Are cytochrome B gene sequencing and polymorphism-specific polymerase chain reaction as reliable as multilocus enzyme electrophoresis for identifying Leishmania spp. from Argentina?

Recently, two techniques, polymerase chain reaction (PCR) amplification and sequencing of cytochrome b gene (cyt b gene sequencing) and polymorphism-specific PCR (PS-PCR) were recommended for Leishmania species identification. Before this study, however, the accuracy of these methods had not been tested against the multilocus enzyme electrophoresis, the current gold standard technique on this task. Therefore, a trial was done for the first time to compare the results obtained by these techniques, using 17 Argentinean Leishmania stocks in independent assays. For all the stocks examined, the same results at species level were obtained by the three techniques. Among them, 14 were assigned to L. (Viannia) braziliensis, and three to L. (V.) guyanensis. The two techniques, cyt b gene sequencing and PS-PCR, were able to distinguish between all the proven species responsible for leishmaniases in Argentina. Thus, both techniques were validated and could be used independently for the species designation of Leishmania parasites in the country.

Multilocus enzyme electrophoresis and cytochrome B gene sequencing-based identification of Leishmania isolates from different foci of cutaneous leishmaniasis in Pakistan.

Seventeen Leishmania stocks isolated from cutaneous lesions of Pakistani patients were studied by multilocus enzyme electrophoresis and by polymerase chain reaction amplification and sequencing of the cytochrome b (Cyt b) gene. Eleven stocks that expressed nine zymodemes were assigned to L. (Leishmania) major. All of them were isolated from patients in the lowlands of Larkana district and Sibi city in Sindh and Balochistan provinces, respectively. The remaining six, distributed in two zymodemes (five and one), isolated from the highland of Quetta city, Balochistan, were identified as L. (L.) tropica. The same result at species level was obtained by the Cyt b sequencing for all the stocks examined. No clear-cut association between the clinical features (wet or dry type lesions) and the Leishmania species involved was found. Leishmania (L.) major was highly polymorphic compared with L. (L.) tropica. This difference may be explained by the fact that humans may act as a sole reservoir of L. (L.) tropica in anthroponotic cycles; however, many wild mammals can be reservoirs of L. (L.) major in zoonotic cycles.

Leishmania isoenzyme polymorphisms in Ecuador: relationships with geographic distribution and clinical presentation.

BACKGROUND: Determinants of the clinical presentation of the leishmaniases are poorly understood but Leishmania species and strain differences are important. To examine the relationship between clinical presentation, species and isoenzyme polymorphisms, 56 Leishmania isolates from distinct presentations of American tegumentary leishmaniasis (ATL) from Ecuador were analyzed. METHODS: Isolates were characterized by multilocus enzyme electrophoresis for polymorphisms of 11 isoenzymes. Patients were infected in four different ecologic regions: highland and lowland jungle of the Pacific coast, Amazonian lowlands and Andean highlands. RESULTS: Six Leishmania species constituting 21 zymodemes were identified: L. (Viannia) panamensis (21 isolates, 7 zymodemes), L. (V.) guyanensis (7 isolates, 4 zymodemes), L. (V.) braziliensis (5 isolates, 3 zymodemes), L. (Leishmania) mexicana (11 isolates, 4 zymodemes), L. (L.) amazonensis (10 isolates, 2 zymodemes) and L. (L.) major (2 isolates, 1 zymodeme). L. panamensis was the species most frequently identified in the Pacific region and was associated with several clinical variants of cutaneous disease (CL); eight cases of leishmaniasis recidiva cutis (LRC) found in the Pacific highlands were associated with 3 zymodemes of this species. Mucocutaneous leishmaniasis found only in the Amazonian focus was associated with 3 zymodemes of L. braziliensis. The papular variant of CL, Uta, found in the Andean highlands was related predominantly with a single zymodeme of L. mexicana. CONCLUSION: Our data show a high degree of phenotypic variation within species, and some evidence for associations between specific variants of ATL (i.e. Uta and LRC) and specific Leishmania zymodemes. This study further defines the geographic distribution of Leishmania species and clinical variants of ATL in Ecuador.

