The identification of sandfly species, from an area of Argentina with endemic leishmaniasis, by the PCR-based analysis of the gene coding for 18S ribosomal RNA.

The area around Río Blanco, in the Orán department in the north of the Argentinian province of Salta, is endemic for American tegumentary leishmaniasis. In an attempt to facilitate the identification of the Lutzomyia species in this area, sequences of the gene coding for the 18S ribosomal RNA (rRNA) of sandflies caught in a Shannon trap were explored, by a combination of PCR and analysis of restriction-fragment-length polymorphism (RFLP). The products from the PCR, which employed two primers developed specifically for this study (Lu.18S 1S and Lu.18S AR), were cloned into a commercial vector (pGEM-T Easy) so that their nucleotide sequences could be investigated. In the RFLP analysis, the products of single and double digestion with the AfaI and HapII restriction enzymes were separated by electrophoresis in 3% or 4% agarose. Taken together with the results of a morphological investigation of the flies, the resultant DNA fragment patterns were sufficient to identify most of the sandflies caught as Lu. neivai. Although two other species, Lu. cortelezzii and Lu. sallesi, were collected, they were relatively rare and only identified morphologically. A single digestion of the 18S-rRNA gene sequences with AfaI or HapII appeared sufficient and useful for the identification of Lu. neivai from the north of Salta province, and for several other Lutzomyia species.

Characterization of human infection by Leishmania spp. in the Northwest of Argentina: immune response, double infection with Trypanosoma cruzi and species of Leishmania involved.

The aims of this study were to characterize human American tegumentary leishmaniasis, which includes cutaneous, mucocutaneous and mucosal leishmaniasis, in Northwest Argentina, to determine the prevalence of double infection with Trypanosoma cruzi and to identify the species of Leishmania in this area. Most of the 330 leishmaniasis patients presented cutaneous ulcers (96.1%), 2.4% mucocutaneous and 1.5% the mucosal form ('espundia'). The aetiological agents, determined by isoenzyme electrophoresis, were identified as Leishmania (Viannia) braziliensis in 16 out of 20 isolates and in the remaining 4 as Leishmania (Leishimania) amazonensis, the first ever-documented in Argentina. Sera analysed by ELISA and IFA using complex antigen from both T. cruzi and L. braziliensis showed a very high percentage of positives (66.3-78.2%). When antigens for specific diagnosis of Chagas' disease were used, 40.9% of the leishmaniasis patients were also found to be infected by T. cruzi. These results indicate that the strong immune response against T. cruzi gave no protection to Leishmania, in spite of the serological cross-reaction between these parasites. In addition, we showed that more than 40% of the patients would be misdiagnosed as chagasic if complex antigens, as epimastigotes or soluble fraction from epimastigotes, were used in IFA or ELISA. This is of paramount importance not only because patients' treatment would be associated to misdiagnosis but the fact that in many countries in Central and South America, a positive test for Chagas' disease means a rejection for those seeking employment.

Clinical status and parasitic infection in a Wichí Aboriginal community in Salta, Argentina.

In a study, carried out in 2000, of the clinical and parasitological status of a Wichi Aboriginal community living in the suburbs of Tartagal, northern Salta, Argentina, 154 individuals were screened for parasitic infections. Ninety-five faecal samples were also obtained from the same population. Ninety-three percent of the subjects were positive for 1 or more of the parasites investigated by direct test and 70.5% of them had parasitic superinfection. The most frequent helminths were Strongyloides stercoralis (50.5%) and hookworm (47.4%). We found low reinfection rates and a long reinfection period after treatment and provision of safe water and sanitation. Serum reactivity of these patients was analysed by enzyme-linked immunosorbent assay and indirect immunofluorescent assay and 22.1% of them had anti-Toxocara antibodies, 16.2% were positive for a complex antigen of Leishmania braziliensis, 29.9% were positive for a complex Trypanosoma cruzi antigen, and 17.5% were positive for a specific Trypanosoma cruzi antigen, Ag 163B6/cruzipain.

