Challenges and solutions in Lab diagnosis of Brucellosis.pptx

Introduction
History
Lab diagnosis of Brucellosis
Culture detection of Brucellae
Serodiagnosis of Human Brucellosis
Diagnosis of Brucellosis by Nucleic Acid Amplification Tests

1. Challenges and solutions in Lab diagnosis of Brucellosis A Comprehensive Overview Dr K Chakrapani Yadav Assistant Professor Department of Microbiology Kurnool Medical College Kurnool

2. objectives Introduction History Lab diagnosis of Brucellosis Culture detection of Brucellae Serodiagnosis of Human Brucellosis Diagnosis of Brucellosis by Nucleic Acid Amplification Tests Take Home Message

3. Introduction Brucellosis is a highly contagious febrile illness, primarily a zoonotic disease affecting various domestic animals, such as sheep, goat or cattle Human infection - associated with occupational or domestic exposure to infected animals or their products. It causes more than 500,000 infections per year worldwide.

4. Many names of Brucellosisis Human Disease Geographical regions Malta Fever Mediterranean Fever Rock Fever of Gibraltar Cyprus fever Animal Disease Bang’s Disease Enzootic Abortion Epizootic Abortion Slinking of Calves Ram Epididymitis Contagious Abortion Human Disease Remittent character of the fever - Undulant fever Resemblance to malaria and typhoid Typhomalarial fever, Intermittent typhoid.

5. History of Brucellosis Jeffery Allen Marston British Army surgeon Contracted Malta fever Described his own case in great detail. Center for Food Security and Public Health, Iowa State University, 2012

6. History of Brucellosis Sir William Burnett (1779-1861) Physician General to the British Navy Differentiated among the various fevers affecting soldiers Center for Food Security and Public Health, Iowa State University, 2012 Professor FEG Cox. The Wellcome Trust, Illustrated History of Tropical Diseases

7. History of Brucellosis Sir David Bruce (1855-1931) British Army physician and microbiologist Discovered Micrococcus melitensis Center for Food Security and Public Health, Iowa State University, 2012 Professor FEG Cox. The Wellcome Trust, Illustrated History of Tropical Diseases

8. Causative agent Brucella spp. are small (0.5–1.5 m), facultative intracellular slow-growing Gram-negative coccobacilli that lack capsules, flagellae , endospores or native plasmids. They are aerobic, do not ferment sugars and are positive in a few oxidative metabolic tests.

9. Causative agent Important species associated with human infections are as follows: B. melitensis - pathogenic to sheep, goat and camel ; most virulent B. abortus - acquired from cattle and buffalo B. suis - infects most often pigs B. canis – causes abortion in dogs .

10. Antigenic Structure Two major types of lipopolysaccharide (LPS) antigens - M & A Present in varying proportion in three major species of Brucella: B. m elitensis - M antigen is predominant B. a bortus - A antigen is predominant B. suis - either M or A antigens

11. Antigenic Structure The O-specific side chains of the LPS molecule are considered the cause of the reported cross-reactions in both the agglutination and complement fixation tests between smooth species of Brucella and Yersinia enterocolitica O:9, Escherichia hermannii , Escherichia coli O:157, Salmonella O:30, Stenotrophomonas maltophilia and Vibrio cholerae O:1

12. MODES OF TRANSMISSION Consumption of contaminated products: Raw & unpasteurized milk and related dairy products. Direct contact- direct contact of abraded skin or mucosa with the infected animal tissue (such as placenta or miscarriage products) and infected animal fluids (such as blood, urine, or milk) Inhalation: aerosols in the infected cowshed or slaughterhouses.