A case of tinea nigra palmaris in Okinawa, Japan.

We report a case of tinea nigra on the left palm of a 13-year-old girl. She had noticed a pigmented, asymptomatic macule on the left palm approximately 4-5 years prior to her first visit to our hospital. The color of the lesion tended to change before and after a bath; it became lighter after a bath and darkened some time later. Physical examination revealed that the macule was 4 cm x 5 cm in size, dark brown in color and irregularly shaped. Direct potassium hydroxide (KOH) microscopic examination from skin scrapings revealed branched brown hyphae with light brown septa. A fungal culture on Sabouraud's agar media produced wet, medium brown, yeast-like colonies, the surface of which later became black and shiny. A slide culture disclosed light brown, elliptic or peanut-shaped conidia comprised of one to two ampullaceous cells. Scanning electron microscopic examination of the conidia showed both annellation conidia with lunate bud scars and sympodial conidiogenesis. Using extracted DNA from separately cultured fungi, we performed polymerase chain reaction with the primers specific to Hortaea werneckii. The results showed positive bands. We performed direct sequencing with the DNA segments from the positive bands. The causative fungus in our case was determined to be type C of H. werneckii on the grounds of the base sequences obtained. The final diagnosis of the present case was made as tinea nigra by H. werneckii. We also report a brief survey of all the cases of tinea nigra reported in Japan to date.

Atypical clinical variants in New World cutaneous leishmaniasis: disseminated, erysipeloid, and recidiva cutis due to Leishmania (V.) panamensis.

In recent times, there has been an increase in the number of reports for new and rare variants of cutaneous leishmaniasis (CL). Here, we describe three unusual clinical forms of CL identified in Ecuadorian children. A total of 131 patients with CL were diagnosed over a 2-year period of active search. In 3 (2.29%), the lesions were very unusual; these included erysipeloid, recidiva cutis (LRC), and disseminated leishmaniasis (DL). The erysipeloid case is characterized by erythematous and indurated plaque seen on the face of a 5-year-old boy; the LRC one is differentiated by slowly progressing red-brown papules around large scars of healed sores in a 6-year-old girl, and the DL case is characterized by dozens of cutaneous ulcers distributed in the whole body of a 1-year-old girl. Leishmania parasites were isolated by lesion aspirate and analyzed by the technique multilocus enzyme electrophoresis (MLEE). All three isolates were identified as Leishmania (Viannia) panamensis. These distinct clinical variants rarely have been reported previously in the American cutaneous leishmaniasis, and for the first time L. (V.) panamensis was identified as the etiologic agent. Our cases extend the spectrum of clinical presentations in New World leishmaniasis.

Detection and identification of Leishmania species within naturally infected sand flies in the andean areas of ecuador by a polymerase chain reaction.

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas.

A case of bullous congenital ichthyosiform erythroderma (BCIE) caused by a mutation in the 1A helix initiation motif of keratin 1.

Bullous congenital ichthyosiform erythroderma (BCIE) is an autosomally dominant inherited disorder characterized by erythematous, erosive, and bullous skin lesions over the entire body at birth and abnormal hyperkeratosis on the palmoplantar sufaces as the patient grows older. BCIE is caused by a mutation in the keratin 1 (K1) and/or keratin 10 (K10) genes, and most pathogenic mutations are found within the helix initiation and termination motifs of the central helical rod domain (K1 and K10) or the upstream H1 homology domain (K10). In addition to inherited cases, sporadic cases due to a new mutation account for approximately half the total cases of BCIE. We report herein a typical sporadic case of BCIE with erythroderma, erosion, and blisters on the entire body surface at birth and palmoplantar and flexuaral areas of hyperkeratosis in the later stage. We found in this case a novel mutation, 559C to T, at amino acid position 187, which resulted in a leucine to phenylalanine substitution within the helix initiation motif of K1.

The attachment and entry of Leishmania (Leishmania) major into macrophages: observation by scanning electron microscope.

