Rapid diagnosis and genotyping of Leishmania isolates from cutaneous and visceral leishmaniasis by microcapillary cultivation and polymerase chain reaction-restriction fragment length polymorphism of miniexon region

We have performed a combination of microcapillary cultivation method and restriction fragment length polymorphism (RFLP) analysis of amplified products by 1 single PCR of miniexon region of Leishmania for molecular diagnosis and genotyping of different Leishmania species isolated from cutaneous leishmaniasis (CL) and visceral leishmaniasis. We have analyzed 10 microcapillary cultivated isolates from cutaneous cases and 5 microcapillary cultivated isolates from visceral cases (totally 15) by polymerase chain reaction-RFLP (PCR-RFLP). Of 10 isolates, 3 (30%) were genotyped as Leishmania infantum and 7 (70%) of 10 isolates were genotyped as Leishmania tropica from the microcapillary cultivated isolates of cutaneous cases. On the other hand, all 5 isolates (100%) were genotyped as L. infantum from microcapillary cultivated visceral cases. Our most interesting finding is the presence of 3 L. infantum isolates in CL cases without kala-azar history. Therefore, we suggest that further investigations must be done about this subject. On the other hand, we suggest the combination of microcapillary culture method and PCR-RFLP of miniexon region of leishmaniae can be used in routine laboratory experimentation because of their simple, cheap, and rapid benefits (within a week), whereas other different approaches offer a multitude of valid taxonomic characters for species identification.     

The use of direct agglutination test (DAT) in serological diagnosis of Ethiopian cutaneous leishmaniasis

Leishmania aethiopica (L.a.) is the main species of Leishmania that causes Ethiopian cutaneous leishmaniasis (ECL). The routine diagnosis of ECL depends on parasitological examination of smear, culture or biopsy. In this study, DAT was set-up and evaluated for its diagnostic performance using defined sera of 45 ECL patients, 18 visceral leishmaniasis (VL) patients, 12 patients with other diseases, and 37 normal controls. The test was also evaluated in 64 patients clinically diagnosed as ECL, leprosy, or other skin diseases. Using L.a. derived antigen, the sensitivity and specificity of the test was determined to be 90.5% and 91.8% respectively. However, using antigen derived from a non-homologous strain, only 4 sera of 21 active ECL patients were positive. Eighteen sera of VL patients were positive irrespective of the different antigen sources. The data show that DAT can be a useful addition to the diagnosis of ECL.     

A urine-based polymerase chain reaction method for the diagnosis of visceral leishmaniasis in immunocompetent patients

In the Mediterranean basin and Middle East, including Iran, visceral leishmaniasis (VL), also known as kala-azar, is caused by Leishmania donovani infantum. For the first time, the use of urine samples for the diagnosis of VL in immunocompetent patients has been used in this study. Based on its high sensitivity and specificity, as well as simplicity, this approach can serve as a valuable tool in the diagnosis of VL. We studied 60 urine samples from 60 individuals, 30 of which were patients with VL confirmed by parasitology, serology, or molecular methods, 5 were from healthy individuals, and 25 were from patients with cutaneous leishmaniasis, malaria, brucellosis, and hydatid cyst. Out of 30 samples from confirmed VL immunocompetent patients, 29 were positive (sensitivity, 96.8%) by polymerase chain reaction (RV1 and RV2 primers), and all the remaining 30 samples either from healthy individuals or patients with other diseases were negative (specificity, 100%). High sensitivity, specificity, and simplicity of the test can serve as a valuable tool in the diagnosis of VL.     

