Evaluation of a canine D-dimer point-of-care test kit for use in samples obtained from dogs with disseminated intravascular coagulation, thromboembolic disease, and hemorrhage

OBJECTIVE: To evaluate a canine D-dimer point-of-care (cD-d POC) test kit for use in healthy dogs and dogs with disseminated intravascular coagulation (DIC), thromboembolic disease (TED), and hemorrhage. ANIMALS: 12 healthy dogs, 18 dogs with DIC, 23 dogs with TED (19 acute and 4 chronic), and 18 dogs with hemorrhage. PROCEDURE: The cD-d POC, canine D-dimer ELISA (cD-d ELISA), human D-dimer latex agglutination (hD-d LA), and fibrin degradation product (FDP) tests were performed on citrated plasma. RESULTS: All healthy dogs had negative cD-d POC test results and mean cD-d ELISA value of 0.2 U/mL. All dogs with DIC had positive cD-d POC test results and mean cD-d ELISA value of 44 U/mL. Dogs with acuteTED had a mean cD-d ELISA value of 34 U/mL, and 17 of 19 had positive cD-d POC test results. Mean cD-d ELISA value in dogs with hemorrhage was 14 units/mL, and 15 of 18 had positive cD-d POC test results. The cD-d ELISA values in dogs with hemorrhage were significantly higher than those of healthy dogs but lower than those of dogs with DIC and acute TED. The cD-d POC, cD-d ELISA, and hD-d LA tests were comparable in differentiating healthy dogs from dogs with DIC, acute TED, or hemorrhage and appeared to be superior to measurement of FDPs. CONCLUSIONS AND CLINICAL RELEVANCE: The cD-d POC test kit can be quickly and easily used and reliably detects dogs with DIC or acute TED. Positive results may also be seen in dogs with internal hemorrhage.     

The utility of plasma D-dimer to identify thromboembolic disease in dogs

This prospective study was designed to investigate D-dimer concentrations in clinically healthy dogs, clinically ill dogs without thromboembolic disease (TE), and dogs with TE. The goals of this study were to determine whether the coagulation cascade is activated in nonembolic metabolic and inflammatory conditions and whether differentiation from TE is possible. Group 1 consisted of 30 clinically healthy dogs presented for routine care. Group 2 consisted of 67 clinically ill dogs without TE. This group was subdivided into the following categories: postoperative surgical procedures, congestive heart failure, renal failure, hepatic disease, and neoplastic disease. Group 3 consisted of 20 dogs diagnosed with TE. A CBC and a measurement of prothrombin time (PT), activated partial thromboplastin time (PTT), fibrinogen degradation product (FDP) concentration, and plasma D-dimer concentration was performed on dogs in all groups. D-dimer concentrations were highest in dogs with TE; next highest was the hepatic disease group. Only these 2 groups had median D-dimer concentrations markedly different from clinically healthy dogs. The frequency of platelet abnormalities was markedly greater for the TE and neoplastic disease groups. The sensitivity of D-dimer concentrations >500 ng/mL for predicting TE was 100%; however, the specificity of D-dimer for TE at that concentration was 70%. The specificity of D-dimer concentrations >1,000 ng/mL to predict TE was 94% (sensitivity, 80%), and the specificity of D-dimer concentrations >2,000 ng/mL was 98.5% (sensitivity, 36%). FDPs were not high in any TE patient; thus, they may be an insensitive indicator of thromboembolism, with or without overt disseminated intravascular coagulation (DIC).     

D-dimer concentrations in healthy dogs and dogs with disseminated intravascular coagulation

