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Infectious Disease Adam Birkenheuer, DVM, DACVIM, PhD North Carolina State University Practical Approach to Molecular Diagnostics: IFA, PCR, WB, WTH? INTRODUCTION There is NO standardization for molecular testing between different laboratories other than commercially available kits. This means that the results between different labs usually cannot be compared directly. When there are discrepancies between the results from different labs, it is often impossible to tell which lab was "right." You MUST remember that no tests are 100% sensitive and/or specific. The results of ALL tests must be interpreted in light of the patient. IFA (INDIRECT FLUORESCENT ANTIBODY, IMMUNOFLUORESCENCE ASSAY) What is it? This is one of the most commonly used techniques used in the aid of diagnosing infectious diseases. This is the methodology that is used to generate titers. Although it is the most common, it is also usually the most difficult to interpret. What does it test for? IFA is usually used to test for the presence of specific antibodies in a biological fluid. Serum, CSF, and aqueous humor are the most common samples. In other cases (i.e., FeLV bone marrow IFA) you are testing for the presence of antigen in a biological sample. In order to perform an IFA test the operator must have the antigen (usually a whole organism) fixed to a glass slide or a source of specific antibodies. How do I interpret the results? Sensitivity? Specificity? Predictive value? IFA results are usually reported as titers (1:20, 1:40, 1:80 etc.) The titer reported is the final dilution in which the operator was able to detect the presence of fluorescence on the slide. The use of "cutoff titers" is usually not recommended unless the lab has established the sensitivity and specificity of a given test. The presence of antibodies is only circumstantial evidence for infection. It doesn't even always guarantee exposure to a particular organism (i.e. false positive results due to cross reactivity). A four-fold change (NOT four dilution) in antibody titer between acute and convalescent samples is considered diagnostic for most infections (i.e., 1:20 to 1:80, 1:640 to 1:180). Each dilution represents a 2-fold change. Acute and convalescent titers should always be run at the same lab, and ideally should be run simultaneously. There is no standardization between labs in regards to antigens, reagents, dilutions and interpretation. The sensitivity of most IFA titers have NOT been established. The specificity of most IFA titers have NOT been established. The predictive value of most IFA titers have NOT been established. If you are doing an IFA looking for antigen (i.e., FeLV bone marrow) the specificity and predictive value are very high. For chronic conditions, a single test is usually adequate to rule-in or rule-out an organism as a potential cause. For acute conditions (less than 2 weeks) paired titers should be run. ELISA (ENZYME LINKED Immunosorbent ASSAY) What is it? It is another commonly used test modality for several infectious disease. It is the technology that is used in most of the "SNAP" type test kits. What does it test for? ELISAs can be used to detect antibodies or antigens. Not all blue dots are created equally. Almost all of the in house "snap" tests are ELISAs. How do I interpret the results? Sensitivity? Specificity? Predictive value? Due to their commercial nature, the majority of snap tests are standardized and the sensitivity and specificity have been determined compared to a gold standard test. Unfortunately the reported results do not always correlate with the result in the real world where infection rates and levels of antigen may be higher or lower. Algorithms for approaching positive Ehrlichia and Lyme snap tests will be presented. A positive antigen test is consistent with infection (i.e. heartworm ag). A positive antibody test is only indicative of exposure to the organism. May have false negative tests in the acute phase of infection due to lack of specific antibodies. Not quantitative so rising/falling titers cannot be detected. WESTERN BLOT What is it? This is a test that is usually run in a reference laboratory. It is often run as a confirmatory test. In order to run a Western blot, the lab must be able to grow the pathogen in large quantities. What does it test for? Western blot is still just an antibody test. It does however test for the presence of antibodies against specific pathogen proteins rather than whole organism. How do I interpret the results? Sensitivity? Specificity? Predictive value? Researchers have determined that the presence of antibodies against particular pathogen proteins are more specific for infection than antibodies detected in a whole pathogen prep IFA. Generally believed to be more sensitive than IFA, but this is not always the case. The predictive value is generally considered to be very high. May have false negative tests in the acute phase of infection due to lack of specific antibodies. Not quantitative so rising/falling titers cannot be detected. PCR (POLYMERASE CHAIN REACTION) What is it? Amplification of a specific (hopefully) piece of DNA in test tube. What does it test for? The presence of a specific piece of pathogen DNA in a small portion of your biological sample. How do I interpret the results? Sensitivity? Specificity? Predictive value? If a PCR test is positive, then it is very likely that that animal has that particular infection (If the appropriate controls are run). The only way I believe that you should have pathogen DNA in your blood is if you are actively infected. If a PCR test is negative, it