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Introduction:Ionized hydrogen peroxide (iHP) is a new technology used for the decontamination of surfaces or laboratory areas. It utilizes a low concentration of hydrogen peroxide (H2O2) mixed with air and ionized through a cold plasma arc. This technology generates reactive oxygen species as a means of decontamination.Objectives:The purpose of this study is to review the effects of iHP on the structure of the spores of Bacillus atrophaeusby observing its effects using transmission electron microscopy (TEM) and also by evaluating the existence of DNA damage by fluorescence-based quantitative polymerase chain reaction (qPCR).Methods:Spore samples of B. atrophaeusdecontaminated using iHP at different exposure times (Control, 1, 2, 6, and 12?h) were fixed for TEM. In addition, DNA was extracted for evaluation of DNA damages using fluorescence-based qPCR assays.Results:Damages to the spore structures of B. atrophaeuscaused by the decontamination process with iHP at different exposure times (Control, 1, 2, 6, and 12?h) can be observed in micrographs. The effects of the decontamination to short DNA segment (132 base pairs [bp]) of the yaaHgene using qPCR present a linear degradation, and for the long DNA segment (680?bp), it presents a biphasic mode.Conclusion:The results of the qPCR analysis show two initial stages of damage to DNA with very noticeable damage at 12?h contact time, which confirms the observations of the TEM micrographs for the B. atrophaeusspores. The study demonstrates damage to the spore core DNA.

Introduction: Ionized Hydrogen Peroxide (iHP) is a new technology used for the decontamination of surfaces or laboratory areas. It utilizes a low concentration of hydrogen peroxide (H2O2) mixed with air and ionized through a cold plasma arc. This technology generates reactive oxygen species (ROS) as a means of decontamination.Objectives: The purpose of this study is to evaluate the diffusion effect of iHP and its decontamination capabilities using biological and enzyme indicators.Methods: A gas-tight fumigation room with a volume of 880 ft3was used for the decontamination trials. During the decontamination process, empty animal cages were placed inside to create fumigant distribution restrictions. Spore and enzyme indicators were placed in eleven locations throughout the decontamination room. Generation of iHP was done with the use of TOMI’s SteraMist Environmental System and the SteraMist Solution, with 7.8% H2O2at a dose of 0.5 ml per ft3.Results: For the decontamination of 1hr, 2hrs, 6hrs, and 12hrs, the biological indicators of B. atrophaeusin Stainless Steel (SS) Disk in Tyvek envelope have an inactivation rate of 94%, 97%, 100%, and 100%, respectively. For G. stearothermophilusin SS disk and Tyvek envelope, it has 82%, 68%, 100%, and 100%, respectively and, for G. stearothermophilusin SS strips it has an effective rate of 88%, 67%, 91%, and 100%, respectively.Conclusion: iHP inactivates spores, and the residual tAK activity indicates a gas-like fumigant diffusion due to the uniformity of the inactivation without the use of oscillating fans as the contact time is extended.

Introduction: During pandemic situations like the one caused by the emergent coronavirus SARS-CoV-2, healthcare systems face the challenge of limited personal protective equipment and impaired supply chains. This problem poses a threat to healthcare workers, first responders, and the public, which demands solutions that can span the gap between institutional shortages and resupplies.Objectives: To examine the efficacy of autoclave-based decontamination for the reuse of single-use surgical masks and N95 filtering facepiece respirators (FFRs). This method is the most readily available form of decontamination in the hospital and laboratory settings.Methods: Three models of N95 FFRs and two procedural masks were evaluated in this study. A moist heat autoclave using four different autoclave cycles: 115°C for one hour, 121.1°C for 30 minutes, 130°C for two minutes, and 130°C for four minutes was used. After the autoclave process, the FFRs were NIOSH fit tested and particle counting was performed for both coarse particles of 5 micrometers (µM) and fine particles from 0.1µM to 1.0µM.Results: We observed negligible alterations in the functionality and integrity of 3M 1805 and 3M 1870/1870+ N95 FFRs after three autoclave cycles. Surgical masks also showed minimal changes in functionality and integrity. The 3M 1860 FFR failed fit test after a single autoclave decontamination cycle.Discussion and Conclusion: The study finds that specific surgical masks and N95 FFR models can withstand autoclave decontamination for up to three cycles. Additionally, the autoclave cycles tested were those that could be readily achieved by both clinical and research institutions.

