**2. Molecular diagnosis**

PCR is being used for the diagnosis of parasitic diseases, including leishmaniasis. PCR is considered to be the most sensitive and specific technique among the methods applied so far for the direct detection and identification of the causative agent. The procedure is rapid and can be applied to a variety of clinical samples. Regarding the efficacy of the assay, it depends on the target selected for amplification (conserved or variable target region), the number of the target copies, the extraction technique used, the biological sample tested and the PCR protocol adapted or developed [1,2].

The PCR-based assays are advantageous over immunological techniques such as enzyme linked immunosorbent assay (ELISA) and immunofluorescence antibody test (IFAT) as host species specific reagents are not required. The increased PCR sensitivity over serology for the detection of infection is of great interest in certain cases such as in patients with cutaneous, muco-cutaneous leishmaniasis (CL or MCL) and the immunocompromised ones (e.g. coin‐ fected with HIV, under chemotherapy etc). The former have low or no concentrations of antibodies against *Leishmania* due to the localized character of the disease while the latter present limited antibody production both resulting in negative serological tests [3]. In particular, in chronic CL patients, who constitute the greater diagnostic challenge due to their low parasite density, PCR assays for the detection of *Leishmania* DNA presented 100% sensitivity. Moreover, the fact that antibodies remain detectable for years after successful treatment makes the application of PCR a necessity[4].

PCR has been also proved to be valuable in the diagnosis of post-kala-azar dermal leishma‐ niasis (PKDL) [5]. Additionally, the detection of parasite DNA has been shown to be a useful prognostic marker for the disease relapse or the development of PKDL even after successful treatment outcome. [6]. Furthermore, persistent infection has been found in apparently healed scars from MCL patients [7], the presence of *Leishmania braziliensis* was reported in patients previously treated by immunotherapy or patients being at different stages of treatment and in subjects who had never presented clinical manifestations but they had lived in endemic areas and migrated to nonendemic regions [8].

Moreover, several studies reported that PCR detects parasitaemia a few weeks before the appearance of clinical manifestations. The detection of asymptomatic infected humans contributes to the prevention of the sand fly infection and the transfusion-transmitted kalaazar especially forthe patients thatrequire multiple transfusions, atleastin endemic areas [3,9].

Regarding canine leishmaniasis, PCR assays constitute useful tools in cases of clinically healthy dogs which harbour infection but may never develop clinical disease. As the PCR positive results indicate infection, these assays could contribute to the prevention of the importation of infected clinically healthy dogs to nonendemic areas where infection may spread via local sand fly vectors and the transmission via blood transfusion [10]. Finally, the parasite detection is crucial in case of negative results obtained by serology. This discrepancy may be attributed to the gap between infection and seroconversion, the transient presence of specific antibodies and the possibility for some infected dogs never to be seroconverted. In contrast, false positive results may be obtained due to the existence of anti-*Leishmania* antibodies for a considerable time after convalescence [11]. On the other hand, a positive PCR result in asymptomatic dogs cannot support decision-making regarding treatment as the parasite DNA may be present for a long time after the parasite has been cleared while also a single negative PCR result in a clinically suspected dog cannot rule out infection. Along with the need for PCR assays simplification, there is also a demand for standardization and optimization due to the lack of a universal PCR assay for the diagnosis of leishmaniasis [12]. Most laboratories perform "inhouse" PCR assays using different primer pairs, DNA targets and PCR protocols [13].

This review aims to critically present current molecular approaches for leishmaniasis diagno‐

PCR is being used for the diagnosis of parasitic diseases, including leishmaniasis. PCR is considered to be the most sensitive and specific technique among the methods applied so far for the direct detection and identification of the causative agent. The procedure is rapid and can be applied to a variety of clinical samples. Regarding the efficacy of the assay, it depends on the target selected for amplification (conserved or variable target region), the number of the target copies, the extraction technique used, the biological sample tested and the PCR

The PCR-based assays are advantageous over immunological techniques such as enzyme linked immunosorbent assay (ELISA) and immunofluorescence antibody test (IFAT) as host species specific reagents are not required. The increased PCR sensitivity over serology for the detection of infection is of great interest in certain cases such as in patients with cutaneous, muco-cutaneous leishmaniasis (CL or MCL) and the immunocompromised ones (e.g. coin‐ fected with HIV, under chemotherapy etc). The former have low or no concentrations of antibodies against *Leishmania* due to the localized character of the disease while the latter present limited antibody production both resulting in negative serological tests [3]. In particular, in chronic CL patients, who constitute the greater diagnostic challenge due to their low parasite density, PCR assays for the detection of *Leishmania* DNA presented 100% sensitivity. Moreover, the fact that antibodies remain detectable for years after successful

