Gaps and inconsistencies in the current knowledge and implementation of biosafety and biosecurity practices for rickettsial pathogens.

INTRODUCTION: Rickettsia spp. and Orientia spp. are the causes of neglected infections that can lead to severe febrile and systemic illnesses in humans. Implementing proper biosafety practices when handling these pathogens is crucial to ensure a safe and sustainable work environment. It is essential to assess the current knowledge and identify any potential gaps to develop effective measures that minimise the risk of exposure to these pathogens. By doing so, we can establish a comprehensive framework that promotes safety, mitigates hazards, and safeguards the well-being of personnel and the surrounding community. METHODS AND RESULTS: This review aimed to synthesise and determine the evidence base for biosafety precautions for Rickettsia spp. and Orientia spp. pathogens. Enhancing our understanding of the relative infectious risk associated with different strains of Rickettsia and Orientia spp. requires identifying the infectious dose of these pathogens that can cause human disease. The application of risk groups for Rickettsia and Orientia spp. is inconsistent across jurisdictions. There is also incomplete evidence regarding decontamination methods for these pathogens. With regards to Orientia spp. most of the available information is derived from experiments conducted with Rickettsia spp. CONCLUSIONS: Rickettsia and Orientia spp. are neglected diseases, as demonstrated by the lack of evidence-based and specific biosafety information about these pathogens. In the case of Orientia spp., most of the available information is derived from Rickettsia spp., which may not be appropriate and overstate the risks of working with this pathogen. The advent of effective antibiotic therapy and a better understanding of the true hazards and risks associated with pathogen manipulation should inform decisions, allowing a sustainable and safe work environment.

Fatal Case of Mediterranean Spotted Fever Associated with Septic Shock, Iran.

A fatal case of Mediterranean spotted fever associated with septic shock was reported in a 61-year-old man living in a village in southeastern Iran. The patient had a history of tick bite a few days before symptom onset. Phylogenetic analysis confirmed infection by Rickettsia conorii subspecies israelensis.

Validation of an Indirect Immunofluorescence Assay and Commercial Q Fever Enzyme-Linked Immunosorbent Assay for Use in Macropods.

Kangaroos are considered to be an important reservoir of Q fever in Australia, although there is limited knowledge on the true prevalence and distribution of coxiellosis in Australian macropod populations. Serological tests serve as useful surveillance tools, but formal test validation is needed to be able to estimate true seroprevalence rates, and few tests have been validated to screen wildlife species for Q fever. In this study, we modified and optimized a phase-specific indirect immunofluorescence assay (IFA) for the detection of IgG antibodies against Coxiella burnetii in macropod sera. The assay was validated against the commercially available ID Screen Q fever indirect multispecies enzyme-linked immunosorbent assay (ELISA) kit (IDVet, Grabels, France) to estimate the diagnostic sensitivity and specificity of each assay, using Bayesian latent class analysis. A direct comparison of the two tests was performed by testing 303 serum samples from 10 macropod populations from the east coast of Australia and New Zealand. The analysis indicated that the IFA had relatively high diagnostic sensitivity (97.6% [95% credible interval [CrI], 88.0 to 99.9]) and diagnostic specificity (98.5% [95% CrI, 94.4 to 99.9]). In comparison, the ELISA had relatively poor diagnostic sensitivity (42.1% [95% CrI, 33.7 to 50.8]) and similar diagnostic specificity (99.2% [95% CrI, 96.4 to 100]) using the cutoff values recommended by the manufacturer. The estimated true seroprevalence of C. burnetii exposure in the macropod populations included in this study ranged from 0% in New Zealand and Victoria, Australia, up to 94.2% in one population from New South Wales, Australia.

The incidence of Orientia tsutsugamushi infection in rural South India.