only means that the pathogen DNA was not detected in that test tube. It does not mean that the pathogen is not present in that patient. Most PCR tests only evaluate 1-10 microliters of blood equivalent (That is 0.001-0.01 cc of whole blood equivalent). The negative predictive value of a PCR test is only known if the test has been evaluated in a population of patients in which the true prevalence of disease is known. Unfortunately this is not the case for ANY commercially available PCR test that I know of. All PCR tests are not created equally so careful questioning and evaluation should be performed about quality control and sensitivity and specificity if available rather than "price shopping" when it comes to choosing a diagnostic lab. Unlike antibody titers PCR can detect infections during any phase of infection as long as the appropriate tissue is tested. However, some organisms have lower numbers of circulating in the bloodstream during chronic infection and false negatives can occur. RT-PCR (REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION) What is it? A specific (hopefully) fragment of RNA in a test tube. What does it test for? The presence of a specific piece of RNA in a small portion of your biological sample. RT-PCR is most often used to detect RNA viruses. It has however been proposed that RT-PCR might me more sensitive that standard PCR for diagnosing other infectious diseases due to higher numbers of RNA transcripts compared to gene copy numbers. RT-PCR is also used to detect the presence of specific mRNA transcripts for gene expression. How do I interpret the results? Sensitivity? Specificity? Predictive value? The same basic principles for PCR apply for the diagnosis of pathogens. Gene expression can be determined as a +/- if that gene is not expressed constitutively. In order to determine up or down regulation, Quantitative competitive RT-PCR must be used and compared to a standard. REAL-TIME PCR "SOMETIMES KNOWN AS THE OTHER RT-PCR" What is it? Amplification of a specific fragment DNA or RNA in a test tube where the results are measured real-time rather than at an endpoint as they are with standard PCR or RT-PCR. What does it test for? The presence of specific (hopefully) DNA or RNA in a test tube. Real-time PCR also has the ability to quantify the amount oftemplate in a sample. How do I interpret the results? Sensitivity? Specificity? Predictive value? The interpretation of real-time PCR for the diagnosis of infectious diseases is essentially the same as standard PCR. Although there is a big push to do real-time PCR, there is little or no data to show that it is clearly superior to standard PCR. In the future the number of organisms present in the bloodstream may be used as prognostic indicators. One of the major utilities of real-time PCR in human medicine is the quantification of HIV transcripts as an assessment of response to anti-viral treatment. PCR Real-time PCR has a major advantage over standard PCR and RT-PCR when it comes to evaluating gene expression. It allows an investigator to determine if animal have up or down regulation of particular genes in given disease states. Emerging Infectious Diseases Babesia: Canine babesiosis is caused by either large (B. canis) or small babesia (B. gibsoni, other small Babesia). Babesia gibsoni commonly infects American Pit Bull Terriers. Babesia canis is most commonly diagnosed in greyhounds. Babesiosis can be acute or chronic. In addition to tick-transmission, infection via blood transfusions, dog-fights and perinatal routes have been documented. Signs: The most common hematological effects are anemia and thrombocytopenia. Babesiosis can look exactly like idiopathic immune mediated hemolytic anemia (IMHA). The anemia can be severe (PCV < 10%), and is often (> 85%) Coombs positive. The severity of signs do not always correlate with the degree of parasitemia, which can be low (< 1%). Thrombocytopenia is suspected to be immune mediated (ITP). The thrombocytopenia is often severe (< 50,000 plt/ul), but bleeding is rare. Some cases have had ITP without anemia. The effects on the leukon are variable and inconsistent. Some cases have a profound leukocytosis with a left shift that often accompanies a strong regenerative response. Experimental cases have had transient leukopenia. Other clinical signs that may be seen include fever, lymphadenopathy, splenomegaly, pigmenturia, hyperglobulinemia, and jaundice. Diagnosis: Microscopy or PCR can easily rule babesiosis in, but it is very difficult to rule out babesiosis completely. At this point in time I recommend that microscopy, PCR and serology are all considered if you want to maximize your chances of identifying the infection. The organisms stain well with a modified Wright's stain (i.e. Diff-quik® or Leukostat®). Evaluation of capillary blood (ear or toenail) may improve parasite recovery. You need to use oil immersion. There is variable seroreactivity, so serology against B. canis, B. gibsoni and canine small babesia sp. is warranted. Acute and convalescent (3-4wks) titers may be helpful. Seroreactivity of > 1:64 is suspicious for exposure in most labs. In one study, a single PCR test identified 85% of B. gibsoni and 2 consecutive PCR tests identified 100%. Treatment: Currently imidocarb diproprionate (Imizol® 6.6mg/kg IM, repeat in 2 wk.) is the only approved treatment for canine babesiosis in the U.S. Atovaquone 13.5mg/kgPO TID (with a fatty meal) and azithromycin 10mg/kg PO Q24 in combination for 10 days has been the only treatment to reduce or eliminate B. gibsoni (Asian) parasitemia. Follow-up: Babesia canis is likely to be cured by imidocarb. Babesia