13. Occupational hazard Veterinarians , workers in clinical research and laboratories is primarily associated with respiratory, conjunctival and skin routes of infection, e.g. through inhalation , sprays and aerosols; abrasions, accidental inoculation or cuts

14. Virulence and pathogenesis Entry Routes : Gastrointestinal tract, Respiratory tract, Conjunctiva, Abraded skin & Bloodstream (transfusion/transplacental) Translocation & Uptake : Crosses epithelial barriers and Endocytosed by mucosal macrophages & dendritic cells Initial Localization : Regional lymph nodes Systemic Spread Disseminates via bloodstream Targets macrophage-rich tissues (liver, spleen, lymph nodes, bone marrow)

15. Virulence and pathogenesis 5. Intracellular Lifestyle Facultative intracellular survival - escaping phagosome–lysosome fusion Evades immune response facilitated by factors including its ability to produce urease , which offers protection from stomach acid, Brucella-containing vacuoles , where the bacteria can survive, LPS and Cu/Zn superoxide dismutase . Resists antibiotic action

16. Why diagnosing brucellosis is challenging? Unusual clinical presentations of the disease pose significant diagnostic challenges. The clinical features are non-specific and can overlap with a wide spectrum of other infectious and non-infectious diseases, leading to brucellosis being labelled the “disease of mistakes” - Misdiagnosis and underdiagnosis Brucella is difficult to cultivate - Low Bacterial Load in Blood & Intermittent Bacteremia Prolonged Incubation and Risk of Laboratory-Acquired Infection People's reaction to brucella is highly variable The immunology of brucellosis is somewhat different. Serologic tests have false positive & negative - Cross-Reactivity and Limitations in Serological Tests Cost of testing

17. Unusual clinical presentations of the disease pose significant diagnostic challenges. The clinical features are non-specific and can overlap with a wide spectrum of other infectious and non-infectious diseases, leading to brucellosis being labelled the “ D isease of mistakes or Major mimicker or G reat imitator ” Misdiagnosis and underdiagnosis Challenge 1

18. Prolonged and variable incubation period , Its frequent presentation as a non-specific febrile syndrome (periodic or undulant fever, including muscle aches, backpain, night sweating and fatigue), the varied evolution (acute, subacute or chronic) the involvement of many organs and tissues , with signs of focal disease Misdiagnosis and underdiagnosis Challenge 1

19. Brucellosis must be differentiated from  Typhoid fever, Malaria, Tuberculosis, Lymphoma, Dengue, Leptospirosis, Rheumatic disease, Epstein- barr virus, Toxoplasmosis, Cytomegalovirus, HIV. Misdiagnosis and underdiagnosis Challenge 1

20. Physicians' awareness of the infection is very poor in many countries, and most cases correctly identified are clinically advanced Overdiagnosis of brucellosis may result in untoward drug effects and, no less importantly, in overlooking other serious infectious or noninfectious illnesses . Misdiagnosis underdiagnosis and Overdiagnosis Challenge 1

21. Increased awareness: Brucellosis significant public health implications, requires increased awareness and education among health professionals , particularly those working in endemic regions. Enhanced understanding of the clinical manifestations and diagnostic challenges associated with the disease is crucial for accurate and timely diagnoses. High index of suspicion is needed to achieve a prompt diagnosis. Misdiagnosis underdiagnosis and Overdiagnosis Solution 1

22. Misdiagnosis underdiagnosis and Overdiagnosis Solution 1 Condition Key Features How to Differentiate from Brucellosis? Tuberculosis (TB) Fever, weight loss, night sweats PPD test, chest X-ray, sputum AFB culture Enteric Fever (Typhoid) Step-ladder fever, abdominal pain Widal test, blood culture for Salmonella Rheumatoid Arthritis (RA) Polyarthritis, morning stiffness Positive RF & anti-CCP Malaria Cyclic fever (tertian, quartan) Blood smear for Plasmodium spp. Leptospirosis Fever, conjunctival suffusion, renal failure Serology for Leptospira Q Fever (Coxiella burnetii) Flu-like symptoms, atypical pneumonia Q fever serology Endocarditis Prolonged fever, cardiac murmur Blood cultures, echocardiogram

23. Lab diagnosis of brucellosis

24. correct diagnosis of brucellosis in humans is very crucial Early and adequate patient management To control outbreaks – reveal exposure to sick animals, consumption of contaminated food (especially dairy products), breach of laboratory safety practices , or the intentional release of brucellae as a biological weapon .

