Peste septicémica rápidamente fatal secundaria a peste bubónica primaria inicialmente no diagnosticada: reporte de caso / Rapidly fatal septicemic plague secondary to undiagnosed primary bubonic plague: case report

RESUMEN La peste es una enfermedad reemergente causada por Yersinia pestis. Los humanos generalmente adquieren la enfermedad por picaduras de pulgas. La peste es una enfermedad sistémica fulminante, siendo la peste neumónica la forma más letal. El diagnóstico tardío es una de las principales causas de mortalidad y diseminación de la enfermedad, dado que limita la efectividad de las medidas de control. Presentamos el caso de un varón de 42 años, que previamente había viajado a una zona endémica de peste, y luego presentó hiperpirexia, hipotensión, y adenopatía inguinal inflamatoria. A pesar del cuadro clínico muy característico, nadie (antes del ingreso a nuestro hospital) sospechó peste. Se inició una combinación antibiótica efectiva y tratamiento intensivo recién al quinto día de enfermedad. El paciente evolucionó con shock séptico, falla respiratoria, y muerte. Se confirmó peste por reacción en cadena de polimerasa (PCR). Este caso enfatiza la importancia de tener un alto índice de sospecha para peste.

ABSTRACT Plague is a re-emerging disease caused by the bacteria Yersinia pestis. Humans usually get the disease through the bite of an infected flea. Plague is a fulminant systemic disease, with pneumonic plague being the most lethal form. Late diagnosis is one of the main causes of mortality and spread of the disease, as it limits the effectiveness of control measures. We present the case of a 42-year-old male, who had previously traveled to an endemic plague area and then presented hyperpyrexia, hypotension, and inflammatory inguinal adenopathy. Despite the very characteristic clinical picture, nobody (before admission to our hospital) suspected plague. An effective combination of antibiotics and intensive treatment was initiated only on the fifth day of illness. The patient went into septic shock, respiratory failure, and death. Plague was confirmed by polymerase chain reaction (PCR). This case emphasizes the importance of having a high suspicion rate for plague.

Seroprevalence and spatial distribution dynamics of Yersinia pestis antibodies in dogs and cats from plague foci in the State of Ceará, Northeastern Brazil

Abstract INTRODUCTION: In Brazil, the plague is established in several foci located mainly in the northeastern part of the country, where it alternates between active and quiescent periods. These foci in the State of Ceará have high epidemiological importance. In addition to other plague detection activities, plague areas can be monitored through serological surveys of dogs and cats (domestic carnivores), which, following feeding on plague-infected rodents, can develop mild to severe forms of the disease and produce long-lasting antibodies. This study aimed to characterize the circulation dynamics and spatial distribution of Yersinia pestis antibodies in dogs and cats in plague foci areas of Ceará. METHODS: An ecological study was conducted to analyze the temporal series and spatial distribution of secondary data obtained from domestic carnivore serum surveillance in Ceará's plague areas from 1990 to 2014. RESULTS: Joinpoint analysis revealed that the overall trend was a reduction in antibody-positive animals. The mean proportion of antibody-positivity during the whole study period was 1.5% (3,023/203,311) for dogs, and 0.7% (426/61,135) for cats, with more than 4% antibody-positivity in dogs in 1997 and 2002. Antibody titers ranging from 1/16 to 1/64 were frequent. Despite fluctuations and a significant reduction, in recent years, there were antibody-positive animals annually throughout the study period, and the localities containing antibody-positive animals increased in number. CONCLUSION: Yersinia pestis is actively circulating in the study areas, posing a danger to the human population.

Aplicação da técnica Loop-mediated isothermal amplification (LAMP) no desenvolvimento de um teste para o diagnóstico da peste / Application of the technique Loop-mediated isothermal amplification (LAMP) in developing a test for the plague diagnosis