OBJECTIVE: To determine sensitivity and specificity of assays of D-dimer concentrations in dogs with disseminated intravascular coagulation (DIC) and healthy dogs and to compare these results with those of serum and plasma fibrin-fibrinogen degradation product (FDP) assays. ANIMALS: 20 dogs with DIC and 30 healthy dogs. PROCEDURE: Semi-quantitative and quantitative D-dimer concentrations were determined by use of latex-agglutination and immunoturbidometry, respectively. Fibrin-fibrinogen degradation products were measured by use of latex-agglutination. A reference range for the immunoturbidometric D-dimer concentration assay was established; sensitivity and specificity of the assay were determined at 2 cutoff concentrations (0.30 microg/ml and 0.39 microg/ml). RESULTS: Reference range for the immunoturbidometric D-dimer concentration assay was 0.08 to 0.39 microg/ml; median concentrations were significantly higher in dogs with DIC than in healthy dogs. Latex-agglutination D-dimer and serum and plasma FDP assays had similar sensitivity (85 to 100%) and specificity (90 to 100%); the immunoturbidometric assay had lower specificity (77%) at the 0.30 microg/ml cutoff and lower sensitivity (65%) at the 0.39 microg/ml cutoff. Sensitivity or specificity of the latex-agglutination D-dimer assay was not significantly improved when interpreted in series or parallel with FDP assays. CONCLUSIONS AND CLINICAL RELEVANCE: Measurement of D-dimer concentrations by latex-agglutination appears to be a sensitive and specific ancillary test for DIC in dogs. Specificity of D-dimer concentrations in dogs with systemic disease other than DIC has not been determined, therefore FDP and D-dimer assays should be performed concurrently as supportive tests for the diagnosis of DIC in dogs.     

Validation of an immunoturbidimetric D-dimer assay in canine citrated plasma

Abstract: D-dimer is a neoantigen formed when thrombin initiates the transformation of fibrinogen to fibrin; it is derived from plasmin digestion of cross-linked fibrin. In human medicine, the usefulness of this analyte in diagnosing disseminated intravascular coagulation (DIC) has been assessed in patients fulfilling the clinical and laboratory requirements for this disorder. In canine medicine, the use of D-dimer is relatively new. Detailed studies are needed to understand the relationship between D-dimer concentration in plasma and DIC status in dogs. We validated a D-dimer immunoturbidimetric assay (Tina-quant [a] D-Dimer, Boehringer Mannheim) in canine citrated plasma samples. Intra-assay and interassay variability (coefficient of variation) was 5.63% and 8.82%, respectively. The assay was linear, using 2 samples with low and high D-dimer concentrations (r = .996 and .998). Accuracy was 102.2% and 95.7% based on a recovery study in which 2 samples were assessed. Reference values for D-dimer were established using 70 healthy dogs that were assessed clinically and evaluated on the basis of a complete laboratory workup. The reference range was set between 0.02 and 0.28 μg/mL (chi-square test for normal distribution, P > .05).     

Hemostatic Abnormalities in Dogs With Hemangiosarcoma

The hemostasis profiles of 24 dogs with histologically confirmed hemangiosarcoma were prospectively evaluated. Microangiopathic hemolysis was defined as the presence of schistocytes; disseminated intravascular coagulation was defined as 1) thrombocytopenia, 2) fibrin(ogen) degradation products greater than 10 micrograms/mL, 3) prolongation of one or more coagulation times (activated partial thromboplastin time or one-stage prothrombin time) by greater than 25% of the control, 4) fragmented red blood cells (greater than or equal to 1+ based on a semiquantitative grading scale), and 5) fibrinogen less than or equal to 80 mg/dL. Three of the five criteria listed above had to be met for disseminated intravascular coagulation to be diagnosed. Fifty percent of the dogs were considered to have disseminated intravascular coagulation at presentation. Thrombocytopenia was present in 75% of the dogs and was the most common abnormality. The mean platelet count was 137,800/microL. Twenty-five percent of the dogs died as a result of the hemostatic abnormalities. Only 12% of the dogs had microangiopathic hemolysis without other evidence of disseminated intravascular coagulation. Hemostatic abnormalities are present in many dogs with hemangiosarcoma at the initial clinical presentation and represent an important clinical finding.     