25. High risk of laboratory-acquired brucellosis ( upto 2% of all human cases of brucellosis globally ) due to its aerosolized nature , making it a Biosafety Level-3 (BSL-3) pathogen . Very low Infectious dose (10 to 100 bacteria) They remain viable on inanimate surfaces for periods of weeks and even months which increases the risk of indirect transmission. Risk of Laboratory-Acquired Infection Challenge 2 Yagupsky P, Baron EJ. 2005. Laboratory-exposures to brucellae and implications for bioterrorism. Emerg Infect Dis 11:1180–1185. https:// doi.org/10.3201/eid1108.041197. Noviello S, Gallo R, Kelly M, Limberger RJ, DeAngelis K, Cain L, Wallace B, Dumas N. 2004. Laboratory-acquired bucellosis . Emerg Infect Dis 10:1848–1850. https://doi.org/10.3201/eid1010.040076.

26. Biosafety Measures Strict BSL-3 Containment : Work with Brucella must occur in a certified BSL-3 laboratory with appropriate engineering controls (negative pressure, HEPA filtration). Aerosol Control : Acc to CDC, all manipulations should be conducted within a Class II or III Biosafety Cabinet (BSC) . Use sealed centrifuge rotors and closed-system pipetting to prevent aerosolization. Risk of Laboratory-Acquired Infection Solution 2 Yagupsky P, Baron EJ. 2005. Laboratory-exposures to brucellae and implications for bioterrorism. Emerg Infect Dis 11:1180–1185. https:// doi.org/10.3201/eid1108.041197. Noviello S, Gallo R, Kelly M, Limberger RJ, DeAngelis K, Cain L, Wallace B, Dumas N. 2004. Laboratory-acquired bucellosis . Emerg Infect Dis 10:1848–1850. https://doi.org/10.3201/eid1010.040076.

27. Biosafety Measures Small GNCB that grow on BA and CA but fail to develop on MacConkey agar should not be processed on an open bench . Decontamination Procedures : Regularly disinfect work surfaces and Fumigate the lab periodically Personal Protective Equipment (PPE) : Wear N95 or PAPR respirators , gloves, lab coats, and eye protection. Risk of Laboratory-Acquired Infection Solution 2 Yagupsky P, Baron EJ. 2005. Laboratory-exposures to brucellae and implications for bioterrorism. Emerg Infect Dis 11:1180–1185. https:// doi.org/10.3201/eid1108.041197. Noviello S, Gallo R, Kelly M, Limberger RJ, DeAngelis K, Cain L, Wallace B, Dumas N. 2004. Laboratory-acquired bucellosis . Emerg Infect Dis 10:1848–1850. https://doi.org/10.3201/eid1010.040076.

28. Early Identification with Molecular & Rapid Serological Methods : Use real-time PCR or multiplex PCR for faster detection (results within hours instead of weeks). Serological tests (IgM/IgG ELISA, Rose Bengal Test, SAT) can provide early evidence of infection while waiting for culture results. Risk of Laboratory-Acquired Infection Solution 2 Yagupsky P, Baron EJ. 2005. Laboratory-exposures to brucellae and implications for bioterrorism. Emerg Infect Dis 11:1180–1185. https:// doi.org/10.3201/eid1108.041197. Noviello S, Gallo R, Kelly M, Limberger RJ, DeAngelis K, Cain L, Wallace B, Dumas N. 2004. Laboratory-acquired bucellosis . Emerg Infect Dis 10:1848–1850. https://doi.org/10.3201/eid1010.040076.

29. Diagnosis Of Human Brucellosis

30. CULTURE DETECTION OF BRUCELLAE

31. Blood cultures Culture, when positive, provides the definitive diagnosis and is considered the gold standard in the laboratory diagnosis of brucellosis.

