Detection of Pneumocystis jirovecii by nested PCR in HIV-negative patients with pulmonary disease / Detección de Pneumocystis jirovecii mediante PCR anidada en pacientes VIH negativos con enfermedad pulmonar

Background. Nested PCR can be used to determine the status of Pneumocystis jirovecii infection in other lung diseases. Aims. This study sought to detect a target DNA fragment (mitochondrial large subunit rRNA or mtL SUrRNA) of P. jirovecii in patients with lung disease who underwent bronchoscopy with collection of bronchoalveolar lavage (BAL). Methods. The results from toluidine blue staining were compared with those obtained using molecular methods that included an «in house» DNA extraction procedure, PCR and nested PCR. Results. Fifty-five BAL samples from patients with atypical chest X-rays were screened for P. jirovecii. None of the samples was positive for P. jirovecii using toluidine blue staining. In contrast, P. jirovecii DNA was detected by nested PCR in BAL samples from 36 of 55 patients (65.5%). The lung diseases in the patients included cancer, pneumonia, tuberculosis, and chronic obstructive pulmonary disease (COPD). Other chronic problems in the patients included hypertension, diabetes, smoking, and alcoholism. Conclusions. Nested PCR showed high sensitivity for detecting P. jirovecii, especially when compared with toluidine blue staining. Using this method, P. jirovecii infection was detected in HIV-negative patients with lung disease (AU)

Antecedentes. El diagnóstico de laboratorio mediante la técnica de PCR anidada permite determinar estados de infección por Pneumocystis jirovecii en otras enfermedades pulmonares. Objetivos. El objetivo de este estudio fue detectar fragmentos de ADN mitocondrial (mtLSU rRNA) de P. jirovecii en muestras de lavado broncoalveolar (LBA) de pacientes con enfermedades pulmonares, sometidos a broncoscopia. Métodos. Se compara la técnica de coloración con azul de toluidina para la microscopia, con los métodos moleculares PCR y PCR anidada; se realizó una extracción in house de ADN para las reacciones moleculares. Resultados. La presencia de P. jirovecii fue estudiada en 55 muestras de LBA de pacientes que presentaron patrones radiográficos de tórax atípicos. Ninguna de las muestras fue positiva para P. jirovecii con la técnica de coloración con azul de toluidina. Por la técnica de PCR anidada se detectó el ADN de P. jirovecii en 36 de los 55 pacientes (65,5%). Las enfermedades pulmonares de los pacientes fueron cáncer, neumonía, tuberculosis y enfermedad pulmonar obstructiva crónica (EPOC). Las otras enfermedades crónicas presentadas por los pacientes fueron hipertensión, diabetes, alcoholismo y tabaquismo. Conclusiones. La PCR anidada mostró ser altamente sensible en la detección de P. jirovecii en comparación con la coloración por azul de toluidina. Este método permite detectar infecciones por P. jirovecii en pacientes VIH negativos con enfermedades pulmonares (AU)

Detection of Pneumocystis jirovecii by nested PCR in HIV-negative patients with pulmonary disease.

BACKGROUND: Nested PCR can be used to determine the status of Pneumocystis jirovecii infection in other lung diseases. AIMS: This study sought to detect a target DNA fragment (mitochondrial large subunit rRNA or mtL SUrRNA) of P. jirovecii in patients with lung disease who underwent bronchoscopy with collection of bronchoalveolar lavage (BAL). METHODS: The results from toluidine blue staining were compared with those obtained using molecular methods that included an "in house" DNA extraction procedure, PCR and nested PCR. RESULTS: Fifty-five BAL samples from patients with atypical chest X-rays were screened for P. jirovecii. None of the samples was positive for P. jirovecii using toluidine blue staining. In contrast, P. jirovecii DNA was detected by nested PCR in BAL samples from 36 of 55 patients (65.5%). The lung diseases in the patients included cancer, pneumonia, tuberculosis, and chronic obstructive pulmonary disease (COPD). Other chronic problems in the patients included hypertension, diabetes, smoking, and alcoholism. CONCLUSIONS: Nested PCR showed high sensitivity for detecting P. jirovecii, especially when compared with toluidine blue staining. Using this method, P. jirovecii infection was detected in HIV-negative patients with lung disease.

