Development, Strategies, and Challenges for Tularemia Vaccine.

Francisella tularensis is a facultative intracellular bacterial pathogen that affects both humans and animals. It was developed into a biological warfare weapon as a result. In this article, the current status of tularemia vaccine development is presented. A live-attenuated vaccine that was designed over 50 years ago using the less virulent F. tularensis subspecies holarctica is the only prophylactic currently available, but it has not been approved for use in humans or animals. Other promising live, killed, and subunit vaccine candidates have recently been developed and tested in animal models. This study will investigate some possible vaccines and the challenges they face during development.

Molecular detection of Coxiella burnetii in tick and blood samples from small ruminants in northwest of Iran.

This survey sought to molecularly detect Coxiella burnetii in Argasidae and Ixodidae ticks attached to small ruminants in the region of West Azerbaijan (Northwest of Iran) and blood samples collected from the same animals. 451 tick samples and 927 blood samples were obtained from sheep (n = 536) and goats (n = 391) and tested by nested PCR for detection of C. burnetii insertion sequence IS1111 or icd gene sequence. The collected ticks were morphologically classified as Rhipicephalus sanguineus, Rhipicephalus turanicus, Hyalomma asiaticum, Hyalomma anatolicum, or Argas reflexus. 14% of ticks (65 in total 43 for IS1111 and 22 for icd gene) tested positive for C. burnetii, none of which were from the Argas genus. Among the 927 blood samples, 218 (23.5%) tested positive for C. burnetii. The positive result from analysis targeting the genes IS1111 and icd were 131 and 87 respectively. As Q fever is a tickborne zoonosis and endemic to Iran, such information is critical for creating effective, coordinated, and strategic tick and pathogen control programs to prevent disease outbreak in domestic animals and humans.

Rapid and automated screening of carbapenemase- and ESBL-producing Gram-negative bacteria from rectal swabs using chromogenic agar media and the ScanStation device.

The ScanSation 100 device (Interscience, France) is an incubator allowing real-time detection of bacterial colony growth by frequently imaging agar plates over time, counting CFU, and detecting colony color. This study evaluated its performance for the early detection of carbapenemase-producing bacteria (CPB) and extended-spectrum ß-Lactamase-producing bacteria (ESBL-PB) from rectal swabs inoculated on CHROMagar mSuperCARBA and ESBL media, respectively. Rectal screening ESwabs collected from patients admitted to Grenoble University Hospital between January and June 2021 were analyzed. After inoculation, chromogenic media were incubated for 24 h in the automaton, with image acquisition every 30 min. ScanStation results were compared to visual observations of the plates after 24 h of incubation. In total, 501 rectal swabs were tested. ScanStation showed 100% positive percent agreement (PPA) for the detection of CPB and ESBL-PB, whereas the PPA of color categorization ranged between 45% and 100%. Negative percent agreement (NPA) ranged between 70% and 98%. Negative predictive values (NPVs) were 100% for both bacterial groups, whereas positive predictive values (PPVs) were 70.3% for CPB and 81.0% for ESBL-PB. Importantly, real-time screening allowed detection of the first suspected colony within 10-14 h of growth, on average, whereas visual observation is usually only performed once a day after 18-24 h of incubation. Our study demonstrates the potential use of early images to accelerate the detection of CPB and ESBL-PB and implement effective and timely infection control measures. IMPORTANCE The ScanStation 100 device is an incubator able to follow the real-time growth of bacterial colonies on agar plates through digital imaging, allowing users to sort plates according to the presence or absence of colonies, and to distinguish their color using four numeric color filters. Real-time screening shows that first colony detection is possible much earlier (after 10-14 h of growth, on average), whereas visual observation is usually performed only once a day after 18-24 h of incubation. The ScanStation device, combined with chromogenic agar media, is an efficient automated screening method to accelerate the detection of Gram-negative multidrug-resistant bacteria in laboratories that do not have access to larger laboratory automation systems. Our study shows that setting the image acquisition to one or two early images may allow for the detection of positive samples that were inoculated in the morning, by the end of the working day.