PCR has been also proved to be valuable in the diagnosis of post-kala-azar dermal leishma‐ niasis (PKDL) [5]. Additionally, the detection of parasite DNA has been shown to be a useful prognostic marker for the disease relapse or the development of PKDL even after successful treatment outcome. [6]. Furthermore, persistent infection has been found in apparently healed scars from MCL patients [7], the presence of *Leishmania braziliensis* was reported in patients previously treated by immunotherapy or patients being at different stages of treatment and in subjects who had never presented clinical manifestations but they had lived in endemic areas

Moreover, several studies reported that PCR detects parasitaemia a few weeks before the appearance of clinical manifestations. The detection of asymptomatic infected humans contributes to the prevention of the sand fly infection and the transfusion-transmitted kalaazar especially forthe patients thatrequire multiple transfusions, atleastin endemic areas [3,9]. Regarding canine leishmaniasis, PCR assays constitute useful tools in cases of clinically healthy dogs which harbour infection but may never develop clinical disease. As the PCR positive results indicate infection, these assays could contribute to the prevention of the importation of infected clinically healthy dogs to nonendemic areas where infection may spread via local sand fly vectors and the transmission via blood transfusion [10]. Finally, the parasite detection is crucial in case of negative results obtained by serology. This discrepancy may be attributed

sis, species identification and phylogenetic analysis.

162 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

treatment makes the application of PCR a necessity[4].

and migrated to nonendemic regions [8].

**2. Molecular diagnosis**

protocol adapted or developed [1,2].

A variety of clinical samples have been used for the detection of *Leishmania* DNA such as whole blood, buffy coat, bone marrow, lymph node, spleen, conjunctival swabs [14,15] and other biological samples such liver, lung, heart, penis, vagina, testis, semen, uterus, placenta, kidney, intestine, milk and urine [16] and more recently nasal, ear and oral swabs [17,18]. Bone marrow, lymph node, spleen and skin are the tissues presenting the highest sensitivity for the diagnosis of canine leishmaniasis [11,19]. The same holds true for the non invasive sampling techniques using conjunctival swabs [15,17]. Whole blood, buffy coat, urine and the other biological samples mentioned above have been shown to be less sensitive.

Several target sequences and different PCR protocols have been described for the detection of *Leishmania* DNA. The most frequently used amplification targets are the Kinetoplast DNA minicircle (kDNA) [20–25] and the small subunit ribosomal RNA (SSU rRNA) [26–29]. There are various gene targets which are also commonly used such as the ribosomal internal transcriped spacer (ITS) [15,30–34], the mini-exon gene (spliced leader) [32,35–40] and a repetitive genomic sequence [41,42].

It is worth mentioning that variable and sometimes conflicting results have been reported by several studies evaluating PCR using different target sequences in different host tissues. These results have been mostly obtained from asymptomatic infected hosts and they may vary depending on the sampling technique, storage method and the PCR protocol employed [1]. Some indicative studies evaluating the most frequently used PCR targets in different tissues are summarized in Table 1.


BM: Bone marrow, WB: whole blood, SB: Skin biopsy, SS: Skin scrapings, DS: Dermal smear, SA: Skin aspirates LA: Lesion aspirates CS: Conjunctival swab, CB: Cultured biopsies, DB: Duodenal biopsy, GB: Gastric biopsy

**Table 1.** Evaluation of the most frequently used PCR targets in different tissues

Real time PCR (or quantitative PCR-qPCR), a molecular technique which has revolutionized the pathogen diagnosis, is considered to be the future reference method for molecular diagnosis. In recent years, qPCR assays based either on SYBR Green or TaqMan chemistries have been developed and evaluated for the detection, quantification and even species differ‐ entiation of *Leishmania spp* in a variety of clinical samples showing high sensitivity and reproducibility [45,46]. qPCR is considered to be a helpful tool for *Leishmania* diagnosis, monitoring during therapy, development of new drugs and diagnostic tools, comparison of drug efficacy or prophylactic schemes, and for epidemiological studies. Regarding diagnosis of leishmaniasis, the kinetic study of parasitemia in the immunocompromised hosts, the diagnosis of relapses and the quantification of the low parasitic load in asymptomatic patients are of great interest [47].

qPCR is highly sensitive especially at the lower parasite loads [48,49], specific and reproducible offering the ability to monitor therapy and to prevent relapses. The applications mentioned above make qPCR an attractive alternative to conventional PCR in routine diagnosis [47,49]. Some of the studies carried out so far and their findings regarding the detection threshold, sensitivity and specificity are summarized in Table 2.


