RESUMO INTRODUÇÃO: A tuberculose pulmonar causada por Mycobacterium tuberculosis é um grave problema de saúde pública que afeta mundialmente milhões de indivíduos. O desenvolvimento de métodos de diagnóstico fáceis e de baixo custo é essencial para o controle da doença nas áreas rurais remotas e de pobreza e entre os grupos vulneráveis. OBJETIVO: Avaliar a acurácia dos métodos laboratoriais para o diagnóstico de tuberculose pulmonar. MATERIAL E MÉTODOS: As amostras de escarro de pacientes com sinais e sintomas clínicos foram analisadas por microscopia após tratamento químico e sedimentação espontânea e comparadas com métodos empregados rotineiramente: baciloscopia direta do escarro, cultura e GeneXpert® MTB/RIF. RESULTADOS: Das amostras analisadas, 16% foram positivas por microscopia nas amostras processadas; 18%, por cultura e Xpert® MTB/RIF; e 13%, por microscopia direta. As amostras processadas apresentaram um aumento de 31% de positividade (57 amostras) em relação à microscopia direta convencional. Na análise dos métodos avaliados, todos os resultados foram estatisticamente significativos, comprovando que não eram positivos ou negativos aleatoriamente e confirmando que há uma tendência para esses resultados. CONCLUSÃO: O tratamento químico e a sedimentação espontânea das amostras de escarro representam uma ferramenta diagnóstica eficaz nas situações em que tecnologias mais avançadas não são viáveis. Além da maior precisão e maior detecção de casos positivos em relação ao esfregaço direto, o procedimento reforça a biossegurança, diminuindo os riscos de contaminação aérea por Mycobacterium tuberculosis para profissionais de laboratório

ABSTRACT INTRODUCTION: Pulmonary tuberculosis caused by Mycobacterium tuberculosis is a serious public health problem affecting millions of people worldwide. The development of easy and low-cost diagnostic methods is crucial for disease control in rural remote and poverty areas and among vulnerable groups. OBJECTIVE: To evaluate the accuracy of laboratory methods for the diagnosis of Pulmonary tuberculosis. MATERIAL AND METHODS: Sputum samples from patients with clinical signs and symptoms were analyzed by microscopy after chemical treatment and spontaneous sedimentation and compared with methods employed routinely: direct sputum smear microscopy, culture, and GeneXpert®MTB/RIF. RESULTS: From the samples analyzed, 16% were positive by microscopy in the processed samples, 18% by both culture and Xpert®MTB/RIF, while 13% in the direct microscopy. The processed samples showed a 31% increase in positivity (57 samples) compared to conventional direct microscopy. In the analysis of the accuracy of the evaluated methods, all the results were statistically significant proving that they were not randomly positive or negative and confirming that there is a tendency for these results. CONCLUSION: Chemical treatment and spontaneous sedimentation of the sputum samples procedure represent an effective diagnostic tool in situations where more advanced technologies are not feasible. Besides the higher accuracy and greater detection of positive cases regarding the direct smear, the procedure strengthens biosafety by decreasing the risks of aerial contamination by Mycobacterium tuberculosis for laboratory professionals.

RESUMEN INTRODUCCIÓN: La tuberculosis pulmonar causada por Mycobacterium tuberculosis es un grave problema de salud pública que afecta millones de individuos en el mundo. El desarrollo de métodos de diagnóstico fáciles y de bajo costo es esencial para el control de la enfermedad en zonas rurales remotas y pobres y entre los grupos vulnerables. OBJETIVO: Evaluar la exactitud de métodos de laboratorio para el diagnóstico de tuberculosis pulmonar. MATERIAL Y MÉTODO: Las muestras del esputo de pacientes con signos y síntomas clínicos fueron analizadas por microscopía luego de tratamiento químico y sedimentación espontánea y comparados con métodos empleados ordinariamente: baciloscopía directa de esputo, cultivo y GeneXpert® MTB/RIF. RESULTADOS: Entre las muestras analizadas, 16% fueron positivas por microscopía en las muestras procesadas; 18% por cultivo y Xpert® MTB/RIF; y 13% por microscopía directa. Las muestras procesadas presentaran un aumento de 31% de positividad (57 muestras) con respecto a la microscopía directa convencional. En el análisis de los métodos, todos los resultados fueron estadísticamente significativos, comprobando que no eran aleatoriamente positivos o negativos y confirmando que hay una tendencia para esos resultados. CONCLUSIÓN: El tratamiento químico y la sedimentación espontánea de las muestras de esputo representan una herramienta diagnóstica eficaz en las situaciones en las cuales tecnologías más avanzadas no son viables. Además de la mayor precisión y mayor detección de casos positivos de lo que hace el frotis directo, el procedimiento fortalece la bioseguridad, disminuyendo los riesgos de contaminación del aire por Mycobacterium tuberculosis para el personal de laboratorio.

