Human Brucellosis.pptx

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1. Human Brucellosis By- Dr. GEETANJALI CHAUDHARI Guide- Dr. VAISHALI WABALE

2. 1. Case 2. Introduction 3. History 4. Epidemiology 5. Brucella and Bioterrorism 6. Etiology 7. Mode of transmission 8. Pathogenesis 9 . Clinical manifestation 10. L aboratory Diagnosis 11. Treatment 12. Prevention

3. Case - 50 yrs old male with - intermittent fever for 3weeks - loss of weight and nausea - no other specific complaints Human Brucellosis ? Lab dianosis - Blood culture –negative Standard agglutination test- Pos.>1:1280 T/t = Doxycycline100 mg BD for 6 wks plus Streptomycin 1gm OD for 2-3 wks - Farmer by occupation and attends cattle and sells milk

4. Introduction Brucellosis (Mediterranean fever/Malta fever/Undulant fever/Cyprus fever/Gibraltar fever/Rock fever) is primarily a disease affecting animals. It can be transmitted to humans. Brucellosis is a major health and economic problem in many parts of the world . It remains the commonest zoonotic disease worldwide. The disease is endemic especially in countries of the Mediterranean basin, the Arabian gulf, the Indian subcontinent and parts of Mexico and Central and South America

5. History Year Discovery 1886 Sir David Bruce- sample from Criemen war of 1850 -named Brucella melitensis as it was discovered in Malta island . 1897 in Danish- Bernhard Bang- Brucella abortus - cattle 1905 Zammit showed that B.melitensis was transmitted to humans by goat’s milk 1910 Alice C. Evans- discovered cocobacillus morphology and share some common biochemical property 1914 USA- Jacob Traum - Brucella suis - aborted swine 1920 Hypothesis was created by american scientist stating brucella disease in human transferred from cattle varies from asymptomatic to fatal And this revolutionised the food safety norms of food industry in USA

6. Epidemeology Globally - More than 500 000 new human cases of brucellosis annually An important cause of travel-associated morbidity . The true incidence of human brucellosis however, is unknown for most countries including India. It has been estimated that the true incidence may be 25 times higher than the reported incidence due to misdiagnosis and underreporting It is an established endemic disease in cattle population with prevalence of 1.8% in 19 of 23 states in Indian subcontinent(1998) & 24.3% in India(2005)

7. Brucella & Bioterrorism CDC has declared Brucella as one of three major bioterrorist agents (anthrax, tularemia, brucella ) the epidemic potential the absence of a human vaccine the drawbacks of current vaccine strains in animals the efficiency of aerosol infection the financial impact of brucellosis in society.

8. Brucella species Fastidious , nonsporing , nonmotile , noncapsulated small gram negative cocobacilli Primarily causes zoonotic disease- sheep , goat & cattles In human it causes brucellosis- aka undulant fever Highly contagious febrile illness A/w occupational and domestic exposure to infected animals and its product Nomen species-based on various properties B.melitensis , B.abortus , B.suis , B.canis , B.ovis , and B.neotomae

9. Antigenic structure Two major type of lipopolysaccharide antigens – M & A Most of the B. melitensis biovars - M antigen predominant Most of the of B.abortus biovars – A antigen predominant B.suis biovars – either M or A antigen

10. Etiological Agents

11. Modes of Transmission

12. Ingestion Consumption of potentially infected unpasteurized milk , milk products, or meat. Unpasteurized cheese, called "village cheese," from endemic areas is a particular risk for tourists .

13. Direct contact with infected animals, aborted fetus, the placenta and the discharge from vagina. High Risk Groups slaughter-house workers, meat inspectors, animal handlers and veterinarians.

14. Inhalation of aerosols containing the bacteria aerosol contamination of the conjunctiva In the lab, transmission is via aerosolization High Risk Groups Laboratory workers.

15. Can brucellosis be spread from person to person? Human-to-human transmission is rare. Bone marrow transplantation from infected donors Blood transfusion Sexual intercourse Neonatal infection; trans- placentally or during delivery Or probably through breast milk.