Comparison of Molecular,Microscopic, and Culture Methods for Diagnosis of Cutaneous Leishmaniasis

Cutaneous leishmaniasis (CL) is endemic in the northwest of Isfahan province, Iran. Increase in the incidence of the disease in Kashan has made it necessary to find out the best method for diagnosis and molecular characterization of Leishmania species. In the present study, 130 patients suspected to cutaneous leishmaniosis referred to health care centers of Kashan were examined. Serosity of lesion was collected for smear preparation and cultured in Novy‐Nicolle‐McNeal medium. DNA was extracted from serosity, and Leishmania species was determined by polymerase chain reaction (PCR) and nested PCR using kinetoplast DNA (kDNA) specific primers. The diagnostic criteria of CL were based on the observation of amastigotes in the smear, promastigotes in culture, presence of expected bands in PCR, or nested PCR. Of 130 specimens, 87 (66.9%), 72 (56.2%), 98 (75.4 %), 96 (73.8%), and 99 (76.2%) were positive for microscopic culture, PCR, nested PCR, and combined PCR and microscopy (proposed method), respectively. Sensitivity, specificity, positive and negative predictive values of PCR were 99%, 100%, 100%, 96.9%, respectively, for microscopy 87.9%, 100%, 100%, 72.1%, for culture 72.7%, 100%, 100%, 53.4 %, and for nested PCR 97%, 100%, 100%, 91.2%, respectively. Based on the results of the study, kDNA‐PCR was the most sensitive method for diagnosis of CL.     

Mucocutaneous leishmaniasis: accuracy and molecular validation of noninvasive procedures in a L. (V.) braziliensis–endemic area

The aim of this study was to evaluate the effectiveness of polymerase chain reaction (PCR) using Kinetoplastid DNA (kDNA) from nasal swabs (NSs), saliva, and oral filter paper imprints (OFPI) in diagnosing mucocutaneous leishmaniasis (ML) and cutaneous leishmaniasis (CL). Seventeen patients with ML, 19 patients with CL, and 33 controls were evaluated. In patients with ML, PCR from NS showed an 86% diagnostic accuracy (95% confidence interval [CI] = 73.81–93.05), followed by saliva 74% (95% CI = 60.45–84.13) and OFPI 68% (95% CI = 54.19–79.24). The highest sensitivity was reached by using the NS 58.82% (95% CI = 36.01–78.39), followed by saliva 23.53% (95% CI = 9.56–47.26) and OFPI 5.88% (95% CI = 1.05–26.98). The specificities of the tests were complete. The NS and OFPI were positive in 2 cases of CL. Mucous membrane samples exhibited a higher specificity compared to the Montenegro skin test and indirect immunofluorescence. NS sensitivity was higher than that of parasitological examinations.     

Consistence of miniexon polymerase chain reaction-restriction fragment length polymorphism and single-copy gene sequence analyses in discriminating Leishmania genotypes

Recently, a new polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP)-based assay had been developed using the miniexon sequences for genotyping Leishmania isolates. We had used this method for rapid diagnosis and genotyping of visceral and cutaneous leishmaniasis with the combination of microcapillary cultivation. In this study, we have evaluated this approach by examining genomic DNAs from 47 independent isolates, which were grouped into 19 genotypes of Leishmania subgenus complexes by sequence polymorphism of single-copy genes. Results obtained provide miniexon RFLP configurations specific to Leishmania enriettii, Leishmania tarentolae, and Leishmania gerbilli for the first time. Altogether, 92% of the results from miniexon PCR-RFLP are in agreement with those based on the sequence database of single-copy genes from the same isolates. The miniexon PCR-RFLP method is simple, sensitive, and specific method useful for routine diagnosis of different Leishmania.     

Accurate and rapid species typing from cutaneous and mucocutaneous leishmaniasis lesions of the New World

The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the Old and New World, using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis. Three new Leishmania-specific hsp70 PCRs were recently described, and we applied 2 of these on 89 clinical samples from a total of 73 Peruvian patients with either cutaneous or mucocutaneous leishmaniasis. The new PCRs on average showed a 2- to 3-fold improved sensitivity in the tested sample types (lesion biopsies, aspirates, and scrapings), for both genus detection and species typing, and were most successful in biopsies. Leishmania braziliensis, L. peruviana, and L. guyanensis were encountered. About one third of the L. braziliensis parasites contained 2 hsp70 alleles. This study is a paradigm for the implementation of a globally applicable upgraded tool for the identification of Leishmania directly on human specimens from cutaneous and mucocutaneous lesions in the New World.     