Sputum handling for rheology.

The rheology of sputum is viewed as a powerful emerging biophysical marker for monitoring muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and non-CF bronchiectasis (NCFB). However, there is no unified practice to process sputa from collection to analysis, which can lead to highly variable, and sometimes inconsistent results. The main objective of this study is to bring light into the handling of sputum samples to establish a standardised and robust protocol before rheological measurements. Sputum collected from 22 CF and 10 NCFB adults, was divided into control (vortexed and fresh: non-heated and non-frozen) and three treated conditions (either non-vortexed, heated or frozen). In addition, 6 CF expectorations were used to study the dynamics of ageing over 24 h. Sputum's mechanical properties were measured with a rotational rheometer to obtain their properties at rest, elastic ([Formula: see text]) and viscous moduli ([Formula: see text]), and at the onset of flow, critical deformation ([Formula: see text]) and critical stress ([Formula: see text]). We demonstrate that heating sputum is completely destructive while freezing sputa at [Formula: see text] has no discernible effect on their rheology. We also show that the variability of rheological measurements largely resulted from the sample's macroscopic heterogeneity, and can be greatly reduced by non-destructive vortex homogenisation. Finally, we observed contrasted ageing effects as a fonction of purulence: while the viscoelasticity of purulent samples reduced by half within 6 h after collection, semi-purulent samples did not evolve. These results guide towards a robust unified protocol for simple sputum handling in rheometry. We therefore suggest to vortex and snap freeze sputum samples immediately after collection when direct testing is not possible.

Case Studies and Literature Review of Francisella tularensis-Related Prosthetic Joint Infection.

Tularemia is a zoonotic infection caused by Francisella tularensis. Its most typical manifestations in humans are ulceroglandular and glandular; infections in prosthetic joints are rare. We report 3 cases of F. tularensis subspecies holarctica-related prosthetic joint infection that occurred in France during 2016-2019. We also reviewed relevant literature and found only 5 other cases of Francisella-related prosthetic joint infections worldwide, which we summarized. Among those 8 patients, clinical symptoms appeared 7 days to 19 years after the joint placement and were nonspecific to tularemia. Although positive cultures are typically obtained in only 10% of tularemia cases, strains grew in all 8 of the patients. F. tularensis was initially identified in 2 patients by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; molecular methods were used for 6 patients. Surgical treatment in conjunction with long-term antimicrobial treatment resulted in favorable outcomes; no relapses were seen after 6 months of follow-up.

Tularemia treatment: experimental and clinical data.

Tularemia is a zoonosis caused by the Gram negative, facultative intracellular bacterium Francisella tularensis. This disease has multiple clinical presentations according to the route of infection, the virulence of the infecting bacterial strain, and the underlying medical condition of infected persons. Systemic infections (e.g., pneumonic and typhoidal form) and complications are rare but may be life threatening. Most people suffer from local infection (e.g., skin ulcer, conjunctivitis, or pharyngitis) with regional lymphadenopathy, which evolve to suppuration in about 30% of patients and a chronic course of infection. Current treatment recommendations have been established to manage acute infections in the context of a biological threat and do not consider the great variability of clinical situations. This review summarizes literature data on antibiotic efficacy against F. tularensis in vitro, in animal models, and in humans. Empirical treatment with beta-lactams, most macrolides, or anti-tuberculosis agents is usually ineffective. The aminoglycosides gentamicin and streptomycin remain the gold standard for severe infections, and the fluoroquinolones and doxycycline for infections of mild severity, although current data indicate the former are usually more effective. However, the antibiotic treatments reported in the literature are highly variable in their composition and duration depending on the clinical manifestations, the age and health status of the patient, the presence of complications, and the evolution of the disease. Many patients received several antibiotics in combination or successively. Whatever the antibiotic treatment administered, variable but high rates of treatment failures and relapses are still observed, especially in patients treated more then 2-3 weeks after disease onset. In these patients, surgical treatment is often necessary for cure, including drainage or removal of suppurative lymph nodes or other infectious foci. It is currently difficult to establish therapeutic recommendations, particularly due to lack of comparative randomized studies. However, we have attempted to summarize current knowledge through proposals for improving tularemia treatment which will have to be discussed by a group of experts. A major factor in improving the prognosis of patients with tularemia is the early administration of appropriate treatment, which requires better medical knowledge and diagnostic strategy of this disease.