[Mucocutaneous leishmaniasis in naturally infected dogs in Salta, Argentina]. / Leishmaniosis mucocutánea en perros naturalmente infectados en Salta, Argentina.

The objective of the present study is to describe two cases of dogs with mucocutaneous lesions caused by Leishmania spp. Both dogs presented destruction of the nasal septum, hyperemia with soft palate edema and barking alteration due to laryngeal compromise. Biopsies were taken from the lesion border and Leishmania spp. amastigotes were seen in the imprints. The dogs presented positive serology when complex soluble antigen from Leishmania mexicana was used. One of the dogs was also suspected to be infected by Trypanosoma cruzi as suggested by its positive reaction with a purified specific antigen, Ag163B6-cruzipain. Most of the studies concerning leishmaniosis in dogs have described the cutaneous form of this disease in close association with human cases of Leishmania infection instead of the mucocutaneous form described herein. The presence of dogs with mucocutaneous leishmaniosis alerts on an increase of the prevalence of this form in humans, which can cause deforming lesions, alterations of the speech and even an inadequate nutrition due to difficulties in deglutition.

Leishmaniosis mucocutánea en perros naturalmente infectados en Salta, Argentina / Mucocutaneous leishmaniasis in naturally infected dogs in Salta, Argentina

The objective of the present study is to describe two cases of dogs with mucocutaneous lesions caused by Leishmania spp. Both dogs presented destruction of the nasal septum, hyperemia with soft palate edema and barking alteration due to laryngeal compromise. Biopsies were taken from the lesion border and Leishmania spp. amastigotes were seen in the imprints. The dogs presented positive serology when complex soluble antigen from Leishmania mexicana was used. One of the dogs was also suspected to be infected by Trypanosoma cruzi as suggested by its positive reaction with a purified specific antigen, Ag163B6-cruzipain. Most of the studies concerning leishmaniosis in dogs have described the cutaneous form of this disease in close association with human cases of Leishmania infection instead of the mucocutaneous form described herein. The presence of dogs with mucocutaneous leishmaniosis alerts on an increase of the prevalence of this form in humans, which can cause deforming lesions, alterations of the speech and even an inadequate nutrition due to difficulties in deglutition.

Leishmaniosis mucocutánea en perros naturalmente infectados en Salta, Argentina / Mucocutaneous leishmaniasis in naturally infected dogs in Salta, Argentina

The objective of the present study is to describe two cases of dogs with mucocutaneous lesions caused by Leishmania spp. Both dogs presented destruction of the nasal septum, hyperemia with soft palate edema and barking alteration due to laryngeal compromise. Biopsies were taken from the lesion border and Leishmania spp. amastigotes were seen in the imprints. The dogs presented positive serology when complex soluble antigen from Leishmania mexicana was used. One of the dogs was also suspected to be infected by Trypanosoma cruzi as suggested by its positive reaction with a purified specific antigen, Ag163B6-cruzipain. Most of the studies concerning leishmaniosis in dogs have described the cutaneous form of this disease in close association with human cases of Leishmania infection instead of the mucocutaneous form described herein. The presence of dogs with mucocutaneous leishmaniosis alerts on an increase of the prevalence of this form in humans, which can cause deforming lesions, alterations of the speech and even an inadequate nutrition due to difficulties in deglutition.(AU)

[Zoonotic parasitosis transmitted by dogs in the Chaco Salteño, Argentina]. / Parasitosis zoonóticas transmitidas por perros en el Chaco Salteño.