Scrub typhus is a common bacterial infection in Asia caused by Orientia tsutsugamushi. This serological cohort study estimated the incidence of infection in a rural population in South India. Participants were enrolled through systematic sampling in 46 villages at baseline, and revisited the following year. Blood samples were tested for IgG antibodies using ELISA, followed by indirect immunofluorescence assays (IFA) in those positive for ELISA at both rounds. A case was defined as sero-conversion (ELISA), or at least a 4-fold titre increase (IFA), between the two time points. In addition to crude incidence rate estimates, we used piecewise linear rates across calendar months, with rates proportional to the monthly incidence of local hospital cases to address seasonality and unequal follow-up times. Of 402 participants, 61.7% were female. The mean age was 46.7 years, (range 13-88). 21 participants showed evidence for serological infection. The estimated incidence was 4.4 per 100 person-years (95% CI 2.8-6.7). The piecewise linear rates approach resulted in a similar estimate of 4.6 per 100 person years (95% CI 2.9-6.9). Considering previous estimates of symptomatic scrub typhus incidence in the same study population, only about 2-5% of infections may result in clinically relevant disease.

Diagnostic evaluation of IgM ELISA and IgM Immunofluorescence assay for the diagnosis of Acute Scrub Typhus in central Nepal.

BACKGROUND: Scrub typhus is an acute febrile illness caused by the obligate intracellular bacterium, Orientia tsutsugamushi. Immunochromatography (ICT) and IgM ELISA are two of the routinely employed antibody based assays for diagnosis of Scrub typhus fever in Nepal, although the recommended gold standard diagnostic test is IgM Immunofluorescence assay (IFA). This study evaluated InBios Scrub Typhus Detect™ Immunoglobulin M (IgM) ELISA and IgM Immunofluorescence assays in single serum sample at the time of admission. METHOD: Study participants (1585 suspected cases), were enrolled based on acute febrile illness with suspected scrub typhus cases in central Nepal. Blood sample was collected from the suspected patients of scrub typhus, presenting with acute febrile illness. IgM antibody to Orientia tsusugamushi was detected by using Scrub Typhus Detect™ Kit and an in-house IgM IFA. The IFA assay was performed with the Gilliam, Karp, Kato strains and O. chuto antigens following the ARRL protocol. RESULT: Statistical analysis of IgM ELISA results when compared to reference test, IgM IFA results demonstrated the following characteristics, sensitivity 84.0% (95%CI: 79.73-87.68%), specificity 94.82% (95% CI: 93.43-95.99%), positive likelihood ratio 16.21% (95% CI: 12.71-20.67%), negative likelihood ratio 0.17% (95% CI: 0.13-0.21%), disease prevalence 22.08% (95% CI: 20.06 -24.21%), positive predictive value 82.12% (95% CI: 78.28-85.42%) and negative predictive value 95.44% (95% CI: 94.27-96.38%) respectively. CONCLUSION: Although IgM IFA is considered the gold standard test for the diagnosis of scrub typhus cases, it is relatively expensive, requires trained personal and a microscope with fluorescence filters. Scrub typhus IgM ELISA may be the best alternative test and possible viable option for resource limited endemic countries like Nepal.

Diagnosis of spotted fever group Rickettsia infections: the Asian perspective.

Spotted fever group rickettsiae (SFG) are a neglected group of bacteria, belonging to the genus Rickettsia, that represent a large number of new and emerging infectious diseases with a worldwide distribution. The diseases are zoonotic and are transmitted by arthropod vectors, mainly ticks, fleas and mites, to hosts such as wild animals. Domesticated animals and humans are accidental hosts. In Asia, local people in endemic areas as well as travellers to these regions are at high risk of infection. In this review we compare SFG molecular and serological diagnostic methods and discuss their limitations. While there is a large range of molecular diagnostics and serological assays, both approaches have limitations and a positive result is dependent on the timing of sample collection. There is an increasing need for less expensive and easy-to-use diagnostic tests. However, despite many tests being available, their lack of suitability for use in resource-limited regions is of concern, as many require technical expertise, expensive equipment and reagents. In addition, many existing diagnostic tests still require rigorous validation in the regions and populations where these tests may be used, in particular to establish coherent and worthwhile cut-offs. It is likely that the best strategy is to use a real-time quantitative polymerase chain reaction (qPCR) and immunofluorescence assay in tandem. If the specimen is collected early enough in the infection there will be no antibodies but there will be a greater chance of a PCR positive result. Conversely, when there are detectable antibodies it is less likely that there will be a positive PCR result. It is therefore extremely important that a complete medical history is provided especially the number of days of fever prior to sample collection. More effort is required to develop and validate SFG diagnostics and those of other rickettsial infections.

