Microbiological Aspect of brucellosisMicrobiological Aspect of brucellosis

introductionintroduction

BrucellaBrucella is a is a genus of of Gram-negative bacteria..

They are small (0.5 to 0.7 by 0.6 to They are small (0.5 to 0.7 by 0.6 to 1.5 µm) non-1.5 µm) non-motile, non-, non-encapsulated coccobacilli, which function as , which function as facultative intracellular parasites.facultative intracellular parasites.

Transmission:Transmission:

ingesting infected foodingesting infected food occupational exposure (e.g. exposure to occupational exposure (e.g. exposure to

cattle, sheep, pigs)cattle, sheep, pigs) consumption of consumption of unpasteurized milk products milk products direct contact with an infected animal, direct contact with an infected animal, inhalation of aerosols.inhalation of aerosols. Transmission from human to humanTransmission from human to human through sexual intercourse through sexual intercourse from mother to childfrom mother to child

BrucellaBrucella Different species of Different species of BrucellaBrucella . . B. melitensis which infects which infects goats and and sheep B. abortus which infects which infects cattle B. suis infects pigs, infects pigs, B. ovis infects infects sheep and and

B. neotomae.. in marine mammals (in marine mammals (B. pinnipedialis and and

B. ceti ), in the common vole ), in the common vole Microtus arvalis ( (B. microti ), and even in a breast implant ( ), and even in a breast implant (B. inopinata ). ).

Fever pattern of BrucellosisFever pattern of Brucellosis

Brucillosis:Microbiological AspectsBrucillosis:Microbiological Aspects associated with many other associated with many other febrile diseases, but diseases, but

with emphasis on muscular pain and sweating.with emphasis on muscular pain and sweating. Duration of the disease: few weeks to many Duration of the disease: few weeks to many

months or even yearmonths or even year In the first stage of the disease, In the first stage of the disease, septicaemia occurs occurs

and leads to the classic triad of fever, sweating and and leads to the classic triad of fever, sweating and migratory migratory arthralgia and and myalgia..

granulomatous hepatitis, , arthritis, , spondylitis, , anaemia, , leukopenia, , thrombocytopenia, , meningitis, , uveitis, , optic neuritis, , endocarditis and various and various neurological disorders collectively known as neurological disorders collectively known as neurobrucellosisneurobrucellosis

endocarditisendocarditis

DiagnosisDiagnosis

BrucellaBrucella is isolated from a is isolated from a blood culture on on Castaneda medium..

Prolonged incubation (up to 6 weeks) Prolonged incubation (up to 6 weeks)

Castaneda bottleCastaneda bottle

Brucillosis:Brucillosis: On On Gram stain they appear as dense they appear as dense

clumps of clumps of Gram-negative coccobacilli and coccobacilli and are exceedingly difficult to seeare exceedingly difficult to see

Treatment and preventionTreatment and prevention Antibiotics like like tetracyclines, , rifampicin and the and the

aminoglycosides streptomycin and and gentamicin are are effective against effective against BrucellaBrucella bacteria. However, the use of bacteria. However, the use of more than one antibiotic is needed for several weeks, more than one antibiotic is needed for several weeks, because the bacteria incubate within because the bacteria incubate within cells..

The gold standard treatment for adults is daily The gold standard treatment for adults is daily intramuscular injections of intramuscular injections of streptomycin 1 g for 14 days 1 g for 14 days and oral and oral doxycycline 100 mg twice daily for 45 days 100 mg twice daily for 45 days

Gentamicin 5 mg/kg by 5 mg/kg by intramuscular injection once daily once daily for seven days is an acceptable substitute when for seven days is an acceptable substitute when streptomycin is not available or difficult to obtainstreptomycin is not available or difficult to obtain

Another widely used regimen is doxycycline plus rifampin Another widely used regimen is doxycycline plus rifampin twice daily for at least six weekstwice daily for at least six weeks

triple therapy of doxycycline, with rifampin and triple therapy of doxycycline, with rifampin and cotrimoxazole, has been used successfully to treat cotrimoxazole, has been used successfully to treat neurobrucellosisneurobrucellosis

Prevention:Prevention: by by pasteurizing all milk that is to be all milk that is to be

ingested by human beings, either in its ingested by human beings, either in its unaltered form or as a derivate, such as unaltered form or as a derivate, such as cheese. .