Toxocara canis and Ancylostoma spp. are geohelmints that parasites dogs and can eventually affect humans, mainly children, causing visceral and cutaneous larva migrans respectively, constituting a serious public health problem. This study was carried out in two towns located in the xerophilous forest Chaco salteño where humans live closely with many animals, including dogs. Hematological values and anti-Toxocara canis antibodies, determined by ELISA in serum, were evaluated in 98 children from this area. Thirty-six children presented with eosinophilia of 10% or higher in peripheral blood. Twenty out of 98 (20.4%) children had antibodies against antigen from L2 larvae of Toxocara canis. A high percentage (55.6%) of the children with eosinophilia presented anti-Toxocara canis antibodies. Nine children had multiple serpiginous lesions typical of cutaneous larva migrans. Feces from dogs were collected in the area where children lived, in order to search for parasite contamination. Three different techniques of stool examination were employed and eggs were counted. Out of the 106 feces examined, parasites were found in 82 samples (77.4%). Ancylostoma spp eggs were found in 74 (69.8%) samples and eggs from Toxocara canis were found in 19 (17.2%). The average number of T. canis and Ancylostoma spp eggs/gr of feces, were 200 and 3,871 respectively. Giardia spp (14.5%), Trichuris vulpis (7.6%), Genus Endamoeba (2.8%) and Taenia spp (1.9%) were also identified in the stools. Sanitary control and health education in order to control these parasitoses are emphasized.

Parasitosis zoonóticas transmitidas por perros en el Chaco Salteño. / [Zoonotic parasitosis transmitted by dogs in the Chaco Salteño, Argentina]

Toxocara canis and Ancylostoma spp. are geohelmints that parasites dogs and can eventually affect humans, mainly children, causing visceral and cutaneous larva migrans respectively, constituting a serious public health problem. This study was carried out in two towns located in the xerophilous forest Chaco salteño where humans live closely with many animals, including dogs. Hematological values and anti-Toxocara canis antibodies, determined by ELISA in serum, were evaluated in 98 children from this area. Thirty-six children presented with eosinophilia of 10

or higher in peripheral blood. Twenty out of 98 (20.4

) children had antibodies against antigen from L2 larvae of Toxocara canis. A high percentage (55.6

) of the children with eosinophilia presented anti-Toxocara canis antibodies. Nine children had multiple serpiginous lesions typical of cutaneous larva migrans. Feces from dogs were collected in the area where children lived, in order to search for parasite contamination. Three different techniques of stool examination were employed and eggs were counted. Out of the 106 feces examined, parasites were found in 82 samples (77.4

). Ancylostoma spp eggs were found in 74 (69.8

) samples and eggs from Toxocara canis were found in 19 (17.2

). The average number of T. canis and Ancylostoma spp eggs/gr of feces, were 200 and 3,871 respectively. Giardia spp (14.5

), Trichuris vulpis (7.6

), Genus Endamoeba (2.8

) and Taenia spp (1.9

) were also identified in the stools. Sanitary control and health education in order to control these parasitoses are emphasized.

Leishmaniosis mucocutánea en perros naturalmente infectados en Salta, Argentina. / [Mucocutaneous leishmaniasis in naturally infected dogs in Salta, Argentina]

The objective of the present study is to describe two cases of dogs with mucocutaneous lesions caused by Leishmania spp. Both dogs presented destruction of the nasal septum, hyperemia with soft palate edema and barking alteration due to laryngeal compromise. Biopsies were taken from the lesion border and Leishmania spp. amastigotes were seen in the imprints. The dogs presented positive serology when complex soluble antigen from Leishmania mexicana was used. One of the dogs was also suspected to be infected by Trypanosoma cruzi as suggested by its positive reaction with a purified specific antigen, Ag163B6-cruzipain. Most of the studies concerning leishmaniosis in dogs have described the cutaneous form of this disease in close association with human cases of Leishmania infection instead of the mucocutaneous form described herein. The presence of dogs with mucocutaneous leishmaniosis alerts on an increase of the prevalence of this form in humans, which can cause deforming lesions, alterations of the speech and even an inadequate nutrition due to difficulties in deglutition.

Polymerase chain reaction reveals Trypanosoma cruzi infection suspected by serology in cutaneous and mucocutaneous leishmaniasis patients.