Francisella tularensis PCR detection in Cape hares (Lepus capensis) and wild rabbits (Oryctolagus cuniculus) in Algeria.

Tularemia is a zoonosis caused by the bacterium Francisella tularensis. Leporids are primary sources of human infections in the northern hemisphere. Africa is classically considered free of tularemia, but recent data indicate that this dogma might be wrong. We assessed the presence of this disease in wild leporids in Algeria. Between 2014 and 2018, we collected 74 leporids carcasses from spontaneously dead or hunted animals. Francisella tularensis DNA was detected by specific real-time PCR tests in 7/36 (19.44%) Cape hares (Lepus capensis) and 5/38 (13.15%) wild rabbits (Oryctolagus cuniculus). Known tularemia arthropod vectors infested half of the PCR-positive animals. At necropsy, F. tularensis-infected animals presented with an enlarged spleen (n = 12), enlarged adrenal glands (12), liver discoloration (12), hemorrhages (11), and pneumonia (11). Immunohistological examination of liver tissue from one animal was compatible with the presence of F. tularensis. Our study demonstrates the existence of tularemia in lagomorphs in Algeria. It should encourage investigations to detect this disease among the human population of this country.

Acute postoperative endophthalmitis: Microbiology from the laboratory to the bedside.

Postoperative endophthalmitis is a dreaded complication of intraocular surgery. Acute presentations need prompt management and good knowledge of differential diagnoses. In the last 10 years, progress in direct microbial detection and identification from intraocular samples included the use of blood culture systems and, more recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, improving the rate of bacterial identification. Whatever the method used, diagnostic sensitivity is better for vitreous samples than for aqueous humor samples. Besides, molecular biology techniques have further improved the identification rate of infectious agents in intraocular samples. They also provide faster results compared to culture-based techniques. Quantitative real-time PCR (qPCR) can also determine the bacterial load in intraocular samples. Several studies have shown that intraocular bacterial loads in endophthalmitis patients are usually high, which helps differentiating infection from contamination. The prognostic value of qPCR remains to be validated. Whole genome DNA sequencing technologies facilitate direct and sequencing of single DNA molecules. They have the potential to increase the rate of microbiological identification. Some antibiotic resistance markers (e.g., methicillin resistance in staphylococci and vancomycin resistance in enterococci) may be detected earlier using molecular techniques (usually real-time PCR tests). Early determination of the involved microorganism and their antibiotic resistances can help establishing an earlier therapeutic strategy.

A case report of ulceroglandular tularemia caused by Francisella tularensis subsp. Holarctica in Iran.

BACKGROUND: Tularemia is a zoonotic disease that has been reported in many countries of the Northern Hemisphere. However, in some countries, such as Iran, this disease has been neglected by the health care system, and it is under-reported. CASE PRESENTATION: Here we report an unusual case of ulceroglandular tularemia occurring in a 35-year-old woman who presented with a skin lesion of the left flank, inguinal lymphadenopathy, and an abdominal abscess. The serological and real-time PCR tests for tularemia were positive for this patient, and infection by Francisella tularensis subsp. holarctica was confirmed. CONCLUSIONS: It is necessary to implement various educational programs to increase the awareness of physicians with tularemia.

Identification of Algerian field-caught mosquito vectors by MALDI-TOF MS.