Development of a Multiplex PCR Test with Automated Genotyping Targeting E7 for Detection of Six High-Risk Human Papillomaviruses.

Cervical cancer is caused by high-risk human papillomaviruses (HPV) and viral detection tests aid in the diagnosis of precursor lesions. In the present study, a molecular test for detection of high-risk HPV DNA, called E7-HPV, was standardized and assessed in samples from women with pre-cancerous lesions. The development of the E7-HPV test for detection and genotyping of six high-risk HPV (types 16, 18, 31, 33, 45 and 52), consisted of evaluating primer quality and adjusting the multiplex PCR conditions. Primer design was based on the E7 region of each HPV, and the fluorochrome 6-FAM was added to PCR primers. Viral detection was performed by capillary electrophoresis in automated sequencer in samples obtained from 60 women (55 with ASC-H/HSIL cytology) from August to September 2013. A non-inferiority analysis was conducted with the cobas HPV test as a reference and following international guidelines for the development of new tests. The two tests had a high concordance rate in HPV16 detection (kappa=0.972), with only one discordant case (cervical intraepithelial neoplasia grade 3, negative with cobas and positive for HPV16 by E7-HPV) and complete agreement in HPV18 detection. When comparing detection of all high-risk HPV, three cases were positive with cobas but negative with E7-HPV, and another three cases were negative with cobas but positive with E7-HPV (HPV16, 31 and 52). When we evaluate the cases initially suspected by cytology, the two tests had the same sensitivity in detection CIN2 or worse. In conclusion, the E7-HPV test has satisfactory initial results, and its development can be continued.

Retrieval of HPV oncogenes E6 and E7 mRNA from cervical specimens using a manual open technology protocol.

BACKGROUND: HPV oncogenes mRNA detection gains momentum as an adjuvant for HPV-related cervical abnormalities diagnosis, but is based on costly detection assays not allowing viral type targeting. OBJECTIVE: To assess detection rate of HPV oncogenes E6/E7 mRNA from cervical specimens using a manual, open technology, fully customizable protocol and determine whether HPV-related epidemiological features influence mRNA retrieval. We reviewed literature and compared our retrieval rate with automated technologies. METHODS: We used 60 samples positive for HPV DNA types 16, 18, 31 and/or 45. We extracted mRNA with a TRizol-based protocol, and tested mRNA purity and concentration using light absorbance. We reverse-transcribed mRNA into cDNA for E6/7 detection. RESULTS: HPV oncogenes E6/E7 mRNA was retrieved from 36 (60%) out of 60 specimens. No HPV load-related clinical or epidemiological feature was significantly associated with mRNA retrieval. Presence of HPV-DNA 16/18 was associated with mRNA retrieval (OR = 9.08; 95% CI 1.26 to 65.32 for HPV 16; and 18.2; IC95% 1.86 to 391.44 for HPV 18). CONCLUSIONS: The open-technology protocol yielded an mRNA detection rate similar to that of automated technologies. Advantages are lower costs and target HPV type customization.

N-RAS and K-RAS gene mutations in Brazilian patients with multiple myeloma.

Point mutations affecting codons 12, 13 (exon 1) and 61 (exon 2) of the N-RAS gene and codons 12 and 13 (exon 1) of the K-RAS gene are identified in approximately 30.0% and 10.0%, respectively, of multiple myeloma (MM) patients living in the northern hemisphere. To date, there are no reports about the prevalence of RAS gene mutations in MM Brazilian patients, and this comprised the aim of the present study. DNA from bone marrow aspirates of 252 patients with MM (139 males and 113 females; aged 59.33 +/- 11.95 years) were investigated for whole exons 1 and 2 of the N-RAS gene and whole exon 1 of the K-RAS gene by direct sequencing of DNA amplified in vitro by the polymerase chain reaction. Fifty-three out of 252 (21.03%) MM patients presented RAS mutations. Heterozygous mutations at codons 4, 10 (exon 1), 61 and 65 (exon 2) of the N-RAS gene were identified in seven out of 252 (2.78%) patients. K-RAS heterozygous mutations at codons 7, 12, 13 (exon 1) were seen in 46 out of 252 (18.25%) patients. To the best of our knowledge, the mutation at codon 7 of K-RAS gene is reported for the first time in MM. Taken together, these results suggest that Brazilian MM patients are characterized by: (i) a low prevalence of RAS mutation and (ii) RAS mutations located at distinct regions of the critical codons of the N-RAS and K-RAS genes.