gibsoni may be cured by atovaquone and azithromycin combination therapy. Other treatments may result in a clinical remission with persistent parasitemia. These dogs are at risk for recrudescence, and may act as a reservoir. We recommend two consecutive blood smear evaluation and PCR 6-8 weeks post treatment. Serology is unlikely to be helpful for short-term follow-up, since antibody titers may persist for months following treatment. Zoonosis: NA Feline babesiosis has not been reported in the U.S. However, cats can be infected by Cytauxzoon felis (see cytauxzoonosis) Ehrlichiosis/Anaplasmosis: Canine ehrlichiosis is caused by several different species of Ehrlichia. Those species reported in the US include E. canis, E. chafeensis, E. ewingii, A. phagocytophilum (E. equi), and A. platys (E. platys). Clinical disease in dogs is most commonly caused by E. canis. Other than E. platys the clinical disease caused by each species is virtually indistinguishable. Ehrlichiosis can either be acute or chronic. Most cases are recognized during the chronic stage. Signs: (E. canis, chafeensis, ewingii, and equi) The hematological effects can be variable, but the most common are thrombocytopenia and a non-regenerative anemia. Ehrlichiosis is only occasionally associated with a secondary IMHA. Ehrlichiosis can also cause pancytopenia. The effects on the leukon are variable. Both leukocytosis and leukopenia have been reported. Lymphocytosis can be seen. The accompanying clinical signs are often vague including; fever, lethargy, anorexia, weight loss, and vomiting. Bleeding tendencies such as epistaxis, petechia, or ecchymosis may also be present. Hyperglobulinemia (polyclonal much more commonly than monoclonal), hypoalbuminemia, lymphadenopathy, proteinuria, polyarthritis (common with E. ewingii), and uveitis may be present. The lack of thrombocytopenia does not rule out ehrlichiosis. Anaplasma platys only causes thrombocytopenia, and does not cause systemic illness. Diagnosis: Serology is helpful in the diagnosis of ehrlichiosis. Acute and convalescent (3-4wks) titers should be performed if the clinical signs are acute. A four-fold change is consistent with ehrlichiosis. If the signs are chronic (> 4wks.), then a single high titer (> 1:80) is consistent with infection. SNAP 3DX and SNAP 4DX have spots that detect antibodies against E. canis alone or E. canis and Anaplasma respectively. Occasionally a morula is identified in a white blood cell on a blood smear, but the parasitemia is often very low. The resolution of clinical signs in response to therapy, along with serologic evidence of infection is diagnostic. PCR is considered a sensitive test, and may also distinguish the species causing the infection. A negative PCR test does not rule out the possibility of ehrlichiosis. Treatment: Doxycycline (10mg/kg/day for 2-3 wk.) is considered the treatment of choice. Other drugs that are reported to be effective include; tetracycline, oxytetracycline, minocycline, and chloramphenicol. Imidocarb diproprionate has had variable success. Follow-up: Resolution of clinical signs after therapy, along with seroreactivity, is diagnostic, and further diagnostics are not indicated. Serology is generally a poor way to assess recovery as antibody titers may persist for months. If the animal is seroreactive, but does not respond to therapy, the PCR should be performed since some species, such as E. chafeensis, may not respond as well to therapy. If the PCR is negative, then an alternative diagnosis should be considered. Zoonosis: Has not been documented for E. canis, but there are genetic similarities between E. ewingii and the HGE agent. Feline ehrlichiosis: The clinical signs appear similar to what is seen in dogs, and it should be considered when more common causes of disease are not apparent. Some cats have been PCR positive for ehrlichiosis. Leishmaniasis: Leishmaniasis is an emerging canine disease in the US. The vector in the US has not been identified. Leishmaniasis typically presents as a chronic disease. To date the majority of cases in the US have been Foxhounds. Signs: The signs are often non-specific and include weight loss, lethargy and anorexia. Skin lesions are common. Non-regenerative anemia and mild thrombocytopenia. Hypoalbuminemia, hyperglobulinemia, proteinuria and azotemia have been noted. Diagnosis: Organisms may be seen in macrophages in tissue or blood. There is an IFA test available, but antibodies are not always detectable in infected dogs. If the signs are acute, then acute and convalescent titers should be performed. A four-fold change is suggestive of active infection. It can take some dogs years to develop a positive titer. If the signs are chronic then a single titer can be performed. If the titer is > 64 or the clinical signs are highly suspicious, then a PCR should be performed. It has been demonstrated that PCR can accurately identify over 85% of dogs with leishmaniasis. Treatment: There is currently no treatment that is known to clear the infection. Antimony compounds are only available through the Center for Disease Control. Chronic treatment with allopurinol and antimony compounds can induce remission, but dogs remain infected. Follow-up: Monitor clinical signs, PCR, and serology. Do not expect a cure. Zoonosis: Casual contact should not pose a major risk, but direct exposure is a potential. Also, common vector transmission is possible (sentinel). Feline leishmaniasis does not appear to be endemic to the US at this time. Bartonellosis: Bartonella vinsonii and B. henselae cause bartonellosis in dogs and cats respectively. Signs: The full spectrum of canine disease