Biosafety and biosecurity requirements for Orientia spp. diagnosis and research: recommendations for risk-based biocontainment, work practices and the case for reclassification to risk group 2.

Scrub typhus is an important arthropod-borne disease causing significant acute febrile illness by infection with Orientia spp.Using a risk-based approach, this review examines current practice, the evidence base and regulatory requirements regarding matters of biosafety and biosecurity, and presents the case for reclassification from Risk Group 3 to Risk Group 2 along with recommendations for safe working practices of risk-based activities during the manipulation of Orientia spp. in the laboratory.We recommend to reclassify Orientia spp. to Risk Group 2 based on the classification for RG2 pathogens as being moderate individual risk, low community risk. We recommend that low risk activities, can be performed within a biological safety cabinet located in a Biosafety Level (BSL) 2 core laboratory using standard personal protective equipment. But when the risk assessment indicates, such as high concentration and volume, or aerosol generation, then a higher biocontainment level is warranted. For, the majority of animal activities involving Orientia spp., Animal BSL 2 (ABSL2) is recommended however where high risk activities are performed including necropsies, Animal BSL (ABSL3) is recommended.

A Concise Review of the Epidemiology and Diagnostics of Rickettsioses: Rickettsia and Orientia spp.

Rickettsioses are globally distributed and caused by the family Rickettsiaceae, which comprise a diverse and expanding list of organisms. These include two genera, Rickettsia and Orientia Serology has been traditionally the mainstay of diagnosis, although this has been limited by cross-reactions among closely related members and diminished sensitivity/utility in the acute phase of illness. Other techniques, such as nucleic acid amplification tests using blood specimens or tissue swabs/biopsy specimens, sequencing, and mass spectrometry, have emerged in recent years for both pathogen and vector identification. This paper provides a concise review of the rickettsioses and the traditional and newer technologies available for their diagnosis.

Scrub Typhus Outbreak in a Remote Primary School, Bhutan, 2014.

Scrub typhus in Bhutan was first reported in 2009. We investigated an outbreak of scrub typhus in a remote primary school during August-October 2014. Delay in recognition and treatment resulted in 2 deaths from meningoencephalitis. Scrub typhus warrants urgent public health interventions in Bhutan.

Rickettsia gravesii sp. nov.: a novel spotted fever group rickettsia in Western Australian Amblyomma triguttatum triguttatum ticks.

A rickettsial organism harboured by Amblyomma triguttatum ticks on Barrow Island, Western Australia, was discovered after reports of possible rickettsiosis among local workers. Subsequent isolation of this rickettsia (strain BWI-1) in cell culture and analysis of its phylogenetic, genotypic and phenotypic relationships with type strains of Rickettsia species with standing in nomenclature suggested that it was sufficiently divergent to warrant its classification as a new species. Multiple gene comparison of strain BWI-1 revealed degrees of sequence similarity with Rickettsia raoultii, its closest relative, of 99.58, 98.89, 97.03, 96.93 and 95.73 % for the 16S rRNA, citrate synthase, ompA, ompB and sca4 genes, respectively. Serotyping in mice also demonstrated that strain BWI-1T was distinct from Rickettsia raoultii. Thus, we propose the naming of a new species, Rickettsia gravesii sp. nov., based on its novel genotypic and phenotypic characteristics. Strain BWI-1T was deposited in the ATCC, CSUR and ARRL collections under reference numbers VR-1664, CSUR R172 and RGBWI-1, respectively.

A longitudinal study of serological responses to Coxiella burnetii and shedding at kidding among intensively-managed goats supports early use of vaccines.

