ABSTRACT Background: Global publications on Q fever have increased after the 2007 epidemic in the Netherlands. However, the epidemiology of Q fever/coxiellosis in Brazil is still poorly understood. Accordingly, there have been few studies investigating the presence of Coxiella burnetii in dairy products around the world, especially in Brazil, where consumption of fresh cheese made from raw-milk is very high. Objective: This study was a random survey to assess the prevalence of C. burnetii by PCR in traditional Minas artisanal cheese from the Serro microregion, Brazil, which is manufactured from bovine raw-milk. Methods: DNA extracted from 53 cheese samples were analyzed by nested PCR with C. burnetii-specific primers and the products confirmed by DNA sequencing. Results: Out of the 53 cheese samples five (9.43%) were C. burnetii DNA-positive, each coming from one of the respective randomly selected manufacturing agroindustries.Based on our results, it is estimated that 1.62 tons/day of ready-to-eat cheese made from raw-milk from a total of 16.2 tons produced daily in the study region are contaminated with C. burnetii. Conclusion: To our knowledge, this is the first report of highly heat-resistant zoonotic pathogen in raw-milk Brazilian artisanal cheese. This food safety hazard has been completely neglected in ready-to-eat raw-milk Brazilian artisanal cheese and could imply potential threats to consumers, since C. burnetii survives in artisanal cheese submitted to long ripening periods. Thus, this work established random and representative baseline prevalence of C. burnetii in this food product in Brazil. Further epidemiological studies, monitoring trends and setting control targets are warranted. Finally, these results point out the importance of including C. burnetii in animal and public health surveillance programs.

ABSTRACT Q fever is a worldwide zoonosis caused by Coxiella burnetii—a small obligate intracellular Gram-negative bacterium found in a variety of animals. It is transmitted to humans by inhalation of contaminated aerosols from urine, feces, milk, amniotic fluid, placenta, abortion products, wool, and rarely by ingestion of raw milk from infected animals. Nested PCR can improve the sensitivity and specificity of testing while offering a suitable amplicon size for sequencing. Serial dilutions were performed tenfold to test the limit of detection, and the result was 10× detection of C. burnetti DNA with internal nested PCR primers relative to trans-PCR. Different biological samples were tested and identified only in nested PCR. This demonstrates the efficiency and effectiveness of the primers. Of the 19 samples, which amplify the partial sequence of C. burnetii, 12 were positive by conventional PCR and nested PCR. Seven samples—five spleen tissue samples from rodents and two tick samples—were only positive in nested PCR. With these new internal primers for trans-PCR, we demonstrate that our nested PCR assay for C. burnetii can achieve better results than conventional PCR.

ABSTRACT The increasing use of illicit drugs imposes a public health challenge worldwide. People who inject drugs (PWID) are more susceptible to health complications due to immunosuppression associated with drug use and non-hygienic self-administration of substances, contaminants, and liquids. PWID are subjected to increased risk of acquiring and transmitting different pathogens (frequently functioning as sentinel cases for (re)emerging pathogens), including those transmitted by arthropods and vertebrate reservoirs in unhealthy environments. A clear association between injection drug use and HIV, HBV, and HCV infections has been described; however, other infectious viral and bacterial agents have been seldomly assessed. In this study, we investigated the seroprevalence of Bartonella spp., Coxiella burnetii, and Hantavirus among 300 randomly selected PWIDs from Rio de Janeiro, as part of a multi-city cross-sectional study carried out in the 1990s. Point seroprevalences and respective 95% CIs are as follows: 9.3% for C. burnetii (95% CI: 6.0%-13.0%), 1.0% for Bartonella spp. (95% CI: 0.0%-3.0%), and 4.0% for Hantavirus (95% CI: 2.0%-7.0%). In addition to the blood-borne pathogens, the results of this study increase our knowledge on other transmissible infectious agents in PWID. The high seroprevalence of C. burnetii and Hantavirus found among PWID is intriguing and suggests the need to carry out prospective studies, including molecular analyses, to confirm these findings and allow a better understanding of the putative relevance of these zoonotic infectious agents among PWID.