Diagnosis of brucellosis relies on:Diagnosis of brucellosis relies on:

Demonstration of the agent: Demonstration of the agent: blood cultures in tryptose blood cultures in tryptose brothbroth, ,

bone marrow cultures. bone marrow cultures. The growth of brucellae is extremely The growth of brucellae is extremely slow .the culture poses a risk to laboratory personnel due to slow .the culture poses a risk to laboratory personnel due to high infectivity of brucellae.high infectivity of brucellae.

Demonstration of antibodies against the agent either with Demonstration of antibodies against the agent either with ELISA ELISA or the 2-mercaptoethanol assay for IgM antibodies or the 2-mercaptoethanol assay for IgM antibodies associated with chronic diseaseassociated with chronic disease

Histologic evidence of granulomatous hepatitis (hepatic Histologic evidence of granulomatous hepatitis (hepatic biopsy)biopsy)

Radiologic alterations in infected vertebrae: the Pedro Pons Radiologic alterations in infected vertebrae: the Pedro Pons sign (preferential erosion of antero-superior corner of lumbar sign (preferential erosion of antero-superior corner of lumbar vertebrae) and marked osteophytosis are suspicious of vertebrae) and marked osteophytosis are suspicious of brucellic spondylitis.brucellic spondylitis.

The disease's The disease's sequelae are highly variable and may include are highly variable and may include granulomatous hepatitis, , arthritis, , spondylitis, , anaemia, , leukopenia, , thrombocytopenia, , meningitis, , uveitis, , optic neuritis, , endocarditis and various neurological disorders and various neurological disorders collectively known as neurobrucellosis.collectively known as neurobrucellosis.

Identification and diagnosisIdentification and diagnosis From humans the organism is most commonly From humans the organism is most commonly

isolated from isolated from blood or or bone marrow, but may be , but may be isolated from the liver, the spleen, cerebrospinal isolated from the liver, the spleen, cerebrospinal fluid or focal fluid or focal abscess. From sheep, goats, and . From sheep, goats, and cattle, the organism is most commonly cultured from cattle, the organism is most commonly cultured from the the reproductive tract or reproductive fluids, or reproductive fluids, including semen, uterine fluids and tissues, and including semen, uterine fluids and tissues, and milk. milk.

Standard blood media may be used for blood or Standard blood media may be used for blood or bone marrow specimens (enriched culture, other bone marrow specimens (enriched culture, other specimens may use Trypticase specimens may use Trypticase soy agar with 5% agar with 5% sheep blood agar, MacConkey agar, or Martin Lewis sheep blood agar, MacConkey agar, or Martin Lewis agar. agar.

Optimal Temperature: 35-37 degrees Celsius. Plate Optimal Temperature: 35-37 degrees Celsius. Plate cultures should be incubated in 5% carbon dioxide. cultures should be incubated in 5% carbon dioxide. It takes 3-7 days to form colonies on plates. It takes 3-7 days to form colonies on plates.

It is crucial to be able to differentiate It is crucial to be able to differentiate BrucellaBrucella from from Salmonella which could also which could also be isolated from be isolated from blood cultures and are and are Gram-negative. Testing for Gram-negative. Testing for urease as it is as it is positive for the positive for the BrucellaBrucella and negative for and negative for the the SalmonellaSalmonella..

Public healthPublic health When brucellosis is suspected, clinicians should note "suspect or When brucellosis is suspected, clinicians should note "suspect or

rule out brucellosis" on the laboratory submissionrule out brucellosis" on the laboratory submission Review laboratory containment methods and microbiological Review laboratory containment methods and microbiological

procedures to ensure compliance with recommendations in the procedures to ensure compliance with recommendations in the Biosafety in Microbiological and Biomedical Laboratories (BMBL). Biosafety in Microbiological and Biomedical Laboratories (BMBL).

