Evaluation of buffy coat 16S rRNA PCR, buffy coat culture and whole blood PCR for detection of bacteraemia

The use of Gram type-specific PCR on buffy coat from clinical specimens for the detection of bacteraemia was evaluated for the first time using whole blood culture as the gold standard. In addition, the established buffy coat culture and whole blood PCR were also compared. Gram-positive bacteria belonging to six species and Gram-negative bacteria from 10 species were isolated and identified by culture and detected using broad-range 16S rDNA primers and Gram-specific primers. Data from the three methods all conferred very high sensitivity, specificity, positive and negative predictive values when compared to whole blood culture. The Kappa coefficients of agreement were 0.9819 (buffy coat PCR), 0.9458 (whole blood PCR) and 1.0 (buffy coat culture), which establishes their validity as alternative methods to routine blood culture in detecting bacteraemia. In addition, results showed that there was a direct correlation of WBC counts greater than 12,000 cells per mm³ to the occurrence of bacteraemia as detected by the four methods (p < 0.05).

Comparación de la observación de leucocitos en el sedimento urinario con el recuento en cámara de Neubauer / Comparison between the observation of white blood cells from centrifuged urine smear and the Neubauer chamber

Se comparó la metodología rutinaria de observación del sedimento urinario entre porta y cubreobjetos con el recuento en cámara de Neubauer, tomado como método de referencia y se verificó su capacidad de predicción de bacteriuria significativa. En un período de dos meses se realizaron 2.287 urocultivos. Sólo 1.153 resultaron evaluables según los criterios de exclusión y en 982 se pudo correlacionar ambos métodos microscópicos con el resultado de los cultivos. La correlación entre los recuentos en cámara y las observaciones del sedimento urinario fue del 96,4%. Las sensibilidades respectivas de la observación del sedimento y del recuento en cámara respecto del urocultivo fueron de 53,5% y 55,5%. Las especificidades respectivas fueron del 90,7% y 91,4%. A partir de los resultados obtenidos se infiere que la observación de leucocituria significativa es predictiva de la bacteriuria significativa. Por el contrario, su ausencia no puede ser un motivo para descartarla. La observación del sedimento entre porta y cubreobjetos resultó prácticamente equivalente al método más tedioso de recuento en cámara de Neubauer.

The current method of centrifuged urine smear was compared to the Neubauer chamber method, using this technique as the gold standard. Their predictive ability for detecting significant bacteriuria was determined. During two months, 2.287 urine cultures were studied. Only 1.153 were evaluable. In 982 samples a comparison between the two microscopic methods could be established. The correlation between them was 96,4%. The sensitivity of the centrifuged urine smear and the Neubauer chamber method was 53.5% and 55.5% respectively. Specificities were 90.7% and 91.4%. Those results pointed out that seeing a significant number of polymorphonuclear neutrophils (PMNs) both in the centrifuged urine smear and in the Neubauer chamber was predictive of significant bacteriuria. On the other hand, absence of PMNs is not an argument for not considering this possibility. Centrifuged urine was equivalent to the Neubauer chamber method in this comparative study.

C-reactive protein and buffy coat smear in early diagnosis of childhood septicemia.

Out of 200 cases of septicemia in children (age group 0-14 years), 111 had positive C-Reactive Protein (CRP > 12 mg/l) and 84 were buffy coat smear positive. Blood culture was positive in 98 cases, with predominant organism being Klebsiella pneumoniae, followed by Staphylococcus aureus. CRP test showed 100% sensitivity and 87.3% specificity, while buffy coat smear showed 76.5% sensitivity and 91.2% specificity. As blood culture reports are not available before 48-72 hours, combination of CRP test and buffy coat smear examination will be very helpful in early diagnosis of childhood septicemia.

Demonstration of acid-fast bacilli in buffy coat and bone marrow smear--a diagnostic tool in pulmonary tuberculosis.