32. Challenges WITH CULTURE

33. Challenges WITH CULTURE Slow growing feature of Brucella, Laboratory safety concerns, Reduced sensitivity in prolonged disease and focal infections The sensitivity of blood culture for diagnosis of brucellosis is 10 to 90 percent

34. Challenge 3 Brucella species are characterized by a long generation time (i.e., several hours), low concentration of circulating bacteria , and reduced levels of CO2 emission (CO2 being the metabolic product monitored by current automated blood culture systems ) Brucella Spp.

35. Solution 3 To maximize recovery of the organism, incubation of inoculated medium for 4 weeks and performance of blind subcultures of apparently negative blood culture media have been advised by the ASM and the WHO. Brucella Spp. Moyer NP, Holcomb LA. 1995. Brucella, p 549–555. In Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH (ed), Manual of clinical microbi ology, 6th ed. American Society for Microbiology, Washington, DC. Alton GG, Jones LM. 1967. Laboratory techniques in brucellosis, p 13–17. World Health Organization, Geneva, Switzerland.

36. Challenge 4 Manual monophasic method Manual biphasic methods. Castañeda flask TUMS medium Hémoline medium Blood culture methods Low Bacterial Load in Blood & Intermittent Bacteremia

37. Challenge 4 3) Lysis-based blood cultures – Lysis-filtration In-house lysis-centrifugation method - separating the bacteria from the blood lysate by centrifugation instead of filtration, followed by seeding of the sediment onto agar plates. Isolator microbial tube. Bactec Myco /F lytic vial. Blood culture methods Low Bacterial Load in Blood & Intermittent Bacteremia

38. Challenge 4 4) Blood clot cultures 5) Automated blood culture systems - BACTEC or BacT /ALERT Blood culture methods Low Bacterial Load in Blood & Intermittent Bacteremia

39. Challenge 4 4) Blood clot cultures 5) Automated blood culture systems - BACTEC or BacT /ALERT Blood culture methods Low Bacterial Load in Blood & Intermittent Bacteremia

40. Factors influencing detection of brucellae by automated blood culture systems The method of measuring the metabolic changes occurring in positive blood culture vials differs among automated blood culture systems: in the Bactec 9000 and Bactec FX, fluorescence levels increase as the CO2 content increases and the O2 concentration decreases; in the BacT /Alert blood culture system (bioMérieux, Marcy l’Etoile , France), a colorimetric sensor monitors the changing CO2 content

41. Factors influencing detection of brucellae by automated blood culture systems The release of CO2 in blood culture bottles depends on multiple factors: the initial quantity of bacteria or fungi inoculated (which is the product of the concentration of circulating microorganisms and the volume of the blood ), the species’ replication time and its intrinsic metabolic activity, the adequacy of the broth to meet the nutritional requirements of the organism, the presence of inhibitory factors, the sensitivity and frequency of the CO2 measurements, and the cutoff levels employed to define positivity.

42. Solution 4 Blood Culture Optimization: Use enriched media (e.g., Castaneda biphasic medium, Brucella Broth with CO₂ enrichment ). Extend incubation time up to 4 weeks to enhance detection. Automated blood culture systems has increased the sensitivity of blood cultures and shortened the time to detection of these fastidious species. Alternative Sample Collection: If blood cultures are negative, consider bone marrow culture , which has higher sensitivity (~90%) than blood cultures (~50-70%). CSF, synovial fluid, and tissue biopsy cultures may be useful in focal infections. Blood culture methods Low Bacterial Load in Blood & Intermittent Bacteremia

43. Solution 4 Drawing a large blood sample increases the sensitivity of the culture, and it is therefore recommended to obtain volumes of 20 to 30 ml in adults , 2 to 4ml in children younger than 3 years , and 10ml in older children Blood culture methods Low Bacterial Load in Blood & Intermittent Bacteremia

44. A total of 122 cultures were drawn, and 28 (22.8%) grew B. melitensis by one or both techniques. The automated system detected all 28 positive cultures, whereas the lysis-centrifugation method detected only 22 (sensitivity, 78.6%; P 0.023). The automated system was also superior in terms of time to positivity, detecting 21 of the 22 (95.5%) cultures positive by both methods within 3 days, compared to only 15 (68.2%) detected by the Isolator microbial tube. Eight of the 22 cultures (36.4%) detected by both culture methods were found to be positive at least 24 h earlier by the Bactec instrument, and the remaining 14 were detected by both methods on the same day Which of the current blood culture systems is superior for recovering brucellae?