Use primary barriers: use safety centrifuge cups, personal Use primary barriers: use safety centrifuge cups, personal protective equipment, and class II or higher Biological Safety protective equipment, and class II or higher Biological Safety Cabinets (BSCs) for procedures with a high likelihood of producing Cabinets (BSCs) for procedures with a high likelihood of producing droplet splashes or aerosols. droplet splashes or aerosols.

Use secondary barriers: restrict access to the laboratory when work Use secondary barriers: restrict access to the laboratory when work is being performed and maintain the integrity of the laboratory’s air is being performed and maintain the integrity of the laboratory’s air handling system by keeping external doors and windows closed. handling system by keeping external doors and windows closed.

Perform all procedures on unidentified isolates carefully to minimize Perform all procedures on unidentified isolates carefully to minimize the creation of splashes or aerosols. the creation of splashes or aerosols.

Prohibit sniffing of opened culture plates to assist in the Prohibit sniffing of opened culture plates to assist in the identification of isolates. identification of isolates.

Manipulate isolates of small gram-negative or gram-variable rods Manipulate isolates of small gram-negative or gram-variable rods within a BSC. within a BSC.

Serology for brucellosisSerology for brucellosis Serology for brucellosis is a blood test to look for Serology for brucellosis is a blood test to look for antibodies against against

BrucellaBrucella, the bacteria that causes the disease brucellosis., the bacteria that causes the disease brucellosis. How the Test is PerformedHow the Test is Performed Blood is typically drawn from a vein, usually from the inside of the elbow or Blood is typically drawn from a vein, usually from the inside of the elbow or

the back of the hand. The site is cleaned with germ-killing medicine the back of the hand. The site is cleaned with germ-killing medicine (antiseptic). The health care provider wraps an elastic band around the (antiseptic). The health care provider wraps an elastic band around the upper arm to apply pressure to the area and make the vein swell with blood.upper arm to apply pressure to the area and make the vein swell with blood.

Next, the health care provider gently inserts a needle into the vein. The Next, the health care provider gently inserts a needle into the vein. The blood collects into an airtight vial or tube attached to the needle. The elastic blood collects into an airtight vial or tube attached to the needle. The elastic band is removed from your arm. Once the blood has been collected, the band is removed from your arm. Once the blood has been collected, the needle is removed, and the puncture site is covered to stop any bleeding.needle is removed, and the puncture site is covered to stop any bleeding.

In infants or young children, a sharp tool called a lancet may be used to In infants or young children, a sharp tool called a lancet may be used to puncture the skin and make it bleed. The blood collects into a small glass puncture the skin and make it bleed. The blood collects into a small glass tube called a pipette, or onto a slide or test strip. A bandage may be placed tube called a pipette, or onto a slide or test strip. A bandage may be placed over the area if there is any bleeding.over the area if there is any bleeding.

The blood is then tested in a laboratory to look for antibodies. For The blood is then tested in a laboratory to look for antibodies. For BrucellaBrucella, , the serum agglutination test (SAT) is the simplest and most widely used the serum agglutination test (SAT) is the simplest and most widely used testing method.testing method.

DIAGNOSTIC WORKUPDIAGNOSTIC WORKUP Routine studies include a Routine studies include a CBCCBC, , sedimentation ratesedimentation rate, , chemistry panelchemistry panel, ,

urinalysisurinalysis, , chest x-rayschest x-rays, VDRL test, and , VDRL test, and tuberculin skin testtuberculin skin test. . Serial Serial blood blood culturescultures should be done on all patients. Febrile agglutinins usually should be should be done on all patients. Febrile agglutinins usually should be done. An done. An ASO titer ASO titer or streptozyme test should be done to exclude rheumatic or streptozyme test should be done to exclude rheumatic fever. RNA, ANA, and DNA tests should be done to look for lupus and other fever. RNA, ANA, and DNA tests should be done to look for lupus and other connective tissue disease. An HIV antibody titer may need to be ordered.connective tissue disease. An HIV antibody titer may need to be ordered.