New world mucosal and cutaneous leishmaniasis: an emerging health problem among British travellers

BACKGROUND: Mucosal leishmaniasis (ML) is an important complication of new world cutaneous leishmaniasis (CL) caused by species of the Leishmania Viannia subgenus. Previous reports of ML among travellers to Latin America are few. AIMS: To determine the annual number of cases of CL due to L. Viannia species diagnosed at this institution and to correlate this with changing patterns of travel. Secondly, to document the clinical presentation, diagnosis, treatment and outcome of ML at this institution. DESIGN: Retrospective observational study. METHODS: Data were collected from a clinical database, laboratory records, patient case notes and an international passenger survey. RESULTS: Between 1995 and 2003, the annual number of cases of CL (total 79) steadily increased from 4 per year to 18 per year; the estimated number of travellers from the UK to Latin America increased 3.5-fold. Six cases of ML were diagnosed among British travellers in 1995 (1), 1997 (1) and 2002 (4). These infections were acquired in Bolivia (3), Colombia (2) and Belize (1). Nasopharyngeal symptoms developed 0-15 months after returning to the UK. Four patients had concurrent CL at diagnosis. Diagnosis of ML was delayed up to 6 months from the onset of symptoms. Mucosal biopsies from all 6 patients were PCR-positive for L. (Viannia) DNA; microscopy and culture were less sensitive. ML relapsed in one patient following treatment. DISCUSSION: Increasing travel to Latin America from the UK was associated with an increasing number of diagnoses of L. Viannia CL. ML is likely to emerge as a more frequently imported infection among such travellers. Familiarity with these diseases is important for prompt diagnosis and optimal management.     

In Vitro and In Vivo Efficacy of Novel Flavonoid Dimers against Cutaneous Leishmaniasis

Treatment of leishmaniasis by chemotherapy remains a challenge because of limited efficacy, toxic side effects, and drug resistance. We previously reported that synthetic flavonoid dimers have potent antipromastigote and antiamastigote activity against Leishmania donovani, the causative agent of visceral leishmaniasis. Here, we further investigate their leishmanicidal activities against cutaneous Leishmania species. One of the flavonoid dimers (compound 39) has marked antipromastigote (50% inhibitory concentrations [IC₅₀s], 0.19 to 0.69 μM) and antiamastigote (IC₅₀s, 0.17 to 2.2 μM) activities toward different species of Leishmania that cause cutaneous leishmaniasis, including Leishmania amazonensis, Leishmania braziliensis, Leishmania tropica, and Leishmania major. Compound 39 is not toxic to peritoneal elicited macrophages, with IC₅₀ values higher than 88 μM. In the mouse model of cutaneous leishmaniasis induced by subcutaneous inoculation of L. amazonensis in mouse footpads, intralesional administration of 2.5 mg/kg of body weight of compound 39.HCl can reduce footpad thickness by 36%, compared with that of controls values. The amastigote load in the lesions was reduced 20-fold. The present study suggests that flavonoid dimer 39 represents a new class of safe and effective leishmanicidal agent against visceral and cutaneous leishmaniasis.     

Leishmania (Viannia) subgenus kDNA amplification for the diagnosis of mucosal leishmaniasis

The utility of 2 polymerase chain reaction (PCR)-based assays amplifying genus or Viannia subgenus Leishmania minicircle kDNA for the diagnostics of ML was assessed. The Viannia subgenus product was yielded after PCR from isolates of L. (Viannia) braziliensis, L. (Viannia) colombiensis, and L. (Viannia) guyanensis, whereas no product was obtained with the non-Viannia-pertaining species: L. (Leishmania) amazonensis, L. (Leishmania) donovani, and L. (Leishmania) chagasi. With both assays, 11 of 13 (86.4%) patients with confirmed ML could be identified, whereas only 2 (16.7%) of these patients were positive by microscopy. All amplified genus-specific products gave a positive signal by hybridization with a Leishmania (Viannia) subgenus-specific radioactive probe. The Viannia subgenus-specific kDNA PCR represents a sensitive and specific tool for the diagnosis of ML, remarkably improving the sensitivity of parasitological methods and offering an alternative for the radioactive-dependent assays for subgenus characterization.     