caused by Bartonella species has yet to be elucidated. Has been shown to be a cause of endocarditis, and has been associated with granulomatous inflammation and hepatic disease in dogs. Although polyarthritis has only been confirmed in a handful of cases of canine bartonellosis, lameness and stiffness were the most common presenting signs for dogs with confirmed Bartonella endocarditis suggesting that it may be more common finding. Anemia and thrombocytopenia have been detected in nearly half of the dogs diagnosed with Bartonella vinsonii. The pathogenicity of feline bartonellosis is unclear, and it has not consistently been found to have any specific or characteristic hematological or biochemical effects. Some studies have detected an association between stomatitis and lymphadenopathy in cats co-infected with Bartonella and FIV, while other studies have not detected associations between Bartonella and clinical diseases in cats. Diagnosis: Serology is suggestive of exposure or infection. PCR is also available and positive test results are indicative of current infection. Culture is available but culture alone seems to have a low clinical sensitivity. A combination of culture followed by PCR appears to be the most sensitive method for the detection of Bartonella spp. Treatment: The optimal treatment for bartonellosis is unknown. Currently patients are being treated with azithromycin (5-10mg/kg PO Q24 for 5 days then every other day for 45 days). Follow-up: Resolution of clinical signs and culture. Since the full spectrum of disease is unknown and a large percentage of normal animals can test positive for Bartonella cautious interpretation of test results is warranted and consideration of alternative diagnoses when patients signs fail to resolve with treatment. Zoonosis: Humans, especially immunodeficient people, have been infected with Bartonella so client education is warranted. Rocky Mountain Spotted Fever (RMSF): RMSF caused by Rickettsia rickettsii is an acute systemic disease of dogs and humans. RMSF is generally seasonal (Apr.-Sept.) correlating with the Dermacentor sp. lifecycle. Signs: Thrombocytopenia is the most common hematological abnormality (>85%). The degree of thrombocytopenia ranges from moderate ( 75,000plt/ul) to severe (< 5,000 plt/ul). The major mechanism is consumption secondary to vasculitis, but there is some evidence for immune mediated destruction. Leukocytosis is the second most common hematological finding. The degree of leukocytosis can be severe (> 50,000WBC/ul), and tends to increase along with the duration of the disease. RMSF is not known to commonly cause immune mediated hemolytic anemia. The anemia associated with RMSF is often mild (PCV 25-30%). The hematological effects are rarely seen without accompanying clinical signs, such as fever, lethargy, anorexia, pain, petechia, jaundice and neurologic signs. Diagnosis: Serology is very helpful in the diagnosis of RMSF. If the signs are acute, then paired acute and convalescent (1-2weeks) titers must be submitted. A four-fold change is diagnostic for an active infection. If the patient is sick > 10-14 days, then a single high titer (> 1:1024) is consistent with an active infection. Positive Immunofluorescence of skin biopsies or positive nested PCR results also indicate active infection. Response to therapy (doxycycline, tetracycline, enrofloxacin, or chloramphenicol) is suggestive, but not diagnostic. Treatment: Doxycycline (5mg/kg BID or 10mg/kg Q24), chloramphenicol (15-30mg/kg TID), and enrofloxacin (5mg/kg BID) for 1-2 weeks are effective treatments. Resistance has not been reported, so if signs persist after treatment an alternative diagnosis should be considered. Follow-up: An accurate diagnosis is important, since the dog can serve as a sentinel for human infections, so a convalescent titer is indicated even if the animal has responded to treatment. Resistant RMSF has not been reported. Zoonosis: Casual contact should not pose a major risk, but direct exposure is a potential. Also, common vector transmission is possible (sentinel). RMSF has not been reported in cats. Cytauxzoonosis: Cyauxzoonosis is an emerging infectious disease of cats in North America caused by the protozoal parasite Cytauxzoon felis. It is transmitted via the tick vector Dermacentor variabilis and possibly other tick species such as Amblyomma americanum. Cats typically present acutely and the mortality rate is very high (over 90%). Over 90% of the cases are diagnosed between April and September. Outdoor cats are at higher risk for infection and there appear to be hyperendemic areas of C. felis transmission. Bobcats appear to be the reservoir host and only rarely develop severe disease. Most cats die within 5-7 days of the onset of clinical signs. The majority of clinical signs are due to obstruction of small vessels with schizont-laden macrophages which results in ischemia and thrombosis. Signs: The most common signs are lethargy, depression and fever. Pancytopenia is the classic hematologic finding for cytauxzoonosis, but there may only be reductions of one or two cell lines in affected cats. Hyperbilirubinemia and increased ALT concentrations are common biochemical findings. Physical examination typically reveals lymphadenopathy, hepato-splenomegaly and fever. Cats are often dyspneic, moribund, hypothermic and neurologic in the end stages of disease. Diagnosis: Cytologic diagnosis is the most common and rapid means of diagnosing cytauxzoonosis. The earliest stage of infection is the multiplication of schizonts in macrophages. These infected macrophages can be identified in tissue aspirates (particularly the