Introduction:During pandemic situations like the one caused by the emergent coronavirus SARS-CoV-2, healthcare systems face the challenge of limited personal protective equipment and impaired supply chains. This problem poses a threat to healthcare workers, first responders, and the public, which demands solutions that can span the gap between institutional shortages and resupplies.Objectives:To examine the efficacy of autoclave-based decontamination for the reuse of single-use surgical masks and N95 filtering facepiece respirators (FFRs). This method is the most readily available form of decontamination in the hospital and laboratory settings.Methods:Three models of N95 FFRs and two procedural masks were evaluated in this study. A moist heat autoclave using four different autoclave cycles: 115°C for one hour, 121.1°C for 30 minutes, 130°C for two minutes, and 130°C for four minutes was used. After the autoclave process, the FFRs were NIOSH fit tested and particle counting was performed for both coarse particles of 5 micrometers (µM) and fine particles from 0.1µM to 1.0µM.Results:We observed negligible alterations in the functionality and integrity of 3M 1805 and 3M 1870/1870+ N95 FFRs after three autoclave cycles. Surgical masks also showed minimal changes in functionality and integrity. The 3M 1860 FFR failed fit test after a single autoclave decontamination cycle.Discussion and Conclusion:The study finds that specific surgical masks and N95 FFR models can withstand autoclave decontamination for up to three cycles. Additionally, the autoclave cycles tested were those that could be readily achieved by both clinical and research institutions.

Introduction:Ionized Hydrogen Peroxide (iHP) is a new technology used for the decontamination of surfaces or laboratory areas. It utilizes a low concentration of hydrogen peroxide (H2O2) mixed with air and ionized through a cold plasma arc. This technology generates reactive oxygen species (ROS) as a means of decontamination.Objectives:The purpose of this study is to evaluate the diffusion effect of iHP and its decontamination capabilities using biological and enzyme indicators.Methods:A gas-tight fumigation room with a volume of 880 ft3was used for the decontamination trials. During the decontamination process, empty animal cages were placed inside to create fumigant distribution restrictions. Spore and enzyme indicators were placed in eleven locations throughout the decontamination room. Generation of iHP was done with the use of TOMI’s SteraMist Environmental System and the SteraMist Solution, with 7.8% H2O2at a dose of 0.5 ml per ft3.Results:For the decontamination of 1hr, 2hrs, 6hrs, and 12hrs, the biological indicators of B. atrophaeusin Stainless Steel (SS) Disk in Tyvek envelope have an inactivation rate of 94%, 97%, 100%, and 100%, respectively. For G. stearothermophilusin SS disk and Tyvek envelope, it has 82%, 68%, 100%, and 100%, respectively and, for G. stearothermophilusin SS strips it has an effective rate of 88%, 67%, 91%, and 100%, respectively.Conclusion:iHP inactivates spores, and the residual tAK activity indicates a gas-like fumigant diffusion due to the uniformity of the inactivation without the use of oscillating fans as the contact time is extended.

Ehrlichia chaffeensisis the causative agent of human monocytotropic ehrlichiosis (HME), a disease that ranges in severity from mild to fatal infection. Ehrlichia chaffeensisis maintained in a zoonotic cycle involving white-tailed deer (Odocoileus virginianus) as the main vertebrate reservoir and lone star ticks (Amblyomma americanum) as its principal vector. Through complete genomic analysis from human ehrlichial isolates and DNA sequences obtained from deer and tick specimens, nine strains of E. chaffeensishave been characterized. Few studies have examined the genetic diversity of E. chaffeensisin ticks, and some of these investigations have identified that the genetic sequences coincide with the circulating strains reported so far. Here, we report the first evidence of E. chaffeensisDNA from an unfed Amblyomma tenellum(formerly Amblyomma imitator) collected in South Texas. We characterized the genetic variation of this E. chaffeensisgenotype using conserved gene markers such as rRNA, dsb, and groEL. We also used gene targets useful to distinguish genotypes, such as the variable length PCR target gene (VLPT) and 120-kDa gene, encoding the tandem-repeat proteins TRP32 and TRP120, respectively. Our results suggest a novel E. chaffeensisgenotype that exhibited greater variability than other genotypes of E. chaffeensisand highlights the role for A. tenellumas a potential vector of E. chaffeensis.