Vector-borne diseases represent a real threats worldwide, in reason of the lack of vaccine and cure for some diseases. Among arthropod vectors, mosquitoes are described to be the most dangerous animal on earth, resulting in an estimated 725,000 deaths per year due to their borne diseases. Geographical position of Algeria makes this country a high risk area for emerging and re-emerging diseases, such as dengue coming from north (Europe) and malaria from south (Africa). To prevent these threats, rapid and continuous surveillance of mosquito vectors is essential. For this purpose we aimed in this study to create a mosquito vectors locale database using MALDI-TOF mass spectrometry technology for rapid identification of these arthropods. This methodology was validated by testing 211 mosquitoes, including four species (Aedes albopictus, Culex pipiens, Culex quinquefasciatus, and Culiseta longiareolata), in two northern wilayahs of Algeria (Algiers and Bejaia). Species determination by MALDI TOF MS was highly concordant with reference phenotypic and genetic methods. Using this MALDI-TOF MS tool will allow better surveillance of mosquito species able to transmit mosquito borne diseases in Algeria.

Ulceroglandular Infection and Bacteremia Caused by Francisella salimarina in Immunocompromised Patient, France.

Although Francisella tularensis is a well-known, highly virulent bacterium that causes tularemia in humans, other Francisella species have been associated with sporadic human infections. We describe a human cutaneous infection with bacteremia caused by F. salimarina, a Francisella species recently identified from seawater and fishes, in an immunocompromised patient in France.

Enterobacter cloacae Complex and CTX-M Extended-Spectrum ß-Lactamases in Algeria.

We evaluated the ß-lactam resistance phenotypes of clinical and environmental strains of the Enterobacter cloacae complex (ECC) isolated from three Algerian hospitals. The first combination of API 20E, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and hsp60 genetic clustering methodologies were carried out for the identification of ECC strains. Our research showed that API 20E and MALDI TOF MS are satisfactory in genus identification of ECC strains, but sequence-based methods are then necessary to discriminate the species and subspecies levels. Among 36 ECC strains, 94.44% belonged to Enterobacter hormaechei species. Twenty-five isolates clustered with the reference strain of E. hormaechei subsp. xiangfangensis, making it the most frequently isolated subspecies. Enterobacter kobei was found only once (2.77%). All ECC isolates were phenotypically extended-spectrum ß-lactamase (ESBL) producers and were resistant to ticarcillin, piperacillin, cefoxitin, cefotaxime, ceftazidime, ceftriaxone, and aztreonam, but susceptible to ertapenem and imipenem. The genetic analyses only allowed the detection of resistance genes of the CTX-M-1 group (32 strains, 88.9%), including CTX-M-15 (30 strains), CTX-M-3 (1 strain), and CTX-M-22 (1 strain). We report for the first time the detection of CTX-M-22 among ECC strains in an Algerian hospital (Tlemcen hospital). None of the isolated strains harbored CTX-M-2, CTX-M-9, or CTX-M-8/25 group genes. In this review, we address recent comparison in the identification methods of multidrug-resistant E. cloacae complex in Algeria, focusing also on the CTX-M ESBLs. This represents a serious public health challenge, which requires the clarification of the current situation and warrants the reinforcement of hygiene measures in the Algerian hospitals.

Reuse of medical face masks in domestic and community settings without sacrificing safety: Ecological and economical lessons from the Covid-19 pandemic.

The need for personal protective equipment increased exponentially in response to the Covid-19 pandemic. To cope with the mask shortage during springtime 2020, a French consortium was created to find ways to reuse medical and respiratory masks in healthcare departments. The consortium addressed the complex context of the balance between cleaning medical masks in a way that maintains their safety and functionality for reuse, with the environmental advantage to manage medical disposable waste despite the current mask designation as single-use by the regulatory frameworks. We report a Workflow that provides a quantitative basis to determine the safety and efficacy of a medical mask that is decontaminated for reuse. The type IIR polypropylene medical masks can be washed up to 10 times, washed 5 times and autoclaved 5 times, or washed then sterilized with radiations or ethylene oxide, without any degradation of their filtration or breathability properties. There is loss of the anti-projection properties. The Workflow rendered the medical masks to comply to the AFNOR S76-001 standard as "type 1 non-sanitory usage masks". This qualification gives a legal status to the Workflow-treated masks and allows recommendation for the reuse of washed medical masks by the general population, with the significant public health advantage of providing better protection than cloth-tissue masks. Additionally, such a legal status provides a basis to perform a clinical trial to test the masks in real conditions, with full compliance with EN 14683 norm, for collective reuse. The rational reuse of medical mask and their end-of-life management is critical, particularly in pandemic periods when decisive turns can be taken. The reuse of masks in the general population, in industries, or in hospitals (but not for surgery) has significant advantages for the management of waste without degrading the safety of individuals wearing reused masks.