Introduction Over the last recent years, the number of Q fever cases have has increased throughout the world. An epidemiological investigation was performed in the area in which the first molecular documentation of Q fever in Brazil was previously reported. Methods Indirect immunofluorescence assay (IFA) and PCR of Coxiella burnetii targeting the htpAB gene were performed in samples from 14 dogs (blood); 1 cat (blood); 10 goats (blood, milk, vaginal swab and anal swab); 3 sheep (blood); and 2 horses (blood). Results Two dogs, two sheep and five goats were seroreactive. DNA was amplified from 6 milk and 2 blood samples from goats and from dogs, respectively. The sequence of the amplicons exhibited 99% sequence similarity with the homologous sequence of the htpAB gene of C. burnetii RSA 331 (GenBank - CP000890). Conclusions The results confirm C. burnetii infection in animals in Rio de Janeiro and reinforce the need for the surveillance of Q fever in Brazil.

Coxiella burnetii is the agent of Q fever , an emergent worldwide zoonosis of wide clinical spectrum. Although C. burnetii infection is typically associated with acute infection, atypical pneumonia and flu-like symptoms, endocarditis, osteoarticular manifestations and severe disease are possible, especially when the patient has a suppressed immune system; however, these severe complications are typically neglected. This study reports the sequencing of the repetitive element IS1111 of the transposase gene of C. burnetii from blood and bronchoalveolar lavage (BAL) samples from a patient with severe pneumonia following methotrexate therapy, resulting in the molecular diagnosis of Q fever in a patient who had been diagnosed with active seronegative polyarthritis two years earlier. To the best of our knowledge, this represents the first documented case of the isolation of C. burnetii DNA from a BAL sample.

Febre maculosa brasileira (FMB) é a mais importante e frequente doença rickettsial no Brasil. Relatamos um caso fatal de FMB em um homem negro de 32 anos de idade que morreu de choque irreversível após cinco dias de febre, cefaléia intensa, dor abdominal, e sem evidência de exantema. Amostras de baço, rim e coração coletadas na necropsia foram positivas para Rickettsia rickettsii por PCR e sequenciamento. Os autores ressaltam a necessidade de um alto índice de suspeita diagnóstica para febre maculosa em pacientes negros. Ausência de exantema não deve dissuadir os clínicos de considerar a possibilidade de FMB e iniciar a terapêutica empírica.

Brazilian spotted fever (BSF) is the most important and frequent rickettsial disease in Brazil. A fatal case of BSF is reported in a 32-year-old black man, who died of irreversible shock after five days of fever, severe headache and abdominal pain with no rash. Spleen, kidney and heart samples collected at autopsy were positive for Rickettsia rickettsii by PCR and sequencing. The authors emphasize the need for a high index of diagnostic suspicion for spotted fever in black patients. Absence of a skin rash should not dissuade clinicians from considering the possibility of BSF and initiating empirical therapy.

