Correction: Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum.

[This corrects the article DOI: 10.1371/journal.pone.0247560.].

The conventional diagnosis challenge: Real-time PCR and nested PCR correlation with the scoring system for individuals at high-risk of Pneumocystis jirovecii pneumonia. / Desafío diagnóstico: PCR anidada y en tiempo real frente a un sistema de puntuación en individuos con gran riesgo de neumonía por Pneumocystis jirovecii

INTRODUCTION: Pneumocystis jirovecii is an opportunistic fungus that affects mainly people living with HIV (CD4 cell count lower than 200 cells/ml) and other immunosuppressed patients. Since P. jirovecii does not grow on routine mycological media, diagnosis of P. jirovecii pneumonia relies on indirect evidence of its presence in respiratory samples. OBJECTIVES: To associate the results of direct immunofluorescence and two molecular methods with a score to predict P. jirovecii pneumonia in patients with AIDS. MATERIALS AND METHODS: A prospective study was conducted with 40 patients. A respiratory sample collected before treatment was subjected to direct immunofluorescence using the Merifluor kit, to nested PCR targeting the mitochondrial large subunit ribosomal RNA, and to the VIASURE real-time PCR kit. RESULTS: These three techniques revealed P. jirovecii in 6, 12, and 15 samples, respectively. All positive samples by direct immunofluorescence were positive by nested PCR, and all positive samples by nested PCR amplified by real-time PCR. There was a statistically significant association between the P. jirovecii pneumonia score and the molecular methods. Two patients were early diagnosed and responded well to treatment. CONCLUSION: Molecular methods, especially real-time PCR, are recommended for early diagnosis of P. jirovecii pneumonia in AIDS patients.

Introducción: Pneumocystis jirovecii es un hongo oportunista que afecta principalmente a personas con HIV (recuento de CD4 menor de 200 células/ml) y a otros pacientes inmunosuprimidos. Como P. jirovecii no crece en los medios micológicos de rutina, el diagnóstico de neumonía por P. jirovecii se basa en la evidencia presente en muestra respiratorias. Objetivos: Asociar los resultados de la inmunofluorescencia directa y los de dos métodos moleculares con un puntaje para predecir la neumonía causada por P. jirovecii en pacientes con sida. Materiales y métodos: Se realizó un estudio prospectivo de 40 pacientes. Se recolectó una muestra respiratoria antes del inicio de tratamiento y se sometió a una prueba de inmunofluorescencia directa con el kit Merifluor, una PCR anidada para la amplificación de la subunidad larga del ribosoma mitocondrial y una PCR en tiempo real usando el kit VIASURE Resultados: Estas tres técnicas evidenciaron la presencia de P. jirovecii en 6, 12 y 15 muestras, respectivamente. Todas las muestras positivas por inmunofluorescencia directa fueron positivas en la PCR anidada y todas las muestras positivas en la PCR anidada amplificaron por PCR en tiempo real. Se encontró una asociación estadística entre los valores de la neumonía causada por P. jirovecii y los métodos moleculares. Dos pacientes con diagnóstico temprano respondieron satisfactoriamente al tratamiento. Conclusión: Se recomiendan los métodos moleculares, especialmente la PCR en tiempo real, para el diagnóstico temprano de neumonía causada por P. jirovecii en pacientes con sida.

The conventional diagnosis challenge: Real-time PCR and nested PCR correlation with the scoring system for individuals at high-risk of Pneumocystis jirovecii pneumonia

Introduction. Pneumocystis jirovecii is an opportunistic fungus that affects mainly people living with HIV (CD4 cell count lower than 200 cells/ml) and other immunosuppressed patients. Since P. jirovecii does not grow on routine mycological media, diagnosis of P. jirovecii pneumonia relies on indirect evidence of its presence in respiratory samples. Objectives. To associate the results of direct immunofluorescence and two molecular methods with a score to predict P. jirovecii pneumonia in patients with AIDS. Materials and methods. A prospective study was conducted with 40 patients. A respiratory sample collected before treatment was subjected to direct immunofluorescence using the Merifluor kit, to nested PCR targeting the mitochondrial large subunit ribosomal RNA, and to the VIASURE real-time PCR kit. Results. These three techniques revealed P. jirovecii in 6, 12, and 15 samples, respectively. All positive samples by direct immunofluorescence were positive by nested PCR, and all positive samples by nested PCR amplified by real-time PCR. There was a statistically significant association between the P. jirovecii pneumonia score and the molecular methods. Two patients were early diagnosed and responded well to treatment. Conclusion. Molecular methods, especially real-time PCR, are recommended for early diagnosis of P. jirovecii pneumonia in AIDS patients.