Leishmania spp. identification by polymerase chain reaction–restriction fragment length polymorphism analysis and its applications in French Guiana

Leishmania (Viannia) guyanensis was for many years the only species commonly identified in French Guiana, but precise species identifications were quite rare. We describe a new restriction fragment length polymorphism–polymerase chain reaction technique using a 615-bp fragment of the RNA polymerase II gene and 2 restriction enzymes, TspRI and HgaI. Seven reference strains (Leishmania (Leishmania) amazonensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) braziliensis, L. (V.) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Leishmania) major, Leishmania (Leishmania) infantum) and 112 clinical samples from positive lesions were used for the development of the technique. The rates of positive species identification were 85.7% for punch skin biopsy specimens, 93.1% for positive Giemsa-stained smears, and 100% for positive culture supernatants. In the framework of cutaneous leishmaniasis species surveillance for the 2006 to 2008 period, parasite identification was carried out for 199 samples from different patients. The prevalence of the various Leishmania spp. was 84.4% for L. (V.) guyanensis, 8.0% for L. (V.) braziliensis, 5.0% for L. (L.) amazonensis, and 2.6% for L. (V.) lainsoni. L. (V.) braziliensis seems to be locally an emerging pathogen.     

Single-step duplex kDNA-PCR for detection of Leishmania donovani complex in human peripheral blood samples

A duplex polymerase chain reaction (PCR) assay was performed using a sense Leishmania genus-specific oligonucleotides to amplify the conserved region of 120 bp of minicircle kDNA and an additional antisense Leishmania donovani oligonucleotide that amplifies a complex-specific fragment of 90 bp. All 12 tested reference isolates of the L. donovani complex yielded a complex-specific amplification product of 90 bp concurrently with a genus-specific 120-bp product. All 17 tested reference isolates pertaining to other complexes presented only the genus-specific 120-bp product. Peripheral blood samples of twenty patients with visceral leishmaniasis positive by the genus-specific PCR presented both fragments with the duplex assay. This duplex PCR can be applied as a 1-step diagnostic tool where discrimination of L. donovani complex species is relevant for clinical and epidemiologic purposes.     

Out-patient parenteral antimicrobial therapy--a viable option for the management of cutaneous leishmaniasis.

Cutaneous infection with Leishmania braziliensis complex requires treatment with parenteral pentavalent antimonials to prevent development of mucocutaneous leishmaniasis. Patients with imported disease are usually managed in hospital because of concerns over drug toxicity. This study describes the clinical features and outcome of infection treated in the UK in an out-patient setting. Thirteen marines (aged 19-35 years) who acquired leishmaniasis in Belize were studied prospectively. Three had at least two lesions (0. 6-3 cm diameter), eight had regional lymphadenopathy and one had localized painless lymphatic thickening. Histology for amastigotes and PCR for Leishmania braziliensis complex was positive in all. Culture was positive in six. Patients received 1.5-2 g (mean 1.7 g) (20 mg/kg) sodium stibogluconate intravenously daily for 20 days. All developed transient musculoskeletal symptoms and asymptomatic hepatitis. Eleven developed biochemical pancreatitis, and one thrombocytopenia. Three developed transient ECG changes and one herpes zoster. There were four device-related infections, two requiring hospitalization (one required surgical drainage of an abscess). All lesions re-epithelialized. A total of 250 bed-days were saved over a 67-day period. These results indicate that in selected patients, out-patient therapy for cutaneous leishmaniasis with parenteral high-dose sodium stibogluconate may be appropriate, provided there is adequate monitoring of therapy.     