Leishmaniasis, a zoonotic protozoan disease, starts with the inoculation of the Leishmania promastigotes into the skin at the time of blood ingestion by a female sandfly. The infection of leishmaniasis is established when the Leishmania organisms start their own intracellular multiplication after having been phagocytized by the host's macrophages. In the earliest stage of the infection, therefore, the attachment of the promastigates to the macrophages is essential. We incubated a mixed culture of macrophages (JM774-1A) and Leishmania (Leishmania) major for 6 hours in vitro and observed the process of the attachment between the parasite and host cell by scanning electron microscope. We found for the first time that the attachment between the two occurred at the site of the parasite body, in addition to the previously reported sites of the flagellar tip, flagellar base, and aflagellar tip (posterior pole).

Inhibition of intracellular proliferation of Leishmania parasites in vitro and suppression of skin lesion development in BALB/c mice by a novel lipid A analog (ONO-4007).

A synthetic lipid A analog (ONO-4007) exhibits antileishmanial activity by activating Leishmania-infected macrophages in experimental leishmaniasis. In the present in vitro study, ONO-4007 at concentrations between 0.01 and 1.00 mg/mL markedly inhibited the proliferation of Leishmania major and L. amazonensis promastigotes. Ultrastructurally, L. major-infected macrophages showed degenerated intracellular amastigotes after exposure to ONO-4007. Leishmania-infected macrophages treated with ONO-4007 showed poorly developed parasitophorous vacuoles. High levels of tumor necrosis factor-alpha were induced by ONO-4007 in Leishmania-infected macrophages. In this in vivo study, L. amazonensis-infected BALB/c mice were treated with a dose of 30 mg/kg of ONO-4007 by perilesional and peritoneal injections. The skin lesion size was assessed before treatment with ONO-4007 and at eight weeks after injection. The lesion size was significantly suppressed in mice perilesionally injected with ONO-4007 (P < 0.01) compared with the controls. The data from our present in vitro and in vivo studies indicate that ONO-4007 has an antileishmanial effect.

Cutaneous ulcerations following subcutaneous interferon beta injection to a patient with multiple sclerosis.

We report a case treated with interferon beta-1b for multiple sclerosis (MS), who developed severe cutaneous ulcers after six months of therapy. Interferon beta-1b had been used in a regimen of 8 million IU administered subcutaneously through oblique direction of the needle, twice a week. The cutaneous ulcers developed at inoculation sites, as a result of penetration of interferon beta into dermis. Other underlying diseases of coagulative or bleeding disorders or secondary infection were excluded. Histological features of non-specific inflammatory reactions including hyperplastic changes of blood vessels without any evidence of vasculitis were the prominent features in this case. Corticosteroid and interferon beta-1b therapy was continued on restricted sites on the extremities with care not to repeat injections at the same sites previously used. The administration of interferon beta into subcutaneous fatty tissues vertically reduced the incidence of dermal penetration of drug and occurrence of ulcerations in this patient. We review other case reports of severe cutaneous reactions associated with interferon beta-1b therapy in MS patients and conclude that local cytokine-mediated, adverse, immune reaction or non-specific cutaneous inflammatory reaction to interferon beta-1b initiated the skin ulceration long after institution of therapy at the injection sites, and the reaction might be related to the depth of injection.

Amyloid deposition is frequently observed in skin lesions of hypertrophic lupus erythematosus.

Four cases of Hypertrophic Lupus Erythematosus (HLE) were reported. The lesions of HLE were observed on the forearms, face and hands in all four cases. Clinically, the lesions were erythematous, hyperkeratotic plaques. The clinical course was marked by chronicity and progression of the lesion. Histologically, marked hyperkeratosis, parakeratosis, acanthosis, degenerative changes of basal cells in H/E stain, and thickened, multilayered basement membrane in PAS stain, were observed. The observations of Dylon stain revealed that localized amyloid deposition was observed in all four cases of HLE lesions, as fluorescent-orange colored amyloid deposits in the papillary dermis and subepidermal areas at near orjust below the dermo-epidermal junction appeared under fluorescent microscope. On the basis of clinical and histological observations, we suggest that chronic irritation, such as sunlight exposure over a long-duration, might have caused the characteristic abnormalities at the dermo-epidermal junction and also initiated the frequency of amyloid deposits locally secondary to the diseases. We compared our HLE cases to other types of lupus erythematosus (LE) skin lesions, as to whether deposition of amyloid materials were frequently observed or not. Amyloid deposition was observed in one case of DLE and none of the SLE cases. Localized amyloid deposition was more frequently observed in skin lesions, secondary to HLE disease, as compared to other types of LE.