Introducción. Pneumocystis jirovecii es un hongo oportunista que afecta principalmente a personas con HIV (recuento de CD4 menor de 200 células/ml) y a otros pacientes inmunosuprimidos. Como P. jirovecii no crece en los medios micológicos de rutina, el diagnóstico de neumonía por P. jirovecii se basa en la evidencia presente en muestras respiratorias. Objetivos. Asociar los resultados de la inmunofluorescencia directa y los de dos métodos moleculares con un puntaje para predecir la neumonía causada por P. jirovecii en pacientes con sida. Materiales y métodos. Se realizó un estudio prospectivo de 40 pacientes. Se recolectó una muestra respiratoria antes del inicio de tratamiento y se sometió a una prueba de inmunofluorescencia directa con el kit Merifluor, una PCR anidada para la amplificación de la subunidad larga del ribosoma mitocondrial y una PCR en tiempo real usando el kit VIASURE. Resultados. Estas tres técnicas evidenciaron la presencia de P. jirovecii en 6, 12 y 15 muestras, respectivamente. Todas las muestras positivas por inmunofluorescencia directa fueron positivas en la PCR anidada y todas las muestras positivas en la PCR anidada amplificaron por PCR en tiempo real. Se encontró una asociación estadística entre los valores de la neumonía causada por P. jirovecii y los métodos moleculares. Dos pacientes con diagnóstico temprano respondieron satisfactoriamente al tratamiento. Conclusión. Se recomiendan los métodos moleculares, especialmente la PCR en tiempo real, para el diagnóstico temprano de neumonía causada por P. jirovecii en pacientes con sida.

Immunohistochemical detection of Treponema pallidum in skin samples with clinical and histopathological correlations and Warthin-Starry staining critical analysis

Abstract Background: Syphilis in its different phases may be a difficult diagnosis in clinical and histopathological grounds. Objectives: The present study objectives were to evaluate the detection and tissue distribution of Treponema pallidum in skin lesions of syphilis. Methods: A blinded diagnostic accuracy study was performed with immunohistochemistry and Warthin-Starry silver staining in skin samples from patients with syphilis and other diseases. Patients attended two tertiary hospitals between 2000 and 2019. Prevalence ratios (PR) and 95% confidence intervals (95% CI) were calculated for the association between immunohistochemistry positivity and clinical-histopathological variables. Results: Thirty-eight patients with syphilis and their 40 biopsy specimens were included in the study. Thirty-six skin samples were used as non-syphilis controls. The Warthin-Starry technique was unable to accurately demonstrate bacteria in all samples. Immunohistochemistry showed spirochetes only in skin samples from patients with syphilis (24/40) with 60% sensitivity (95% CI 44.8-75.2). Specificity was 100% and accuracy, 78.9% (95% CI 69.8-88.1). Most cases had spirochetes in both dermis and epidermis and there was a high bacterial load. Study limitations: Correlation between immunohistochemistry and clinical or histopathological characteristics was observed but was limited statistically due to the small sample size. Conclusions: Spirochetes were promptly seen in an immunohistochemistry protocol, which can contribute to the diagnosis of syphilis in skin biopsy samples. On the other hand, the Warthin-Starry technique showed to be of no practical value.

Immunohistochemical detection of Treponema pallidum in skin samples with clinical and histopathological correlations and Warthin-Starry staining critical analysis.