BM: Bone marrow, WB: whole blood, SB: Skin biopsy, CS: Conjunctival swab, LN: Lymph node, S: Spleen, L: Liver, LU: Lung, K: Kidney, BC: Buffy coat, BS: Biopsy specimen, p/r:parasites/reaction, p/s: parasite/sample TRYP: tryparedoxin peroxidase gene

**Table 2.** Detection threshold, sensitivity and specificity of qPCR using various targets in different tissues

Given that PCR is restricted to well equiped laboratory settings, and that there is a need for simplification of the PCR assay and a demand for standardization and optimization [56], the described tools below may represent a good alternative for rapid and simple diagnosis of leishmaniasis in endemic areas and epidemiological studies [12,57].

Quantitative nucleic acid sequence-based amplification (QT-NASBA) has proven to be a very sensitive and specific assay in diagnostic microbiology which is based on the amplification of single-stranded RNA sequences. In fact, this technique detects RNA in a background of DNA [13]. Several QT-NASBA assays have been developed for the detection of *Leishmania* parasites including QT-NASBA combined with electro-chemiluminescence (ECL) [57,58] and QT-NASBA combined with oligochromatographic technology (OC) [12,59] for the detection of NASBA products. The QT-NASBA assays developed, are commonly based on amplification of single-stranded 18S ribosomal RNA sequences [12,57,58,60,61]. This target is considered to be highly efficient for the diagnosis of leishmaniasis as each parasite contains a large number of copies of the 18SrRNA gene [62] while also the cytoplasm is assumed to contain approxi‐ mately 104 rRNA copies [62]. Moreover the target is present in all *Leishmania* species and it does not vary between different species allowing high sensitivity and quantification of all species in a similar manner [12,57,58]. However, this target shows high similarity with the 18S rRNA gene sequence of *Endotrypanum*, *Crithidia, Wallacein*a, and *Leptomonas* organisms which may result in false positive results especially in the case of immunocompromised patients [12]. The fact that NASBA detects RNA, makes it a molecular tool of great importance for the measurement of viable parasites. As a consequence, its application makes possible the assessment of the efficacy of drug therapies, the prediction of treatment outcome and the monitoring of the emergence of drug resistance. As it is well known, the DNA is still detected for a long time after parasite death, thus making RNA a preferable amplification target for the demonstration of parasite viability [13,56,58]. Moreover, when targeting RNA, the starting number of the template molecules is much higher resulting in increased assay sensitivity and decreased sample volume required [56]. The latter, makes also QT-NASBA a highly sensitive assay as it is able to detect very low target levels on clinical samples.

Loop-mediated isothermal amplification (LAMP), a novel method of DNA amplification under isothermal condition [63], has been developed to detect *Trypanosoma spp, Plasmodium spp, Mycobacterium spp and Filaria spp* [64]. Recently a reverse transcriptase step has been developed to specifically amplify RNA so as to amplify RNA viruses such as HIV and avian influenza viruses and to increase the assay sensitivity [65]. The recently developed LAMP seems to be a promising diagnostic tool. The results obtained from several studies are encour‐ aging as this assay is much faster than conventional or nested PCR, it may be applied in field conditions, it shows high specificity and sensitivity [63,64,66–69].

In the context of a generalized effort for simplification of the parasite detection, assays including PCR-ELISA and PCR-OC have been developed and evaluated. Several studies reported that PCR-ELISA showed high sensitivity. In a study, PCR-ELISA in blood samples from HIV negative VL patients was evaluated and presented higher sensitivity (83.9% and 73.2%) and specificity (100% and 87.2%) than conventional PCR [70]. Other investigators have also evaluated the use of the assay in blood samples from HIV co-infected VL patients and PCR-ELISA found to be highly sensitive [23,71,72]. Basiye et al, reported that PCR-OC is highly sensitive for *Leishmania* diagnosis on blood samples from VL patients (sensitivity 96,4% and specificity 88.8%) compared to NASBA-OC which was shown to be more specific (specificity 100%) [60]. In another study the repeatability and reproducibility of the assay was studied and found to be 95.9% and 98.1% in purified nucleic acid specimens and 87.1% and 91.7% in blood specimens spiked with parasites respectively [73].