A peste, infecção causada pela bactéria Yersinia pestis, é uma zoonose primária de roedores, geralmente transmitida por pulgas, que infecta humanos e outros mamíferos. O diagnóstico bacteriológico tradicional da peste pode ser comprometido pela qualidade das amostras coletadas em áreas remotas, transportadas inadequadamente e recebidas no laboratório semanas após a coleta. Técnicas de diagnóstico molecular dispensam o cultivo e são exequíveis quando as bactérias estão inviáveis ou em amostras multicontaminadas. Métodos moleculares convencionais (PCR, qPCR, Nested-PCR) requerem equipamentos sofisticados para amplificação e visualização dos resultados, impedindo seu uso em laboratórios menos equipados. A amplificação isotérmica mediada por loop (Loop-mediated isothermal amplification - LAMP), uma variação da PCR convencional, utiliza enzima que permite amplificação isotérmica, apresenta alta especificidade, sensibilidade, rapidez e custo reduzido, sendo utilizada na detecção de diversos patógenos. Esta técnica emprega de quatro a seis primers e a enzima Bst DNA polimerase, que além da atividade de síntese, atua abrindo a fita dupla de DNA. O resultado da amplificação é visualizado no próprio tubo, a olho nu. O objetivo deste projeto foi aplicar a técnica LAMP no desenvolvimento de um teste para o diagnóstico da peste. Foram construídos cinco primers específicos para amplificação do gene caf1, exclusivo de Y. pestis, utilizados em ensaios com o DNA da cepa Y. pestis A1122, para determinar as concentrações ótimas dos reagentes, temperatura de incubação (60°C, 63°C e 65°C) e tempo de duração da reação (15, 30, 45, 60 e 90 minutos). As reações foram incubadas em banho maria. A amplificação foi visualizada diretamente nos tubos contendo SYBR® Safe ou SYBR® Green I. Foi observado que a partir de 45 minutos de reação ocorre amplificação do gene caf1, nas três temperaturas testadas. Além disso, a técnica mostrou-se específica e sensível, detectando até 10 pg de DNA de Y. pestis.

Human plague outbreak in two villages, Yunnan Province, China, 2005.

Plague is still a serious public health problem in Asia. On July 5, 2005, a suspected outbreak of human plague in two Chinese villages was reported to Yunnan Institute of Endemic Disease Control and Prevention (YIEDC). Active case finding, laboratory investigation, environmental inspection, and control measures were conducted by provincial and local health authorities. A suspected case was an individual who resided in one of the two villages and developed fever and painful swollen lymph nodes in the groin, axilla, and neck between June 26 and July 11, 2005. Confirmation was by indirect hemagglutination test (IHA) for plague F1 antibody. A confirmed animal plague case was an animal that tested positive for one of the following tests: IIA, reverse indirect hemagglutination, or bacterial culture. There were three confirmed and one suspected case of human plague. Of nine retrieved rats, three were confirmed cases. Most surveyed houses had poor sanitation, and there was a history of dead rats observed in the villages. After control measures were implemented, the rat density and flea index decreased to acceptable levels and no new cases occurred. The cause of this outbreak was likely due to rat die off in the villages, such that rat flea populations migrated to humans under environmentally favorable conditions. The outbreak was controlled after implementing environmental and educational control measures.

Contribuição para o diagnóstico de peste / Contribution towards plague diagnosis

Apesar de sua fundamentação clínico-epidemiológica, numerosos casos suspeitos de peste nos focos brasileiros têm sido descartados por serem negativos pelo teste de hemaglutinação para detecção de anticorpos contra o antígeno F1 da Yersinia pestis. A transcendência da peste justifica estudar se tais resultados decorrem da falta de resposta ao F1, e se outras proteínas da Yersinia pestis poderiam ser reconhecidas nos soros suspeitos, sendo desta forma candidatas como alvo diagnóstico alternativo ao F1. Assim sendo, cepas de Yersinia pestis e de Yersinia pseudotuberculosis, uma proteína YopH recombinante e a F1 foram utilizadas para analisar soros de pacientes e soros imunes de coelhos. A F1 e a YopH não foram reconhecidas pelos soros humanos HA- e nenhuma proteína majoritária, comum a todos os soros humanos e coelhos, foi identificada, o que permite concluir que os casos suspeitos devem ser submetidos a uma avaliação clínico-laboratorial mais rigorosa, aprofundando a investigação epidemiológica em busca de outras etiologias.

Despite the clinical-epidemiological features of plague, numerous suspected cases in Brazilian outbreaks have been discarded because of negative results from the hemagglutination test for antibodies against the Yersinia pestis F1 antigen. The transcendence of plague justifies studying whether such results are due to unresponsiveness to F1, and whether other Y. pestis proteins might be recognized in suspect serum. These would therefore be candidates to be alternative diagnostic targets to the F1 antigen. Thus, strains of Y. pestis and Y. pseudotuberculosis, a recombinant YopH protein and the F1 antigen were used to analyze serum from patients and immune serum from rabbits. F1 and YopH were not recognized by HA-negative human serum and no major protein common to all the human and rabbit serum samples was identified. This allows the conclusion that suspected cases must be subjected to more rigorous clinical-laboratory evaluation, with strengthening of epidemiological investigations in the search of other etiologies.

Prompt laboratory diagnosis in timely containment of a plague outbreak in India.