Serological studies on rK39 negative Visceral Leishmaniasis in an endemic focus of Leishmania donovani induced Cutaneous Leishmaniasis

Sri Lanka reports a focus of L. donovani induced cutaneous leishmaniasis (CL). Our more recent parasite and clinical studies and historical evidence point towards long term existence of Leishmania in the country, indicating a possible evolution leading to antigenic heterogenicity as well. In-house enzyme-linked immunosorbent assay (ELISA) that was developed during phase 1 study indicated >80% sero-positivity in local CL, while visceral leishmaniasis (VL) remained very rare with majority being negative when tested with rK39 assay. A novel serological tool was developed and sero-positivity of VL was assessed for the first time. The assay showed 100.0% sensitivity and 98.3% specificity for detection of VL. Samples were showed less positivity with established direct agglutination test (DAT) and rK39 strip test. The assay was less expensive than that of established rapid diagnostic tests (RDTs), culture and PCR assays. This assay may be useful in diagnosing clinical VL infections, detection of light microscopy (LM) negative patients, tracking post treatment stages, field screening of asymptomatic cases and in further serological studies.     

In vitro and in vivo studies of the utility of dimethyl and diethyl carbaporphyrin ketals in treatment of cutaneous leishmaniasis

Carbaporphyrin ketals are porphyrinoid compounds in which a pyrrole ring of a typical porphyrin macrocycle has been replaced by a ketal-substituted indene ring. It was recently demonstrated that these compounds are effective in vitro against Leishmania tarentolae. Their in vitro effectiveness is increased when they are exposed to visible light; they act as photosensitizers capable of mediating the production of reactive oxygen species (ROS). Following on this evidence, the effectiveness and cytotoxicity of the dimethyl and diethyl carbaporphyrin ketals (CKOMe and CKOEt, respectively) were determined in vitro using pathogenic Leishmania species with and without exposure to visible light (2 and 4 h). The effectiveness against various pathogenic Leishmania species was determined to be in a micromolar range. Additionally, the effect of encapsulating the carbaporphyrin ketals in liposome formulations was tested. Liposomal delivery diminished their toxicity, while the effectiveness was enhanced upon exposure to visible light (photodynamic effect). The cytotoxicity levels for human U937 cells and hamster peritoneal macrophages were in the ranges of 0.3 to 9 μM and 7 to 330 μM, respectively. When tested in vivo, using a hamster (Mesocricetus auratus) model of cutaneous leishmaniasis, CKOMe was active even in the dark, suggesting that the compound, once metabolized in the animal tissue, produces an active ingredient that does not seem to be photosensitive. Reduction in lesion size, histopathologic analyses, and smears confirmed the in vivo effectiveness of the compound, since the parasitic load was diminished without noticeable toxic effects.     

Applications of molecular methods for Leishmania control

This article reviews the recent advances made in the field of human leishmaniasis. Special emphasis is placed upon the application of various molecular tools for accurate and rapid diagnosis, understanding the mechanisms of drug resistance and identification of vaccine candidates. The focus will be on the major role played by recombinant antigens in the immunoserodiagnosis and progress of the Leishmania genome project, which has enabled researchers to design better PCR primers and molecular probes for microarrays. A special interest is placed on the recombinant antigen (rK39) cloned from the Leishmania chagasi kinesin gene and a very recently cloned recombinant antigen (KE16) from the Old World Leishmania donovani species with high sensitivity and specificity. Advances made in the specific PCR primer designed to diagnose and differentiate various species and strains of Leishmania causing visceral and post-kala-azar-dermal leishmaniasis have been covered. Molecular methods (e.g., DNA and protein microarrays) applied to understanding the pathobiology of the parasite, mechanism of host invasion, drug interaction and drug resistance to develop effective therapeutic molecules, gene expression profiling studies that have opened doors to understand many host-parasite relations, effective therapy and vaccine candidates are extensively covered in this review.     