Presence of Francisella tularensis subsp. holarctica DNA in the Aquatic Environment in France.

In 2018, the incidence of tularemia increased twofold in the west of France, with many pneumonic forms, suggesting environmental sources of infection. We investigated the presence of Francisellatularensis subsp. holarctica and other Francisella species DNA in the natural aquatic environment of this geographic area. Two sampling campaigns, in July 2019 and January 2020, allowed the collection of 87 water samples. Using a combination of real-time PCR assays, we tested the presence of either Francisella sp., F. tularensis/F. novicida, and F. tularensis subsp. holarctica, the latter being the only tularemia agent in Europe. Among 57 water samples of the first campaign, 15 (26.3%) were positive for Francisella sp., nine (15.8%) for F. tularensis and/or F. novicida, and four (7.0%) for F. tularensis subsp. holarctica. Ratios were 25/30 (83.3%), 24/30 (80.0%), and 4/30 (13.3%) for the second campaign. Among the thirty sites sampled during the two campaigns, nine were positive both times for Francisella sp., seven for F. tularensis and/or F. novicida, and one for F. tularensis subsp. holarctica. Altogether, our study reveals a high prevalence of Francisella sp. DNA (including the tularemia agent) in the studied aquatic environment. This aquatic environment could therefore participate in the endemicity of tularemia in the west of France.

Francisella tularensis human infections in a village of northwest Iran.

BACKGROUND: Recent seroepidemiological studies have suggested that tularemia could be an endemic bacterial zoonosis in Iran. METHODS: From January 2016 to June 2018, disease cases characterized by fever, cervical lymphadenopathy and ocular involvement were reported in Youzband Village of Kaleybar County, in the East Azerbaijan Province, northwestern Iran. Diagnostic tests included Francisella tularensis serology (including tube agglutination test and ELISA), PCR, and culture. RESULTS: Among 11 examined case-patients, the tularemia tube agglutination test was positive in ten and borderline in one. PCR detected the F. tularensis ISFtu2 elements and fopA gene in one rodent and a spring water sample from the same geographic area. CONCLUSIONS: Based on the clinical manifestations of the disease suggesting an oropharyngeal form of tularemia, serology results in case patients, and F. tularensis detection in the local fauna and aquatic environment, the water supply of the village was the likely source of the tularemia outbreak. Intervention such as dredging and chlorination of the main water storage tank of the village and training of villagers and health care workers in preventive measures and treatment of the illness helped control the infection.

Mink, SARS-CoV-2, and the Human-Animal Interface.

Mink are small carnivores of the Mustelidae family. The American mink is the most common and was imported to Europe, Asia, and Latin America for breeding, as its fur is very popular. Denmark, the Netherlands, and China are the biggest producers of mink. Mink farms with a high population density in very small areas and a low level of genetic heterogeneity are places conducive to contagion. The mink's receptor for SARS-CoV-2 is very similar to that of humans. Experimental models have shown the susceptibility of the ferret, another mustelid, to become infected with SARS-CoV-2 and to transmit it to other ferrets. On April 23, 2020, for the first time, an outbreak of SARS-CoV-2 in a mink farm was reported in the Netherlands. Since then, COVID-19 has reached numerous mink farms in the Netherlands, Denmark, United States, France, Greece, Italy, Spain, Sweden, Poland, Lithuania, and Canada. Not only do mink become infected from each other, but also they are capable of infecting humans, including with virus variants that have mutated in mink. Human infection with variant mink viruses with spike mutations led to the culling in Denmark of all mink in the country. Several animals can be infected with SARS-CoV-2. However, anthropo-zoonotic outbreaks have only been reported in mink farms. The rapid spread of SARS-CoV-2 in mink farms raises questions regarding their potential role at the onset of the pandemic and the impact of mutants on viral fitness, contagiousness, pathogenicity, re-infections with different mutants, immunotherapy, and vaccine efficacy.