The existence of patients suffering a double infection caused by Trypanosoma cruzi and Leishmania spp. has been suggested by several authors. Since the conventional serological tests now available for the diagnosis of Chagas' disease lack specificity due to the cross-reactivity between these two parasites, a serological confirmation of a T. cruzi infection cannot be made unless specific antigens are used. An enzyme linked immunosorbent assay (ELISA) to detect antibodies against a specific T. cruzi antigen, named Ag163B6, and immunoblotting using T. cruzi epimastigotes, are non-conventional serological techniques that could be employed for specific diagnosis of Chagas' disease. Using these two methods 34 cutaneous or mucocutaneous leishmaniasis patients were classified into two groups: (A) patients with serological evidence of T. cruzi infection, i.e. those who tested positive in at least one assay (18/34); and (B) patients with no serological evidence of T. cruzi infection, i.e. those who were negative for both assays (16/34). Taking into account the difficulties of xenodiagnosis and its low sensitivity (less than 50%) for a direct diagnosis in the chronic period of the disease, we used polymerase chain reaction (PCR) to confirm a T. cruzi infection in those leishmaniasis patients who presented positive results with the non-conventional serological techniques. Of the 18 patients with serological evidence of T. cruzi infection, 17 gave positive results when genomic DNA primers were used. Using minicircle primers, 15/18 of that group were positive. Nevertheless, all the patients suspected of being double infected were positive in at least one PCR test. Just one patient with no serological evidence of T. cruzi infection gave a positive PCR result when amplifying the minicircle sequence. The proof of the existence of a T. cruzi infection by PCR in leishmaniasis patients suspected to be chagasic when non-conventional serology was used, strongly supports the use of the specific Ag163B6 and immunoblotting with epimastigotes as specific serological diagnostic tools to determine a T. cruzi infection.

Trypanosoma cruzi and Leishmania spp. human mixed infection.

The occurrence of leishmaniasis patients carrying a double infection with Trypanosoma cruzi has been suspected but not proved. In this study, we analyzed sera of leishmaniasis patients from a region endemic for both parasites by using immunoblotting with epimastigotes and a purified antigen specific for T. cruzi (Ag 163B6). Seven of 12 patients showed a pattern of bands characteristic of chagasic patients reacting with antigens with molecular weights of 131, 125, 116, 111, 51-45, and 43 kD, and positive reactivity with Ag 163B6. Xenodiagnosis for T. cruzi was carried out in all patients; this technique has a positivity rate of 50% in chronic chagasic patients. The presence of T. cruzi trypomastigotes was shown in the blood of three, thus confirming the existence of a double infection in humans. Since the two parasites possess cross-reacting antigens, it may be assumed that previous infection with one of the parasites may affect the course of subsequent infection with the other. Nevertheless, T. cruzi infection did not prevent the appearance of typical leishmaniasis lesions. Therefore, antigenic cross-reactivity is unable to induce a sterilizing immune response against Leishmania.

[Study of cases of leishmaniasis in the Province of Salta: evidences of mixed infection with Trypanosoma cruzi and Leishmania spp]. / Estudio de casos de leishmaniasis en la provincia de Salta. Evidencias de infección mixta por trypanosoma cruzi y leishmania spp.