[Persistent neonatal hyperbilirubinemia resulting from Gilbert's syndrome in association with RhD hemolytic disease]. / Hiperbilirrubinemia neonatal prolongada devido à associação entre síndrome de Gilbert e doença hemolítica por incompatibilidade RhD.

OBJECTIVE: To report on an infrequent association of pathologies causing considerable increase in bilirubin production and a significant decrease in its excretion. DESCRIPTION: The third pregnancy of an RhD negative woman. Her first child was normal and delivered to term and did not receive Rhogam. The second pregnancy was problematic due to Rh isoimmunization. She delivered a child to term, who required three exchange transfusions, but died on the 8th day of life. The third child was delivered at term, ORh positive, direct Coombs positive and had stump bilirubin of 6.5 mg/dl and hematocrit at 44%. Five hours after birth the child was jaundiced. Phenobarbital and intensive phototherapy were introduced. Hyperbilirubinemia was soon controlled, but relapsed whenever phototherapy was discontinued. On the 10th day of life the child received a transfusion for significant anemia. As jaundice persisted to the 13th day, associated Gilbert syndrome was considered and DNA sequence analysis was requested. The test demonstrated a mutant homozygote genotype UDPT1A1[TA]7TAA. Phototherapy remained necessary until the 17th day of life and she was discharged from hospital the following day, after bilirubinemia had been controlled. She returned for follow-up and exhibited normal growth and neurological development. COMMENTS: This case demonstrates the significance of increased bilirubin production/decreased bilirubin excretion causing intense hyperbilirubinemias and, in the absence of vigorous treatment, kernicterus. The effectiveness of intense phototherapy has also been demonstrated, reducing the risks of more aggressive treatments such as exchange transfusion. It also provides evidence of the importance of bilirubinemia follow-up until complete resolution.

Hiperbilirrubinemia neonatal prolongada devido à associação entre síndrome de Gilbert e doença hemolítica por incompatibilidade RhD / Persistent neonatal hyperbilirubinemia resulting from Gilbert's syndrome in association with RhD hemolytic disease

OBJETIVO: Relatar associação infreqüente de patologia que cause aumento considerável de produção de bilirrubina e outra diminuição importante na sua excreção. DESCRIÇÃO: Mãe tercigesta, Rh negativo. Na primeira gestação, gerou recém-nascido normal, de termo, não tendo recebido imunoglobulina humana anti-RhD. A segunda gestação complicou-se por isoimunização Rh, dando à luz neonato de termo, o qual necessitou três exsanguinotransfusões e faleceu com 8 dias de vida. Na gestação atual, conseguiu dar à luz a termo recém-nascido tipo ORh positivo, Coombs direto positivo, bilirrubina de cordão 6,5 mg/dl e hematócrito 44 por cento. Com 5 horas de vida, estava ictérico, tendo sido iniciados fenobarbital (por 3 dias) e fototerapia intensiva. A hiperbilirrubinemia foi logo controlada, porém ascendia rapidamente sempre que a fototerapia era suspensa. No 10° dia de vida, a criança foi transfundida por anemia importante. Em vista da persistência da icterícia, no 13° dia de vida pensou-se em associação com síndrome de Gilbert, e o seqüenciamento de DNA foi solicitado. O resultado mostrou genótipo mutante homozigoto UDPT1A1[TA]7TAA. Permaneceu em fototerapia até o 17° dia de vida. Recebeu alta no dia seguinte, após controle de bilirrubinemia. Voltou para acompanhamento ambulatorial e apresentou desenvolvimentos pondo-estatural e neurológico normais. COMENTARIOS: O caso ressalta a importância da associação do aumento de produção/diminuição de excreção de bilirrubina na gênese de hiperbilirrubinemias prolongadas, intensas e passíveis de causar kernicterus, se não tratadas vigorosamente. Demonstra, ainda, a eficácia da fototerapia intensiva, reduzindo os riscos de tratamentos mais agressivos. Ressalta, também, a importância do acompanhamento das icterícias neonatais até a completa remissão dos sintomas.

