Lyophilization for bacteria preservation: a promising approach for Yersinia pestis strains from an unique collection in Brazil (Fiocruz-CYP).

Yersinia pestis, the causative agent of plague, is a highly virulent bacterium that poses a significant threat to human health. Preserving this bacterium in a viable state is crucial for research and diagnostic purposes. This paper presents and evaluates a simple lyophilization protocol for the long-term storage of Y. pestis strains from Fiocruz-CYP, aiming to explore its impact on viability and long-term stability, while replacing the currently used methodologies. The lyophilization tests were conducted using the non-virulent Y. pestis strain EV76, subjected to the lyophilization process under vacuum conditions. Viability assessment was performed to evaluate the effects of lyophilization and storage conditions on Y. pestis under multiple temperature conditions (- 80 °C, - 20 °C, 4-8 °C and room temperature). The lyophilization protocol employed in this study consistently demonstrated its efficacy in maintaining high viability rates for Y. pestis samples in a up to one year follow-up. The storage temperature that consistently exhibited the highest recovery rates was - 80 °C, followed by - 20 °C and 4-8 °C. Microscopic analysis of the post-lyophilized cultures revealed preserved morphological features, consistent with viable bacteria. The high viability rates observed in the preserved samples indicate the successful preservation of Y. pestis using this protocol. Overall, the presented lyophilization protocol provides a valuable tool for the long-term storage of Y. pestis, offering stability, viability, and functionality. By refining the currently used methods of lyophilization, this protocol can improve long-term preservation for Y. pestis strains collections, facilitating research efforts, diagnostic procedures, and the development of preventive and therapeutic strategies against plague.

Performance assessment of a new indirect rapid diagnostic test for plague detection in humans and other mammalian hosts.

Plague is a flea-borne zoonosis that affects a wide range of mammals and still causes outbreaks in human populations yearly across several countries. While crucial for proper treatment, early diagnosis is still a major challenge in low- and middle-income countries due to poor access to laboratory infrastructure in rural areas. To tackle this issue, we developed and evaluated a new Fraction 1 capsular antigen (F1)-based rapid diagnostic test (RDT) as an alternative method for plague serological diagnosis and surveillance in humans and other mammals. In this study, 187 serum samples from humans, dogs, rodents and rabbits were retrospectively assessed using the plague RDT method. To calculate its performance, results were compared to those obtained by traditional hemagglutination (HA) and ELISA, which are well-established methods in the plague routine serodiagnosis. Remarkably, the results from RDT were in full agreement with those from the ELISA and HA assays, resulting in 100% (CI 95% = 95.5-100%) of sensitivity and 100% (CI 95% = 96.6-100%) of specificity. Accordingly, the Cohen's Kappa test coefficient was 1.0 (almost perfect agreement). Moreover, the RDT showed no cross-reaction when tested with sera from individuals positive to other pathogens, such as Y. pseudotuberculosis, Yersinia enterocolitica, Anaplasma platys, Ehrlichia canis and Leishmania infantum. Although preliminary, this study brings consistent proof-of-concept results with high performance of the Plague RDT when compared to HA and ELISA. Although further human and animal population-based studies will be necessary to validate these findings, the data presented here show that the plague RDT is highly sensitive and specific, polyvalent to several mammal species and simple to use in field surveillance or point-of-care situations with instant results.

Spatial and Temporal Distribution of Rodents during the Epizootic and Enzootic Periods of Plague, with a Focus on Exu, Northeastern Brazil.

The plague caused by the Yersinia pestis bacterium is primarily a flea-transmitted zoonosis of rodents that can also be conveyed to humans and other mammals. In this work, we analyzed the spatial and temporal distribution of rodent populations during epizootic and enzootic periods of the plague in the municipality of Exu, northeastern Brazil. The geospatial analyses showed that all the rodent species appeared through the whole territory of the municipality, with different occurrence hotspots for the different species. Important fluctuations in the rodent populations were observed, with a reduction in the wild rodent fauna following the end of a plague epizootic period, mostly represented by Necromys lasiurus and an increase in the commensal species Rattus rattus. A higher abundance of rats might lead to an increased exposure of human populations, favoring spillovers of plague and other rodent-borne diseases. Our analysis highlights the role of wild rodent species as amplifier hosts and of commensal rats (R. rattus) as preserver hosts in the enzootic period of a specific transmission infection area.

Spatiotemporal analysis of bubonic plague in Pernambuco, northeast of Brazil: Case study in the municipality of Exu.