45. Which of the current blood culture systems is superior for recovering brucellae? Automated blood culture systems are most effective ; biphasic (solid and liquid) blood culture ( Ruiz-Castaneda technique ) is still used in some resource-limited settings

46. Blood versus bone marrow cultures as the diagnostic gold standard. In order to improve detection, it was advised to culture alternate sources such as bone marrow aspirates, liver biopsy specimens, or lymph nodes. The rationale behind obtaining these specimens was that Brucella organisms multiply and concentrate inside the reticuloendothelial system, and thus, culturing of these macrophage-rich tissues may increase bacterial recovery

47. Blood versus bone marrow cultures as the diagnostic gold standard. Gotuzzo et al. found that bone marrow aspirates were positive in 46 of 50 (92.0%) patients and that peripheral blood cultures were positive in only 35 (70.0%). detection times for bone marrow cultures were shorter, indicating a higher bacterial inoculum The improved performance of bone marrow cultures was noted in patients with acute disease as well as in those with a long-standing infection

48. From Detection to Identification Gram stain of the bacteria developing on the agar surface or in the culture broth is initially performed. Very small, faintly stained Gram-negative coccobacilli that resemble fine sand and may appear as microcolonies suspended in the liquid blood culture medium are usually observed

49. From Detection to Identification Identification: By automated identification systems such as MALDI-TOF or VITEK; or by conventional biochemical tests Brucella is catalase and oxidase positive Urease test is rapidly positive for B. suis and B. canis Presumptive identification of brucellae is based on the typical Gram staining appearance, capnophilia , positive oxidase, catalase, and urease activity, no fermentation of sugars, and lack of motility.

50. CHALLENGE 5 The main drawbacks of the phenotypic identification of brucellae are the long turnaround time and the exposure of laboratory technicians to a highly transmissible bacterium. In addition, commercial systems may misidentify brucellae as the closely related Ochrobactrum anthropic, a serious mistake that has already caused outbreaks of laboratory-acquired disease. IDENTIFICATION

51. SOLUTION 5 The presumptive identification of brucellae should be confirmed by a molecular method (NAAT) or by a positive slide agglutination reaction with specific antiserum against the bacterial O-lipopolysaccharide (O-LPS) MALDI-TOF technology - fast, accurate, reproducible, and cost-effective identification of isolates to the species level IDENTIFICATION

52. SOLUTION In patients with a longer evolution of infection and/or focal complications, prolonged incubation and performance of blind subcultures are still required. MALDI-TOF technology and nucleic acid amplification assays and hybridization tests enables a rapid, precise, and safe identification and determination of the species of recovered Brucella isolates. CULTURES

53. SEROLOGICAL TESTING

54. CHALLENGE 5 The serodiagnosis of brucellosis does not provide direct evidence of the presence of the microorganism. Since antibody production varies among individuals due to medical history and immune response differences, serological test results often require careful interpretation and may be inconclusive . Cross-reactivity with other Gram-negative bacteria (e.g., Yersinia enterocolitica , Francisella tularensis , Vibrio cholerae ) affects specificity. SEROLOGY Cross-Reactivity and Limitations in Serological Tests

55. CHALLENGE 5 Diagnostic criteria for serological tests, such as agglutination titres, ELISA cutoff values, or lateral flow test bands, are widely used but frequently debated due to variations in clinical and epidemiological factors . IgG isotype antibodies may persist for many months after the conclusion of successful antibiotic therapy In endemic regions, asymptomatic infections and lingering antibodies after treatment contribute to high seroprevalence, complicating the distinction between past and active infections. SEROLOGY Cross-Reactivity and Limitations in Serological Tests