BACKGROUND: Syphilis in its different phases may be a difficult diagnosis in clinical and histopathological grounds. OBJECTIVES: The present study objectives were to evaluate the detection and tissue distribution of Treponema pallidum in skin lesions of syphilis. METHODS: A blinded diagnostic accuracy study was performed with immunohistochemistry and Warthin-Starry silver staining in skin samples from patients with syphilis and other diseases. Patients attended two tertiary hospitals between 2000 and 2019. Prevalence ratios (PR) and 95% confidence intervals (95% CI) were calculated for the association between immunohistochemistry positivity and clinical-histopathological variables. RESULTS: Thirty-eight patients with syphilis and their 40 biopsy specimens were included in the study. Thirty-six skin samples were used as non-syphilis controls. The Warthin-Starry technique was unable to accurately demonstrate bacteria in all samples. Immunohistochemistry showed spirochetes only in skin samples from patients with syphilis (24/40) with 60% sensitivity (95% CI 44.8‒75.2). Specificity was 100% and accuracy, 78.9% (95% CI 69.8‒88.1). Most cases had spirochetes in both dermis and epidermis and there was a high bacterial load. STUDY LIMITATIONS: Correlation between immunohistochemistry and clinical or histopathological characteristics was observed but was limited statistically due to the small sample size. CONCLUSIONS: Spirochetes were promptly seen in an immunohistochemistry protocol, which can contribute to the diagnosis of syphilis in skin biopsy samples. On the other hand, the Warthin-Starry technique showed to be of no practical value.

Comparison between Colorimetric In Situ Hybridization, Histopathology, and Immunohistochemistry for the Diagnosis of New World Cutaneous Leishmaniasis in Human Skin Samples.

New world cutaneous leishmaniasis (NWCL) is an anthropozoonosis caused by different species of the protozoan Leishmania. Colorimetric in situ hybridization (CISH) was shown to satisfactorily detect amastigote forms of Leishmania spp. in animal tissues, yet it was not tested for the diagnosis of human NWCL. The aim of this study was to compare CISH, histopathology (HP), and immunohistochemistry (IHC) techniques to diagnose NWCL in human cutaneous lesions. The sample comprised fifty formalin-fixed, paraffin-embedded skin biopsy specimens from patients with NWCL caused by L. (V.) braziliensis. These specimens were analyzed by CISH, using a generic probe for Leishmania, IHC, and HP to assess the sensitivity of these methods by using a parasitological culture as a standard reference. Additional specimens from three patients diagnosed with cutaneous mycoses were also included to evaluate cross-reactions between CISH and IHC. The sensitivities of IHC, CISH, and HP for detecting amastigotes was 66%, 54%, and 50%, respectively. IHC, unlike CISH, cross-reacted with different species of fungi. Together, these results demonstrate that CISH may be a complementary assay for the detection of amastigote in the laboratorial diagnosis routine of human NWCL caused by L. (V.) braziliensis.

The outcomes of polyparasitism in stray cats from Brazilian Midwest assessed by epidemiological, hematological and pathological data.

We evaluated the epidemiological, hematological, and pathological data of Leishmania spp., Toxoplasma gondii, Platynosomum illiciens, feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV) infections and the coinfections in stray cats of an endemic area for leishmaniasis. The diagnosis was performed by serological tests and necropsy. We described gross lesions and histopathological findings. We used immunohistochemistry and chromogenic in situ hybridization for L. infantum detection. We found infection in 27 out of 50 sampled cats, among them, 14 presented coinfections. A strong correlation between splenomegaly and lymphadenomegaly with FeLV, and an association between hepatic lesions and cachexia with parasitism due to P. illiciens were observed. Moreover, we found a significant increase in the monocyte count in the FeLV-infected and a decrease in the red blood cell count in the FIV-infected animals. Amastigote forms of Leishmania spp. and tissue changes were detected in lymphoid organs of an animal coinfected with P. illiciens, T. gondii, and FIV. Polyparasitism recorded in stray cats of the Brazilian Midwest should be considered in effective control strategies for public health diseases. Moreover, stray cats of Campo Grande may be a source of infection of FIV, FeLV and P. illiciens for populations of domiciled cats.

Frequency of detection and load of amastigotes in the pancreas of Leishmania infantum-seropositive dogs: clinical signs and histological changes.