Along with other countries in America, plague reached Brazil through the sea routes during the third pandemic. A brief ports phase was followed by an urban phase that took place in smaller inland cities and finally, it attained the rural area and established several foci where the ecological conditions were suitable for its continued existence. However, the geographic dispersion of plague in Brazil is still poorly studied. To better understand the disease dynamics, we accessed satellite-based data to trace the spatial occurrence and distribution of human plague cases in Pernambuco, Northeastern Brazil and using the municipality of Exu as study case area. Along with the satellite data, a historical survey using the Plague Control Program files was applied to characterize the spatial and temporal dispersion of cases in the period of 1945-1976. Kernel density estimation, spatial and temporal clusters with statistical significance and maximum entropy modeling were used for spatial data analysis, by means of the spatial analysis software packages. The use of geostatistical tools allowed evidencing the shift of the infection from the urban to the wild-sylvatic areas and the reemergence of cases after a period of quiescence, independent of the reintroduction from other plague areas.

Rodent hosts and flea vectors in Brazilian plague foci: a review.

Plague, caused by the Yersinia pestis bacterium, has several foci scattered throughout a large area from the Brazilian territory that ranges from the Northeastern State of Ceará to the Southeastern State of Minas Gerais and another separated area at the State of Rio de Janeiro. This review gathers data from plague control and surveillance programs on the occurrence and geographic distribution of rodent hosts and flea vectors in the Brazilian plague areas during the period of from 1952 to 2019. Furthermore, we discuss how the interaction between Y. pestis and some rodent host species may play a role in the disease dynamics. The absence of human cases nowadays in Brazil does not mean that it was eradicated. The dynamics of plague in Brazil and in other countries where it was introduced during the 3rd pandemic are quite alike, alternating epidemics with decades of quiescence. Hence, it remains an important epidemic disease of global concern. The existence of a large animal reservoir and competent vectors demonstrate a need for continuous surveillance to prevent new outbreaks of this disease in humans.

Rodents and other small mammal reservoirs in plague foci in northeastern Brazil.

INTRODUCTION: Plague is an acute, infectious zoonotic disease, primarily of wild rodents and their fleas, that affects humans and other mammals. In Brazil, several plague foci are located in the northeast region. Plague surveillance based on monitoring of rodents was discontinued in 2007, and the current information on rodent populations is unsatisfactory. Our purpose was to update the information on rodents and other small mammals in plague foci in northeastern Brazil. METHODOLOGY: Nine surveys in the historically most important northeastern plague areas were conducted in 2013-2015. RESULTS: In this study, 393 animals (13 rodent and four marsupial species) were entrapped. The plague bacterium Yersinia pestis was not detected in tissue sample cultures from the 225 animals that were analyzed. Eighty sera samples were analyzed for anti-F1 antibodies by hemagglutination (HA) and protein A ELISA tests, and all were negative, except for one marsupial, Monodelphis domestica, which was HA positive. CONCLUSIONS: Qualitative and quantitative differences in the animal populations were observed in the areas surveyed, and the antibody positive marsupial indicated that plague continues to circulate in the wild.

Zoonoses As Ecological Entities: A Case Review of Plague.

As a zoonosis, Plague is also an ecological entity, a complex system of ecological interactions between the pathogen, the hosts, and the spatiotemporal variations of its ecosystems. Five reservoir system models have been proposed: (i) assemblages of small mammals with different levels of susceptibility and roles in the maintenance and amplification of the cycle; (ii) species-specific chronic infection models; (ii) flea vectors as the true reservoirs; (iii) Telluric Plague, and (iv) a metapopulation arrangement for species with a discrete spatial organization, following a source-sink dynamic of extinction and recolonization with naïve potential hosts. The diversity of the community that harbors the reservoir system affects the transmission cycle by predation, competition, and dilution effect. Plague has notable environmental constraints, depending on altitude (500+ meters), warm and dry climates, and conditions for high productivity events for expansion of the transmission cycle. Human impacts are altering Plague dynamics by altering landscape and the faunal composition of the foci and adjacent areas, usually increasing the presence and number of human cases and outbreaks. Climatic change is also affecting the range of its occurrence. In the current transitional state of zoonosis as a whole, Plague is at risk of becoming a public health problem in poor countries where ecosystem erosion, anthropic invasion of new areas, and climate change increase the contact of the population with reservoir systems, giving new urgency for ecologic research that further details its maintenance in the wild, the spillover events, and how it links to human cases.

The aquatic environment as a reservoir of Vibrio cholerae O1 in hydrographic basins of the state of Pernambuco, Brazil.