Effect of cytokines and chemokines on sickle neutrophil adhesion to fibronectin.

A role for leukocytes in sickle cell vaso-occlusive crisis is becoming increasingly recognized. Neutrophil counts are higher in sickle cell patients and neutrophils from these patients demonstrate increased adhesion to endothelial monolayers under certain circumstances. The effects of selected cytokines on the adhesion mechanisms of normal neutrophils and neutrophils from sickle cell anaemia patients (SCA neutrophils) were investigated. Neutrophils were separated from the blood of homozygous (HbSS) SCA patients and healthy controls. Following pre-incubation (25 min, 37 degrees C) of the cells with cytokines, the adhesion of the cells to fibronectin (FN)-coated plates (20 micro) was determined (60 min, 37 degrees C, 5% CO2). Basal adhesion of normal and SCA neutrophils to FN was not statistically different. Pretreatment of normal neutrophils with either IL-6 (10-100 pg/ml), GCSF (1- 10 ng/ml) or IL-8 (1-100 ng/ml) had no significant effect upon their adhesion to FN. In contrast, SCA neutrophil adhesion to FN was increased significantly following pre-incubation with IL-6, G-CSF and IL-8 (p < 0.01). RANTES (1-100 ng/ml) had no significant effect on either normal or SCA neutrophil adhesion to FN. Flow-cytometric analyses demonstrated that IL-8 (10 ng/ml) significantly augments CD11b (Mac-1 integrin subunit) expression on SCA neutrophils, but not normal neutrophils. IL-6 and G-CSF (10 pg/ml and 10 ng/ml, respectively), however, had no effect on SCA neutrophil adhesion molecule expression. In conclusion, SCA neutrophil adhesion mechanisms may increase in the presence of certain cytokines, in vivo, and this activation may contribute to the physiopathology of sickle cell disease.

Aspectos nutricionais, psicológicos e sociais de mäes de crianças desnutridas / Nutritional, psychological and social aspects of mothers of malnourished children

Mäes de desnutridos foram avaliadas por psicólogo, nutricionista, assistente social e pediatra, com o objetivo de verificar de que maneira os aspectos psicológicos, sociais e de nutriçäo estäo interligados. Todas as mäes pertenciam ao baixo nível sócio-econômico, com renda familiar extremamente baixa; precária escolaridade materna, com 57,6 por cento menor ou igual a 4 anos; 27,3 por cento das mäes eram solteiras, 48,5 por cento solteiras com companheiro, 18,2 por cento casadas e 6 por cento separadas. Na personalidade dessas mäes destacaram-se imaturidade, dificuldade de contato, baixa auto-estima, sentimento de insuficiência/inferioridade, agressividade. O nível intelectual mostrou que 82,6 por cento situavam-se entre médio e médio inferior. No preparo dos alimentos, observou-se que 80 por cento eram desorganizadas e com higiene comprometida, 64 por cento näo tinham interesse, 77 por cento causavam desperdício, 61,9 por cento mostraram insegurança e 37 por cento preparavam alimentos com aparência e consistência inadequadas. Os resultados possibilitaram verificar que os aspectos sociais constituem-se realmente em fator de risco. Apesar disso, outros aspectos säo extremamente relevantes, já que certas características de personalidade da mäe dificultam o exercício do papel materno e podem determinar desorganizaçäo externa e falta de interesse durante o preparo de alimentos.

Ambulatório de adolescência clínica: desenvolvimento puberal e estado nutricional. Dados preliminares / Adolescence outpatient clinic puberal development and nutritional status

Estudo transversal de 35 adolescentes (10 anos a 19 anos, 11 meses e 29 dias) acompanhados no Ambulatório de Adolescên