Identification of Pneumocystis jirovecii with Fluorescence In-Situ Hybridization (FISH) in Patient Samples-A Proof-of-Principle.

In resource-limited settings, where pneumocystosis in immunocompromised patients is infrequently observed, cost-efficient, reliable, and sensitive approaches for the diagnostic identification of Pneumocystis jirovecii in human tissue samples are desirable. Here, an in-house fluorescence in situ hybridization assay was comparatively evaluated against Grocott's staining as a reference standard with 30 paraffin-embedded tissue samples as well as against in-house real-time PCR with 30 respiratory secretions from immunocompromised patients with clinical suspicion of pneumocystosis. All pneumocystosis patients included in the study suffered from HIV/AIDS. Compared with Grocott's staining as the reference standard, sensitivity of the FISH assay was 100% (13/13), specificity was 41% (7/17), and the overall concordance was 66.7% with tissue samples. With respiratory specimens, sensitivity was 83.3% (10/12), specificity was 100% (18/18), and the overall concordance was 93.3% as compared with real-time PCR. It remained unresolved to which proportions sensitivity limitations of Grocott's staining or autofluorescence phenomena affecting the FISH assay accounted for the recorded reduced specificity with the tissue samples. The assessment confirmed Pneumocystis FISH in lung tissue as a highly sensitive screening approach; however, dissatisfying specificity in paraffin-embedded biopsies calls for confirmatory testing with other techniques in case of positive FISH screening results. In respiratory secretions, acceptable sensitivity and excellent specificity were demonstrated for the diagnostic application of the P. jirovecii-specific FISH assay.

Evaluation of fluorescence in situ hybridisation (FISH) for the detection of fungi directly from blood cultures and cerebrospinal fluid from patients with suspected invasive mycoses.

The aim of this study was to evaluate the diagnostic performance of in-house FISH (fluorescence in situ hybridisation) procedures for the direct identification of invasive fungal infections in blood cultures and cerebrospinal fluid (CSF) samples and to compare these FISH results with those obtained using traditional microbiological techniques and PCR targeting of the ITS1 region of the rRNA gene. In total, 112 CSF samples and 30 positive blood cultures were investigated by microscopic examination, culture, PCR-RFLP and FISH. The sensitivity of FISH for fungal infections in CSF proved to be slightly better than that of conventional microscopy (India ink) under the experimental conditions, detecting 48 (instead of 46) infections in 112 samples. The discriminatory powers of traditional microbiology, PCR-RFLP and FISH for fungal bloodstream infections were equivalent, with the detection of 14 fungal infections in 30 samples. However, the mean times to diagnosis after the detection of microbial growth by automated blood culture systems were 5 hours, 20 hours and 6 days for FISH, PCR-RFLP and traditional microbiology, respectively. The results demonstrate that FISH is a valuable tool for the identification of invasive mycoses that can be implemented in the diagnostic routine of hospital laboratories.

Fluorescent in situ hybridization of pre-incubated blood culture material for the rapid diagnosis of histoplasmosis.

Fluorescence in situ hybridization (FISH) has been shown to be useful for the detection of Candida and Cryptococcus species in blood culture materials. FISH procedures for the detection of Histoplasma capsulatum var. capsulatum have not been reported so far. This study describes the development and evaluation of fluorescently labeled rRNA-targeting FISH probes to detect and identify H. capsulatum in blood cultures. All three analyzed H. capsulatum reference strains and clinical isolates showed positive signals with the newly designed specific oligonucleotide probes for H. capsulatum, whereas negative reactions were observed for all three nontarget yeast species and the two nontarget bacteria. The assay was also successfully applied for detections of H. capsulatum cells in pre-incubated blood culture samples of patients with clinical suspicion of histoplasmosis (n = 33). The described FISH-based assay was shown to be easy to apply, sensitive, and specific (compared to polymerase chain reaction) for the detection and identification of H. capsulatum in this proof-of-principle analysis. Larger multicentric assessments are recommended for a thorough diagnostic evaluation of the procedure.