BACKGROUND: Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and is highly lethal in humans and dogs if left untreated. The frequency of this parasite and associated histological changes in the pancreas of dogs are poorly studied. Therefore, the objectives of this study were to evaluate the frequency of detection and load of amastigotes in the pancreas of L. infantum-seropositive dogs and to identify the clinical signs and histological changes associated with parasitism of this organ. METHODS: One hundred forty-three dogs from an endemic area in Brazil that tested seropositive for L. infantum were studied. The dogs were clinically examined, killed, and necropsied between 2013 and 2014. One fragment of the pancreas was randomly collected for histopathology and immunohistochemistry, and spleen and bone marrow were collected for culture. RESULTS: Leishmania amastigotes were detected in the pancreas of 22 dogs (15.4%) by immunohistochemistry, all exhibiting L. infantum parasitism in the spleen and/or bone marrow. Poor body condition and cachexia were only associated with infection of the pancreas with Leishmania spp. (p = 0.021) and were found in 40.9% of dogs with pancreatic infection. Anorexia, vomiting, and/or diarrhea were observed in 9.2% of dogs with pancreatitis. The median parasite load in the pancreas was 1.4 infected macrophages/mm2. Pancreatic histological changes and their frequencies were: granulomatous pancreatitis (28.0%), lymphoplasmacytic pancreatitis (23.8%), acinar cell degeneration (6.3%), fibrosis (5.6%), hemorrhage (2.1%), eosinophilic pancreatitis (0.7%), suppurative pancreatitis (0.7%), and necrosis (0.7%). CONCLUSIONS: The present results demonstrate that L. infantum is one of the etiological agents of chronic pancreatitis in dogs; however, the frequency of detection and parasite load are low in this organ. The lack of an association of poor body condition and cachexia with pancreatitis and the low frequency of clinical signs commonly associated with pancreatitis suggest that a significant portion of the organ is not affected by this parasite. On the other hand, the association of poor body condition and cachexia with concomitant infection of the pancreas, spleen, and/or bone marrow with this parasite suggests that these manifestations are the result of a more advanced stage of canine visceral leishmaniasis.

Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum.

In canine leishmaniosis caused by the protozoan Leishmania infantum, little is known about how co-infections with or co-seropositivities for other pathogens can influence aggravation of this disease. Therefore, the objectives of this study were to evaluate the frequency of co-infections with or co-seropositivities for certain pathogens in dogs seropositive for L. infantum and their relationship with clinical signs, histological changes and L. infantum load. Sixty-six L. infantum-seropositive dogs were submitted to clinical examination, collection of blood and bone marrow, culling, and necropsy. Antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato, Ehrlichia spp. and Toxoplasma gondii and Dirofilaria immitis antigens were investigated in serum. Samples from different tissues were submitted to histopathology and immunohistochemistry for the detection of Leishmania spp. and T. gondii. Quantitative real-time PCR was used to assess the L. infantum load in spleen samples. For detection of Coxiella burnetii, conventional PCR and nested PCR were performed using bone marrow samples. All 66 dogs tested positive for L. infantum by qPCR and/or culture. Fifty dogs (76%) were co-seropositive for at least one pathogen: T. gondii (59%), Ehrlichia spp., (41%), and Anaplasma spp. (18%). Clinical signs were observed in 15 (94%) dogs monoinfected with L. infantum and in 45 (90%) dogs co-seropositive for certain pathogens. The L. infantum load in spleen and skin did not differ significantly between monoinfected and co-seropositive dogs. The number of inflammatory cells was higher in the spleen, lung and mammary gland of co-seropositive dogs and in the mitral valve of monoinfected dogs. These results suggest that dogs infected with L. infantum and co-seropositive for certain pathogens are common in the region studied. However, co-seropositivities for certain pathogens did not aggravate clinical signs or L. infantum load, although they were associated with a more intense inflammatory reaction in some organs.

Genetic and histopathological characterization of Toxoplasma gondii genotypes isolated from free-range chickens reared in the metropolitan region of Rio de Janeiro state, Brazil.

This study aimed to genetically characterize Toxoplasma gondii isolates obtained from free-range chickens reared in the metropolitan region of the state of Rio de Janeiro, Brazil, and to evaluate the morbidity and histological changes associated with these isolates in mice. A mouse bioassay was used to isolate T. gondii from a pool of tissue samples (brain, heart, and thigh muscles) collected from 163 chickens. The 36 isolates obtained were genetically characterized by restriction fragment polymorphism (PCR-RFLP) analysis of the SAG1, 5'-3'SAG2, aSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3 genomic regions. Seventeen atypical genotypes were identified and nine of them were reported for the first time. All identified genotypes caused clinical signs and histological changes in mice, with the majority being associated with high cumulative morbidity (65%) and severe or very severe histological changes (76%). The exclusive identification of atypical genotypes, with a predominance of new genotypes, indicates great genetic diversity of T. gondii in the region studied. In addition, the finding that all identified genotypes caused clinical signs and often severe histological changes in mice suggests potentially relevant virulence of these strains.