ABSTRACTIncreasing entomologic and epidemiologic evidence suggests that spotted fever group rickettsiae (SFGR) other than Rickettsia rickettsiiare responsible for spotted fever rickettsioses in the United States. A retrospective seroepidemiologic study was conducted on stored acute- and convalescent-phase sera that had been submitted for Rocky Mountain spotted fever testing to the North Carolina State Laboratory of Public Health. We evaluated the serologic reactivity of the paired sera to R. rickettsii, Rickettsia parkeri, and Rickettsia amblyommiiantigens. Of the 106 eligible pairs tested, 21 patients seroconverted to one or more antigens. Cross-reactivity to multiple antigens was observed in 10 patients, and seroconversions to single antigens occurred in 11 patients, including 1 against R. rickettsii, 4 against R. parkeri, and 6 against R. amblyommii. Cross-absorption of cross-reactive sera and/or Western blots identified two presumptive cases of infection with R. parkeri, two presumptive cases of infection with R. rickettsii, and one presumptive case of infection with R. amblyommii. These findings suggest that species of SFGR other than R. rickettsiiare associated with illness among North Carolina residents and that serologic testing using R. rickettsiiantigen may miss cases of spotted fever rickettsioses caused by other species of SFGR.

Rickettsia massiliae, belonging to the spotted fever group of Rickettsia, is a human pathogen causing a similar course of disease to that caused by R. conorii, the originally recognized etiologic agent of Mediterranean spotted fever. In view of this similarity, we performed an ultrastructural study of R. massiliaein organs of Rhipicephalus sanguineusticks, in order to advance knowledge of the complex dynamics at the tick-pathogen interface in rickettsioses.

Spotted fever group rickettsioses are emerging and reemerging infectious diseases, some of which are life-threatening. In order to understand how dendritic cells (DCs) contribute to the host resistance or susceptibility to rickettsial diseases, we first characterized the in vitro interaction of rickettsiae with bone marrow-derived DCs (BMDCs) from resistant C57BL/6 (B6) and susceptible C3H/HeN (C3H) mice. In contrast to the exclusively cytosolic localization within endothelial cells, rickettsiae efficiently entered and localized in both phagosomes and cytosol of BMDCs from both mouse strains. Rickettsia conorii-infected BMDCs from resistant mice harbored higher bacterial loads compared to C3H mice. R. conorii infection induced maturation of BMDCs from both mouse strains as judged by upregulated expression of classical major histocompatibility complex (MHC) and costimulatory molecules. Compared to C3H counterparts, B6 BMDCs exhibited higher expression levels of MHC class II and higher interleukin-12 (IL-12) p40 production upon rickettsial infection and were more potent in priming naïve CD4+T cells to produce gamma interferon. In vitro DC infection and T-cell priming studies suggested a delayed CD4+T-cell activation and suppressed Th1/Th2 cell development in C3H mice. The suppressive CD4+T-cell responses seen in C3H mice were associated with a high frequency of Foxp3+T regulatory cells promoted by syngeneic R. conorii-infected BMDCs in the presence of IL-2. These data suggest that rickettsiae can target DCs to stimulate a protective type 1 response in resistant hosts but suppressive adaptive immunity in susceptible hosts.

ABSTRACTSpotted fever group rickettsioses are emerging and reemerging infectious diseases, some of which are life-threatening. In order to understand how dendritic cells (DCs) contribute to the host resistance or susceptibility to rickettsial diseases, we first characterized the in vitro interaction of rickettsiae with bone marrow-derived DCs (BMDCs) from resistant C57BL/6 (B6) and susceptible C3H/HeN (C3H) mice. In contrast to the exclusively cytosolic localization within endothelial cells, rickettsiae efficiently entered and localized in both phagosomes and cytosol of BMDCs from both mouse strains. Rickettsia conorii-infected BMDCs from resistant mice harbored higher bacterial loads compared to C3H mice. R. conoriiinfection induced maturation of BMDCs from both mouse strains as judged by upregulated expression of classical major histocompatibility complex (MHC) and costimulatory molecules. Compared to C3H counterparts, B6 BMDCs exhibited higher expression levels of MHC class II and higher interleukin-12 (IL-12) p40 production upon rickettsial infection and were more potent in priming naïve CD4+T cells to produce gamma interferon. In vitro DC infection and T-cell priming studies suggested a delayed CD4+T-cell activation and suppressed Th1/Th2 cell development in C3H mice. The suppressive CD4+T-cell responses seen in C3H mice were associated with a high frequency of Foxp3+T regulatory cells promoted by syngeneic R. conorii-infected BMDCs in the presence of IL-2. These data suggest that rickettsiae can target DCs to stimulate a protective type 1 response in resistant hosts but suppressive adaptive immunity in susceptible hosts.