Risk factors associated with short-term outcome and development of perioperative complications in dogs undergoing surgery because of gastric dilatation-volvulus: 166 cases (1992-2003)

OBJECTIVE: To evaluate risk factors associated with death and development of perioperative complications in dogs undergoing surgery for treatment of gastric dilatation-volvulus (GDV). DESIGN: Retrospective case series. ANIMALS: 166 dogs. PROCEDURES: Records of dogs with confirmed GDV that underwent surgery were reviewed. Logistic regression was performed to identify factors associated with development of complications (ie, hypotension, arrhythmias, gastric necrosis necessitating gastrectomy, disseminated intravascular coagulation, peritonitis, sepsis, postoperative dilatation, postoperative vomiting, and incisional problems) and with short-term outcome (ie, died vs survived to the time of suture removal). RESULTS: Short-term mortality rate was 16.2% (27/166). Risk factors significantly associated with death prior to suture removal were clinical signs for > 6 hours prior to examination, combined splenectomy and partial gastrectomy, hypotension at any time during hospitalization, peritonitis, sepsis, and disseminated intravascular coagulation. Partial gastrectomy was not a significant risk factor for death but was for peritonitis, disseminated intravascular coagulation, sepsis, and arrhythmias. Age, gastrectomy, and disseminated intravascular coagulation were risk factors for development of hypotension. Use of a synthetic colloid or hypertonic saline solution was associated with a significantly decreased risk of hypotension. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the prognosis for dogs undergoing surgery because of GDV is good but that certain factors are associated with an increased risk that dogs will develop perioperative complications or die.     

Pulmonary thromboembolism in dogs: 47 cases (1986-1987)

Medical records of 47 dogs with pulmonary thromboembolism were reviewed. Middle-aged to older dogs predominated and dyspnea and arterial hypoxemia were consistent clinical findings. Thoracic radiographic findings were variable. Cardiac disease, neoplasia, hyperadrenocorticism, disseminated intravascular coagulation, and sepsis were identified most frequently. Multiple disease processes were identified in 64% of the dogs.     

Hemostatic abnormalities in dogs with primary immune-mediated hemolytic anemia

Hemostatic parameters were prospectively measured in 20 dogs with primary immune-mediated hemolytic anemia. Eight of 20 dogs had received prior treatment with prednisone. Activated partial thromboplastin time was increased in nine dogs; one-stage prothrombin time was increased in two dogs; fibrinogen concentration was increased in 17 dogs; and antithrombin activity was decreased in 10 dogs. Fibrin(ogen) degradation products concentration was increased in 12 dogs, and D-dimer concentration was increased in 16 dogs. Four or more laboratory criteria of disseminated intravascular coagulation (DIC) were present in nine dogs, and three criteria of DIC were found in four additional dogs. Thromboembolism was the most common finding in the dogs that died. In this study population, mortality was not significantly associated with any clinical finding or laboratory variable.     

Cardiac hemangiosarcoma in the dog: a review of 38 cases

During the period 1975 to 1984, a histopathologic diagnosis of primary cardiac hemangiosarcoma was made in 38 dogs at Angell Memorial Animal Hospital. The diagnosis was confirmed by exploratory thoracotomy in 16 cases and at necropsy in 22 cases. At the time of exploratory thoracotomy, 7 dogs were euthanatized because of nonresectability of the primary tumor and/or gross metastatic disease. In 9 dogs, the tumor was resected by removing part of the right atrium. Complications included atrial and ventricular arrhythmias, anemia, disseminated intravascular coagulation, and pneumonia. Prolonged and multiple hospitalizations were a common feature of the postoperative period. Adjuvant therapy was not utilized in any case. The mean survival time was 4 months (2 days to 8 months).     

Serum and plasma latex agglutination tests for detection of fibrin(ogen) degradation products in clinically ill dogs

Abstract: An increased concentration of fibrin(ogen) degradation products (FDPs) commonly is used in conjunction with other hemostatic test abnormalities to identify patients with disseminated intravascular coagulation (DIC). Positive FDP results, however, have been observed in dogs without clinical evidence of DIC. The purpose of this study was to evaluate FDP concentrations in a group of clinically ill dogs with a variety of disorders. Dogs included in the study had the following hemostatic parameters evaluated: prothrombin time, activated partial thromboplastin time, fibrinogen concentration, platelet count, and FDP concentration. Two rapid latex agglutination methods were compared for detecting FDP in serum samples (Thrombo-Wellcotest, International Murex Technologies Corp) and plasma samples (FDP Plasma, American Bioproducts Inc). Results of the serum FDP method were positive in 8% (4/50) of the dogs tested: 3 with DIC and 1 with immune-mediated hemolytic anemia and liver disease. Results of the plasma FDP test were positive in 60% (30/50) of the animals tested: 6 with DIC, 3 with confirmed thrombosis, and 21 with a variety of conditions, including neoplasia, immune-mediated hemolytic anemia, pancreatitis, gastric dilatation-volvulus, heat stroke, severe trauma, sepsis, protein-losing nephropathy, liver disease, hyperadrenocorticism, and chronic heart failure. Because the plasma FDP test was positive more frequently than the serum FDP test in ill dogs, it may be more sensitive for the detection of canine FDP.     