Frequency, active infection and load of Leishmania infantum and associated histological alterations in the genital tract of male and female dogs.

Visceral leishmaniasis caused by the protozoan Leishmania infantum is a zoonosis. The domestic dog is the primary reservoir in urban areas. This study aimed to evaluate the frequency, active infection and load of L. infantum in the genital tract of male and female dogs seropositive for this parasite, as well as to identify histological genital alterations associated with this protozoan. We studied 45 male and 25 female L. infantum-seropositive noncastrated dogs from the same endemic area in Brazil. Tissue samples from the testis, epididymis, prostate, vulva, vagina, and uterus were examined by singleplex qPCR and parasitological tests (histopathology, immunohistochemistry, and parasitological culture). The latter were performed for the detection of active infection (parasites able to multiply and to induce lesions). Forty-four (98%) males and 25 (100%) females were positive for L. infantum in the genital tract (epididymis: 98%; vulva: 92%; vagina: 92%; testis: 91%; uterus: 84%; prostate: 66%). Active infection in the genital tract was confirmed in 69% of males and 64% of females (32% in the uterus). Parasite loads were similar in the testis, vulva, epididymis and vagina and lower in the prostate. Only the parasite load in the vagina was significantly associated with the number of clinical signs. Granulomatous inflammation predominated in all organs, except for the prostate. Only in the testis and epididymis was the inflammatory infiltrate significantly more intense among dogs with a higher parasite load in these organs. The high frequency, detection of active infection and similarity of L. infantum loads in the genital tract of infected males and females suggest the potential of venereal transmission of this parasite by both sexes and of vertical transmission by females in the area studied. Additionally, vertical transmission may be frequent since active L. infantum infection was a common observation in the uterus.

Validation of an algorithm based on clinical, histopathological and immunohistochemical data for the diagnosis of early-stage mycosis fungoides

Abstract Background: Diagnosis of mycosis fungoides is challenging due to the non-specificity of clinical and histopathological findings. The literature indicates an average delay of 4-6 years for a conclusive diagnosis. Refinement of the histopathological criteria for the diagnosis of patients in early stages of the disease is considered of interest. Objectives: To study the histopathological aspects of early-stage mycosis fungoides and the applicability, in a retrospective form, of the diagnostic algorithm proposed by Pimpinelli et al. Methods: Observational, retrospective, transversal study based on revision of histopathological exams of patients with suspected mycosis fungoides. Medical records were reviewed, and complementary immunohistochemistry performed. Results: Sixty-seven patients were included. The most frequent histopathological features were superficial perivascular lymphoid infiltrate (71.6%), epidermotropism (68.7%), lymphocytic atypia (63.8%), hyperkeratosis (62.7%) and acanthosis (62.7%). Forty-three patients scored 4 points at the algorithm, by clinical and histological evaluation. Immunohistochemistry was performed on 23 of the 24 patients with less than 4 points. Of those 23, 22 scored 1 point, allowing a total of 61 patients (91%) with the diagnosis of early-stage mycosis fungoides. Study limitations: Its retrospective character, reduced sample size and incomplete application of the algorithm. Conclusions: Application of the Pimpinelli et al. algorithm, even in an incomplete form, increased the percentage of cases diagnosed as mycosis fungoides. Routine application of the algorithm may contribute to earlier and specific management and improvement of the patients' outcome.

Validation of an algorithm based on clinical, histopathological and immunohistochemical data for the diagnosis of early-stage mycosis fungoides.