liver, lung and spleen) or on the feathered edge of peripheral blood smears. These infected macrophages are frequently mistaken for platelet clumps and can measure nearly 100 microns in diameter. In endemic areas hepatic aspirates may be warranted in highly suspicious cases. The parasite may also be identified in red blood cells on wright-giemsa stained blood smears as the classic signet ring. There are no commercially available serologic tests. PCR is now available, is sensitive, specific and can be performed rapidly to aid in the diagnosis or confirmation of cytauxzoonosis. Treatment: Supportive care with IV fluids and anti-coagulants are the standard of care for the treatment of cytauxzoonosis. Heparin is my anti-coagulant of choice (100-300 U/kg SQ TID). Anti-protozoal therapies have been administered to cats with cytauxzoonosis but the effect on outcome is not clear since no controlled studies have been performed. Imidocarb diproprionate (2mg/kg IM once every two weeks) has been recommended. Pre-treatment with atropine (0.05mg/kg SQ once) appears to reduce the cholinergic side-effects associated with imidocarb. Anti-biotics are frequently administered to cats with cytauxzoonosis presumably to prevent secondary infections as many cats are neutropenic, although some antibiotics (doxycycline and clindamycin) do have anti-protozoal activity. We are investigating new treatments with promising preliminary data. Follow-up: If cats survive more than 7 days, the prognosis improves. Since there appears to be hyperendemic areas client education regarding their other cats and tick prevention are warranted. Prospective testing of cats in the same household as infected cats has identified carrier cats. Haemobartonellosis (Haemoplasmas AKA Hemotropic Mycoplasmas): Mycoplasma sp. are distinct from Bartonella sp. Feline haemobartonellosis is caused by M. haemofelis, and is thought to be transmitted by fleas. A second form of feline haemoplasmosis is casued by M. haemominutum which is generally believed to be less pathogenic. Canine haemobartonellosis is caused by M. haemocanis, and has been experimentally transmitted by ticks. Clinical disease is more common in cats. One group has recently made an association between canine haemoplasmosis and babesiosis suggesting a similar route of transmission. Signs: Anemia is the most common hematological sign in cats affected by M. haemofelis. Many cases may not show any signs at all including anemia. When present, other accompanying clinical signs are vague and may include depression, anorexia, weight loss, pale mm, icterus, and splenomegally. Infection in dogs is most often silent, unless the dog is splenectomized or immunosuppressed in some other way. Diagnosis: Epicellular organisms may be seen on peripheral blood smears. However, the lack of visualized organisms does not rule out haemobartonellosis. Several PCR tests have recently been developed and are more sensitive and specific than microscopic examination of blood smears. There is no serologic test for Mycoplasma. Treatment: Doxycyline (5mg/kg PO BID for 3 wk.) is considered the treatment of choice, as an alternative fluoroquinolones can be used. Follow-up: If there is complete resolution of signs, then serial rechecks of peripheral blood smears may be adequate. PCR may be more sensitive. Almost all studies have demonstrated that treatment with antibiotics will reduce Zoonosis: NA Hepatozoonosis: Chronic systemic disease of dogs caused by Hepatozoon canis or Hepatozoon americanum. Transmission of Hepatozoon occurs via the ingestion of an infected tick. H. americanum is the species identified in the US and has been diagnosed most frequently in the SE. Signs: Leukocytosis (20,000-200,000 WBC's/ul) is common in dogs infected with H. americanum. Some studies have shown and eosinophilia. A mild non-regenerative anemia is often present. The platelet counts have been variable, with some cases displaying a thrombocytosis and others (w/ concurrent E. canis) having thrombocytopenia. Dogs infected with H. canis are almost always systemically ill. Displaying signs such as fever, malaise, anorexia, emaciation, stiffness, ocular discharge and pain. Periosteal reaction on longbones. Diagnosis: Organisms are occasionally seen in leukocytes on peripheral blood smears. Infected dogs commonly have a periosteal reaction on multiple long bones. A serum antibody test is available, but H. americanum might not seroreact. Organism recovery is high in muscle biopsies, some consider this the test of choice. Treatment and Follow-up: Currently, there is no treatment that is effective in eliminating the infection. Some treatments can induce a remission (trimethoprim sulfa/clindamycin/pyramethamie combination therapy followed by decoquinate), and the improvement of clinical signs, but relapses are common after cessation of therapy. Zoonosis: NA Hepatozoonosis has rarely been reported in cats, most of which have been co-infected with a feline retrovirus. Lyme disease: Lyme disease is generally regionally specific. Lameness is the most commonly reported sign. In endemic areas up to 80% of all dogs have been exposed to Lyme. Signs: Canine Lyme disease caused by the spirochete, Borrelia burgdorferi, and usually does not have any specific or characteristic hematological or biochemical effects (except possibly "lyme-associated nephritis). "Lyme-associated nephritis" is a form of glomerulonephritis, but the cause and effect association with lyme disease is tenuous at best. The majority of patients have been young retrievers and the prognosis is poor. Diagnosis: Serology is helpful if there is no history of vaccination. The