Dual proteomics of infected macrophages reveal bacterial and host players involved in the Francisella intracellular life cycle and cell to cell dissemination by merocytophagy.

Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.

Francisella tularensis-specific antibody levels in sera from Swedish patients with suspected tularemia during a 13-year period.

Introduction: For a majority of tularemia patients, serology is the basis for the diagnosis. The aim of this study was to perform an analysis of the samples analyzed at a Swedish reference laboratory for the presence of Francisella tularensis-specific antibody levels in sera from individuals with suspected tularemia. Annual and monthly variations of the total number of samples and proportions of positive samples were analyzed, as well as the influence of age and gender. Methods: We performed a retrospective analysis of the presence of F. tularensis-specific antibodies in serological samples from patients with suspected tularemia analyzed during the period 2010 - 2022 at the University Hospital of Umeå in Sweden, a national reference laboratory, by use of various statistical methods. In total, some 15,100 serum samples had been analyzed for the presence of IgG and IgM antibodies by ELISA during the 13-year period. Results: Overall, there were higher number of samples with IgG positive or borderline titers, 2,522 and 921, respectively, than with IgM positive or borderline titers, 1,802 and 409, respectively. Repeated samples were obtained from some 1,930 individuals and approximately a third of the cases, which were initially seronegative, had seroconverted when resampled. Peak number of monthly samples were recorded in August and September, > 3,000. Annual numbers varied greatly and peak numbers were observed in 2015 and 2019, 1,832 and 2,250, respectively, whereas some other years the numbers were 700 - 800. There was also much variation in the annual and monthly percentages of positive samples and they varied between less than 10% to greater than 20%. The highest percentages of positive samples were recorded in September and October. IgG and IgM titers declined with age and these differences were highly significant for IgG titers, with decreasing average titers for each 20-year interval. Discussion: Collectively, the data demonstrate the marked annual and seasonal variations in tularemia sampling occurring in Sweden. Also, the proportion of positive samples increased during months and years with peak number of samples. Another notable finding was that average antibody titers decreased with increased age.

Functional characterization of Francisella tularensis subspecies holarctica genotypes during tick cell and macrophage infections using a proteogenomic approach.

Tularemia is a vector-borne disease caused by the Gram-negative bacterium Francisella tularensis. Known hosts and vectors in Europe are hare and ticks. F. tularensis is transmitted from ticks and animals, but also from the hydrotelluric environment and the consumption of contaminated water or food. A changing climate expands the range in which ticks can live and consequently might contribute to increasing case numbers of tularemia. Two subspecies of F. tularensis are human pathogenic. Francisella tularensis tularensis (Ftt) is endemic in North America, while Francisella tularensis holarctica (Fth) is the only subspecies causing tularemia in Europe. Ft is classified as a category A bioterrorism agent due to its low infectious dose, multiple modes of transmission, high infectivity and potential for airborne transmission and has become a global public health concern. In line with the European survey and previous phylogenetic studies, Switzerland shows the co-distribution of B.6 and B.12 strains with different geographical distribution and prevalence within the country. To establish itself in different host environments of ticks and mammals, F. tularensis presumably undergoes substantial changes on the transcriptomics and proteomic level. Here we investigate the transcriptomic and proteomic differences of five strains of Fth upon infection of rabbit macrophages and tick cells.

Identification of Francisella tularensis in ascites in the context of typhoidal tularaemia.

Tularaemia is a highly infectious, zoonotic disease caused by Francisella tularensis, which has become increasingly prevalent over the past decade. Depending on the route of infection, different clinical manifestations can be observed. We report a case of typhoidal tularaemia presenting as a febrile illness with gastrointestinal symptoms in a patient in her mid-80s. During the acute illness phase and in the context of alcohol-related liver cirrhosis, the patient developed progressive ascites. During paracentesis, spontaneous bacterial peritonitis was consistently reported. Blood culture revealed Gram-negative bacilli identified as F. tularensis upon microscopic examination. Immediate clinical improvement was observed after adaptation to a pathogen-specific antibiotic regime. Typhoidal tularaemia presents general, non-specific symptoms without the local manifestations seen in other forms of the disease, thus representing a diagnostic challenge. In the case of protracted fever and if the epidemiological context as well as possible exposure are compatible, tularaemia should be considered in the differential diagnosis.

Current vaccine strategies and novel approaches to combatting Francisella infection.