Vaccination against Coxiella burnetii, the cause of Q fever, is reportedly the only feasible strategy of eradicating infection in ruminant herds. Preventive vaccination of seronegative goats is more effective in reducing shedding of C. burnetii than vaccinating seropositive goats. The age at which goats born on heavily-contaminated farms first seroconvert to C. burnetii has not yet been documented. In a 16-month birth cohort study, the age at which goats seroconverted against C. burnetii was investigated; 95 goats were bled every 2 weeks and tested for antibodies against C. burnetii. Risk factors for seroconversion were explored and goats shedding C. burnetii were identified by testing vaginal swabs taken at the goats' first kidding using a com1 polymerase chain reaction assay. The first surge in the number of goats with IgM to C. burnetii was observed at week 9. Thus, a first vaccination not later than 8 weeks of age to control C. burnetii in highly contaminated environments is indicated. The odds of seroconversion were 2.0 times higher [95% confidence interval (CI) 1.2, 3.5] in kids born by does with serological evidence of recent infection (IgM seropositive) compared to kids born by IgM seronegative does, suggesting either in utero transmission or peri-parturient infection. The rate of seroconversion was 4.5 times higher (95% CI 2.1, 9.8) during than outside the kidding season, highlighting the risk posed by C. burnetii shed during kidding, even to goats outside the kidding herd. Shedding of C. burnetii at kidding was detected in 15 out of 41 goats infected before breeding.

Tick-borne infectious diseases in Australia.

Tick bites in Australia can lead to a variety of illnesses in patients. These include infection, allergies, paralysis, autoimmune disease, post-infection fatigue and Australian multisystem disorder. Rickettsial (Rickettsia spp.) infections (Queensland tick typhus, Flinders Island spotted fever and Australian spotted fever) and Q fever (Coxiella burnetii) are the only systemic bacterial infections that are known to be transmitted by tick bites in Australia. Three species of local ticks transmit bacterial infection following a tick bite: the paralysis tick (Ixodes holocyclus) is endemic on the east coast of Australia and causes Queensland tick typhus due to R. australis and Q fever due to C. burnetii; the ornate kangaroo tick (Amblyomma triguttatum) occurs throughout much of northern, central and western Australia and causes Q fever; and the southern reptile tick (Bothriocroton hydrosauri) is found mainly in south-eastern Australia and causes Flinders Island spotted fever due to R. honei. Much about Australian ticks and the medical outcomes following tick bites remains unknown. Further research is required to increase understanding of these areas.

Novel genotypes of Coxiella burnetii identified in isolates from Australian Q fever patients.

Coxiella burnetii, the causative agent of Q fever, was first discovered in Australia in 1937. However, little is known about the strains of C. burnetii present in this country. In this study, six published genotyping methods were applied to 42 isolates from Australian patients with acute (n=39) and chronic (n=3) Q fever. All the isolates contained the plasmid QpRS and lacked the acute disease antigen A (adaA) gene. Two methods of genotyping based on single nucleotide polymorphisms (SNPs) also failed to discriminate between the isolates. However, results from the method based on SNPs within the multi-spacer sequence typing (MST) loci determined a novel MST genotype, with the Australian isolates forming a unique phylogenetic clade. Multi-locus variable number of tandem repeats (VNTR) analysis (MLVA) determined 14 genotypes, all of which were novel compared with those previously identified in strains from other countries. Many of these were single locus variants, differing from each other at just one of the 15 loci tested. Our results show that the Australian isolates exhibit significant diversity from previously characterised strains, but are genetically closely related to each other, supporting a model of evolution by clonal expansion in a geographically isolated location.

Update on tick-borne rickettsioses around the world: a geographic approach.