Members of the genus Rickettsia possess the ability to invade host cells and promptly escape from phagosomal vacuoles into the host cell cytosol, thereby avoiding destruction within the endosomal pathway. The mechanism underlying rickettsial phagosomal escape remains unknown, although the genomic sequences of several rickettsial species have allowed for the identification of four genes with potential membranolytic activities (tlyA, tlyC, pat1, and pld). This study was undertaken to determine which of the selected genes of Rickettsia prowazekii mediate the escape process. Quantitative ultrastructural analyses indicated that the period of active phagosomal escape was between 30 and 50 min postinfection. Reverse transcriptase PCR analyses determined that tlyC and pld were transcribed during the period of active phagosomal escape but that tlyA and pat1 were not. The functionality of both tlyC and pld was determined by complementation studies of Salmonella, which replicates within endosomes. Complementation of Salmonella organisms with either tlyC or pld resulted in the escape of transformants from endosomal vacuoles into the host cell cytosol demonstrated by quantitative ultrastructural analyses. These data suggest a role for tlyC and pld in the process of phagosomal escape by R. prowazekii.

ABSTRACTMembers of the genus Rickettsiapossess the ability to invade host cells and promptly escape from phagosomal vacuoles into the host cell cytosol, thereby avoiding destruction within the endosomal pathway. The mechanism underlying rickettsial phagosomal escape remains unknown, although the genomic sequences of several rickettsial species have allowed for the identification of four genes with potential membranolytic activities (tlyA, tlyC, pat1, and pld). This study was undertaken to determine which of the selected genes of Rickettsia prowazekiimediate the escape process. Quantitative ultrastructural analyses indicated that the period of active phagosomal escape was between 30 and 50 min postinfection. Reverse transcriptase PCR analyses determined that tlyCand pldwere transcribed during the period of active phagosomal escape but that tlyAand pat1were not. The functionality of both tlyCand pldwas determined by complementation studies of Salmonella, which replicates within endosomes. Complementation of Salmonellaorganisms with either tlyCor pldresulted in the escape of transformants from endosomal vacuoles into the host cell cytosol demonstrated by quantitative ultrastructural analyses. These data suggest a role for tlyCand pldin the process of phagosomal escape by R. prowazekii.

Owing to the potential role of the tick Amblyomma cooperi in the enzootic cycle of Rickettsia rickettsii, the etiologic agent of Brazilian spotted fever (BSF), this study evaluated infection by Rickettsia species in A. cooperi ticks collected from an area in Brazil where BSF is endemic. Among a total of 40 A. cooperi adult ticks collected in an area of BSF endemicity in the state of São Paulo, PCR analysis detected DNA of Rickettsia bellii in 16 ticks (40%), and 3 other ticks (7.5%) were positive for a previously unidentified spotted-fever-group (SFG) rickettsia. Cultivation in Vero cell cultures by the shell vial technique with individual A. cooperi ticks resulted in two isolates of R. bellii and one isolate genotypically characterized as an SFG rickettsia. The two R. bellii isolates were established in Vero cell cultures in the laboratory and were confirmed to be R. bellii by molecular analysis of the gltA and 17-kDa protein-encoding genes and by electron microscopic analysis. The SFG rickettsial isolate could not be stably passaged in cell culture in the laboratory, but molecular analysis of early passages suggested that it was closely related to Rickettsia parkeri, Rickettsia africae, and Rickettsia sibirica. These results do not support the role of A. cooperi in the ecology of R. rickettsii in the area studied, but they add two more species of rickettsiae to the poorly developed list of species occurring in ticks in South America.