Tularemia is caused by subspecies of Francisella tularensis and can manifest in a variety of disease states, with the pneumonic presentation resulting in the greatest mortality. Despite decades of research, there are no approved vaccines against F. tularensis in the United States. Traditional vaccination strategies, such as live-attenuated or subunit vaccines, are not favorable due to inadequate protection or safety concerns. Because of this, novel vaccination strategies are needed to combat tularemia. Here we discuss the current state of and challenges to the tularemia vaccine field and suggest novel vaccine approaches going forward that might be better suited for protecting against F. tularensis infection.

Identification of an N-terminal tag (580N) that improves the biosynthesis of fluorescent proteins in Francisella tularensis and other Gram-negative bacteria.

Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFPFt that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.

Mutation of wbtJ, a N-formyltransferase involved in O-antigen synthesis, results in biofilm formation, phase variation and attenuation in Francisella tularensis.

Two clinically important subspecies, Francisella tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) are responsible for most tularaemia cases, but these isolates typically form a weak biofilm under in vitro conditions. Phase variation of the F. tularensis lipopolysaccharide (LPS) has been reported in these subspecies, but the role of variation is unclear as LPS is crucial for virulence. We previously demonstrated that a subpopulation of LPS variants can constitutively form a robust biofilm in vitro, but it is unclear whether virulence was affected. In this study, we show that biofilm-forming variants of both fully virulent F. tularensis subspecies were highly attenuated in the murine tularaemia model by multiple challenge routes. Genomic sequencing was performed on these strains, which revealed that all biofilm-forming variants contained a lesion within the wbtJ gene, a formyltransferase involved in O-antigen synthesis. A ΔwbtJ deletion mutant recapitulated the biofilm, O-antigen and virulence phenotypes observed in natural variants and could be rescued through complementation with a functional wbtJ gene. Since the spontaneously derived biofilm-forming isolates in this study were a subpopulation of natural variants, reversion events to the wbtJ gene were detected that eliminated the phenotypes associated with biofilm variants and restored virulence. These results demonstrate a role for WbtJ in biofilm formation, LPS variation and virulence of F. tularensis.

Systematic Review: Clinical Features, Antimicrobial Treatment, and Outcomes of Human Tularemia, 1993-2023.

BACKGROUND: Francisella tularensis, the causative agent of tularemia, is endemic throughout the Northern Hemisphere and requires as few as 10 organisms to cause disease, making this potential bioterrorism agent one of the most infectious bacterial pathogens known. Aminoglycosides, tetracyclines, and, more recently, fluoroquinolones are used for treatment of tularemia; however, data on the relative effectiveness of these and other antimicrobial classes are limited. METHODS: Nine databases, including Medline, Global Health, and Embase, were systematically searched for articles containing terms related to tularemia. Articles with case-level data on tularemia diagnosis, antimicrobial treatment, and patient outcome were included. Patient demographics, clinical findings, antimicrobial administration, and outcome (eg, intubation, fatality) were abstracted using a standardized form. RESULTS: Of the 8878 publications identified and screened, 410 articles describing 870 cases from 1993 to 2023 met inclusion criteria. Cases were reported from 35 countries; more than half were from the United States, Turkey, or Spain. The most common clinical forms were ulceroglandular, oropharyngeal, glandular, and pneumonic disease. Among patients treated with aminoglycosides (n = 452 [52%]), fluoroquinolones (n = 339 [39%]), or tetracyclines (n = 419 [48%]), the fatality rate was 0.7%, 0.9%, and 1.2%, respectively. Patients with pneumonic disease who received ciprofloxacin had no fatalities and the lowest rates of thoracentesis/pleural effusion drainage and intubation compared to those who received aminoglycosides and tetracyclines. CONCLUSIONS: Aminoglycosides, fluoroquinolones, and tetracyclines are effective antimicrobials for treatment of tularemia, regardless of clinical manifestation. For pneumonic disease specifically, ciprofloxacin may have slight advantages compared to other antimicrobials.

Efficacy of Doxycycline and Ciprofloxacin for Treatment of Pneumonic Tularemia in Cynomolgus Macaques.