A Febre Maculosa Brasileira (FMB) é uma zoonose causada por Rickettsia rickettsii e transmitida por carrapatos do gênero Amblyomma, mais freqüentemente pela espécie Amblyomma cajennense. Este trabalho tem como objetivo relatar a primeira detecção molecular de R. rickettsii em Rhipicephalus sanguineus naturalmente infectado no Rio de Janeiro, Brasil. Carrapatos foram coletados de cães, procedentes de uma região rural do município de Resende, estado do Rio de Janeiro, Brasil (22º30'9.46"S, 44º42'44.29"WO), onde ocorreram cinco casos humanos de FMB em 2006. Todos os carrapatos foram identificados segundo chave dicotômica, utilizando-se lupa estereoscópica e separados de acordo com estágio, espécie e sexo. Para a extração de DNA utilizou-se o kit comercial QIAamp DNA (QIAGEN ®). O DNA foi submetido à técnica de PCR utilizando 04 conjuntos de iniciadores para a amplificação dos genes: Rr190.70p/Rr190.602n (OmpA, 532bp), BG1-21/BG2-20 (OmpB, 650bp), Tz15/Tz16 (17 kDa gene que codifica a proteína, 246bp) e RPCs .877p/RpCS.1258n (gltA, 381bp). Os produtos da PCR foram separados por eletroforese em gel agarose 1% corados com brometo de etídio e visualizados sob luz ultravioleta e, aqueles que apresentaram bandas amplificadas foram purificados utilizando-se o kit comercial QIAquick ® e seqüenciados pelo ABI PRISM®. As seqüências nucleotídicas foram geradas usando Bioedit®, editado em software e comparados os correspondentes homólogos com as sequências disponíveis através GenBank, utilizando Discontiguous Mega Blast (http://www.ncbi.nlm.nih.gov). Confirmou-se R. rickettsii (GenBank FJ356230) no seqüenciamento de apenas um espécime, adulto de carrapato R. sanguineus. A caracterização molecular de R. rickettsii em exemplar de carrapato R. sanguineus confirma que esta espécie pode ter importante papel na transmissão de R. rickettsii para humanos no território brasileiro.

The Brazilian Spotted Fever (BSF) is a zoonotic disease caused by Rickettsia rickettsii and transmitted by ticks of the genus Amblyomma, more frequently, Amblyomma cajennense. The aim of this paper was to report the first molecular detection of R. rickettsii on R. sanguineus naturally infected in Rio de Janeiro, Brazil. Ticks were collected from dogs in a rural region of Resende municipality, Rio de Janeiro State, Brazil (22º30'9.46"S, 44º42'44.29"WO), where occurred five human cases of BSF in 2006. The ticks were identified under a stereoscopic microscope and separated in pools by stages, species and sex. DNA extraction was carried out using QIAamp DNA Mini Kit (QIAGEN®). The DNA was submitted to PCR amplification using 04 set of primers: Rr190.70p/Rr190.602n (OmpA, 532bp), BG1-21/BG2-20 (OmpB, 650bp), Tz15/Tz16 (17 kDa protein-encoding gene, 246bp) and RpCS.877p/RpCS.1258n (gltA, 381bp). PCR products were separated by electrophoresis on 1% agarose gels and visualized under ultraviolet light with ethidium bromide. PCR products of the expected sizes were purified by QIAquick® and sequenced by ABI PRISM®. The generated nucleotide sequences were edited with using Bioedit® software and compared with the corresponding homologous sequences available through GenBank, using Discontiguous Mega Blast (http://www.ncbi.nlm.nih.gov). It was confirmed R. rickettsii by sequencing of the material (GenBank FJ356230). The molecular characterization of R. rickettsii in the tick R. sanguineus emphasizes the role of dogs as carriers of ticks from the environment to home. Moreover, this result suggests that there is a considerable chance for active participation of R. sanguineus as one of tick species in the transmission of R. ricketsii to human being in the Brazilian territory.

A lethal case of Brazilian spotted fever (BSF) is presented. Clinical features were initially of gastrointestinal involvement and evolved with progression to septic shock, meningoencephalitis and death on the 6th day of illness. Indirect immunofluorescence assay (IFA) for spotted fever group rickettsia (SFGR) was non-reactive. Diagnosis was confirmed by the polymerase chain reaction (PCR) and the nucleotide sequencing of a fragment of the ompA gene showed 100% homology to Rickettsia rickettsii. BSF has not been reported in the city of Rio de Janeiro in the last three decades, and the present description should alert the clinicians to its presence in urban Rio de Janeiro, and to the differential diagnosis with dengue fever, gastroenteritis, leptospirosis and bacterial septic shock, among others.

