ABSTRACT
The aim of the present work was to evaluate the usefulness of a simplified method for DNA extraction coupled to a nested-PCR protocol, based on the amplification of pneumolysin gene fragments for the diagnosis of pneumococcal pneumonia in pediatric patients with clinical and radiological evidence of bacterial infection. Bacterial DNA was extracted from sera by boiling and used without further purification in the PCR for the pneumolysin gene. None toxic reagents were used and the necessary steps to obtain the DNA were left at a minimum; furthermore, it overcomes the use of expensive commercial kits for DNA purification. The total procedure can be completed the same day of sampling and, most important, it avoids the use of sophisticated technology. Both in vitro analytical specificity and sensitivity (10 CFU/ml) of the assay were similar to those previously reported. When clinical samples were tested, the rate of positivity was shown to be 83.3% and 71% in pediatric patients with positive (group a) and negative blood cultures (group b), respectively. In group a, DNA detection was successful in samples from children without treatment or with less than 48 h of antibiotic therapy. None amplification was obtained from sera patients with viral infection or in samples from healthy controls. The application of the strategy described in this paper substantially seems to improve the diagnostic process in a determinate group: blood culture-negative children with pneumonia.
El objetivo del presente trabajo fue evaluar la utilidad de un método simplificado para extracción de ADN, acoplado a un protocolo de PCR anidada, basada en la amplificación de fragmentos del gen de la neumolisina para el diagnóstico de neumonía neumocócica en niños con evidencias clínicas y radiológicas de infección bacteriana. El ADN bacteriano fue extraído del suero por calentamiento y utilizado en la PCR para el gen de la neumolisina sin purificación posterior. Para la obtención de ADN no se utilizan reactivos tóxicos ni costosos "kits" comerciales. El procedimiento completo puede ser realizado en el día y lo que es más importante, evita el uso de tecnología sofisticada. La especificidad analítica in vitro y la sensibilidad (10 UFC/ml) del ensayo fueron similares a lo hallado en publicaciones anteriores. El porcentaje de muestras positivas fue del 83,3% y del 71% en los pacientes con hemocultivos positivos (grupo a) y negativos (grupo b), respectivamente. En el grupo a, sólo se obtuvieron resultados positivos mediante la PCR anidada en los pacientes no tratados o con menos de 48 hs de tratamiento antibiótico. No se obtuvieron señales de amplificación en los sueros de los pacientes con infecciones virales ni en las muestras del grupo control. La aplicación de la estrategia descripta incrementa la posibilidad diagnóstica de neumonía neumocócica en niños con hemocultivos negativos.
Subject(s)
Child, Preschool , Female , Humans , Infant , Male , Community-Acquired Infections/microbiology , DNA, Bacterial/isolation & purification , Pneumonia, Pneumococcal/microbiology , Polymerase Chain Reaction/methods , Streptococcus pneumoniae/isolation & purification , Bacteremia/microbiology , Community-Acquired Infections/diagnosis , Diagnosis, Differential , DNA, Bacterial/blood , DNA, Bacterial/genetics , Pneumonia, Bacterial/diagnosis , Pneumonia, Pneumococcal/diagnosis , Pneumonia, Viral/diagnosis , Sensitivity and Specificity , Streptococcus pneumoniae/geneticsABSTRACT
The aim of the present work was to evaluate the usefulness of a simplified method for DNA extraction coupled to a nested-PCR protocol, based on the amplification of pneumolysin gene fragments for the diagnosis of pneumococcal pneumonia in pediatric patients with clinical and radiological evidence of bacterial infection. Bacterial DNA was extracted from sera by boiling and used without further purification in the PCR for the pneumolysin gene. None toxic reagents were used and the necessary steps to obtain the DNA were left at a minimum; furthermore, it overcomes the use of expensive commercial kits for DNA purification. The total procedure can be completed the same day of sampling and, most important, it avoids the use of sophisticated technology. Both in vitro analytical specificity and sensitivity (10 CFU/ml) of the assay were similar to those previously reported. When clinical samples were tested, the rate of positivity was shown to be 83.3% and 71% in pediatric patients with positive (group a) and negative blood cultures (group b), respectively. In group a, DNA detection was successful in samples from children without treatment or with less than 48 h of antibiotic therapy. None amplification was obtained from sera patients with viral infection or in samples from healthy controls. The application of the strategy described in this paper substantially seems to improve the diagnostic process in a determinate group: blood culture-negative children with pneumonia.