16. Pathogenesis Brucella spp. - facultative intracellular bacteria - evade host defense mechanism by its ability of intracellular existence Incubation period - 1week to 2-3months

17. Pathogenesis Phagocytised bacteria multiply in macrophages Carried to liver ,spleen, bone marrow, lymph nodes ,kidney Multiply in the cells of reticuloendothelial system Formation of granulomas and release of bacteria in systemic circulation Virulence factors Survival and replication inside macrophages -adenine & guanine monophosphtase system inhibition of phagosome lysosome fusion Release of myloperoxidase &TNF production -A superoxide dismutase detoxifies reactive oxygen intermediates Urease protects B.abortus & B.suis from gastic acid Endotoxins- Smooth Lipopolysaccharide

18. Immune response Adaptive immune responses play a crucial role in controlling the infection. interferon-γ (IFN-γ ) and interleukin-2 (IL-2) secreted by CD8+ T cells preventing the progression of the disease. During infection, γδ T cells carrying Vγ9δ T receptors . These cells not only increase bactericidal activities of macrophages but also eliminate the infected cells by cytotoxic effects. Secreted cytokines such as IL-12 , IFN-γ , and TNF-α molecules play an essential role in a natural and adaptive immune response. Cellular immunity has a fundamental role in controlling the disease.

19. Undulant/waxing &waning fever is a/w periodic release of bacteria and their components from phagocytic cells Cell mediated immunity plays important role in controlling the infection with Brucella spp. Cytokines produced by activated macrophages like TNF alpha, TNF gamma, IL1&2-influence macrophage mediated antibrucella activity

20. Virulence Cell wall Lipopolysaccharide (LPS) Brucella spp undergo antigenic variation on s/c Colonies switch from smooth to rough morphology- results in Smooth phase Have smooth type LPS Resistant to intracellular killing by PMN Rough phase loss of virulence and diminished reactivity with Brucella specific antibodies

21. Virulence Virulence factor Functions Outer membrane protein 25 down regulator of TNF alpha Stressed induced proteins limiting antibiotic action Urease enzyme protect Brucellae in their passage through the stomach

22. Clinical manifestations Human brucellosis usually presents as an acute febrile illness . Most cases are caused by B. melitensis . All age groups are affected . Complications may affect any organ system . The disease may persist as relapse, chronic localized infection or delayed convalescence.

23. Human brucellosis Acute k/a undulant fever a/w prolonged bacteremia and irregular fever Muscular & articular pain,drenching sweats,anorexia,constipation Complications articular,osseous,visceral,neurological Chronic Non bacteremic,low grade infection with periodic exacerbations Symptoms due to hypersensitivity c/f sweating, lassitude,joint pain,minimal or no pyrexia i llness lasts for years

24. Differential diagnosis of Brucellosis Typhoid fever Malaria Tuberculosis Lymphoma Dengue Leptospirosis Rheumatic disease Epstein- barr virus Toxoplasmosis Cytomegalovirus HIV

25. Laboratory diagnosis

26. Isolation of Brucella spp. Samples- Blood Bone marrow Tissues, biopsy sample, CSF, synovial fluid, urine, abscess Strict aerobes Capnophilic , requires 5-10%of CO2 for their growth PH 6.6 - 7.4 Grow slowly on blood & chocolate agar Good growth on Buffered charcoal yeast extract agar Serum dextrose, Serum potato infusion, trypticase soy agar

27. Blood culture methods Conventional blood culture - Most definitive method Cultures are positive in 30-50% cases Bone marrow cultures yeild higher rate of isolation and remain positive for long period B.melitensis and B.suis isolated more rapidly than B.abortus Tryptic soy broth or Brucella broth Biphasic blood culture bottle having liquid and solid medium in the same bottle Advantage-method minimises material and manipulation reducing the chances of contamination Reduced risk of infection to lab workers Castenedas medium

28. Automated blood cultures Detect positive growth in 5-7days Bactec 9240- blood culture + within 7 days - synovial fluid + within 3-5days BacT /alert- standard aerobic, FAN, enhanced aerobic media

29. Septi check system Uses standard blood culture bottle with BHI Bottle connected to second plastic chamber having a paddle with agar surfaces Trisurface paddle is faced with chocolate, MacConkey , malt strip agar First s/c made after 4-6hrs of incubation by inverting the bottle and allowing the broth to enter the second chamber

