninfected patients: a single-center, 8-year experience in Italy and review of the literature

Molecular diagnosis of leishmaniasis: current status and future applications

Real-time PCR in clinical microbiology: applications for routine laboratory testing

The high sensitivity of a PCR-ELISA in the diagnosis of cutaneous and visceral leishmaniasis caused by Leishmania infantum

A new PCR-ELISA for diagnosis of visceral leishmaniasis in blood of HIV-negative subjects

Rapid identification of emerging pathogens: coronavirus

One step oligochromatographic device and method of use

Molecular dipstick test for diagnosis of sleeping sickness

DNA extraction and PCR assays for detection of Toxoplasma gondii

Detection of SARS-CoV by oligochromatography of RT-PCR amplicons

Structures antigéniques de Trypanosoma brucei (Protozoa, Kinetoplastida)

Use of polymerase chain reaction (PCR) and hybridization assays to detect Trypanosoma cruzi in chronic chagasic patients treated with itraconazole or allopurinol

Rapid and simple method for purification of nucleic acids

Probable inference, the law of succession, and statistical inference

Relation between variation in copy number of ribosomal RNA encoding genes and size of harbouring chromosomes in Leishmania of subgenus Viannia

Phylogeny of Trypanosomatidae and Bodonidae (Kinetoplastida) based on 18S rRNA: evidence for paraphyly of Trypanosoma and six other genera

Lower trypanosomatids in HIV/AIDS patients

Case of leishmaniasis in the Congo

Autochtonous visceral leishmaniasis in Zaire

Diagnosis of Leishmania via the polymerase chain reaction a simplified procedure for field work

American tegumentary leishmaniasis: direct species identification of Leishmania in non-invasive clinical samples

Leishmaniasis in Sudan. Visceral leishmaniasis

Evaluation of PCR for diagnosis of visceral leishmaniasis

Molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification

Kala-azar in displaced people from southern Sudan: epidemiological, clinical and therapeutic findings

Diagnostic tests for kala-azar: a multi-centre study of the freeze-dried DAT, rK39 strip test and KAtex in East Africa and the Indian subcontinent

Diagnostic accuracy of a new Leishmania PCR for clinical leishmaniasis in Nepal and its role in diagnosis of disease

Detection of PCR products using self probing amplicons and fluorescence

NASBA and other transcription based amplification methods for research and diagnostic microbiology

Quantitative nucleic acid sequence-based assay as a new molecular tool for detection and quantification of Leishmania parasites in skin biopsy samples

Loop-mediated isothermal amplification of DNA

We kindly acknowledge Drs. C. Grevelding, F. Portaels, D. Mumba, U. D'Alessandro, and A. Solari for providing DNA from Schistosoma mansoni, Mycobacterium tuberculosis, Trypanosoma brucei gambiense, Plasmodium falciparum, and Trypanosoma cruzi, respectively. We thank Dr. H. Schallig and G. Schoone for providing the material for the extraction method of Boom et al. and Dr. Alejandro Llanos-Cuentas and Hamad El-Neel El-Taib for assistance with the projects in Peru and Sudan, respectively.