Randomized controlled clinical trial to access efficacy and safety of miltefosine in the treatment of cutaneous leishmaniasis Caused by Leishmania (Viannia) guyanensis in Manaus, Brazil.

Miltefosine has been used in the treatment of several new world cutaneous leishmaniasis (CL) species with variable efficacy. Our study is the first evidence on its clinical efficacy in Leishmania (Viannia) guyanensis. In this phase II/III randomized clinical trial, 90 CL patients were randomly allocated (2:1) to oral miltefosine (2.5 mg/kg/day/28 days) (N = 60) or parenteral antimony (15-20 mg/Sb/kg/day/20 days) (N = 30) according to age groups: 2-12 y/o and 13-65 y/o. Patients were human immunodeficiency virus (HIV) noninfected parasitological proven CL without previous treatment. Definitive cure was accessed at 6 months follow-up visit. No severe adverse events occurred. Vomiting was the most frequent adverse event (48.3%) followed by nausea (8.6%) and diarrhea (6.7%). Cure rates were 71.4% (95% confidence interval [CI] = 57.8-82.7) and 53.6% (95% CI = 33.9-72.5) (P = 0.05) for miltefosine and antimonial, respectively. There were no differences in cure rates between age groups within the same treatment arms. Miltefosine was safe and relatively well tolerated and cure rate was higher than antimony.

Combining diagnostic procedures for the management of leishmaniasis in areas with high prevalence of Leishmania guyanensis.

BACKGROUND: The Amazon region corresponds to approximately 40% of the cases of leishmaniasis in Brazil. We report a prospective study with 180 patients conducted in a health care unit that diagnoses 10% of the cases of leishmaniasis in the Brazilian Amazon. The study addresses how a combination of procedures improves diagnosis in areas with high prevalence of Leishmania guyanensis. OBJECTIVES: to evaluate diagnostic methods in areas with high prevalence of Leishmania guyanensis. METHODS: All subjects were amastigote-positive by direct microscopic examination of lesion scarifications. We conducted skin biopsy and histopathology, polymerase chain reaction and parasite cultivation. RESULTS: Polymerase chain reaction detected almost ninety percent of infections when two amplification protocols were used (mini-exon and HSP-70). HSP-70 specific polymerase chain reaction matched the sensitivity of parasite cultivation plus histopathology. CONCLUSION: The best combination was polymerase chain reaction plus histopathology, which increased diagnostic sensitivity to 94%. Species discrimination by polymerase chain reaction disclosed prevalence of human infections with Leishmania guyanensis of 94% and with Leishmania braziliensis of 6% for this region.

Borrelia Burgdorferi "sensu lato" in Brazil: Occurrence confirmed by immunohistochemistry and focus floating microscopy.

In the present study, we report the occurrence of Lyme's borreliosis in patients from the Brazilian Amazon Region. Borreliosis was investigated by immunohistochemistry and focus floating microscopy for Borrelia burgdorferi in skin biopsy samples from 22 patients with both clinical and histopathology evidences compatible with Erythema Migrans. Spirochetes were detected by specific immunohistochemistry and focus floating microscopy for B. burgdorferi in samples from five patients. Clinical cure of the cutaneous lesions was observed in all the patients after treatment with doxycycline regimen as proposed by the Center Disease Control guidelines. A limitation of our study was the fact that we were not able to isolate and culture these organisms. These are the first known Brazilian cases of borreliosis to have Focus Floating Microscopy confirmation.

Post-ART epidermodysplasia verruciformis in a patient with AIDS.