Buffy coat smears prepared from peripheral blood and bone marrow aspirate obtained from proved 60 untreated cases of pulmonary tuberculosis were stained with Ziehl-Neelsen method and thoroughly screened for presence of tubercle bacilli. Acid-fast bacilli were detected in 55% cases in buffy coat, 48.3% cases in bone marrow, 38.3% cases both in buffy coat and bone marrow and 65% cases either in buffy coat or in bone marrow or in both. Considering the fact that demonstration of acid-fast bacilli may not be possible in more than 25-50% of the suspected cases of tuberculosis, these techniques are recommended for evaluation of their utility in establishing diagnosis of tuberculosis, particularly in reference to sputum negative cases of pulmonary tuberculosis and tuberculosis of inaccessible extrapulmonary sites.

Alteraciones de la concentración de leucocitos y subpoblaciones de linfocitos de cerdos que sobrevivieron al desafío con el virus de la fiebre porcina clásica / Changes in leucocyte and lymphocyte subpopulations of pigs that survived classical swine fever virus challenge

La infección con el virus de la fiebre porcina clásica (FPC) provoca la disminución de todas las subpoblaciones de leucocitos circulantes y una inmunosupresión marcada en los cerdos que se enferman y mueren. Sin embargo, no se ha informado del efecto del virus sobre los leucocitos circulantes en cerdos que sobreviven a la infección. Con objeto de conocer este efecto se estudiaron 6 cerdos que, después de ser desafiados experimentalmente con el virus de la FPC, desarrollaron la enfermedad, pero posteriormente se recuperaron. Los parámetros evaluados en estos cerdos fueron los signos clínicos, la temperatura rectal, la biometría hemática y las subpoblaciones del linfocitos determinadas por rosetas de eritrocitos. Los resultados mostraron que hubo un incremento en la temperatura rectal desde el día siete haste el día doce posinoculación, asociada a anorexia, decaimiento y diarrea ligera. En los primeros siete días hubo una disminución marcada de la concentración de leucocitos totales, linfocitos, leucocitos polimorfonucleares segmentados y en banda, monocitos, linfocitos T totales (TE), T de alta afinidad (Taa). B con receptor fc (Bfc) y Null. Al mismo tiempo se observó un incremento de T autólogos (Taut), que son células inmaduras, y no hubo alteración de los linfocitos B con receptor de complemento (Bc). Sin embargo, a partir del séptimo día se incrementaron los valores de la mayoría de las células y disminuyeron los Taut. Estos resultados indican que la recuperación de los animales estuvo asociada al incremento de los leucocitos circulantes

Experimental transmission of bovine leukosis virus by leucocytes recovered from the stable fly Stomoxys calcitrans L

1. Wild stable flies (stomoxys calcitrans) feeding on heifers infected with bovine leukosis virus (BLV) carried viable bovine leucocytes in the midgut and proboscis that, when inoculated by the subcutaneous route into lambs aged 5 to 60 days, elicited the development of antibodies to glycoprotein (gp51) and polipeptide 25 (p25). 2. Antibodies were detected as early as one month later and persisted for an experimental period of 24 or 36 months. Uninoculated control lambs reared to gether with the experimental animals did not acquire the infection, indicating the lack of horizontal transmission. 3. S. calcitrans reared in the laboratory were intermittently allowed to feed on the skin of BLV-infected heifers and on five lambs over a period of 3-10 months. Although some of these lambs were bitten about 500 times, none developed antibodies to BLV (gp51 or p25) over observation periods of 30 or 36 months

Valor diagnóstico de leucocitos fecales y coprocultivo en el síndrome diarreico agudo / Diagnostic value of the faecal leucocytes and culture in the acute diarrheic syndrome