BACKGROUND: The incidence of pneumonic tularemia is very low; therefore, it is not feasible to conduct clinical efficacy testing of tularemia medical countermeasures (MCMs) in humans. The US Food and Drug Administration's Animal Model Qualification Program under the Drug Development Tools Program is a regulatory pathway for animal models used in MCM efficacy testing and approval under the Animal Rule. The National Institute of Allergy and Infectious Diseases and Biomedical Advanced Research and Development Authority worked together to qualify the cynomolgus macaque model of pneumonic tularemia. METHODS: Using the model parameters and end points defined in the qualified model, efficacy of the antibiotics doxycycline and ciprofloxacin was evaluated in separate studies. Antibiotic administration, aimed to model approved human dosing, was initiated at time points of 24 hours or 48 hours after onset of fever as an indicator of disease. RESULTS: Upon aerosol exposure (target dose of 1000 colony-forming units) to Francisella tularensis SchuS4, 80% of vehicle-treated macaques succumbed or were euthanized. Ciprofloxacin treatment led to 10 of 10 animals surviving irrespective of treatment time. Doxycycline administered at 48 hours post-fever led to 10 of 10 animals surviving, while 9/10 animals survived in the group treated with doxycycline 24 hours after fever. Selected surviving animals in both the placebo and doxycycline 48-hour group showed residual live bacteria in peripheral tissues, while there were no bacteria in tissues from ciprofloxacin-treated macaques. CONCLUSIONS: Both doxycycline and ciprofloxacin were efficacious in treatment of pneumonic tularemia, although clearance of bacteria may be different between the 2 drugs.

Systematic Review of Tularemia During Pregnancy.

BACKGROUND: Tularemia is caused by the gram-negative bacterium Francisella tularensis. Although rare, tularemia during pregnancy has been associated with pregnancy complications; data on efficacy of recommended antimicrobials for treatment are limited. We performed a systematic literature review to characterize clinical manifestations of tularemia during pregnancy and examine maternal, fetal, and neonatal outcomes with and without antimicrobial treatment. METHODS: We searched 9 databases, including Medline, Embase, Global Health, and PubMed Central, using terms related to tularemia and pregnancy. Articles reporting cases of tularemia with ≥1 maternal or fetal outcome were included. RESULTS: Of 5891 articles identified, 30 articles describing 52 cases of tularemia in pregnant patients met inclusion criteria. Cases were reported from 9 countries, and oropharyngeal and ulceroglandular tularemia were the most common presenting forms. A plurality (46%) of infections occurred in the second trimester. Six complications were observed: lymph node aspiration, lymph node excision, maternal bleeding, spontaneous abortion, intrauterine fetal demise, and preterm birth. No deaths among mothers were reported. Of 28 patients who received antimicrobial treatment, 1 pregnancy loss and 1 fetal death were reported. Among 24 untreated patients, 1 pregnancy loss and 3 fetal deaths were reported, including one where F. tularensis was detected in placental and fetal tissues. CONCLUSIONS: Pregnancy loss and other complications have been reported among cases of tularemia during pregnancy. However, risk of adverse outcomes may be lower when antimicrobials known to be effective are used. Without treatment, transplacental transmission appears possible. These data underscore the importance of prompt recognition and treatment of tularemia during pregnancy.

Francisella tularensis Bone and Joint Infections: United States, 2004-2023.

Tularemia is caused by the highly infectious bacterium Francisella tularensis, which is recognized as a Tier 1 bioterrorism agent. Tularemia has a range of recognized clinical manifestations, but fewer than 20 bone or joint infections from 6 countries have been reported in the literature to date. This series includes 13 cases of F. tularensis septic arthritis or osteomyelitis in the United States during 2004-2023 and describes exposures, clinical presentation, diagnosis, and outcomes for this rare but severe form of tularemia. Clinicians should consider F. tularensis in patients with compatible exposures or a history of joint replacement or immunosuppression.

Tularemia Clinical Manifestations, Antimicrobial Treatment, and Outcomes: An Analysis of US Surveillance Data, 2006-2021.

BACKGROUND: Tularemia, a potentially fatal zoonosis caused by Francisella tularensis, has been reported from nearly all US states. Information on relative effectiveness of various antimicrobials for treatment of tularemia is limited, particularly for newer classes such as fluoroquinolones. METHODS: Data on clinical manifestations, antimicrobial treatment, and illness outcome of patients with tularemia are provided voluntarily through case report forms to the US Centers for Disease Control and Prevention by state and local health departments. We summarized available demographic and clinical information submitted during 2006-2021 and evaluated survival according to antimicrobial treatment. We grouped administered antimicrobials into those considered effective for treatment of tularemia (aminoglycosides, fluoroquinolones, and tetracyclines) and those with limited efficacy. Logistic regression models with a bias-reduced estimation method were used to evaluate associations between antimicrobial treatment and survival. RESULTS: Case report forms were available for 1163 US patients with tularemia. Francisella tularensis was cultured from a clinical specimen (eg, blood, pleural fluid) in approximately half of patients (592; 50.9%). Nearly three-quarters (853; 73.3%) of patients were treated with a high-efficacy antimicrobial. A total of 27 patients (2.3%) died. After controlling for positive culture as a proxy for illness severity, use of aminoglycosides, fluoroquinolones, and tetracyclines was independently associated with increased odds of survival. CONCLUSIONS: Most US patients with tularemia received high-efficacy antimicrobials; their use was associated with improved odds of survival regardless of antimicrobial class. Our findings provide supportive evidence that fluoroquinolones are an effective option for treatment of tularemia.