Current Status of Putative Animal Sources of SARS-CoV-2 Infection in Humans: Wildlife, Domestic Animals and Pets.

SARS-CoV-2 is currently considered to have emerged from a bat coronavirus reservoir. However, the real natural cycle of this virus remains to be elucidated. Moreover, the COVID-19 pandemic has led to novel opportunities for SARS-CoV-2 transmission between humans and susceptible animal species. In silico and in vitro evaluation of the interactions between the SARS-CoV-2 spike protein and eucaryotic angiotensin-converting enzyme 2 (ACE2) receptor have tentatively predicted susceptibility to SARS-CoV-2 infection of several animal species. Although useful, these data do not always correlate with in vivo data obtained in experimental models or during natural infections. Other host biological properties may intervene such as the body temperature, level of receptor expression, co-receptor, restriction factors, and genetic background. The spread of SARS-CoV-2 also depends on the extent and duration of viral shedding in the infected host as well as population density and behaviour (group living and grooming). Overall, current data indicate that the most at-risk interactions between humans and animals for COVID-19 infection are those involving certain mustelids (such as minks and ferrets), rodents (such as hamsters), lagomorphs (especially rabbits), and felines (including cats). Therefore, special attention should be paid to the risk of SARS-CoV-2 infection associated with pets.

Amoebae can promote the survival of Francisella species in the aquatic environment.

Francisella tularensis, a tier 1 select agent, is the causative bacterium of tularemia, a zoonosis with a large animal reservoir. However, F. tularensis, like many other Francisella species, is assumed to have an aquatic reservoir. The mechanisms of Francisella species persistence in surface water remain poorly characterized. In this study, we deeply investigated the long-term interactions of the tularemia agent F. tularensis subsp. holarctica, F. novicida or F. philomiragia with amoebae of the Acanthamoeba species. In amoeba plate screening tests, all the Francisella species tested resisted the attack by amoebae. In in vitro infection models, intra-amoebic growth of Francisella varied according to the involved bacterial species and strains, but also the amoeba culture medium used. In co-culture models, the amoebae favoured Francisella survival over 16 days, which was likely dependent on direct contact between bacteria and amoebae for F. novicida and on amoeba-excreted compounds for F. novicida and for F. tularensis. In a spring water co-culture model, amoebae again enhanced F. novicida survival and preserved bacterial morphology. Overall, our results demonstrate that amoebae likely promote Francisella survival in aquatic environments, including the tularemia agent F. tularensis. However, bacteria-amoebae interactions are complex and depend on the Francisella species considered.

High-resolution melting PCR assay as a powerful tool for the epidemiological surveillance of tularemia in Western Europe.

In Europe, tularemia is caused by Francisella tularensis subsp. holarctica and is a sporadic disease affecting mainly wildlife animals and humans. Classification of this species relies on canonical single nucleotide polymorphisms (canSNPs). Four main clades have been described for F. tularensis subsp. holarctica: B.4, B.6, B.12 and B.16. Phylogeographic studies have shown that clade B.6 is predominant in Western Europe and B.12 in Eastern and Central Europe. Based on this global phylogeny, we aimed to design a molecular typing assay for all genetic subclades of subclade B.11, which is the predominant subclade in clade B.6. We designed high-resolution melting (HRM) primers for the screening of 109 canSNPs divided in seven orders of discrimination for the molecular epidemiology analysis and tracking of Francisella tularensis subsp. holarctica in Western Europe.