An assessment of the malignant potential of actinic keratoses and Bowen's disease: p53 and PCNA expression pattern correlate with the number of desmosomes.

Actinic keratoses (AK) and Bowen's disease (BD), both intraepidermal skin tumors, have a potential progression to squamous cell carcinoma (SCC). To evaluate the malignant potential of AK and BD, the expression pattern of p53 protein and proliferating cell nuclear antigen (PCNA) were examined in five types of AK and BD by immunohistochemistry. The ultrastructural difference of epidermal cells between AK and BD lesions was investigated. In the study of p53 and PCNA expression, the atrophic and acantholytic types of AK showed lower positive rates compared to others. These two types did not demonstrate all layers expression pattern. The number of desmosomes of the epidermal cells was significantly reduced in BD, and in the bowenoid and hypertrophic types of AK compared with other types of AK The number of hemi-desmosomes showed greatest reduction in BD and the bowenoid type of AK On the basis of our findings, it is hypothesized that atrophic and acantholytic types of AK may have the lowest, and the bowenoid type of AK and BD may have the highest, malignant potential.

Identification of endotrypanum species from a sloth, a squirrel and Lutzomyia sandflies in ecuador by PCR amplification and sequencing of the mini-exon gene.

PCR amplification and nucleotide sequencing of the mini-exon gene revealed that four strains isolated from a sloth (Choloepus hoffmanni), a squirrel (Sciurus granatensis) and two sandflies (Lutzomyia hartmanni) in Ecuador were indistinguishable from Endotrypanum monterogeii. Another strain isolated from Lu. hartmanni showed the high sequence similarity to E. schaudinni. Since three of these strains have been previously identified as Leishmania (Viannia) equatorensis, the results demonstrate that L. (V.) equatorensis is genetically closely related to the genus Endotrypanum. The present study also indicates that Endotrypanum species are distributed in arboreal animals and sandflies in Ecuador, and that mini-exon gene amplification is useful for epidemiological studies of Leishmania and Endotrypanum in the New World.

Leishmaniasis recidiva cutis due to Leishmania (Viannia) panamensis in subtropical Ecuador: isoenzymatic characterization.

BACKGROUND: Information regarding leishmaniasis recidiva cutis (LRC), a clinical variant of cutaneous leishmaniasis, in the New World is scarce. LRC is characterized by slowly progressing lesion(s) that appear after a variable period of time, from months to years, in or around the scar of an apparently clinically healed sore. PATIENTS AND METHODS: Six patients are reported who presented with crusted, papular lesions located on the edge of a healed scar, with a mean of 18.2 months of slowly progressive evolution. The isolated strains of Leishmania parasites were characterized by enzyme electrophoresis. Eleven enzyme systems were assayed. Skin biopsies from the active border of the lesions were taken for histopathology. RESULTS: Tissue sections showed a granulomatous, lymphohistiocytic, dermal infiltrate containing Langhans' giant cells. The anamnestic data, together with the clinical and histopathologic findings, support the diagnosis of LRC. The isoenzyme profile of Leishmania parasites isolated from five of the six patients identified them as Leishmania (Viannia) panamensis. CONCLUSIONS: These findings are the first reported evidence of LRC within the clinical spectrum of American tegumentary leishmaniasis in Ecuador, and of its causative agent. The existence of LRC has future implications for both disease treatment and vaccine development.

Efficacy of vaccination with a combination of Leishmania amastigote antigens and the lipid A-analogue ONO-4007 for immunoprophylaxis and immunotherapy against Leishmania amazonensis infection in a murine model of New World cutaneous leishmaniasis.