new C6 peptide-based antiobody tests are useful for discriminating vaccinal and natural exposures. Otherwise Western immunoblotting or possibly PCR may be helpful in diagnosing exposure to or infection with Borrellia. Unfortunately since 95% of dogs exposed to Borrellia do not develop clinical disease associations between infection/exposure and disease are difficult. Treatment: Tetracyclines or Clavamox are considered the treatments of choice. Follow-up: Resolution of clinical signs is probably the best follow-up. There is no evidence to suggest that treatment of naturally infected asymptomatic dogs will prevent clinical disease. Zoonosis: NA General Information Serology: Serology for all of the above mentioned organisms only test for the presence of antibodies, not the organism. The presence of antibodies only indicates exposure, it does not confirm active infection unless acute and convalescent titers show a four-fold change. All antibody titers are performed on serum (red top), and require 1ml. We recommend that an EDTA anti-coagulated whole blood sample is drawn at the same time (ideally pre-treatment) as the serum for serology and stored in the refrigerator (4 C) in case PCR is indicated. Polymerase Chain Reaction: Test specifically for the presence of the DNA of the organism. PCR tests are usually performed on EDTA anti-coagulated whole blood 1ml (purple top), but for some organisms other tissue or fluid samples may be preferred. Antibiotic therapy may lower parasitemia, thereby yielding a false negative PCR. Immune-Mediated Hemolytic Anemia Signalment: Dogs with IMHA are usually older than 1 year of age. There does not appear to be a significant gender predisposition. Several breeds have a higher incidence of IMHA, such as cocker spaniels, miniature poodles, old English sheepdogs, and Doberman pinschers. Hereditary erythrocyte disease (PK and PFK deficiency osmotic fragility). History: Dogs are usually presented for weakness, lethargy, anorexia, pale mucous membranes, jaundice, or discolored urine. There is an association between recent vaccination and IMHA. Assessment of historical tick attachment, drugs (sulfas), toxins (zinc, onions), other systemic signs may make infectious or neoplastic causes more likely. Some studies suggest a seasonal increase of IMHA cases in the spring and summer. Babesiosis has been associated with a recent dog bite by an American Pit Bull Terrier. Physical Examination: Pallor, weakness, depression, or jaundice. Fever may be present and does not always indicate and infectious etiology. Systolic heart murmur may be present due to decrease viscosity of the blood. Tachycardia and tachypnea may be present due to low oxygen delivery. Animals with concurrent ITP may have petechia or ecchymoses. Ocular exam may reveal uveitis or petechia. CHECKLIST OF TESTS TO CONSIDER FOR ANEMIA
HOW I TREAT UNCOMPLICATED IMHA Criteria
HOW I TREAT COMPLICATED IMHA Criteria
Adjunctive treatments: Cyclosporine (Neoral): 7-15mg/kg/day orally. Use of the Neoral formulation results in more stable serum concentrations. Monitoring drug levels is still recommended. Therapeutic serum concentrations are 200-400ng/ml. Many people use as a first choice treatment for complicated IMHA cases. Mycophenolate mofetil (Cellcept®): 10-20mg/kg PO BID. Very little information in veterinary medicine for use in IMHA/ITP, but anecdotal evidence is promising. Human gamma globulin: 0.5-1.0 gram/kg intravenously over 4-12 hours. May be repeated within 48 Hrs. Danazol: 5mg/kg PO BID. Limited to no proven benefit for the immune mediated component, but may decrease embolism. Leflunomide: a very expensive treatment that has shown some efficacy for the treatment of immune mediated disease. I have not yet used this drug. Cyclophosphamide: Due to recent reports of decreased survival, I am not using or recommending this drug for IMHA. Splenectomy: Absolute "last resort" for me. I have never performed a splenectomy for IMHA. Some recent studies may show benefit. Plasmapheresis: Special facilities and exorbitantly wealthy clients required, with unknown benefits. Supportive Therapy: Intravenous fluid therapy: IV fluid therapy should be avoided if possible since IV catheterization increases the risk of thromboembolism. Blood transfusion: I don't have a "magic" number as to when I will administer a transfusion. I base it on clinical signs and the rate at which the PCV is dropping. Ideally, cross-matched blood will be administered, but at a minimum, donors should be screened for infectious diseases. Oxyglobin: Polymerized bovine hemoglobin. Can be given safely without cross matching. Interferes with serum chemistry analysis and total protein measurement. The PCV and RBC count are no longer representative of the animal's oxygen carrying capacity. I monitor the hemoglobin concentration. Antibiotics: Antibiotics are not indicated for idiopathic IMHA, but antimicrobials such as doxycycline or imidocarb diproptionate are administered pending the results of infectious disease testing. Prognosis: Favorable prognostic criteria