Foi desenvolvido um procedimento para diagnóstico rápido da peste que ao mesmo tempo permite a identificação de marcadores de virulência de Y. pestis, com múltiplos iniciadores, em uma só reação de PCR. Foram usados quatro pares de iniciadores direcionados a genes presentes nos três plasmídeos de virulência e na ilha de patogenicidade dessa bactéria. A técnica permitiu a discriminação de cepas que perderam um ou mais genes conhecidos, usando como molde DNA total obtido de culturas de Y. pestis e também de hemoculturas simuladas contendo concentrações conhecidas de Y. pestis. Também se mostrou eficiente para a confirmação da Y. pestis na cultura de sangue de um paciente suspeito de peste. Como os resultados são obtidos em poucas horas, esta técnica poderá ser útil na metodologia do Programa de Controle da Peste.

We have developed a procedure for the rapid diagnosis of plague that also allows the identification of prominent virulence markers of Y. pestis strains. This procedure is based upon the use of a single polymerase chain reaction with multiple pairs of primers directed at genes present in the three virulence plasmids as well as in the chromosomal pathogenicity island of the bacterium. The technique allowed the discrimination of strains which lacked one or more of the known pathogenic loci, using as template total DNA obtained from bacterial cultures and from simulated blood cultures containing diluted concentration of bacteria. It also proved effective in confirming the disease in a blood culture from a plague suspected patient. As the results are obtained in a few hours this technique will be useful in the methodology of the Plague Control Program.

Foi realizada a caracterização genotípica e fenotípica de fatores de patogenicidade em 16 amostras de Yersinia enterocolitica O:3 isoladas de suínos sadios do Rio de Janeiro. Foi observado que apenas 6 cepas possuíam o plasmídio de virulência, pYV (+ 70 kb) e apresentavam dependência ao cálcio no meio MOX a 37C. Um plasmídio críptico de cerca de 8,6 kb foi encontrado em uma cepa. Doze cepas revelaram sensibilidade à pesticina enquanto que apenas três se revelaram capazes de hidrolisar a esculina. Através de PCR com "primers" específicos, foi constatada a presença dos genes ail em 14 cepas, irp2, em 1 cepa e a ausência de psaA em todas as cepas analisadas. Quanto aos quimioterápicos, a quase totalidade das cepas mostrou-se ao mesmo tempo resistente à ampicilina e carbenicilina e sensível ao sulfazotrin e à cefoxitina. As respostas foram variadas frente ao cloranfenicol, tetraciclina, kanamicina, gentamicina e ácido nalidíxo.

Sixteen Yersinia enterocolitica serotype O:3 strains, isolated from pigs from Rio de Janeiro, have been analyzed for genetic and phenotypic markers of pathogenicity. It was observed that only 6 strains harbored the pYV (+70 kb) plasmid and one strain harbored a small cryptic plasmid of about 8.6 kb. Accordingly only strains harboring pYV were calcium dependent in the MOX medium at 370C. Twelve strains showed pesticin sensitivity and the esculin reaction was negative in 13 strains. PCR analysis of pathogenicity genes using specific primers showed the presence of the ail gene in 14 strains, the irp2 gene in one and the psaA in none. Most of the strains were resistant to ampicillin and carbenicillin, although they were susceptible to sulfazotrin and cefoxitin. For chloramphenicol, tetracycline, kanamycin, gentamicin and nalidixic acid the results varied among the strains.

The irp2 gene codes for a 190 kDa protein (HMWP2) synthesidez when highly pathogenic Yersinia are grown under conditions of iron starvation. In this work, the presence of irp2 in strains of Y. pestis isolated from different hosts during several plague outbreaks in the foci of Northeast Brazil wasstudied. For this purpose, 53 strains were spotted onto nylon filters and their DNA was hybridized with the A13 probe which is a 1 kb fragment of the irp2 coding sequence. All strains except two hybridized with the probe. However, when the initial stock culture of these two strains were analyzed, they both proved to bepositive with the A13 probe, indicating that the locus was lost after subculturein vitro but was always present in vivo. To examine the degree of conservation of the chromosomal fragment carrying irp2 among Brazilian strains, the hybridization profiles of 15 strains from different outbreaks, different hosts and different foci were compared. The hybridization profiles of these strains were all identical when their DNA was digested with either EcoRI, EcorRV or AvaII, indicatingthat the restriction sites surrounding the irp2 locus are very well conserved among Northeast Brazilian strains of Y. pestis. Altogether, these results suggest that the irp2 chromosomal region should be of prime importance for the bacteria during their multiplication in the host.