Evidence of hypercoagulability in dogs with parvoviral enteritis

OBJECTIVE: To determine whether dogs with naturally occurring canine parvoviral (CPV) enteritis have laboratory evidence of hypercoagulability. DESIGN: Case-control study. Animals-9 dogs with naturally occurring CPV enteritis and 9 age-matched control dogs. PROCEDURE: Blood was collected from all dogs within 24 hours of admission for thromboelastography (TEG) and determination of activated partial thromboplastin time (aP-TT), prothrombin time (PT), antithrombin III (AT) activity, and fibrinogen concentration. Fibrin-fibrinogen degradation product (FDP) concentration, D-dimer concentration, and platelet count were obtained in dogs with CPV enteritis only. Records were reviewed for evidence of thrombosis or phlebitis. RESULTS: All 9 dogs with CPV enteritis had evidence of hypercoagulability, determined on the basis of significantly increased TEG maximum amplitude and decreased AT activity. Fibrinogen concentration was significantly higher in dogs with CPV enteritis than in control dogs. The aPTT was moderately prolonged in dogs with CPV enteritis, and FDP concentration was < 5 mg/ml in 7 of 9 dogs. No dogs had a measurable D-dimer concentration. Platelet counts were within reference range. Four of 9 dogs had clinical evidence of venous thrombosis or phlebitis associated with catheters. One dog had multifocal splenic thrombosis identified at necropsy. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs with CPV enteritis have a high prevalence of clinical thrombosis or phlebitis and laboratory evidence of hypercoagulability without disseminated intravascular coagulopathy. Thromboelastography may help identify hypercoagulable states in dogs.     

Concurrent Diseases and Conditions in Dogs with Splenic Vein Thrombosis

Background: Splenic venous thrombosis (SVT) is usually considered an incidental finding on abdominal ultrasound examination but can indicate the presence of underlying disease. Concurrent disease processes and conditions in dogs with SVT have not been identified previously. Objectives: To identify concurrent diseases and conditions in dogs with SVT. Animals: Eighty dogs with SVT. Methods: Retrospective review. Medical records from 1994 through 2008 were searched for dogs with SVT identified by ultrasound examination. These records were then reviewed for signalment, medical history, clinicopathologic testing, diagnostic imaging, and clinical diagnosis. Results: The most common concurrent conditions were neoplasia (54%), exogenous corticosteroid administration (43%), systemic inflammatory response syndrome (26%), disseminated intravascular coagulation (20%), pancreatitis (18%), and immune‐mediated disease (16%). The most common neoplastic disease was lymphoma, and the most common immune‐mediated disease was immune‐mediated hemolytic anemia. Protein‐losing nephropathy and naturally occurring hyperadrenocorticism were identified in <10% of the dogs. Concurrent splenic infarcts were identified in 33% of dogs, and concurrent portal vein thrombi were found in 18% of dogs. Conclusions: SVT is a sonographic finding of clinical importance, and dogs with SVT can have 1 or more coexisting diseases.     