56. CHALLENGE 5 The sensitivity and specificity of serological tests not only depend on laboratory variables but also on population characteristics and local epidemiological conditions, affecting diagnostic accuracy. Despite their limitations, serological tests remain widely used in endemic regions because they are affordable and easy to perform, making them practical for areas with limited medical resources. SEROLOGY Cross-Reactivity and Limitations in Serological Tests

57. Solution 5 Combination of Serological Tests for Improved Sensitivity : Rose Bengal Test (RBT) for screening (high sensitivity, low specificity). Standard Agglutination Test (SAT) for confirmation (significant at titers ≥1:160). ELISA (IgM & IgG) for differentiating acute vs. chronic infections. Coombs Test in chronic brucellosis to detect incomplete antibodies. Use of Lateral Flow Assays (LFAs) for Rapid Testing Rapid serodiagnostic tools like Brucella LFA can provide quick, point-of-care results. SEROLOGY Cross-Reactivity and Limitations in Serological Tests

58. Rose Bengal Plate Test ( RBPT) Principle of the test : The RBPT antigen is a suspension of pure, smooth killed cells of Brucella abortus strain 99 stained with Rose Bengal dye. The stained bacterial suspension agglutinates when mixed with patients serum containing specific antibodies.

59. Procedure

60. Result : Any degree of agglutination is taken as positive and no agglutination is taken as negative. Rose Bengal Plate Test ( RBPT) RBPT Reactive RBPT Non-reactive

61. Advantages Technically simple. Used as a screening tool for brucellosis. High sensitivity(99%) Disadvantages Low specificity Cross-reactivity and false positivity in areas of endemic brucellosis. Cross-reaction in case of infection with Y.enterocolitica (O:9)

62. Standard Agglutination Test (SAT) Gold standard test against which other serological tests are compared Principle - Tube agglutination test Significant titer >1:160 in non-endemic areas In endemic areas demonstration of rising titre by repeating the test after 2–4 weeks is more reliable

63. Standard Agglutination Test Interpretation: SAT detects antibodies against antigens of smooth LPS SAT measures total quantity of agglutinating antibodies but does not differentiate between immunoglobulin isotypes In acute infection, IgM antibodies appear early and are followed by IgG and IgA antibodies. As the disease progresses, IgM levels decline, and the avidity and subclass distribution of IgG and IgA change. Thus, SAT may become negative.

64. Standard Agglutination Test False-negative SAT may occur due to: Prozone phenomenon – overcome by serial dilution of sera ‘Blocking' or nonagglutinating IgG or IgA : Can be removed by: Prior heating of serum at 55°C for 30 min 4% saline to dilute patient’s sera Coombs antiglobulin test (most reliable). False-positive SAT - due to antigenic cross reactions - Escherichia coli O157, Francisella tularensis, Salmonella enterica group N, Stenotrophomonas maltophilia and Vibrio cholerae.

65. Purpose : Quantitative test to assess the titer of antibodies in the serum. Principle: Antibody in serum, produced in response to exposure to Brucella antigen will visibly agglutinate. This is a tube agglutination test. Standard Tube Agglutination Test  ( STAT)

66. Procedure

67. Result : Positive reaction shows matt formation with clear supernatant and negative reaction shows a cloudy suspension with button formation. Interpretation : A SAT titer in ≥1:160 is read as a positive result. Standard Tube Agglutination Test  ( STAT)

68. Advantages Direct agglutination of bacterial antigens by specific antibodies .Hence it improves specificity of the test. Most widely used serological test for the confirmation of human brucellosis. Disadvantages Lack of seropositivity in the course of early illness. Presence of blocking antibodies. Prozone phenomenon

69. Brucella IgM ELISA Principle : Qualitative immunoenzymatic determination of IgM class antibodies against Brucella in human serum or plasma( citrate,heparin ).

70. Reagents needed : Microtiter plate Sample dilution buffer Washing buffer Conjugate TMB Substrate solution Stop solution Cut-off control Positive control Negative control Sample Dilution : Dispense 10μl sample in 1mL (1:100) of Sample Diluent into tubes. Washing solution preparation : 10ml washing buffer + 190ml distilled water.