BACKGROUND: Diagnosis of mycosis fungoides is challenging due to the non-specificity of clinical and histopathological findings. The literature indicates an average delay of 4-6 years for a conclusive diagnosis. Refinement of the histopathological criteria for the diagnosis of patients in early stages of the disease is considered of interest. OBJECTIVES: To study the histopathological aspects of early-stage mycosis fungoides and the applicability, in a retrospective form, of the diagnostic algorithm proposed by Pimpinelli et al. METHODS: Observational, retrospective, transversal study based on revision of histopathological exams of patients with suspected mycosis fungoides. Medical records were reviewed, and complementary immunohistochemistry performed. RESULTS: Sixty-seven patients were included. The most frequent histopathological features were superficial perivascular lymphoid infiltrate (71.6%), epidermotropism (68.7%), lymphocytic atypia (63.8%), hyperkeratosis (62.7%) and acanthosis (62.7%). Forty-three patients scored 4 points at the algorithm, by clinical and histological evaluation. Immunohistochemistry was performed on 23 of the 24 patients with less than 4 points. Of those 23, 22 scored 1 point, allowing a total of 61 patients (91%) with the diagnosis of early-stage mycosis fungoides. STUDY LIMITATIONS: Its retrospective character, reduced sample size and incomplete application of the algorithm. CONCLUSIONS: Application of the Pimpinelli et al. algorithm, even in an incomplete form, increased the percentage of cases diagnosed as mycosis fungoides. Routine application of the algorithm may contribute to earlier and specific management and improvement of the patients' outcome.

First description of parasite load and clinicopathological and anatomopathological changes in a dog naturally coinfected with Dioctophyme renale and Leishmania infantum in Brazil.

This article reports the case of a domestic dog naturally coinfected with the nematode Dioctophyme renale and with the protozoan Leishmania infantum. The dog exhibited no clinical signs but had normocytic hypochromic anemia, hyperproteinemia, hyperglobulinemia, hypoalbuminemia, and hematuria. Necropsy revealed eight D. renale specimens in the abdominal cavity and in right kidney whose parenchyma was atrophied. Histopathological analysis showed glomerular atrophy, fibrosis and a marked diffuse pyogranulomatous inflammatory infiltrate in the right kidney. Moderate multifocal granulomatous peritonitis was observed in the greater omentum. Several Dioctophyme renale eggs were present amidst the inflammatory infiltrate of the right kidney and greater omentum. Leishmania infantum parasites were detected in perirenal adipose tissue of the right kidney, greater omentum, spleen, bone marrow, and popliteal lymph node. The high D. renale load and the severe and uncommon histological alterations associated with the eggs of this parasite may have been influenced by coinfection with L. infantum.

Gait variability and symmetry assessment with inertial sensors for quantitative discrimination of Trendelenburg sign in total hip arthroplasty patients: a pilot study based on convenience sampling

Abstract Introduction The aim of this pilot study based on convenience sampling was to analyze the feasibility to quantitatively discriminate Trendelenburg sign (TS), a characteristic drop in pelvic position during gait in hip disfunctions, in patients with total hip arthroplasty (THA), by assessing gait variability and symmetry using inertial sensors. Methods Thirteen patients with right THA, divided into two groups with (GTS, n=4) and without TS (GnTS, n=9) assessed by experienced physician, were enrolled in the study. Harris Hip Score was applied for specific evaluation of THA. The protocol consisted in walking on a level treadmill during 3 minutes with two inertial sensors attached at anterior superior iliac spine of both sides. For each left and right step, features were extracted from the Y-axis gyroscope signals: peak value, mean absolute value, standard deviation and range. For each feature, a symmetry ratio was calculated as the ratio between left and right side. Results No significant differences were found in Harris Hip Score between groups. The variability assessed by standard deviation for left step, contralateral to the replaced side, was significantly larger for GTS group (p<0.001). Significant differences in the symmetry ratios were found between GTS and GnTS for all features extracted from gyroscopes Y-axis (W=144, p<0.001). The symmetry ratios for GnTS group were approximately equal one (except for range), whereas for the GTS group they exceed the 10% criterion. Conclusion The variability and symmetry ratios of gait features extracted from inertial sensors were successful to discriminate TS in THA patients.

Occurrence of Leishmania infantum in the central nervous system of naturally infected dogs: Parasite load, viability, co-infections and histological alterations.

Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and little is known about the occurrence and pathogenesis of this parasite in the CNS. The aims of this study were to evaluate the occurrence, viability and load of L. infantum in the CNS, and to identify the neurological histological alterations associated with this protozoan and its co-infections in naturally infected dogs. Forty-eight Leishmania-seropositive dogs from which L. infantum was isolated after necropsy were examined. Cerebrospinal fluid (CSF) samples were analyzed by parasitological culture, quantitative real-time PCR (qPCR) and the rapid immunochromatographic Dual Path Platform test. Brain, spinal cord and spleen samples were submitted to parasitological culture, qPCR, and histological techniques. Additionally, anti-Toxoplasma gondii and anti-Ehrlichia canis antibodies in serum and distemper virus antigens in CSF were investigated. None of the dogs showed neurological signs. All dogs tested positive for L. infantum in the CNS. Viable forms of L. infantum were isolated from CSF, brain and spinal cord in 25% of the dogs. Anti-L. infantum antibodies were detected in CSF in 61% of 36 dogs. Inflammatory histological alterations were observed in the CNS of 31% of the animals; of these, 66% were seropositive for E. canis and/or T. gondii. Amastigote forms were associated with granulomatous non-suppurative encephalomyelitis in a dog without evidence of co-infections. The highest frequency of L. infantum DNA was observed in the brain (98%), followed by the spinal cord (96%), spleen (95%), and CSF (50%). The highest L. infantum load in CNS was found in the spinal cord. These results demonstrate that L. infantum can cross the blood-brain barrier, spread through CSF, and cause active infection in the entire CNS of dogs. Additionally, L. infantum can cause inflammation in the CNS that can lead to neurological signs with progression of the disease.

Occurrence of Leishmania infantum and associated histological alterations in the genital tract and mammary glands of naturally infected dogs.

The objectives of this study were to evaluate the occurrence of Leishmania infantum in the male and female genital tract and female mammary glands of dogs and the parasite burden and to identify histological alterations associated with this protozoan. Twenty male and 20 female Leishmania-seropositive dogs with isolation of L. infantum were examined. Tissue samples of the prepuce, glans, epididymis, testes, prostate, vulva, vagina, uterus, uterine tubes, and mammary glands were analyzed by immunohistochemistry and histopathology. For parasitological culture and in situ hybridization, samples were collected from the testis, epididymis, and uterus. Additionally, seminal fluid was aspirated from the epididymis for parasitological culture. In the genital tract, 34 (85 %) dogs, including 18 males and 16 females, were positive for Leishmania. Of these, 27 (79 %) animals were symptomatic. Leishmania was detected in the mammary glands of 13 (65 %) females. L. infantum was isolated for the first time from the seminal fluid and uterus of naturally infected dogs. The parasite burden and intensity of the inflammatory reaction were greater in the prepuce and glans of males and in the vulva and mammary glands of females. In addition to inflammation, testicular degeneration, atrophy, absence of spermatogenesis, and necrosis were observed. Detection of amastigote forms in the mammary gland lumen indicates possible elimination of this parasite in milk. The frequent parasitism observed in the genital tract of infected males and females and the viability of L. infantum in seminal fluid and uterus suggest the possibility of bidirectional venereal and vertical transmission.

Gastrointestinal parasites of swine raised in different management systems in the State of Rio de Janeiro, Brazil / Parasitos gastrintestinais de suínos criados sob diferentes sistemas de manejo no Estado do Rio de Janeiro

From 2012 to 2013 were surveyed gastrointestinal parasites from pig farms located in different municipalities in the state of Rio de Janeiro. Fecal samples from 790 pigs were collected from the rectum on 88 family farms and 702 farms with industrial production. The samples were subjected to Faust et al., Sheather, Ritchie, Lutz and direct examination faecal techniques. The estimated parasite prevalence was 93.1% in family farms and 59.1% in industrial farms. Balantidium coli, coccidia and Entamoeba sp. were the parasites with the highest frequencies, and the male and female reproductive categories and fatteners pigs the most infected (p<0.05). Trophozoites of B. coli were most evident in stool samples from semi-solid followed by solid and diarrheal consistencies. Strongyles eggs and Trichuris suis have been detected exclusively in family farms. Ascaris suum eggs and Strongyloides ransomi showed low frequency. The high degree of parasitism, especially protozoa, indicates the need to reassess the management of pigs in both types of production...