A focal outbreak of pneumonic plague occurred in a hamlet of village Hatkoti, district Shimla, Himachal Pradesh in the first fortnight of February, 2002. A total of 16 cases with 4 deaths were reported. Diagnosis of plague was confirmed by the laboratory in 10 (63%) cases. Y. pestis was isolated from clinical samples of 3 cases and confirmed by bacteriophage lysis. Molecular tests confirmed the presence of Y. pestis specific pla and F1 genes in 4 cases; DNA fingerprinting had identity with the known sequence of plague bacilli. Paired samples from 5 cases showed more than 4 fold rise and 1 case showed more than 4 fold fall in antibodies against F1 antigen of Y. pestis. The present communication emphasises that timely and systematic laboratory investigations give confirmatory diagnosis in shortest possible time which forms the backbone of the outbreak control in a timely fashion and prevents confusion and controversy.

Surat plague of 1994 re-examined.

A plague episode in Surat in 1994, and its spread to other cities in India, lasted only a little over 2 weeks, but it created an unprecedented panic that had global repercussions. At first, the Surat hospital doctors could not diagnose the disease, but when they did, immediate intervention, in the form of preventation and treatment (administration of antibodies) prevented the disease from spreading beyond Surat, Delhi, Calcutta, Bombay and their vicinities. Fewer than 1,200 people were diagnosed with plague. A DNA-based study in 2000 decisively concluded that the Surat episode was a plague, but the Indian isolates were genetically more heterogeneous compared to others in the world.

Plague: a decade since the 1994 outbreaks in India.

The severe 1994 plague outbreaks in Surat and Beed drew attention to plague as a continuing source of both natural and potentially manmade disease. This article written a decade later reviews various aspects of Plague not only as a disease but also as an infectious disaster.

Cutaneous reactions in nuclear, biological and chemical warfare.

Nuclear, biological and chemical warfare have in recent times been responsible for an increasing number of otherwise rare dermatoses. Many nations are now maintaining overt and clandestine stockpiles of such arsenal. With increasing terrorist threats, these agents of mass destruction pose a risk to the civilian population. Nuclear and chemical attacks manifest immediately while biological attacks manifest later. Chemical and biological attacks pose a significant risk to the attending medical personnel. The large scale of anticipated casualties in the event of such an occurrence would need the expertise of all physicians, including dermatologists, both military and civilian. Dermatologists are uniquely qualified in this respect. This article aims at presenting a review of the cutaneous manifestations in nuclear, chemical and biological warfare and their management.

Avaliação da técnica Nested-PCRTbU para aplicação no diagnóstico da peste / Evaluation of Nested-PCRTbU technique for application in the diagnose of pestis

O diagnóstico de certeza da peste baseia-se na identificação e isolamento da Y. pestis pelo cultivo de material de pacientes humanos, de roedores e suas pulgas. No Brasil, os casos humanos e as epizootias de peste ocorrem em locais distantes dos centros de diagnóstico. A competição com a flora cadavérica das carcaças de roedores e procedimentos inadequados de conservação e transporte das amostras aos laboratórios pode resultar na morte do bacilo e resultados falso-negativos. As técnicas moleculares apresentam-se como alternativas para estas situações, particularmente técnicas baseadas em PCR. No presente trabalho foi otimizada para o diagnóstico da peste a técnica N-PCRTbU que se baseia na separação física dos primers por imobilização dos primers internos na tampa do microtubo, dispensando a abertura do mesmo entre as duas etapas de amplificação, o que reduz a possibilidade de falsopositivos pela contaminação cruzada durante a manipulação dos amplicons no Nested-PCR convencional. Dois pares de primers, dirigidos ao gene estrutural (caf1) do antígeno F1 de Y. pestis (um par com homologia a uma região mais externa ao alvo e outro com homologia a uma região mais interna), foram inicialmente testados individualmente por PCR e depois por N-PCR tendo sido obtidos segmentos de tamanho esperado. Foram estabelecidas as concentrações de Taq DNA polimerase, dNTPs e MgCl2, bem como a proporção entre os primers externos e internos para o N-PCRTbU. Nos ensaios para estabelecimento do limiar de detecção a partir de DNA total e da cultura da cepa Y. pestis P. PB 881 a N-PCRTbU foi capaz de detectar respectivamente 10 pg de DNA e 20 bactérias viáveis. Baços de camundongos "Swiss Webster" infectados experimentalmente com a cepa Y. pestis P. PB 881 e amostras de baço, fígado, pulmão e coração conservadas no meio de Cary-Blair por 11 meses foram analisadas em paralelo por cultivo em gelose peptonada e pela técnica NPCRTbU. O fragmento de tamanho esperado (460pb) foi amplificado em todas as amostras mesmo nas que se revelaram multicontaminadas ou negativas pela cultura. Nos ensaios com DNA de outras espécies do gênero Yersinia: Y. pseudotuberculosis e Y. enterocolitica e com baços de camundongos não infectados com a Y. pestis não houve amplificação. Além da especificidade e sensibilidade, como os resultados podem ser obtidos rapidamente, em menos de 24 horas, a N-PCRTbU poderá ser mais uma opção em situações emergenciais [...]