Antimony uptake systems in the protozoan parasite Leishmania and accumulation differences in antimony-resistant parasites

The first line drug against leishmaniasis consists of pentavalent antimony [Sb(V)], but there is general belief that the active form of the metal is the trivalent form [Sb(III)]. In this study, we have quantified the accumulation of Sb(V) and Sb(III) in Leishmania by using inductively coupled plasma mass spectrometry. The accumulation was studied in three Leishmania species at various life stages, sensitive or resistant to antimony. Both Sb(III) and Sb(V) are accumulated in promastigote and amastigote parasites, but through competition experiments with arsenite, we found that the routes of entry of Sb(V) and Sb(III) are likely to differ in LEISHMANIA: The level of accumulation of either Sb(III) or Sb(V), however, was not correlated with the susceptibility of wild-type Leishmania cells to antimony. This suggests that other factors may also be implicated in the mode of action of the drugs. In contrast to metal susceptibility, resistance to Sb(III) correlated well with decreased antimony accumulation. This phenotype was energy dependent and highlights the importance of transport systems in drug resistance of this protozoan parasite.     

Early Curative Applications of the Aminoglycoside WR279396 on an Experimental Leishmania major-Loaded Cutaneous Site Do Not Impair the Acquisition of Immunity

Topical therapy is an attractive approach for the treatment of Leishmania major cutaneous leishmaniasis (CL). WR279396, an expanded-spectrum aminoglycoside ointment, is now in phase 3 trials. Because the application of a cream is easier than the injection of pentavalent antimony, many patients with CL will likely be treated with WR279396 soon after the onset of a lesion. However, this new therapeutic approach may impair the acquisition of immunity. We evaluated the impact of early topical therapy on acquired immunity in an optimized mouse model of L. major-induced CL. The efficacy of the WR279396 ointment in this model has been established previously. Acquired immunity was defined as the absence of lesions upon reinoculation of the same parasite isolate at a different skin site. Bioluminescence-based follow-up of luciferase-expressing L. major loads was also performed. In this model, the control of L. major loads at the initial inoculation site and the acquisition of immunity are simultaneous (day 22 postinoculation). The clinical and parasitological efficacies of WR279396 applied as early as day 11 postinoculation, i.e., during the L. major multiplication phase, did not impair the acquisition of immunity to a second L. major challenge. This is reassuring from the perspective of the wide deployment of WR279396-based therapy in foci where L. major is endemic.Cutaneous leishmaniasis (CL) is a dermatological disease caused by protozoan Leishmania parasites that are transmitted to their mammalian hosts by sand fly bites. The topical treatment of CL is a practical and safe therapeutic option (5). One strong argument for the topical treatment of CL is the simpler implementation of application procedures at primary health care centers, thus, allowing the earlier treatment of the majority of patients.WR279396, a formulation of paromomycin and gentamicin in a hydrophilic base, is efficacious in patients with Leishmania major CL and has a satisfactory safety profile (3, 18). A phase 3 study is ongoing in Tunisia. To identify the parameters that influence the efficacy of WR279396, we have optimized a C57BL/6 mouse-based model of CL that mimics important features of Leishmania metacyclic promastigote transmission to mammals, including humans (10, 12). Real-time bioluminescence-based determination of the parasitic load in mice treated with WR279396 showed an accelerated and greater than 2-log reduction of the parasitic load during a 10-day topical application. These curative applications resulted in a long-term stable cure, with a very low number of parasites persisting in the healed skin (12).Questions about the risk of secondary episodes in topically treated and cured patients, when and if those patients are reexposed to Leishmania isolates of the same species, persist. The optimized C57BL/6 mouse-based model (12) offers a relevant experimental system with which to study the impact of early therapeutic intervention against CL on the development of skin-protective processes (referred to here for simplicity as acquired immunity), which spontaneously develop in the absence of therapeutic intervention (14). The clinical and parasitological efficacies of WR279396 applied as early as day 11 postinoculation, i.e., during the multiplication phase of infection, did not impair the acquisition of immunity to a second L. major challenge.