Activation of innate immunity using adjuvants that activate Toll-like receptor 4 pathways have great potential for improving the protection induced by parasite vaccines. We investigated protective and therapeutic effects of a vaccine against leishmaniasis containing a combination of an adjuvant synthetic lipid A-analogue, ONO-4007 and Leishmania amazonensis antigens. ONO-4007 was co-injected with soluble and membrane-enriched L. amazonensis-amastigote antigens into BALB/c mice that had either already been infected with 1 x 10(6) L. amazonensis promastigotes (immunotherapy study) or before challenge with the same infectious dose (immunoprophylaxis study). Sixty percent of mice vaccinated before infectious challenge controlled their Leishmania infections - defined by the absence of footpad-swelling and negative Leishmania cultures - compared to 0% of controls, and 40% of mice vaccinated after infection resolved their infections compared to 0% of controls. Protective immunity in both immunoprophylaxis and immunotherapy models was associated with increased protein production of IL-12 and IFN-gamma. These data suggest that vaccination with a combination of ONO-4007 and amastigote antigens of L. amazonensis may be useful for the prevention and treatment of leishmaniasis, and that the protective immunity induced is associated with the production of type-1 cytokines.

A genotype distribution of human papillomaviruses detected by polymerase chain reaction and direct sequencing analysis in a large sample of common warts in Japan.

There have been no large-scale epidemiological studies of human papillomavirus (HPV) genotype distribution of common warts in Japan. A total of 213 patients with common warts (104 males and 109 females) in Japan were studied to detect HPV genotype distribution by polymerase chain reaction (PCR) and direct sequencing analysis. The results were as follows: 94 HPV-1a (44.1%), 35 HPV-4 (16.4%), 30 HPV-65 (14.1%), 13 HPV-27 (6.1%), 13 HPV-2a (6.1%), 9 HPV-57b (4.22%), 3 HPV-16 (1.41%), 2 HPV-6a (0.94%), 2 HPV-63 (0.94%), and 1 case for each of HPV-3, -5, -5b, -7, -10, -21, -29, -47, -56, -57, -62, and -92 (0.47%, respectively). Four cases (1.88%) were found in which two different HPV types were detected within the lesions: one case of HPV-1a with HPV-16, one case of HPV-1a with HPV-65, one case of HPV-6a with HPV-8, and one case of HPV-65 with HPV-16. There were seven cases of mucosal types (3.3%), that is, two HPV-6a, three HPV-16, one HPV-56, and one HPV-62, and three cases of epidermodysplasia verruciformis (EV)-related types (1.41%), that is, one HPV-5, one HPV-5b (both of which belonged to a high-risk group), and one HPV-47 (which belonged to a low-risk group). To date, this is the largest sequencing-based study of HPV for common warts in Japan. It is said that common warts are induced predominantly by HPV-2, -27, and -57 in European population. However, the present results showed that in Japan they were induced mostly by HPV-1, -4, and -65. This suggests that regional differences in HPV genotype distribution may exist between European and Japanese populations.

Clinical and epidemiological observations of cutaneous tuberculosis in Larkana, Pakistan.

BACKGROUND: Cutaneous tuberculosis is widespread in Pakistan but has not been fully documented. This study was conducted to determine the clinical pattern, nature and existence of the disease in Larkana, Sindh province, Pakistan. METHODS: We are reporting 153 cases of patients with cutaneous tuberculosis who visited our department from 1996 to 1999. All cases were diagnosed at the clinic, and the biopsies were examined for histopathological evidence. The patients received three antituberculous treatments during a 9 month course. RESULTS: Clinically, 63 (41.2%) cases of lupus vulgaris, 54 (35.3%) of scrofuloderma, 29 (19.59%) of lupus verrucosa cutis, six (3.92%) of tuberculosis cutis orificialis and one (0.64%) case of disseminated cutaneous tuberculosis were observed in our department from 1996 to 1999. All patients were aged between 3 and 50 years and had experienced the present complaints for 1 to 12 years. Sixty-nine (45.1%) cases were children aged under 10 years, 50 cases (37.25%) were aged between 10 and 20 years, and 27 cases (17.65%) were aged over 20 years. There was no considerable ratio difference of the disease between male and female patients. Histopathologically, all the specimens showed chronic granulomatous changes; the majority was infiltrated with epitheloid cells, langhans giant cells, plasma cells and other inflammatory cells, such as lymphocytes, eosinophils and neutrophils in ulcerated lesions. Increased numbers of mast cells were seen in upper and lower dermis in two-thirds of the specimens. Caseating necrosis was visible in half of the specimens while Ziehl-Neelsen stain was negative in all the sections. CONCLUSIONS: The observed number of patients was moderately large, thus indicating a high incidence of cutaneous tuberculosis in Larkana. Lupus vulgaris, a form of cutaneous tuberculosis, was widespread in this area and prevalent in adults, while scrofuloderma was prevalent in children. Moreover, the existing rate of the disease was higher in children aged under 10 years and lower in adults. This indicates that children are more prone to this disease than adults.