Epidermodysplasia verruciformis (EV) is a rare disorder characterized by persistent human papillomavirus (HPV) infection. Here, we describe a 48-year-old, black, married male with AIDS, presenting a 1-year history of asymptomatic hypopigmented lesions that appeared 3 years after antiretroviral therapy (ART) initiation. Pre-ART, the initial CD4 count was 32 cells/mm(3) and the skin lesions appeared when the CD4 count reached 122 cells/mm(3). Dermatological examination demonstrated thin, scaly, slightly verrucous hypopigmented macules and papules, isolated or presenting with a linear aspect (Köbner phenomenon) in some areas, distributed on the neck, trunk, and superior and inferior members. Skin biopsy of a macular lesion revealed epidermal acanthosis with vacuolated keratinocytes presenting blue-gray pallor, arranged in clusters at the granular and upper spinous layer. Immunohistochemistry revealed expression of p16( INK4a) with diffuse positivity in the upper third of the epithelium, corresponding to the vacuolated keratinocytes. Polymerase chain reaction (PCR) was positive for type 12 HPV, and a diagnosis of EV-like associated to AIDS was made. EV-like is a rare disease and in this patent might be a manifestation of immune reconstitution inflammatory syndrome.

Tegumentary leishmaniasis as the cause of immune reconstitution inflammatory syndrome in a patient co-infected with human immunodeficiency virus and Leishmania guyanensis.

We report a case of immune reconstitution inflammatory syndrome (IRIS) in a 32-year-old man infected with human immunodeficiency virus and Leishmania guyanensis. Three months after initiation of highly active anti-retroviral therapy (HAART), the patient had disseminated cutaneous leishmaniasis and started anti-leishmanial therapy. The patient's leishmaniasis manifestations during HAART ranged form an anergic response (46 CD4+ T cells/microL) to a disseminated cutaneous leishmaniasis (112 CD4+ T cells/microL). Eight weeks later (168 CD4+ T cells/microL, skin biopsy specimens showed inflammatory infiltrates with no detectable amastigotes. The patient then became comatose. Prednisone therapy (60 mg/day) was initiated with a significant improvement within 48 hours. Three months later (CD4+ T cell count = 184 cell/microL), localized, classic, cutaneous leishmaniasis developed in the patient and anti-leishmanial treatment was re-introduced. On that occasion, frequency of T regulatory cells was 1.82% of all CD4+ cells. Our data suggest a pivotal role for CD4+ T cells in the onset of IRIS and lesion ulceration and their association with a low frequency of T regulatory cells.

Mortalidade pos-neonatal segundo o peso ao nascer / Mortality post-neonatal in conformity with the weight at born

Foram estudadas 3.739 criancas nascidas de partos ocorridos na Maternidade Prof. Monteiro de Moraes - Recife/Pe, no ano de 1987, das quais 79 foram a obito no periodo pos-neonatal. Os resultados demonstram haver maiorfrequencia de obitos no grupo de baixo peso ao nascer em relacao aos grupos de peso adequado. As criancas nascidas com peso baixo apresentaram risco relativo deobito no periodo pos-neonatal de 2,49 em relacao as de peso ao nascer igual ou superior a 2.500g. .

Contribuicao a epidemiologia da omissao de vacinas / Contribution at the epidemiology of the omission of vaccines: jnowledge of the pediatry of assistence public services

Os autores relatam o estudo de aspectos do conhecimentode pediatras de servicos publicos ambulatoriais da cidade do Recife, em relacaoa vacinacao, no periodo de 1/7/88 a 31/1/89. Foram avaliados 27,5% dos pediatraslotados na referida rede, selecionados aleatoriamente. A pesquisa constatou que,apenas a terca parte dos pediatras estudados, demonstrou conhecimento correto sobre imunizacao. Nao se observou relacao entre o conhecimento demonstrado e variaveis como tempo de formatura, reciclagens anteriores e acesso a normas sobre va-cinas no local de trabalho. Os autores recomendam a melhoria do conhecimento dospediatras em relacao a imunizacao, avaliando-se melhor a qualidade das recicla -gens e treinamentos que sao realizados, alem de melhor supervisao, assegurando ocumprimento das normas existentes .