After the worldwide cholera epidemic in 1993, permanent environmental monitoring of hydrographic basins was established in Pernambuco, Brazil, where cholera is endemic. After a quiescent period, 4 rfbN (serogroup O1) positive water samples that were culture negative were detected by multiplex single-tube nested PCR (MSTNPCR); 2 of these were also ctxA (cholera toxin) positive. From May to June 2012, 30 V. cholerae O1 isolates were obtained by culturing samples. These isolates were analyzed for the presence of virulence genes by PCR, intergenic spacer region 16S-23S PCR (ISR-PCR), and pulsed field gel electrophoresis (PFGE). The isolates were positive for the rfbN gene and negative for the assessed pathogenic genes and were classified into 2 groups by ISR and the same profile by PFGE. Close genetic similarity was observed between them (2012) and environmental strains from 2004 to 2005, indicating the permanence of endemic V. cholerae O1 in the region.

An assessment of whole blood and fractions by nested PCR as a DNA source for diagnosing canine ehrlichiosis and anaplasmosis.

Ehrlichiosis and anaplasmosis are tick-borne diseases. Ehrlichia canis and Anaplasma platys infect mainly white cells and platelets, respectively. The main DNA source for PCR is peripheral blood, but the potential of blood cell fractions has not been extensively investigated. This study aims at assessment of whole blood (WB) and blood fractions potential in nested PCR (nPCR) to diagnose canine ehrlichiosis and anaplasmosis. The 16S rRNA gene was amplified in 71.4, 17.8, 31.57, and 30% of the WB, granulocyte (G), mononuclear cells (M), and buffy coat (BC) samples. Compared to the WB, the sensitivity of the PCR was 42.86% for the M, and BC fractions, 21.43% for the G, and 33.33% for the blood clot (C). There was fair agreement between the WB and M, BC and C, and slight with the G. Fair agreement occurred between the nPCR and morulae in the blood smear. One animal was coinfected with A. platys and E. canis. This study provided the first evidence of A. platys infection in dogs in Paraíba, Brazil, and demonstrated that WB is a better DNA source than blood fractions to detect Ehrlichia and Anaplasma by nPCR, probably because of the plasma bacterial concentration following host cell lysis.

Cutaneous leishmaniasis in northeastern Brazil: a critical appraisal of studies conducted in State of Pernambuco.

American cutaneous leishmaniasis (ACL) is a complex disease with clinical and epidemiological features that may vary from region to region. In fact, at least seven different Leishmania species, including Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) naiffi, Leishmania (Viannia) shawi, Leishmania (Viannia) lindenbergi, and Leishmania (Leishmania) amazonensis, have been implicated in the etiology of ACL in Brazil, and numerous phlebotomine sandfly species of the genus Lutzomyia have been regarded as putative or proven vectors. Because ACL is a focal disease, understanding the disease dynamics at the local level is essential for the implementation of more effective control measures. The present paper is a narrative review about the ACL epidemiology in Pernambuco, northeastern Brazil. Furthermore, the need for more effective diagnosis, treatment, control and prevention strategies for the affected populations is highlighted. This paper will provide researchers with a critical appraisal of ACL in Pernambuco. Hopefully, it will also be helpful for public health authorities to improve current control strategies against ACL at the state and country levels.

[Biosafety, environmental protection and health: completing the puzzle]. / Biossegurança, Proteção Ambiental e Saúde: compondo o mosaico.

This work reflects on the development of the field of Biosafety. The scope of this field is presented, as well as its complex themes and its interdisciplinary perspective. The scope of this field is to propose actions capable of preventing and controlling the risk of worsening human and environmental health. This is done in order to provide alternatives to the theoretical and practical challenges posed by the rapid changes in the world, resulting from human intervention in nature and mediated by scientific and technological advances. We address questions that place Biosafety as a tool in the search for a model of sustainable development, establishing the relationship between environmental degradation, precarious health conditions, and control of the emergence and reemergence of diseases in populations.

Characterization of Vibrio cholerae isolated from the aquatic basins of the State of Pernambuco, Brazil.

Through a continuous bacteriological monitoring programme carried out by the Health Secretariat of the State of Pernambuco, Brazil, two isolates of Vibrio cholerae O1 El Tor Ogawa were discovered in an endemic area in 2001, during a cholera inactive period, along with six V. cholerae non-O1/non-O139 strains and two Aeromonas veronii biovar sobria strains showing an unusual characteristic of agglutination with O1 antiserum. Between that time and 2005, eight other O1 isolates were found. The virulence genes present in the V. cholerae differed among strains, with only three O1 strains harboring the ctxA gene. The O1 and some non-O1/non-O139 strains displayed identical patterns of amplification of the 16S-23S intergenic spacer region. RAPD of the 10 V. cholerae O1 strains, with the two primers used, revealed heterogeneity. The presence of V. cholerae carrying virulence genes in the aquatic basins examined confirms that they constitute a vibrio reservoir during a cholera inactive period, thus strengthening the argument for a continuous monitoring programme and preventative measures for cholera, mainly in the areas where the supply of drinking water is deficient.

[Contribution towards plague diagnosis]. / Contribuição para o diagnóstico de peste.

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.