Objetivo: análisis de resultados de cultivos fecales y leucocitos fecales como diagnóstico etiológico de diarrea aguda y su relación entre ambos. Material y Método: estudio retrospectivo de 200 resultados de leucocitos fecales y coprocultivos tomados de la misma muestra en menores de 15 años sin tratamiento antibiótico previo. No se estudia Yersinia, Campilobacter ni Clostridium. Resultados: de 200 muestras, fue negativo el 63% de leucocitos fecales y 83% de los coprocultivos. De 74 muestras con leucocitos fecales positivos, en un 71,6% no se aisló germen, presentando coprocultivos positivos un 28,3%. De 126 muestras con leucocitos fecales negativos un 89,6% presenta coprocultivos negativos y un 10,3% presenta coprocultivos positivos. En 34 muestras con coprocultivo positivo un 61,7% tiene leucocitos fecales positivos y un 38,2% leucocitos fecales negativos. En 166 muestras no se aisló germen, de ellas en un 31,9% se encontraron leucocitos fecales y un 68% no tenían leucocitos en heces. Conclusiones: la presencia de leucocitos fecales en las muestras fue baja y en menos casos se logró aislar un enteropatógeno. Parecería que no se justifica la solicitud de coprocultivos y leucocitos fecales por su baja positividad y poca importancia en el tratamiento de la diarrea aguda

Bacillaemia and bone marrow involvement in leprosy.

Fifty patients (24 new and 26 receiving specific treatment) of leprosy were investigated to study the concentration and morphological index (MI) of the lepra bacilli in skin, peripheral blood and bone marrow. The organisms were detected in 28 cases on skin slit smear examination, in 38 cases on bone marrow examination and in 38 cases on examination of smears made from buffy coat of peripheral blood. Out of 22 cases negative for the bacilli on skin slit smears, 15 had the organisms either in buffy coat or bone marrow or both. Acid fast bacilli in peripheral blood and bone marrow with skin smear negativity were mainly observed in patients with paucibacillary type of the disease and in those who were receiving treatment. Examination of buffy coat and bone marrow for presence of lepra bacilli is suggested to establish the diagnosis in doubtful cases.

Coprocultura: espécies patogênicas prevalentes em laboratório privado de Porto Alegre, RS / Stool culture: prevatence of pathogenic microrganisms in a private laboratory of Porto Alegre, Brazil

Os autores apresentam os resultados de 1175 coproculturas direcionadas para a pesquisa de E. coli enteropatogênica e êntero-invasiva, Salmonella spp. Shigella spp. e Campylobacter spp., realizadas no período de um ano. Procurou-se avaliar os seguintes parâmetros: tipo de coleta, estaçäo do ano, faixa etária e presença de leucócitos fecais. Näo houve diferença significativa entre a coleta por swab retal ou emissäo espontânea das fezes. Ocorreu associaçäo significativa entre incidência de E. coli enteropatogênica e as estaçöes primavera e veräo, Shigella sonnei e outono e veräo, sendo Campylobacter spp. e Shigella flexneri mais freqüentes apenas no veräo. Quanto à faixa etária, observou-se maior incidência de Shigella sonnei, Campylobacter coli e jejuni até 10 anos de idade. Näo foi possível correlacionar os outros microrganismos isolados devido a suas baixas incidências. A análise da presença de leucócitos fecais evidenciou que, nos microrganismos E. coli enteropatogênica e Campylobacter spp., a proporçäo de amostras leucócito-negativas é maior do que a de positivas. Para os microrganismos Shigella flexneri, Salmonella spp. e Salmonella typhimurium a proporçäo de casos positivos para leucócitos fecais foi a mesma dos casos negativos

Dengue virus replication in cultures of peripheral blood leukocytes during the course of dengue haemorrhagic fever.

A preliminary study was made of the ability of cultured human peripheral blood leukocytes (PBL) to support dengue 2 infection. Leukocyte donors, children hospitalized with primary (one case) or secondary dengue infections (12 cases), were studied during the acute and convalescent stages of illness. D2 virus replication occurred infrequently in PBL cultures obtained from children before the 10th day after onset of symptoms (2/23 samples), but frequently thereafter (8/13 samples). The absence of virus permissive cells during the acute stage of illness might be explained by several different mechanisms, including the possibility that permissive PBL are exhausted as the result of a severe dengue infection. An unexpected finding was the appearance of dengue hemagglutination-inhibition antibodies in cultures of PBL obtained from children during the acute stage of secondary dengue infections.