Detection of new endemic areas of cutaneous leishmaniasis in Pakistan: a 6-year study.

BACKGROUND: Cutaneous leishmaniasis (CL) is endemic in Pakistan and is widely spreading. Recently, an outbreak of the disease was observed in the region. We report some new endemic areas of CL in the country. METHODS: A total of 1210 cases of CL who visited our department from 1996 to 2001 are reported. Among them, 760 were residents of the Jacobabad, Larkana, and Dadu districts of Sindh province and had never previously traveled to endemic areas. These districts have never been reported/recognized as endemic for CL. Others were residents of endemic areas of Balochistan province. Diagnosis was made on clinical presentation; a giemsa-stained smear test and histopathological results. All the cases were treated with the meglumine antimoniate 600 mg/day (adults) and 15 mg/kg/day (children) intramuscularly for 20 consecutive days. RESULTS: All the patients were aged between 2.5 months and 65 years. Three hundred and ninety-two patients were females and 368 were males. Duration of the disease ranged from 2 to 18 months. Most of the patients had a single lesion on the face and/or extremities. Clinically, the disease was classified as: dry papular type, 407 cases; dry ulcerative type, 335 cases; and wet ulcerative type, 18 cases. No cases of muco-cutaneous or visceral leishmaniasis were found during this period. Smear testing was positive in 845 cases, while 365 cases were histopathologically positive. An ultrastructural study was performed using specimens of a few of the cases. Leishmania parasites were detected in the dermal tissues as well as in the macrophages. CONCLUSIONS: We propose that the Jacobabad, Larkana and Dadu districts could be considered endemic for CL. Wet- and dry-type lesions indicate the presence of both Leishmania tropica and L. major in this tropical region.

The Traf2- and Nck-interacting kinase as a putative effector of Rap2 to regulate actin cytoskeleton.

Rap2 belongs to the Ras family of small GTP-binding proteins, but its specific roles in cell signaling remain unknown. In the present study, we have affinity-purified from rat brain a Rap2-interacting protein of approximately 155 kDa, p155. By liquid chromatography tandem mass spectrometry, we have identified p155 as Traf2- and Nck-interacting kinase (TNIK). TNIK possesses an N-terminal kinase domain homologous to STE20, the Saccharomyces cerevisiae mitogen-activated protein kinase kinase kinase kinase, and a C-terminal regulatory domain termed the citron homology (CNH) domain. TNIK induces disruption of F-actin structure, thereby inhibiting cell spreading. In addition, TNIK specifically activates the c-Jun N-terminal kinase (JNK) pathway. Among our observations, TNIK interacted with Rap2 through its CNH domain but did not interact with Rap1 or Ras. TNIK interaction with Rap2 was dependent on the intact effector region and GTP-bound configuration of Rap2. When co-expressed in cultured cells, TNIK colocalized with Rap2, while a mutant TNIK lacking the CNH domain did not. Rap2 potently enhanced the inhibitory function of TNIK against cell spreading, but this was not observed for the mutant TNIK lacking the CNH domain. Rap2 did not significantly enhance TNIK-induced JNK activation, but promoted autophosphorylation and translocation of TNIK to the detergent-insoluble cytoskeletal fraction. These results suggest that TNIK is a specific effector of Rap2 to regulate actin cytoskeleton.