Neuroinvasive Francisella tularensis Infection: Report of 2 Cases and Review of the Literature.

BACKGROUND: Neuroinvasive infection with Francisella tularensis, the causative agent of tularemia, is rare. Establishing clinical suspicion is challenging if risk factors or clinical features classically associated with tularemia are absent. Tularemia is treatable with antibiotics; however, there are limited data to inform management of potentially fatal neuroinvasive infection. METHODS: We collected epidemiologic and clinical data on 2 recent US cases of neuroinvasive F. tularensis infection, and performed a literature review of cases of neuroinvasive F. tularensis infection published after 1950. RESULTS: One patient presented with focal neurologic deficits and brain lesions; broad-range molecular testing on resected brain tissue detected F. tularensis. The other patient presented with meningeal signs; tularemia was suspected based on animal exposure, and F. tularensis grew in cerebrospinal fluid (CSF) culture. Both patients received combination antibiotic therapy and recovered from infection. Among 16 published cases, tularemia was clinically suspected in 4 cases. CSF often displayed lymphocytic pleocytosis. Among cases with available data, CSF culture was positive in 13 of 16 cases, and F. tularensis antibodies were detected in 11 of 11 cases. Treatment typically included an aminoglycoside combined with either a tetracycline or a fluoroquinolone. Outcomes were generally favorable. CONCLUSIONS: Clinicians should consider neuroinvasive F. tularensis infection in patients with meningitis and signs suggestive of tularemia or compatible exposures, lymphocyte-predominant CSF, unrevealing standard microbiologic workup, or lack of response to empiric bacterial meningitis treatment. Molecular testing, culture, and serologic testing can reveal the diagnosis. Favorable outcomes can be achieved with directed antibiotic treatment.

Antimicrobial Susceptibility of Francisella tularensis Isolates in the United States, 2009-2018.

Francisella tularensis is the causative agent of tularemia. We tested the susceptibility of 278 F. tularensis isolates from the United States received during 2009-2018 to 8 antimicrobial drugs (ciprofloxacin, levofloxacin, doxycycline, tetracycline, gentamicin, streptomycin, chloramphenicol, and erythromycin). All isolates were susceptible to all tested drugs.

Confirmed Case of Longstanding Respiratory Francisella tularensis holarctica Infection: Nebraska, 2022.

A male patient with distant history of extensive rabbit contact and pulmonary nodules for 6 years developed empyema. Francisella tularensis holarctica was isolated from thoracentesis fluid. Retrospective immunohistochemical examination of a pulmonary nodule, biopsied 3 years prior, was immunoreactive for F. tularensis. These findings suggest the potential for chronic tularemia.

Tularemia From Veterinary Occupational Exposure.

Tularemia is a disease caused by Francisella tularensis, a highly infectious bacteria that can be transmitted to humans by direct contact with infected animals. Because of the potential for zoonotic transmission of F. tularensis, veterinary occupational risk is a concern. Here, we report on a human case of tularemia in a veterinarian after an accidental needlestick injury during abscess drainage in a sick dog. The veterinarian developed ulceroglandular tularemia requiring hospitalization but fully recovered after abscess drainage and a course of effective antibiotics. To systematically assess veterinary occupational transmission risk of F. tularensis, we conducted a survey of veterinary clinical staff after occupational exposure to animals with confirmed tularemia. We defined a high-risk exposure as direct contact to the infected animal's body fluids or potential aerosol inhalation without use of standard personal protective equipment (PPE). Survey data included information on 20 veterinary occupational exposures to animals with F. tularensis in 4 states. Veterinarians were the clinical staff most often exposed (40%), followed by veterinarian technicians and assistants (30% and 20%, respectively). Exposures to infected cats were most common (80%). Standard PPE was not used during 80% of exposures; a total of 7 exposures were categorized as high risk. Transmission of F. tularensis in the veterinary clinical setting is possible but overall risk is likely low. Veterinary clinical staff should use standard PPE and employ environmental precautions when handling sick animals to minimize risk of tularemia and other zoonotic infections; postexposure prophylaxis should be considered after high-risk exposures to animals with suspected or confirmed F. tularensis infection to prevent tularemia.