30. Isolator lysis centrifugation method Special tube containing Saponin ( lysis of RBC and WBC) + 7.5-10ml of blood Thoroughly mixed by inverting the tube several times to enhance the lysis Tube placed in angle centrifuge and spun at 300 rpm for 15min to concentrate any organism present Sediment aspirated and s/c on appropriate media

31. Identification A presumptive identification of Brucella isolates at genus level can be made on the basis of colonial morphology, appearance of Gram stain smears the results of oxidase and agglutination tests with Brucella -specific antisera. Morphology Gram negative cocco bacilli Arranged singly or in short chains Too small so can be mistaken for cocci Non motile, non capsulated, nonsporing Gram stain from patient’s blood. Gram stain from culture.

32. Biochemical tests Test Result Catalase + Oxidase + Indole - Methyl red - Vogues- Proskeur - Citrate - Urease + Nitrate +

33. Serological tests   Rose Bengal test, Standard agglutination test, A ntiglobulin or Coombs test , C omplement fixation test I mmunocapture test.

34. Rose B engal test Used as a screening test and Positive results are confirmed by the serum agglutination tests . B ased on the reactivity of antibodies against the smooth lipopolysaccharide . Rose Bengal Plate (RBPT) agglutination test -the sensitivity is high (>99%) and false negative results are rarely observed. To increase the specificity the test may be applied to a serial dilution (1:2 through 1:64) of the serum samples. Negative Positive

35. The Standard Tube Agglutination Test (SAT) D eveloped by Wright and colleagues remains the most popular and easy test to perform . C an measure the total quantity of the agglutinating antibodies (IgG and IgM). Tube agglutination test , Killed suspension of standard strain of B. abortus The quantity of specific IgG is determined by treatment of the serum with 0.005M 2 mercaptoethanol (2ME), which inactivates the agglutinability of the IgM . SAT titers above 1 : 160 are considered diagnostic in conjunction with a compatible clinical presentation, however, in endemic areas the titer of 1 : 320 is taken as the cut off.

36. Standard agglutination test M any patients have low levels of agglutinating IgG antibodies and the results can easily be misinterpreted . Usually positive in acute infection but weakly positive or even negative in chronic cases Drawback- Effect of blocking or incomplete antibodies exhibiting Prozone phenomenon

37. Coomb’s test Most reliable and sensitive test for confirmation in relapsing patients with persisting disease I t is complex and demands technique. 

38. Enzyme linked immunosorbent assay(ELISA) Has become increasingly popular, as well as a standardized assay for brucellosis . It measures IgG , IgM , and IgA, which allows a better interpretation of the clinical situation. The specificity of ELISA, however, seems to be less than the agglutination tests. As the diagnosis of  Brucella  is based on the detection of antibodies against smooth LPS, the cut-off value needs to be adjusted, to optimize the specificity when used in endemic areas . ELISA can also be applied in the diagnosis of CNS brucellosis with varying success.

39. Fluorescence polarization assay (FPA) The offers a valuable alternative to conventional serological tests . Measures the size of a fluorescent tagged molecule such as an antigen — ideally antigens selected for this technique should be small (20 Kda ). The utilization of the O-side chain of LPS from  Brucella   spp has shown encouraging results. The sensitivity of test at the selected cut-off value is 96% for culture-confirmed brucellosis and the specificity is 98%.

40. Immunochromatographic   Brucella   IgM / IgG lateral flow assay (LFA) A simplified version of ELISA great potential as a rapid point-of-care assay . H as high sensitivity and specificity for  Brucella  IgM and IgG. U ses a drop of blood obtained by a finger prick, which is used by the bedside and easy to interpret. A rapid and simple diagnostic test for confirmation of brucellosis in an endemic area.

41. Immunocapture agglutination for anti- Brucella  ( Brucella   Capt BCAP) New test, developed to detect agglutinating and non-agglutinating antibodies with high sensitivity . It has been suggested as a possible substitute for Coombs test and a better marker for disease activity

42. Molecular methods Polymerase chain reaction (PCR)-based tests are proving to be faster and more sensitive than the traditional methods . V alidated molecular identification method is available, such as PCR with primers for the genus specific insertion sequences IS 711 or IS 650, or sections of the 16S-23S rRNA , BCPS31 and omp 2a genes PCR-based assays are proposed for the direct detection of Brucella organisms in blood. Before any such assay is introduced into the routine laboratory tests, it must be validated for sensitivity, specificity and reproducibility.