ABSTRACTOwing to the potential role of the tick Amblyomma cooperiin the enzootic cycle of Rickettsia rickettsii, the etiologic agent of Brazilian spotted fever (BSF), this study evaluated infection by Rickettsiaspecies in A. cooperiticks collected from an area in Brazil where BSF is endemic. Among a total of 40 A. cooperiadult ticks collected in an area of BSF endemicity in the state of Sa~o Paulo, PCR analysis detected DNA of Rickettsia belliiin 16 ticks (40%), and 3 other ticks (7.5%) were positive for a previously unidentified spotted-fever-group (SFG) rickettsia. Cultivation in Vero cell cultures by the shell vial technique with individual A. cooperiticks resulted in two isolates of R. belliiand one isolate genotypically characterized as an SFG rickettsia. The two R. belliiisolates were established in Vero cell cultures in the laboratory and were confirmed to be R. belliiby molecular analysis of the gltAand 17-kDa protein-encoding genes and by electron microscopic analysis. The SFG rickettsial isolate could not be stably passaged in cell culture in the laboratory, but molecular analysis of early passages suggested that it was closely related to Rickettsia parkeri, Rickettsia africae, and Rickettsia sibirica.These results do not support the role of A. cooperiin the ecology of R. rickettsiiin the area studied, but they add two more species of rickettsiae to the poorly developed list of species occurring in ticks in South America.

This study evaluates the rickettsial presence in Amblyomma ticks from eight areas of the Amazon forest in Rondônia, Brazil. The following tick species (number in parentheses) were examined: Amblyomma ovale Koch (121), Amblyomma cajennense (F.) (41), Amblyomma naponense (Packard) (36), Amblyomma scalpturatum Neumann (35), Amblyomma oblongoguttatum Koch (30), Amblyomma incisum Neumann (27), Amblyomma rotundatum Koch (16), Amblyomma coelebs Neumann (10), and Amblyomma humerale Koch (6). Ticks were examined individually or in pools (2–10 ticks) by polymerase chain reaction (PCR) targeting the gltA gene. The PCR-determined minimal infection rate for each tick species was A. ovale 28%, A. cajennense 27%, A. naponense 0%, A. scalpturatum 11%, A. oblongoguttatum 3%, A. incisum 0%, A. rotundatum 87%, A. coelebs 10%, and A. humerale 50%. Partial sequences of the gltA gene of Rickettsia from A. ovale, A. scalpturatum, A. oblongoguttatum, A. rotundatum, and A. humerale were 99.9% (349/350) identical to Rickettsia bellii. DNA sequences of PCR products from A. cajennense and A. coelebs were 100% (350/350) identical to Rickettsia amblyommii. R. bellii organisms were isolated in Vero cells from A. scalpturatum, A. ovale, A. rotundatum, and A. oblongoguttatum, but only one of the isolates, cultured from A. scalpturatum, was established in continuous cell culture passage. R. amblyommii was isolated from A. cajennense and was successfully established in continuous passage in cell culture. R. amblyommii infection of Vero cells was analyzed by transmission electron microscopy. This study adds South America to the known geographic distribution of R. amblyommii and reports rickettsiae in six Amblyomma species for the first time.

Two Amblyomma longirostre adult male ticks were collected from a Brazilian porcupine Coendou prehensilis L. in the state of Rondonia, Western Amazon, Brazil. The two ticks were pooled for DNA extraction and tested for the presence of rickettsial DNA by amplifying portions of the gltA, 17-kDa, ompA, and ompB rickettsial genes by polymerase chain reaction (PCR). Portions of the four genes were amplified from the sample and subsequently sequenced. These results indicated the presence of a Rickettsia strain infecting A. longirostre, which was designated as strain Aranha. Compared with homologous ompA rickettsial sequences, "Rickettsia amblyommii" ompA seemed to be the closest relative to Aranha (similarity values: 99.0–99.3%). Phylogenetic analyses of more conserved genes including 17-kDa and gtlA partial sequences indicated that this Rickettsia sp. is a spotted fever group rickettsia. The partial ompB sequence of strain Aranha was distinct from all homologous sequences available in GenBank. Although our ompA analysis suggested a very close molecular phylogenetic relationship of Aranha with "R. amblyommii," we cannot at this time determine if Aranha is a new strain of "R. amblyommii" or a new Rickettsia species in South America.