A dot enzyme linked immunosorbent assay (dot-ELISA) was previously developed to detect specific antibodies in rabbits sera immunized against FIA protein obtained from Yersina pestis. This antigen was covalently linked onto the surface of dacron (polyethyleneterephthalate). Here, standard conditions are described for the optimization of this procedure: an amount of 20 ng of FIA protein was fixed onto dacron; anti-rabbit IgG peroxidase conjugate diluted 1:8,000 and 30% non-fat instant milk as blocking substance were used throughout the method. This procedure was compared with that employing nitrocellulose as solid-phase which showed to be more sensitive. However, the method based on dacron did not show false positive reactions against non-immunized rabbits sera at low antigen amount and diluted anti-IgG peroxidase conjugate.

Dacron (polyethylenetherephthalate) is proposed as a matrix for dot-ELISA procedures, as an alternative to nitrocellulose. Plates of dacron were partially hydrazinolyzed and hydrazide groups introduced were converted to azide groups. The derivative dacron-antigen was covalently linked on to the plates through these azide groups. The derivative dacron-antigen was exaustively washed according to CROOK and antigen was still fixed onto the plates. Protein F1A purified from Yersinia pestis was used as a model. Triration of sera from immunized and non immunized rabbits against this protein was carried out by employing the dot-ELISA method. No significant difference was observed using dacron-antigen and nitrocellulose-antigen preparations. However, both procedures showed to have a significant better performance in comparasion with the passive hemagglutination method. The specificity and reproductibility of the dot-ELISA assay using both preparations showed a similar behaviour. Nitrocellulose preparation was stable at 4ºC, 28ºC and -20ºC for 90 days, whereas the dacron-antigen derivative was stable only when stored at 4ºC. Dacron-antigen derivative could be re-used when the spot developing was proceeded using 4-chloro-1-naphtol as substrate.

O presente trabalho mostra a histopatologia da infecção pela Yersinia pestis, entre as diferentes espécies de roedores silvestres e comensais (cricetídeos, equimídeos, murídeos e cavídeos) que ocorrem na zona endêmica de peste do Nordeste do Brasil. Estes roedores foram encontrados naturalmente infectados nos campos ou inoculados experimentalmente no laboratório (vias percutânea, subcutânea ou picada de pulgas) com cepas locais e/ou estrangeiras de Yersiniapestis. Quase todos os animais, exceto alguns dos cavídeos, desenvolveram a forma bubosepticêmica da peste. Entre as lesões encontradas, a necrose coagulativa multifocal do fígado, a pneumonite intersticial aguda difusa e a atrofia linfoide do baço, podem, por sua constância, ser consideradas como os principais indicadores histológicos da infecção pestosa, embora estas lesões não sejam exclusivas da peste. A diversidade e a intensidade das lesões entre os Zygodontomys lasiurus pixuna, podem explicar a mortalidade elevada desta espécie e a disseminação da peste nos focos naturais do Nordeste brasileiro. Cricetídeos e murídeos mostraram alterações histopatológicas qualitativamente semelhantes. A resistência dos cavídeos à infecção pestosa foi evidenciada pela sobrevida desses roedores à fase aguda da infecção e pelo desenvolvimento de uma reação histiocitária interna, delimitando as áreas abscedadas. è possível que estas lesões crônicas abriguem bacilos virulentos, que permitirão a reinfecção periódica das pulgas e conseqüente reativação do processo epizoótico.

In this paper, the histopathological aspects of plague infection in different species of wild and domestic rodents (cricetidae, echymidae, muridae and cavidae) are described. All of them had been trapped in endemic plague areas and harboured natural infection, while others were laboratory infected by different routes (percutaneous, subcutaneous rout, fleas bite). Several national and foreign strains of Yersinia pestis have been studied. All the rodents, except the group of cavidae, had a bubosepticemic plague. From all the lesions described, liver multifocal coagulative necrosis, diffuse acute interstitial pneumonia and lymphoid atrophy of the spleen were the most frequent and so can be considered, for practical purposes, as histological indicators of plague infection, although they are not exclusively found in plague. The variety and intensiveness of lesions found in Zigodontomys L. pixuna, may explain the high mortality of this species of rodents, making easier the dissemination of plague in the natural foci of northeast Brazil. Similar histological lesions were detected in cricetidae and muridae. The resistance of cavidae to plague infection has become evident, since they survived to the acute phase of infection and developped an intensive histiocytic granulomatous reaction surrounding necrotic areas (abscesses). It is suggested thad these chronic lesions may harbour virulent bacilli and so explain periodic plague infection in local fleas, leading to reactivation of epizootics.