43. Detection in milk Pooled milk can be subjected to Culture Antibody detection- Milk ring test- sample of whole milk + a drop of stained brucella antigen (concentrated suspension of killed B.abortus stained with hematoxyline ) Incubated in water bath at 70 for 40-50 minutes If antibodies are present in the milk,bacilli are agglutinated and rise with cream to form a blue ring at the top leaving milk unstained If antibodies are absent,no coloured ring formed ,milk remains uniformly blue

44. Key points for the diagnosis of brucellosis in humans In acute brucellosis, isolation of Brucella from blood or other tissues is definitive . Culture is often negative, especially in long-standing disease. Serology is the most generally useful diagnostic approach . The RBT, tube agglutination and ELISA procedures are recommended . Methods which differentiate IgM and IgG can distinguish active and past infection . False positive serological reactions may occur. Skin test reactions indicate past exposure not active infection.

45. Treatment Category Treatment Adults and children eight years of age and older: Doxycycline + Streptomycin/ Rifampicin For children <8yrs of age- Cotrimoxazole+Streptomycin /gentamicin Cotrimoxazole + rifampicin For pregnant females Cotrimoxazole /Rifampicin The essential element in the treatment of all forms of human brucellosis is the administration of effective antibiotics for an adequate length of time.

46. Preventive Measures Avoid unpasteurized dairy foods….. Cook meat thoroughly….. Wear gloves…. Take safety precautions in high -risk workplaces…. Vaccinate domestic animals….

47. Vaccination ??? Licenced human Brucella vaccine does not exist . For animals: all are live-attenuated. Currently, B. abortus RB51 is used to immunize cattle. B. melitensis REV.1 is used to immunize goats and sheep.

48. Vaccine Type Route of administration Efficacy B. abortus strain 19-BA Live attenuated epicutaneous route Upto 1 year availability and use is now quite restricted. 2. B. abortus 104M Live attenuated epicutaneous route. - availability and use is now quite restricted. 3. B. abortus strain 19 cells .- Peptidoglycan fraction Sub cellular fraction subcutaneous Upto 2 years not at present in production 4. Brucella chemical vaccine” (BCV),Cell wall fraction Subcellular fraction Intramuscular 1 year most promosing

49. Pathogen- Brucellae : Small, GN cocobacilli , non-motile, non-spore forming. Brucellae grow aerobically. Some spp. Require supplemental carbon dioxide for primary isolation. Any high-quality peptone-based media enriched with blood or serum serve for in vitro cultivation. Isolation form clinical specimens require prolonged (≥ 30 days) incubation. Brucella strains always catalse -positive; but oxidase and urease and H 2 S production vary. PUO without specific signs, H/O animal contact

50. The essential element in the treatment of all forms of human brucellosis is the administration of effective antibiotics for an adequate length of time. Treatment of uncomplicated cases in adults and children eight years of age and older: Doxycycline 100 mg twice a day for six weeks + Streptomycin 1 g daily for two to three weeks. OR Doxycycline 100 mg twice a day for six weeks + Rifampicin 600– 900 mg daily for six weeks.

51. For children <8yrs of age- TMP/SMZ (8/40 mg/kg/day twice daily orally) administered for six weeks plus streptomycin ( 30mg/kg/day once daily intramuscularly) administered for three weeks or gentamicin (5 mg/kg/day once daily intravenously or intramuscularly) administered for 7 to 10 days. Alternatives include TMP/SMZ plus rifampicin (15 mg/kg/day orally) each administered for six weeks, or rifampicin plus an aminoglycoside .

52. Prevention (cont’d.) - B. abortus strain RB51 (stable rough mutant) largely replaced strain 19 as preferred bovine vaccine (USA). - Strain RB51, has advantage of protecting cattle without inducing antibody response, and RB51 appears to lack virulence for humans and despite accidents, no proven cases of human RB51 infection. - No licensed human vaccine again on brucellosis to deal.