In many regions of South America there are overlapping endemic areas for American Trypanosomiasis (Chagas' disease) and Leishmaniasis. T. cruzi and Leishmania spp, the causative agents of these parasitoses belong to the Trypanosomatidae family and share various antigens that cause cross-reactivity in serological diagnosis when complex antigenic mixtures are used. We studied patients who sought medical attention because of cutaneous or mucocutaneous lesions typical of leishmaniasis infection. These patients were from the province of Salta where Trypanosomiasis and Leishmaniasis are endemic diseases. Sixty-two patients gave a positive Montenegro skin test and, of these, 53 (85, 48%) showed the presence of amastigotes in Giemsa stained smears of dermal scrapings. Seven patients were not included because they were negative for both assays. We analyzed the leishmaniasic sera against homologous antigens to study the immune response and against complex heterologous antigens from T. cruzi to evaluate cross-reactivity phenomena. We also tested these sera against specific antigens for diagnosis of Chagas' disease in order to search for mixed infections. When complex antigens from leishmania were used, the sera showed an unusually strong antibody response 100% positive by IFA, 88.7% by ELISA and 80.6% by immunoblotting. Furthermore, significant cross-reactivity was found when conventional antigens for the serodiagnosis of Chagas' disease were used: 74.19% by IHA, 91.93% by IFA, and 76.80% by ELISA. We have previously purified by immunoaffinity, using a monoclonal antibody, an antigen termed Ag163B6 which is not present in L. mexicana. This antigen has shown the ability to specifically differentiate sera of chronic chagasic patients from those of leishmaniasic patients in ELISA. Furthermore, recent studies from our laboratory by immunoblotting, have demonstrated that chronic chagasic patients exhibit a specific reactivity pattern against T. cruzi epimastigotes that can be distinguished from those presented by leishmaniasic patients in spite of cross-reactive antigens. According to the results obtained in these assays, we classified the patients in two groups: 1) Patients with evidence of T. cruzi infection, those who tested positive in at least one assay: 2) Patients with no evidence of T. cruzi infection who were negative for both assays. More than 50% (32/62) of the patients showed strong evidence of mixed infection with T. cruzi. On the other hand, high cross-reactivity between these two parasitoses was shown in the second group without any evidence of T. cruzi infection since 18 out of 30 were positive in at least two conventional serological reactions. This implies that they would be misdiagnosed as chagasics if conventional reactions were used. These results emphasize the importance of the use of defined antigens and appropriate techniques for the differential diagnosis of these parasitoses, which is more important in areas endemic for both of them.

[Strongyloides stercoralis. Report of a case and review of the literature]. / Strongyloides stercoralis. Reporte de un caso y revisión de la literatura.

A fatal case of strongyloidiasis in a boy aged 8 months, referred to our hospital, with severe malnutrition, dehydration, brownish fluid through the naso-gastric tube and relapsing chronic diarrhea with bloody mucus. Through all symptoms and observation of rabditoid larvae, eggs and even adult females, parasitologic diagnosis was achieved. In spite of medical intensive care the patient died after 18 days of hospitalization. Disease's pathophysiology is discussed as well as a review about the trustfulness of the parasite biological cycle as is traditionally described in the literature. Ability is questioned of rabditoid larvae to follow the alternative way as free living adults or parasitic living in adults.

Strongyloides stercoralis. Reporte de un caso y revisión de la literatura. / [Strongyloides stercoralis. Report of a case and review of the literature]

A fatal case of strongyloidiasis in a boy aged 8 months, referred to our hospital, with severe malnutrition, dehydration, brownish fluid through the naso-gastric tube and relapsing chronic diarrhea with bloody mucus. Through all symptoms and observation of rabditoid larvae, eggs and even adult females, parasitologic diagnosis was achieved. In spite of medical intensive care the patient died after 18 days of hospitalization. Diseases pathophysiology is discussed as well as a review about the trustfulness of the parasite biological cycle as is traditionally described in the literature. Ability is questioned of rabditoid larvae to follow the alternative way as free living adults or parasitic living in adults.

Cross-reactivity studies and differential serodiagnosis of human infections caused by Trypanosoma cruzi and Leishmania spp; use of immunoblotting and ELISA with a purified antigen (Ag163B6).

Results of our studies on the reactivity of chagasic and leishmaniasic sera with the purified T. cruzi-specific antigen 163B6, as assessed by ELISA, and with complex antigenic mixtures from T. cruzi and Leishmania mexicana, by immunoblotting, are presented here. Our objective was to identify the antigens responsible for the exhibited cross-reactivity between trypanosomiasis and leishmaniasis, and to find a specific reactivity pattern corresponding to each parasitosis. In spite of the high cross-reactivity observed with the immunoblotting, the use of 7.5% A-B gels made it possible to identify a characteristic pattern for each parasitosis, that could be distinguished by the naked eye. The characteristic pattern corresponding to chagasic patients was ascribed to reactivity with T. cruzi bands of mol. wts 131, 125, 116, 111, 51-45 and 43 kD, that were not recognized by leishmaniasic sera. Trypanosoma cruzi antigens of mol. wts 85, 81, 70, 65-60, 37 and 32 kD were considered as crossing antigens, since they were recognized by leishmaniasis sera. With L. mexicana, most of the chagasic patients presented reaction with antigen of mol. wts 124, 107, 92, 59 and 32 kD, while bands of mol. wts 155, 140, 73, 56 and 48 kD were recognized only by leishmaniasic sera. In this study we found 12 out of 45 sera of patients with leishmaniasis, from a region endemic for both parasitoses, which exhibited a pattern of bands very similar to those corresponding to chagasic individuals, strongly suggesting a mixed infection. This hypothesis was verified by using a purified specific antigen of T. cruzi, Ag163B6, which would be the major cysteine proteinase of this specie (cruzipain). By ELISA, these 12 sera showed a positive reaction with this purified antigen, as those of chagasic patients, thus leading to the confirmation of the presence of a mixed infection.