Infectious Hemolytic Anemias Background: Hemolytic anemias represent an important and often frustrating component of any internal medicine practice. The majority of hemolytic anemias (both idiopathic as well as those associated with an underlying disease) are either documented or presumed to be immune-mediated with a smaller percentage mediated through oxidative damage, red blood cell (RBC) fragility or microangiopathic destruction. The majority of immune-mediated hemolytic anemia (IMHA) cases are associated with IgG antibodies attached to the RBC surface and subsequent extravascular hemolysis by the reticuloendothelial system. Less frequently, hemolysis is mediated through the complement system resulting in intravascular hemolysis characterized by hemoglobinemia and hemoglobinuria. Despite the frequency with which we recognize IMHA, an underlying cause is identified in only a minority of cases, including those cases without restrictions on the types or amounts of diagnostics that are pursued. The inability to identify an underlying cause for IMHA means that immune suppression remains the mainstay treatment. Concerns about potential negative effects of this immune suppression exist especially if an occult underlying infectious cause of IMHA was missed during the diagnostic workup. Recent advances in technology have improved our ability to rapidly and accurately identify several infectious diseases that can cause IMHA. Infectious Causes of Feline Hemolytic Anemias Idiopathic IMHA is diagnosed less frequently in cats than in dogs. Therefore it is of utmost importance to search for an underlying cause. Bacterial, viral and protozoal causes of IMHA have been described in cats. Hemotropic Mycoplasma Hemotropic Mycoplasma spp. (AKA HaemoBartonella spp.) are the most commonly diagnosed infectious cause of IMHA in cats. There are several species that can infect cats.1 The majority of clinical information exists regarding Mycoplasma haemofelis and candidatus M. haemominutum. Mycoplasma haemofelis is the larger of these two species and is considered to be the more pathogenic of the two.2 It is presumed to be transmitted via the cat flea, Ctenocephalides felis, although recent transmission studies have failed to demonstrate this. Unlike M. haemofelis, M. haemominutum is not considered to be highly virulent and is not usually associated with clinical disease unless there is concurrent retroviral infection. These organisms have never been cultured in vitro and there are no commercially available serologic tests. The diagnostic modalities available include microscopy and molecular techniques such as the polymerase chain reaction (PCR). Microscopy is generally considered to have poor sensitivity since the number of organisms present in circulation can wax and wane. It is important that PCR-based tests are able to differentiate between the M. haemofelis and M. haemominutum; a positive result with the latter species appears to be less clinically significant. The treatments of choice are doxycycline (5mg/kg PO BID), enrofloxacin (10mg/kg PO Q24) or marbofloxacin (2.5mg/kg PO Q24) for 14-21 days. Since the anemia is immune-mediated, concurrent treatment with prednisone (2-4mg/kg PO per day) may be indicated as well. To date, no treatments appear to result in complete clearance of the organisms. Therefore, infected cats are at risk of recurrence and may serve as reservoirs of infection. Flea prevention may be important for the prevention of disease transmission. Viral Infections The retroviral infections feline leukemia and the feline immunodeficiency virus have been associated with anemia in infected cats. The anemia caused by retroviruses in cats can be due to several underlying mechanisms including immune-mediated hemolysis, bone marrow suppression, chronic inflammation and neoplasia. Of these, IMHA appears to be an uncommon cause of anemia. The diagnosis is usually made using commercially available in-house ELISA tests and can be confirmed in some cases by either PCR or Western blot assays. At least one study has found that feline infectious peritonitis (FIP) was associated with IMHA in cats. The antemortem diagnosis of FIP remains a challenge and is often presumptively based on a combination of antibody titers, clinical, hematological and biochemical signs and in some cases biopsies. Treatment of IMHA in these cases is still directed towards the immune response and typically consists of prednisone (2-4mg/kg PO per day). Protozoal Infections The primary protozoal diseases associated with IMHA in cats are Cytauxzoon felis and Babesia felis. Cytauxzoon felis is a tick-transmitted infection that is endemic to the southeastern, midwestern and mid-Atlantic regions of the United States.3 It is typically characterized by an acute febrile illness that is associated with decreases in one or more cell lines. The disease typically occurs between the months of April and September which correlates with the peak tick activity in most regions. The diagnosis is typically made via microscopic identification of the parasites infecting red blood cells or macrophages. Since macrophages are the first cell line infected, tissue aspirates of affected organs such as the liver, spleen and lymphnodes may aid in the early diagnosis when the RBC forms are not present. Recently, a PCR test was developed that can accurately and rapidly identify C. felis in affected cats.4 Historically, the disease has been considered uniformly fatal in affected cats, but more recently there have been reports of cats surviving infection. In a current study, researchers are comparing the efficacy of atovaquone and azithromycin combination therapy to imidocarb dipropionate for the treatment of C. felis in naturally infected cats. Feline babesiosis caused by Babesia felis has not been identified in the U.S., but is a common cause of anemia in cats in South Africa.5 The disease is characterized by a regenerative hemolytic anemia, elevations in alanine aminotranferase (ALT) and total bilirubin. Fever does not appear to be a common presenting sign. The diagnosis of feline babesiosis is dependent upon microscopy, but PCR can be performed on feline samples using broad-range primers designed to amplify most Babesia