De 2012 a 2013 foram pesquisados parasitos gastrintestinais de suínos de granjas localizadas em diferentes municípios do estado do Rio de Janeiro. Amostras fecais de 790 suínos foram coletadas da ampola retal, sendo 88 de propriedades familiares e 702 de granjas com produção industrial. As amostras foram submetidas às técnicas de Faust et al., Sheather, Ritchie, Lutz e exame direto. A prevalência estimada foi de 93,1% nas granjas familiares e 59,1% nas granjas industriais. Balantidium coli, coccídios e Entamoeba sp. foram os parasitos que apresentaram as maiores frequências, sendo as categorias machos e fêmeas reprodutoras e leitões de terminação as mais infectadas (p<0,05). Trofozoítas de B. coli foram mais evidenciados nas amostras fecais de consistências semi-sólida, seguida pelas fezes com consistências sólida e diarreica. Ovos de estrongilídeos e Trichuris suis foram detectados exclusivamente em criações familiares. Ovos de Ascaris suum e de Strongyloides ransomi apresentaram baixa frequência. O alto grau de parasitismo, principalmente de protozoários, indica a necessidade de reavaliação do manejo dos suínos em ambos os tipos de produção...

Topical application of Acheflan on rat skin injury accelerates wound healing: a histopathological, immunohistochemical and biochemical study.

BACKGROUND: Dermal wound healing involves a cascade of complex events including angiogenesis and extracellular matrix remodeling. Several groups have focused in the study of the skin wound healing activity of natural products. The phytomedicine Acheflan®, and its main active constituent is the oil from Cordia verbenacea which has known anti-inflammatory, analgesic and antimicrobial activities. To our knowledge, no investigation has evaluated the effect of Acheflan® in an experimental model of skin wound healing. The present study has explored the wound healing property of Acheflan® and has compared it with topical effectiveness of collagenase and fibrinolysin by using Wistar rat cutaneous excision wound model. METHODS: Animals were divided into four groups: untreated animals are negative control (NC), wounds were treated topically every day with Collagenase ointment (TC), with Fibrinolysin ointment (TF) and with cream Acheflan (TAc). Skin samples were collected on zero, 8th and 15th days after wounding. The healing was assessed by hematoxylin-eosin (HE), picrosirius red, hydoxyproline content and immunohistochemical analysis of the vascular endothelial growth factor (VEGF) and matrix metalloprotease-9 (MMP-9). Statistical analysis was done by ANOVA and Student t-test (p < 0.05). RESULTS: The histological analysis HE of wound in the TAc group was more efficient because it was possible to observe the complete remodeling of the epidermis indicating the regression of lesions compared with the NC. The evaluation of picrosirius staining has demonstrated a significant increase of collagen distribution in the TC and TAc treatments compared with NC and TF groups. These results are corroborated with hydroxyproline content. Skin TC and TAc treated rats have showed an increase of VEGF and MMP-9 compared with NC and TF groups. All parameters were significant (P < 0.05). CONCLUSION: The phytomedicine Acheflan® (oil of Cordia verbenacea) and TC possess higher therapeutic properties for wound healing compared with TF. These ointments seem to accelerate wound healing, probably due to their involvement with the increase of angiogenesis and dermal remodeling.

Mapping of the environmental contamination of Toxoplasma gondii by georeferencing isolates from chickens in an endemic area in Southeast Rio de Janeiro State, Brazil.

The environmental contamination of Toxoplasma gondii in an endemic area in Brazil was mapped by georeferencing isolates from chickens in farms in the Southeast of the state of Rio de Janeiro. Tissue samples obtained from 153 adult chickens were analyzed by the mouse bioassay for T. gondii infection. These animals were reared free-range on 51 farms in the municipalities of Rio Bonito and Maricá. The ArcGIS kernel density estimator based on the frequency of T. gondii-positive chickens was used to map the environmental contamination with this parasite. A questionnaire was applied to obtain data on the presence and management of cats and the type of water consumed. Of the farms studied, 64.7% were found to be located in areas of low to medium presence of T. gondii, 27.5% in areas with a high or very high contamination level and 7.8% in non-contaminated areas. Additionally, 70.6% kept cats, 66.7% were near water sources and 45.0% were in or near dense vegetation. Humans used untreated water for drinking on 41.2% of the farms, while all animals were given untreated water. The intensity of environmental T. gondii contamination was significantly higher on farms situated at a distance >500 m from water sources (P=0.007) and near (≤500 m) dense vegetation (P=0.003). Taken together, the results indicate a high probability of T. gondii infection of humans and animals living on the farms studied. The kernel density estimator obtained based on the frequency of chickens testing positive for T. gondii in the mouse bioassay was useful to map environmental contamination with this parasite.