Subject(s)
Community-Acquired Infections/microbiology , DNA, Bacterial/isolation & purification , Pneumonia, Pneumococcal/microbiology , Polymerase Chain Reaction/methods , Streptococcus pneumoniae/isolation & purification , Bacteremia/microbiology , Child, Preschool , Community-Acquired Infections/diagnosis , DNA, Bacterial/blood , DNA, Bacterial/genetics , Diagnosis, Differential , Female , Humans , Infant , Male , Pneumonia, Bacterial/diagnosis , Pneumonia, Pneumococcal/diagnosis , Pneumonia, Viral/diagnosis , Sensitivity and Specificity , Streptococcus pneumoniae/geneticsABSTRACT
The aim of the present work was to evaluate the usefulness of a simplified method for DNA extraction coupled to a nested-PCR protocol, based on the amplification of pneumolysin gene fragments for the diagnosis of pneumococcal pneumonia in pediatric patients with clinical and radiological evidence of bacterial infection. Bacterial DNA was extracted from sera by boiling and used without further purification in the PCR for the pneumolysin gene. None toxic reagents were used and the necessary steps to obtain the DNA were left at a minimum; furthermore, it overcomes the use of expensive commercial kits for DNA purification. The total procedure can be completed the same day of sampling and, most important, it avoids the use of sophisticated technology. Both in vitro analytical specificity and sensitivity (10 CFU/ml) of the assay were similar to those previously reported. When clinical samples were tested, the rate of positivity was shown to be 83.3
and 71
in pediatric patients with positive (group a) and negative blood cultures (group b), respectively. In group a, DNA detection was successful in samples from children without treatment or with less than 48 h of antibiotic therapy. None amplification was obtained from sera patients with viral infection or in samples from healthy controls. The application of the strategy described in this paper substantially seems to improve the diagnostic process in a determinate group: blood culture-negative children with pneumonia.
ABSTRACT
A case of a healthy girl with a trauma in her right leg is described. Twelve hours later she developed fever and pain in the inguinal region. Two days later, she was admitted to the Hospital, and there, an aspiration of pus from the lymph node was performed, and this sample was sent for culture. The Gram smear showed gram-positive filamentous bacilli and when Kinyoun was used they were observed as weakly acid fast rods. After four days of incubation under aerobic conditions, white and hemolitic colonies were seen on human blood agar plates. Biochemical tests as urea hydrolysis and gelatin liquefaction were performed. To complete the identification it was sent to a reference laboratory, INEI-ANLIS Dr. Carlos G. Malbrán, where the isolate was confirmed as Nocardia asteroides sensu stricto. The patient was treated with intravenous cephalotin with good evolution. Therapy was followed with oral trimethoprim-sulfamethoxazole. This case is the first report of a Nocardia infection in the Hospital de Niños de Santa Fe. The fact that N. asteroides was isolated from an immunologically competent girl should be highlighted. This species is uncommon in children and it is rare in our region. In addition, N. asteroides infections are usually seen in pulmonary disease and rarely producing cutaneous infections.