Treatment Outcome of Severe Respiratory Type B Tularemia Using Fluoroquinolones.

BACKGROUND: Fluoroquinolones lack approval for treatment of tularemia but have been used extensively for milder illness. Here, we evaluated fluoroquinolones for severe illness. METHODS: In an observational study, we identified case-patients with respiratory tularemia from July to November 2010 in Jämtland County, Sweden. We defined severe tularemia by hospitalization for >24 hours and severe bacteremic tularemia by Francisella tularensis subsp. holarctica growth in blood or pleural fluid. Clinical data and drug dosing were retrieved from electronic medical records. Chest images were reexamined. We used Kaplan-Meier curves to evaluate time to defervescence and hospital discharge. RESULTS: Among 67 case-patients (median age, 66 years; 81% males) 30-day mortality was 1.5% (1 of 67). Among 33 hospitalized persons (median age, 71 years; 82% males), 23 had nonbacteremic and 10 had bacteremic severe tularemia. Subpleural round consolidations, mediastinal lymphadenopathy, and unilateral pleural fluid were common on chest computed tomography. Among 29 hospitalized persons with complete outcome data, ciprofloxacin/levofloxacin (n = 12), ciprofloxacin/levofloxacin combinations with doxycycline and/or gentamicin (n = 11), or doxycycline as the single drug (n = 6) was used for treatment. One disease relapse occurred with doxycycline treatment. Treatment responses were rapid, with median fever duration 41.0 hours in nonbacteremic and 115.0 hours in bacteremic tularemia. Increased age-adjusted Charlson comorbidity index predicted severe bacteremic tularemia (odds ratio, 2.7 per score-point; 95% confidence interval, 1.35-5.41). A 78-year-old male with comorbidities and delayed ciprofloxacin/gentamicin treatment died. CONCLUSIONS: Fluoroquinolone treatment is effective for severe tularemia. Subpleural round consolidations and mediastinal lymphadenopathy were typical findings on computed tomography among case-patients in this study.

Functional promiscuity of small multidrug resistance transporters from Staphylococcus aureus, Pseudomonas aeruginosa, and Francisella tularensis.

Small multidrug resistance transporters efflux toxic compounds from bacteria and are a minimal system to understand multidrug transport. Most previous studies have focused on EmrE, the model SMR from Escherichia coli, finding that EmrE has a broader substrate profile than previously thought and that EmrE may perform multiple types of transport, resulting in substrate-dependent resistance or susceptibility. Here, we performed a broad screen to identify potential substrates of three other SMRs: PAsmr from Pseudomonas aeruginosa; FTsmr from Francisella tularensis; and SAsmr from Staphylococcus aureus. This screen tested metabolic differences in E. coli expressing each transporter versus an inactive mutant, for a clean comparison of sequence and substrate-specific differences in transporter function, and identified many substrates for each transporter. In general, resistance compounds were charged, and susceptibility substrates were uncharged, but hydrophobicity was not correlated with phenotype. Two resistance hits and two susceptibility hits were validated via growth assays and IC50 calculations. Susceptibility is proposed to occur via substrate-gated proton leak, and the addition of bicarbonate antagonizes the susceptibility phenotype, consistent with this hypothesis.

Identifying key soil characteristics for Francisella tularensis classification with optimized Machine learning models.

Francisella tularensis (Ft) poses a significant threat to both animal and human populations, given its potential as a bioweapon. Current research on the classification of this pathogen and its relationship with soil physical-chemical characteristics often relies on traditional statistical methods. In this study, we leverage advanced machine learning models to enhance the prediction of epidemiological models for soil-based microbes. Our model employs a two-stage feature ranking process to identify crucial soil attributes and hyperparameter optimization for accurate pathogen classification using a unique soil attribute dataset. Optimization involves various classification algorithms, including Support Vector Machines (SVM), Ensemble Models (EM), and Neural Networks (NN), utilizing Bayesian and Random search techniques. Results indicate the significance of soil features such as clay, nitrogen, soluble salts, silt, organic matter, and zinc , while identifying the least significant ones as potassium, calcium, copper, sodium, iron, and phosphorus. Bayesian optimization yields the best results, achieving an accuracy of 86.5% for SVM, 81.8% for EM, and 83.8% for NN. Notably, SVM emerges as the top-performing classifier, with an accuracy of 86.5% for both Bayesian and Random Search optimizations. The insights gained from employing machine learning techniques enhance our understanding of the environmental factors influencing Ft's persistence in soil. This, in turn, reduces the risk of false classifications, contributing to better pandemic control and mitigating socio-economic impacts on communities.