Cat scratch disease (CSD) is a self limited condition characterized by fever, lymph node enlargement and less often eye involvement. Central nervous system involvement by Bartonella henselae infection is possibly an important cause of morbidity; its role as an agent of aseptic meningitis is unknown. We report a case of a 40 years-old man with CSD accompanied by aseptic meningitis and neuroretinitis. Serum indirect immmunofluorescence (IFI) assays for B. henselae were positive and the cerebrospinal fluid (CSF) analysis showed mononuclear pleocytosis and increased level of protein. Serological tests for other etiologies were negative. The patient responded well to antibiotic therapy with oral doxycicline plus rifampin and in the 12th day of hospitalization evolved to total regression of the headache and partial regression of the visual loss. Clinicians should consider CSD as a differential diagnosis when assessing previously healthy patients with aseptic meningitis associated with regional lymphadenopathy and epidemiological history of feline contact.

Descreve-se um caso de febre maculosa brasileira numa paciente adulta, previamente saudável, que evoluiu para o óbito apresentando um quadro de exantema petequial associado à insuficiência respiratória e renal após 12 dias de doença caracterizada por febre, cefaléia, mialgia e diarréia. Teste de imunofluorescência indireta realizado em amostra de sangue foi reativo para IgM e IgG anti-Rickettsia rickettsii.

We describe a case of Brazilian spotted fever in a previously healthy young woman who died with petechial rash associated to acute renal and respiratory insufficiency 12 days following fever, headache, myalgia, and diarrhea. Serologic test in a serum sample, using an immunofluorescence assay, revealed reactive IgM/IgG.

De fevereiro a setembro de 1999, foram realizadas, semanalmente, coletas de carrapatos de cães no Município de Piraí/RJ. Quatrocentos e setenta e quatro ixodídeos foram taxonomicamente identificados, 103 Amblyomma cajennense, sete Amblyomma ovale, 209 Rhipicephalus sanguineus e 155 Amblyomma sp. O teste de hemolinfa associado à coloração de Giemsa revelou que duas espécies de 163 carrapatos testados (R. sanguineus e A. sp.) continham microrganismos com morfologia semelhante à rickettsia do grupo da febre maculosa. No teste de imunofluorescência direta, mais específico, foi verificada a presença de rickettsia do grupo da febre maculosa em uma espécie de R. sanguineus. Considerando que informações sobre rickettsioses no Brasil são limitadas, principalmente com relação aos vetores envolvidos na perpetuação da doença, estes resultados preliminares nos mostram a necessidade da realização deste tipo de estudo, permitindo, desta forma, aumentar nossos conhecimentos a respeito desta zoonose.

Ticks were obtained from dogs from February to September of 1999 at weekly intervals, in the County of Piraí, State of Rio de Janeiro. Four hundred seventy four ixodids were taxonomically identified, 103 Amblyomma cajennense, seven Amblyomma ovale, 209 Rhipicephalus sanguineus, and 155 Amblyomma sp. An hemolymph test associated with Giemsa's stain revealed two specimens in 163 ticks tested (R. sanguineus and Amblyomma sp), containing rickettsia-like organisms. Direct immunofluorescence verified the presence of spotted fever group rickettsia in one specimen of R. sanguineus. Considering the limited information on rickettsiosis in Brazil, principally in relation to the vectors involved in perpetuating it in foci, these preliminary results give us an idea on the importance of infection in ticks, allowing to expand our knowledge on this zoonosis.

This preliminary report describes human and cow cases of poxvirus that recently ocurred in the State of Rio de Janeiro. The electron microscopic findings were consistent with parapoxviral and orthopoxviral infection. Orthopoxvirus strains were isolated from human and cow cases. Detailed viral characterization by means of genetical techniques is under investigation. Based on these informations, poxviral diseases should be also considered an emerging viral zoonosis that can affect human beings.