71. ELISA assay summary

72. RBPT STAT IgM ELISA INTERPRETATION _ _ _ NON-REACTIVE + > 1:160 + REACTIVE + > 1:160 _ REACTIVE + _ _ NON-REACTIVE + _ + NON-REACTIVE (Based on low, non-reactive STAT titres) + 1:80 + RESAMPLING AFTER 45 DAYS + 1:80 _ RESAMPLING AFTER 45 DAYS Interpretation of results

73. Serological Tests to Detect IgG Antibody ™ 2-mercaptoethanol (2ME) agglutination test: Destroys agglutinability of IgM, not altering IgG SAT performed in 2ME treated serum detect only IgG Molecular Methods PCR assay using primers for 16s rRNA operon ( rrs-rrl gene), Omp2 gene ( outermembrane protein), insertion sequence IS711and protein BCSP31. Blood and tissues ideal samples for PCR assays FilmArray BioThreat panel is an automated multiple PCR, useful for simulataneous detection of 17 human pathogens causing bioterrorism including Brucella .

74. CHALLENGE 6 PCR-based diagnosis has limitations, including: PCR tests are highly sensitive , but their accuracy varies across laboratories due to Variability in primer selection , and they cannot distinguish between live and dead bacteria, making it difficult to confirm active infections. Potential contamination causing false positives. Limited availability in low-resource settings. Molecular Diagnostic Challenges

75. SOLUTION 6 Use of Multiple Gene Targets in PCR Real-time PCR using IS711, bcsp31, and omp2 genes improves specificity. Multiplex PCR panels distinguish between B. abortus , B. melitensis , and B. suis . Digital PCR ( dPCR ) for Higher Sensitivity dPCR is a novel technique that allows quantification of low bacterial loads, making it useful in chronic and relapse cases. Loop-Mediated Isothermal Amplification (LAMP) for Low-Resource Settings LAMP assays provide a cost-effective, rapid, and field-friendly molecular diagnostic tool , requiring minimal infrastructure. Molecular Diagnostic Challenges

76. CHALLENGE 7 Serological tests (e.g., SAT, ELISA) may remain positive for months to years after treatment. In endemic areas, distinguishing previous exposure from active disease is difficult. Differentiation Between Active and Past Infection

77. solution 7 PCR for Active Infection Detects active Brucella DNA, unlike serology, which may persist post-treatment. Serum Brucella IgM/IgG Ratio Interpretation IgM predominance suggests acute infection. IgG dominance with negative PCR suggests past infection. Use of Advanced Biomarkers Brucella LPS-based assays help detect ongoing infection. Procalcitonin (PCT) & IL-6 levels may indicate active disease in severe brucellosis case Differentiation Between Active and Past Infection

78. CHALLENGE 8 Brucella spp. are intracellular bacteria, making standard AST unreliable. There is no standardized CLSI/EUCAST breakpoint for Brucella . Antibiotic Susceptibility Testing (AST) Challenges

79. solution 8 Use of Intracellular AST Models Test antibiotics in macrophage culture systems to assess intracellular activity. Genotypic Resistance Testing Whole-genome sequencing (WGS) can predict resistance mutations in rifampin and doxycycline . Antibiotic Susceptibility Testing (AST) Challenges

80. Take home message Despite major advancements, laboratory diagnosis of brucellosis remains challenging due to its slow growth, biosafety risks, serological limitations, and difficulty distinguishing active from past infections . However, integrating automated blood culture systems, molecular diagnostics, novel serological tools, and biosafety practices significantly enhances diagnostic accuracy and safety.

81. Take home message Key Recommendations for Improved Diagnosis Use a multi-modal approach combining blood culture, PCR, and serology. Enhance biosafety protocols to prevent laboratory-acquired infections. Incorporate rapid diagnostics like LAMP and lateral flow assays for early detection . Employ digital PCR and whole-genome sequencing for challenging cases.