Clinical healing of antimony-resistant cutaneous or mucocutaneous leishmaniasis following the combined administration of interferon-gamma and pentavalent antimonial compounds.

In an open trial, longer courses of pentavalent antimonials (Sbv) at sub-optimal doses (10 mg/kg body weight), in association with recombinant human interferon-gamma (IFN-gamma) (100 micrograms/m2 of body surface area) were administered, by daily intramuscular injections, to 13 patients with diagnoses of cutaneous or mucocutaneous leishmaniasis unresponsive to Sbv. Four patients presented with large skin ulcers, and 9 had mucosal involvement as the main manifestation, the latter affecting the nose (3 cases), nose and septum (2 cases), nose and oral cavity (1 case), and nose, pharynx and larynx (3 cases). Except for one case with severe involvement of the upper respiratory tract, the lesions were fully resolved by the end of therapy (mean duration 40 +/- 12 [SD] d, range 30-60 d) in the 11 patients who completed therapy. The main side effects were headache and fever (7 cases), together with leucopenia and eosinophilia (4 cases). It is concluded that combined administration of low doses of Sbv plus IFN-gamma may provide a novel therapeutic approach for the treatment of antimony-resistant cutaneous or mucocutaneous leishmaniasis. The possible mechanisms by which IFN-gamma contributes to resolution of the disease are discussed.

[Prevalence of Strongyloides stercoralis infection in childhood. Orán, Salta, Argentina]. / Prevalencia de la infección por Strongyloides stercoralis en la primera infancia. Orán, Salta, Argentina.

An enteroparasitological, clinical and epidemiological study was carried out in 36 infants who attended the Hospital San Vicente de Paul from Orán for medical assistance. The age of the children varied between 0 and 4 years with an average of 28 months. The main clinical causes for consultation were bronchitis (52.8%), inappetence (52.8%) and paleness (27.8%). The general characteristics of the stool samples were: formed 44.4% and diarrheic 55.6%. The frequency of intestinal helminthic and protozoan elements was the following: S. stercoralis 30(83.3%) N. americanus 5(13.9%), A. lumbricoides 7(19.4%), T. trichiura 3(8.3%), H. nana 5(13.9%), G. intestinalis 10(27.8%), I. belli 1 (2.8%) and E. coli 4 (11.1%). The nutritional condition found in the infants, according to the relation high/weight was: eutrophic 17(42.2%), lightly undernourished 9(20.0%), moderately undernourished 9(25.0%) and severely undernourished 1(2.8%). The mean values of hematocrit and hemoglobin found were under those established as minima by the W.H.O. The white blood cells and eosinophils countings were over the normal mean values.

[Detection of a focus of microfilariasis in northeastern Argentina]. / Detección de un foco de microfilariasis en el noroeste argentino.

A focus of microfilariasis was detected in El Oculto, Orán Department, 300 km northern from Salta city. These results prove the prevalence of this parasitosis in Argentina, despite the lack of cases registered during the last 35 years. The incidence detected was 20.7%; similar values were reported in 1917 by Biglieri and Aráoz in a wooded area located in the oriental face of Aconquija mountain, in Tucuman province, approximately 600 km southern from El Oculto. The results presented suggest the interruption in the transmission of this parasitosis.