The next step is to culture any discharge or various body fluids that might be The next step is to culture any discharge or various body fluids that might be suspect. Thus, a urinalysis and urine culture should be done. A nose and throat suspect. Thus, a urinalysis and urine culture should be done. A nose and throat culture should be done. A sputum smear and culture may need to be done. The culture should be done. A sputum smear and culture may need to be done. The next consideration is to do various next consideration is to do various serologic testsserologic tests. A heterophile antibody titer . A heterophile antibody titer should be done in teenagers. Febrile agglutinin tests may need to be done. should be done in teenagers. Febrile agglutinin tests may need to be done. Acute and convalescent phase sera for viral studies may need to be done.Acute and convalescent phase sera for viral studies may need to be done.

Next one should do skin testing. Thus, histoplasmin, coccidioidin, and Next one should do skin testing. Thus, histoplasmin, coccidioidin, and blastomycin skin testing should be done on patients with a cough. blastomycin skin testing should be done on patients with a cough. TrichinellaTrichinella skin testing may need to be done, as well as brucellin skin testing. A Kveim test skin testing may need to be done, as well as brucellin skin testing. A Kveim test might need to be done for suspected sarcoidosis.might need to be done for suspected sarcoidosis.

The next step is to do plain x-rays of suspected areas. For instance, x-rays of The next step is to do plain x-rays of suspected areas. For instance, x-rays of the teeth may disclose an abscessed tooth. X-rays of the long bones may the teeth may disclose an abscessed tooth. X-rays of the long bones may disclose a metastatic carcinoma.disclose a metastatic carcinoma.

The next step is contrast x-ray studies of various organ systems. An intravenous The next step is contrast x-ray studies of various organ systems. An intravenous pyelogram may show a hypernephroma. A cholecystogram may show pyelogram may show a hypernephroma. A cholecystogram may show gallstones. An upper GI series and barium enema may show chronic gallstones. An upper GI series and barium enema may show chronic pancreatitis or diverticulitis. Angiography may disclose pancreatitis or diverticulitis. Angiography may disclose periarteritis nodosaperiarteritis nodosa, , aortitis or giant cell arteritis.aortitis or giant cell arteritis.

The next step is to do a CT scan of the abdomen and pelvis. If this is negative, The next step is to do a CT scan of the abdomen and pelvis. If this is negative, consider a CT scan of the chest and mediastinum. Echocardiography may consider a CT scan of the chest and mediastinum. Echocardiography may disclose valvular vegetations or an atrial myxoma.disclose valvular vegetations or an atrial myxoma.

Next, consider biopsying various organ systems. For instances, a lymph node Next, consider biopsying various organ systems. For instances, a lymph node biopsy may disclose a lymphoma or sarcoidosis. A muscle biopsy may disclose biopsy may disclose a lymphoma or sarcoidosis. A muscle biopsy may disclose periarteritis nodosa, polymyositis, or trichinella.periarteritis nodosa, polymyositis, or trichinella.

Next one should do bone scans and gallium scans for possible metastasis, Next one should do bone scans and gallium scans for possible metastasis, osteomyelitis, or localized abscesses.osteomyelitis, or localized abscesses.

If all these procedures fail to turn up a lesion, then an exploratory laparotomy If all these procedures fail to turn up a lesion, then an exploratory laparotomy may need to be done. A fibrin test may indicate Mediterranean fever, or urine for may need to be done. A fibrin test may indicate Mediterranean fever, or urine for etiocholanolone may also indicate a relapsing type of fever. A urine test for etiocholanolone may also indicate a relapsing type of fever. A urine test for porphobilinogen may diagnose porphyria.porphobilinogen may diagnose porphyria.

The wisest move is to conduct this investigation with the help of an infectious The wisest move is to conduct this investigation with the help of an infectious disease specialist or a specialist in the body organ system most likely suspected disease specialist or a specialist in the body organ system most likely suspected of harboring the infectionof harboring the infection