spp. The treatment of choice is primaquine, but the margin of safety for the use of this drug in cats is narrow. At least one other species of Babesia has been identified in cats and novel Babesia species have been found in cougars. Clinicians should maintain vigilance for feline babesiosis in the U.S. Rickettsial Infections Recently cats with Ehrlichia infections have been described.6 Some of these infections have been associated with anemia. While IMHA has not been clearly defined in cats with ehrlichiosis, clinicians should consider testing cats with IMHA for ehrlichiosis since the full spectrum of disease associated with ehrlichiosis in cats has not been defined. Infectious Causes of Canine Hemolytic Anemia In most hospitals idiopathic IMHA is still the most common diagnosis for dogs presenting with hemolytic anemia. However, there are a number of infectious diseases that can be associated with IMHA in dogs. Protozoal infections Canine babesiosis is an important cause of canine IMHA worldwide. At least eight genetically distinct piroplasms have been amplified by PCR from the blood of dogs. Babesia gibsoni and the three subspecies of Babesia canis remain the most important of these pathogens. Canine babesiosis is characterized by hemolytic anemia, thrombocytopenia and hyperglobulinemia. It is important to note that anemia is not present in every case. Up to 85% of dogs with babesiosis will have a positive Coomb's test attesting to the immune-mediated nature of the anemia. In most areas babesiosis is transmitted by ticks and dogs that have exposure to ectoparasites are at risk. More recently, several studies have implicated dog bites as risk factors for B. gibsoni infections and the authors have speculated that disease transmission may occur via this route. Other studies have shown that peri-natal transmission of Babesia can occur as well. Additionally, there have been several documented cases of babesiosis in dogs that have been recipients of blood transfusions from infected dogs. There are breed associations with canine babesiosis (greyhounds and B. canis, American pit bull terriers and B. gibsoni) that should prompt clinicians to have an increased incidence of suspicion.7 The three diagnostic tests available for babesiosis include microscopy, serology and PCR. None of these tests have 100% sensitivity or specificity and in many cases more than one modality is needed. Of the three tests available, PCR is the only method to accurately differentiate which species is present in a given patient. Since there can be substantial genetic variation between species, it is important to know which species will be detected by individual labs. Accurate differentiation is important because the treatment will vary among species. Imidocarb dipropionate (6.6mg/kg IM once, repeat in 2 weeks) is the treatment of choice for B. canis and this treatment appears to be effective in clearing the parasite. For B. gibsoni a combination of atovaquone (13.5mg/kg PO TID with a fatty meal) and azithromycin (10mg/kg PO Q24) administered simultaneously for ten days appears effective. Treatment failures appear most common in dogs that have been splenectomized or that have undergone prolonged (weeks to months) immune suppression prior to the diagnosis of babesiosis. If the diagnosis is made promptly, I do not recommend the concurrent use of immune suppression. Instead, reserve immune suppression for cases that are not rapidly responding (3-5 days) to anti-protozoal therapy. Rickettsial infections Canine rickettsial infections are important causes of morbidity and mortality worldwide. They can be caused by Rickettsia rickettsii, Ehrlichia spp. and Anaplasma spp. Of these, E. canis appears to be the organism most commonly associated with IMHA. Although anemia is one of the most common clinical signs that accompanies ehrlichiosis, non-regenerative anemias secondary to bone marrow suppression appear to be more common than IMHA. Occasionally, dogs will have acute hemolytic crisis that is immune mediated and an associated regenerative anemia. Dogs with ehrlichiosis will frequently have concurrent thrombocytopenia and hyperglobulinemia. Diagnoses are primarily based on serology and PCR. Microscopic identification of morulae can be helpful when present, but microscopy is not sensitive for the diagnosis of infection. Some species of Ehrlichia such as E. ewingii have never been cultured in vitro; consequently, no specific serologic tests exist. PCR and microscopy are the only tests available for the diagnosis of E. ewingii infection. Treatment with doxycycline (10mg/kg/PO daily) for three weeks is the treatment of choice for canine ehrlichiosis. IMHA appears to be a rare complication of the other rickettsial infections in dogs. Bacterial infections A few bacterial diseases are associated with anemia in dogs. Definitive associations between these diseases and IMHA have not been made at this time. Candidatus Mycoplasma haemocanis (HaemoBartonella canis) and at least one other species of haemoplasma have been identified in anemic dogs. It does not appear however that these species are virulent pathogens in the absence of concurrent diseases. Bartonella spp. are emerging as an important cause of disease in dogs. While endocarditis remains the primary clinical disease recognized at this time, there is a growing body of evidence that bartonellosis is associated with IMHA in dogs.8 Leptospirosis has been associated with IMHA in dogs, however renal and hepatic failure continue to be the primary clinical problems that need to be addressed in dogs with leptospirosis.
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