Subject(s)
Lymphadenitis/microbiology , Nocardia Infections/diagnosis , Nocardia asteroides/isolation & purification , Child, Preschool , Female , Humans , Immunocompetence , Leg Injuries/complications , Necrosis , Nocardia Infections/etiologyABSTRACT
A case of a healthy girl with a trauma in her right leg is described. Twelve hours later she developed fever and pain in the inguinal region. Two days later, she was admitted to the Hospital, and there, an aspiration of pus from the lymph node was performed, and this sample was sent for culture. The Gram smear showed gram-positive filamentous bacilli and when Kinyoun was used they were observed as weakly acid fast rods. After four days of incubation under aerobic conditions, white and hemolitic colonies were seen on human blood agar plates. Biochemical tests as urea hydrolysis and gelatin liquefaction were performed. To complete the identification it was sent to a reference laboratory, INEI-ANLIS Dr. Carlos G. Malbrán, where the isolate was confirmed as Nocardia asteroides sensu stricto. The patient was treated with intravenous cephalotin with good evolution. Therapy was followed with oral trimethoprim-sulfamethoxazole. This case is the first report of a Nocardia infection in the Hospital de Niños de Santa Fe. The fact that N. asteroides was isolated from an immunologically competent girl should be highlighted. This species is uncommon in children and it is rare in our region. In addition, N. asteroides infections are usually seen in pulmonary disease and rarely producing cutaneous infections.
ABSTRACT
A case of a healthy girl with a trauma in her right leg is described. Twelve hours later she developed fever and pain in the inguinal region. Two days later, she was admitted to the Hospital, and there, an aspiration of pus from the lymph node was performed, and this sample was sent for culture. The Gram smear showed gram-positive filamentous bacilli and when Kinyoun was used they were observed as weakly acid fast rods. After four days of incubation under aerobic conditions, white and hemolitic colonies were seen on human blood agar plates. Biochemical tests as urea hydrolysis and gelatin liquefaction were performed. To complete the identification it was sent to a reference laboratory, INEI-ANLIS Dr. Carlos G. Malbrán, where the isolate was confirmed as Nocardia asteroides sensu stricto. The patient was treated with intravenous cephalotin with good evolution. Therapy was followed with oral trimethoprim-sulfamethoxazole. This case is the first report of a Nocardia infection in the Hospital de Niños de Santa Fe. The fact that N. asteroides was isolated from an immunologically competent girl should be highlighted. This species is uncommon in children and it is rare in our region. In addition, N. asteroides infections are usually seen in pulmonary disease and rarely producing cutaneous infections.
ABSTRACT
This article describes a combination of methods--a solid-phase enzyme-linked immunosorbent assay (ELISA) combined with an ultramicroanalytical system (UMAS)--that can be used to measure tetanus antitoxin activity in human sera or plasma. The test, which is rapid and permits analysis of 78 samples of serum per reaction plate with a volume of 10 microL of diluted serum per sample, is proposed as an alternative to the traditional biologic assay in mice based on seroneutralization of a known dose of tetanus toxin. The study reported here compared these two procedures, using them both to evaluate 100 sera from the Clinical Laboratory of the General Calixto García Hospital in Havana, Cuba. The two sets of results showed a high degree of correlation (r = 0.99) when subjected to linear regression analysis (95% CI = 0.985-0.993). These and other findings indicate that the cheap and rapid ultramicroELISA method can perform certain tasks for which the slower and costlier traditional assay is not well suited, such as field evaluation of tetanus toxoid vaccines and identification of hyperimmune plasmas appropriate for use in producing specific antitetanus immunoglobulin.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Tetanus Antitoxin/blood , Cost-Benefit Analysis , Enzyme-Linked Immunosorbent Assay/economics , Enzyme-Linked Immunosorbent Assay/standards , Humans , Linear Models , Reproducibility of Results , Time FactorsABSTRACT
A solid-phase enzyme immunoassay (ELISA) for measuring tetanus antitoxin activity in human serum is described; the assay is based on a combination of the indirect method and ultramicro analysis. This rapid test, which has the capacity to analyze 78 blood samples per reagent plate (at a volume of 10 microL of diluted serum per sample), is proposed as an alternative to the traditional mouse bioassay system based on the neutralization of a known dose of tetanus toxin. Results from both tests showed a high correlation in the lineal regression analysis (r = 0.99; CI95%: 0.985 to 0.993). It is recommended that the ultramicro ELISA assay be used in the field to evaluate tetanus toxoid vaccines and to identify hyperimmune plasmas suitable for producing antitetanus immunoglobulin.