Actualités épidémiologiques de la peste à Madagascar

La peste est entrée à Madagascar en 1898, à partir du port de Toamasina, suite à l'escale d'un bâteau venant d'Inde. En 1921, elle arrive à Antananarivo et s'étend sur les Hautes Terres Centrales en y provoquant des épidémies sans précédent pendant près de 20 ans. Jusqu'au début des années 1980, elle a persisté à bas bruit surtout en milieu rural, avant de connaître une reviviscence au point de constituer un problème de santé publique de nos jours. La peste urbaine existe surtout dans la ville d'Antananarivo (réémergence en 1978 après 28 ans de silence apparent) et dans le port de Mahajanga (réémergence en 1991 après 63 ans de silence apparent). La re-dynamisation du programme national de lutte contre la peste, à partir de 1994, a permis une meilleure surveillance de la maladie. Cette analyse a pour objectif une actualisation des données épidémiologiques de la peste humaine à Madagascar, à partir des cas déclarés (16 928 cas suspects dont 3 500 confirmés ou probables) obtenus au Laboratoire Central à l'Institut Pasteur de Madagascar de 1980 à 2001. La saison pesteuse sur les hautes terres se situe d'octobre à mars et celle de la ville de Mahajanga de juillet à novembre. Le sex-ratio homme/femme est de 1,3/1, la tranche d'âge la plus touchée est celle des sujets âgés de 5 à 25 ans. Le taux de létalité de 40% au début des années 1980 a diminué à 20% vers la fin des années 1990, le pourcentage de formes pulmonaires est passé de 15% à moins de 5%, indiquant une amélioration de la prise en charge des cas. Par contre, on a assisté à une extension géographique de l'endémie pesteuse dans le pays : 4 districts confirmés en 1980, un pic de 30 districts en 1999 et 21 districts en 2001. En 2002, la diffusion d'un nouveau test de diagnostic rapide de la peste (bandelettes), dans les centres de santé de base des 42 districts endémiques, devrait contribuer à diminuer la morbidité et la létalité due à la peste, et améliorer sa surveillance au niveau national

The use of filter paper plasticized with polyvinyl alcohol-glutaraldehyde in ELISA

F1-antigen purified from Yersinia pestis was covalently linked to 5-mm diameter filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde. These discs were used both for ELISA and dot-ELISA for the detection of anti-F1 IgG in rabbits. The best conditions were achieved using 1.25 µg of F1 antigen/disc, 3 percent w/v skim milk in PBS as blocking agent, anti-IgG peroxidase conjugate diluted 12,000 times, and serum from rabbits immunized or not against Y. pestis, diluted 6,400 times. The absorbance values obtained from the comparative study between this procedure and conventional ELISA were not significantly different but the low cost of the reagents employed in ELISA using the filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde makes this method economically attractive.

Cellulose acetate as solid phase in ELISA for plague

Antigen from Yersinia pestis was adsorbed on cellulose acetate discs (0.5 cm of diameter) which were obtained from dialysis membrane by using a paper punch. ELISA for human plague diagnosis was carried out employing this matrix and was capable to detect amount of 1.3 µg of antigen, 3,200 times diluted positive serum using human anti-IgG conjugate diluted 1:4,000. No relevant antigen lixiviation from the cellulose acetate was observed even after washing the discs 15 times. The discs were impregnated by the coloured products from the ELISA development allowing its use in dot-ELISA. Furthermore, cellulose acetate showed a better performance than the conventional PVC plates.

La peste dans le port de Mahajanga : 6 habitants sur 1000 porteurs d'anticorps anti-F1 en 1999

Les auteurs rapportent les résultats d'une enquête séroépidémiologique destinée à évaluer l'importance de la peste dans la ville de Mahajanga en juillet 1999. 656 sérums issus d'un échantillon de sujets de 2 ans et plus tirés au sort dans la population par sondage en grappes ont été étudiés en utilisant une technique ELISA pour la recherche des anticorps anti-F1. Le taux de prévalence des anticorps anti-F1 est de 6,1 p. 1000, proche de la prévalence attendue dans cette ville où la peste est réapparue en 1991 après 62 ans de silence. L'enquête a également montré que la musaraigne endémique, Suncus murinus, joue sans doute un rôle comme réservoir de la peste à Mahajanga