Tick-borne rickettsioses are caused by obligate intracellular bacteria belonging to the spotted fever group of the genus Rickettsia. These zoonoses are among the oldest known vector-borne diseases. However, in the past 25 years, the scope and importance of the recognized tick-associated rickettsial pathogens have increased dramatically, making this complex of diseases an ideal paradigm for the understanding of emerging and reemerging infections. Several species of tick-borne rickettsiae that were considered nonpathogenic for decades are now associated with human infections, and novel Rickettsia species of undetermined pathogenicity continue to be detected in or isolated from ticks around the world. This remarkable expansion of information has been driven largely by the use of molecular techniques that have facilitated the identification of novel and previously recognized rickettsiae in ticks. New approaches, such as swabbing of eschars to obtain material to be tested by PCR, have emerged in recent years and have played a role in describing emerging tick-borne rickettsioses. Here, we present the current knowledge on tick-borne rickettsiae and rickettsioses using a geographic approach toward the epidemiology of these diseases.

A serological assay using Tropheryma whipplei antigens for the presumptive exclusion of Whipple disease.

Whipple disease (WD) is a rare infection in genetically susceptible people caused by the bacterium Tropheryma whipplei. An indirect immunofluorescence serological assay (IFA), detecting patient antibodies to the bacterium, was developed using T. whipplei as antigen. We hypothesised that this assay could be used to rule out WD in patients in whom the diagnosis was being considered, based on high immunoglobulin (Ig) G titres to T. whipplei. In this study, 16 confirmed WD patients and 156 age-matched controls from across Australia were compared serologically. WD patients mostly underproduced IgG antibody to T. whipplei, with titres of ≤1:32 being common. While at an antibody titre of <1:64 the assay sensitivity for WD was only 69% [95% confidence interval (CI) 41-89%], its specificity for excluding WD was 91% (95% CI 85-95%). This specificity increased to 95% (95% CI 90-98%) at an antibody titre of <1:16. Patients with antibody titres of >1:64 were unlikely to have WD. At this titre, the seroprevalence of T. whipplei IgG antibody was 92% (223/242) in Australian blood donors. Unlike other serological assays, which are used to confirm a specific infection, this novel assay is designed to rule out WD infection with a specificity in Australia of 91%. Further validation of this assay, by trialling in other countries, should now be undertaken, as its usefulness is dependent on there being a high background seropositivity to T. whipplei in the general population at the location in which the assay is being used.

Detection of Various Rickettsial Species in Ticks Collected from Small Ruminants in Western Iran.

Background: Most of the rickettsioses are transmitted by ticks, and often overlooked by the medical profession, but are clinically important as they cause major human diseases. Recent studies have shown the existence of some rickettsial species in Iran, but very little information is available about the status of rickettsial epidemiology and ecology. This study investigated the presence of Rickettsia spp. in ticks and ruminants in western of Iran by molecular methods. Materials and Methods: 250 blood samples were collected from sheep and goats, as well as 244 ticks were collected opportunistically from ruminants in the Kurdistan province. The collected samples were tested using a real-time quantitative PCR (qPCR) assay targeting the Rickettsia 16SrRNA gene. Rickettsia spp. positive by the qPCR were further amplified by conventional PCR of the gltA and OmpA genes. These ampliqons were further analyzed by sequencing. Results: The ticks species collected in this study included Rhipicephalus sanguineus, Rh. turanicus, Haemaphysalis concinna, and Dermacentor marginatus. In total, DNA of Rickettsia spp. was detected in 131 collected ticks (53.7%). Of the positives, Rickettsia slovaca (59.2%) and Ri. hoogstraalii (16.3%) were the most common species identified followed by Ri. raoultii, Ri. massiliae, Ri. sibirica, and Ri. conorii subsp. israelensis. In contrast, there were no positives observed in the blood samples collected from ruminants. Conclusion: The results indicate the presence of rickettsial species in ticks. The detection of these pathogens is significant because they cause clinical disease in humans. The results support the notion that the Iranian public health system needs to be more aware of these diseases.

Synthetic Particulate Subunit Vaccines for the Prevention of Q Fever.

Coxiella burnetti is an intracellular bacterium that causes Q fever, a disease of worldwide importance. Q-VAX® , the approved human Q fever vaccine, is a whole cell vaccine associated with safety concerns. Here a safe particulate subunit vaccine candidate is developed that is ambient-temperature stable and can be cost-effectively manufactured. Endotoxin-free Escherichia coli is bioengineered to efficiently self-assemble biopolymer particles (BPs) that are densely coated with either strings of 18 T-cell epitopes (COX-BP) or two full-length immunodominant antigens (YbgF-BP-Com1) all derived from C. burnetii. BP vaccine candidates are ambient-temperature stable. Safety and immunogenicity are confirmed in mice and guinea pig (GP) models. YbgF-BP-Com1 elicits specific and strong humoral immune responses in GPs with IgG titers that are at least 1 000 times higher than those induced by Q-VAX® . BP vaccine candidates are not reactogenic. After challenge with C. burnetii, YbgF-BP-Com1 vaccine leads to reduced fever responses and pathogen burden in the liver and the induction of proinflammatory cytokines IL-12 and IFN-γ inducible protein (IP-10) when compared to negative control groups. These data suggest that YbgF-BP-Com1 induces functional immune responses reducing infection by C. burnetii. Collectively, these findings illustrate the potential of BPs as effective antigen carrier for Q fever vaccine development.

Characterising Eastern Grey Kangaroos (Macropus giganteus) as Hosts of Coxiella burnetii.

Macropods are often implicated as the main native Australian reservoir hosts of Coxiella burnetii (Q fever); however, the maintenance and transmission capacity of these species are poorly understood. The objective of this cross-sectional study was to describe the epidemiology of C. burnetii in a high-density population of eastern grey kangaroos (Macropus giganteus) in a peri-urban coastal nature reserve in New South Wales, Australia. Blood, faeces and swabs were collected from forty kangaroos as part of a population health assessment. Frozen and formalin-fixed tissues were also collected from 12 kangaroos euthanised on welfare grounds. Specimens were tested for C. burnetii using PCR, serology, histopathology and immunohistochemistry. A total of 33/40 kangaroos were seropositive by immunofluorescence assay (estimated true seroprevalence 84%, 95% confidence interval [CI] 69% to 93%), with evidence of rising titres in two animals that had been tested four years earlier. The PCR prevalence was 65% (95% CI 48% to 79%), with positive detection in most sample types. There was no evidence of pathology consistent with C. burnetii, and immunohistochemistry of PCR-positive tissues was negative. These findings indicate that kangaroos are competent maintenance hosts of C. burnetii, likely forming a significant part of its animal reservoir at the study site.

Tissue distribution of Coxiella burnetii and antibody responses in macropods co-grazing with livestock in Queensland, Australia.

Coxiella burnetii, the causative agent of Q fever, is a zoonotic bacteria of global public health significance. The organism has a complex, diverse, and relatively poorly understood animal reservoir but there is increasing evidence that macropods play some part in the epidemiology of Q fever in Australia. The aim of this cross-sectional survey was to estimate the animal- and tissue-level prevalence of coxiellosis amongst eastern grey (Macropus giganteus) and red (Osphranter rufus) kangaroos co-grazing with domestic cattle in a Q fever endemic area in Queensland. Serum, faeces and tissue samples from a range of organs were collected from 50 kangaroos. A total of 537 tissue samples were tested by real-time PCR, of which 99 specimens from 42 kangaroos (84% of animals, 95% confidence interval [CI], 71% to 93%) were positive for the C. burnetii IS1111 gene when tested in duplicate. Twenty of these specimens from 16 kangaroos (32%, 95% CI 20% to 47%) were also positive for the com1 or htpAB genes. Serum antibodies were present in 24 (57%, 95% CI 41% to 72%) of the PCR positive animals. There was no statistically significant difference in PCR positivity between organs and no single sample type consistently identified C. burnetii positive kangaroos. The results from this study identify a high apparent prevalence of C. burnetii amongst macropods in the study area, albeit seemingly with an inconsistent distribution within tissues and in relatively small quantities, often verging on the limits of detection. We recommend Q fever surveillance in macropods should involve a combination of serosurveys and molecular testing to increase chances of detection in a population, noting that a range of tissues would likely need to be sampled to confirm the diagnosis in a suspect positive animal.