Use of a test for proteins induced by vitamin K absence or antagonism in diagnosis of anticoagulant poisoning in dogs: 325 cases (1987-1997)

OBJECTIVE: To determine usefulness of the test for proteins induced by vitamin K absence or antagonism (PIVKA) to identify anticoagulant-poisoned dogs, compared with one-stage prothrombin time (OSPT) and activated partial thromboplastin time (APTT) tests. DESIGN: Retrospective study. ANIMALS: 325 dogs. PROCEDURE: Comparisons of results of PIVKA, OSPT, and APTT measurements in dogs with anticoagulant poisoning, hepatic disease, disseminated intravascular coagulation, other blood-related disorders, immune-mediated diseases, or other chronic and acute diseases were performed. Median, quartile, and range values were determined. RESULTS: PIVKA tests with a 150-second critical value had > 98% specificity and > 90% sensitivity for diagnosis of anticoagulant poisoning versus > 99% specificity and > 79% sensitivity with a 300-second critical value. Comparison of PIVKA values among diagnostic groups revealed significant differences between dogs with anticoagulant poisoning and all other groups. CONCLUSIONS AND CLINICAL RELEVANCE: The PIVKA test with a 150-second critical value is diagnostically useful for distinguishing anticoagulant poisoning from other coagulopathies. Severe liver disease can cause false-positive results. Administration of vitamin K1 or early evaluation (within a few hours of ingesting anticoagulant) may cause false-negative results. Dogs with PIVKA test values > 150 seconds and clinical signs of anticoagulant poisoning can confidently be considered to have anticoagulant poisoning because of the high test sensitivity and specificity.     

Evaluation of plasma antithrombin activity and D-dimer concentration in populations of healthy cats, clinically ill cats, and cats with cardiomyopathy

BACKGROUND: Current coagulation tests lack sensitivity and detect disseminated intravascular coagulation (DIC) only when it is severe. Measurement of antithrombin (AT) activity and D-dimer concentration permits early diagnosis and more precise classification of coagulopathies in some species. OBJECTIVES: The objectives of this study were to validate and determine the diagnostic utility of a chromogenic AT assay and an immunoturbidimetric D-dimer assay for the diagnosis of DIC in cats. METHODS: Citrated plasma samples were collected from 30 healthy cats, 30 ill cats, and 13 cats with cardiomyopathy. Partial thromboplastin time, prothrombin time, fibrin(ogen) degradation products, platelet concentration, and erythrocyte morphology were determined on all samples to document the presence or the absence of DIC. AT activity and D-dimer concentration were then measured. RESULTS: The chromogenic AT assay was linear and precise. Mean AT activity was higher in ill cats and cats with cardiomyopathy compared with healthy cats, but the difference was only significant in ill cats (P = .003). Seven cats met the criteria for DIC. Of the cats with DIC, 2 had decreased AT activity, 1 had increased AT activity, and 4 had AT activities within normal limits. The immunoturbidimetric D-dimer assay did not appear to accurately measure feline D-dimer. CONCLUSIONS: The chromogenic AT assay appeared to measure AT in cats but was not useful for the diagnosis of DIC. AT may be an acute phase reactant in cats. The immunoturbidimetric D-dimer assay was not useful for the diagnosis of DIC in cats.     

Clinico-pathological findings and coagulation disorders in 45 cases of canine babesiosis in Spain

A retrospective study of clinical cases of babesiosis in dogs examined at the Veterinary Teaching Hospital, Rof Codina, from January 2003 to October 2004 is presented. The diagnosis was confirmed by direct observation of large piroplasms in stained blood smears. Dogs with concurrent diseases were excluded from the study. Clinical signs, complete blood count, serum biochemistry and hemostasis profiles were obtained. The observed clinical signs were due to hemolytic anemia and inflammatory responses but the most relevant clinico-pathological findings were related to alterations in hemostasis. All dogs presented with thrombocytopenia and 20% had disseminated intravascular coagulation syndrome. Anemia of variable severity was observed in most of the dogs.     

Platelet Aggregation and Adenosine Triphosphate Secretion in Dogs with Untreated Multicentric Lymphoma

Whole-blood platelet aggregation and adenosine triphosphate secretion were measured in 15 dogs with untreated multicentric lymphoma and 10 normal control dogs to determine if dogs with lymphoma have altered platelet function. Dogs with quantitative platelet disorders (ie, thrombocytopenia or thrombocytosis) or with clinical evidence of a bleeding disorder were excluded from the study. Platelets from affected dogs had significantly greater maximum aggregation than those from control dogs, suggesting that platelets from dogs with lymphoma are hyperactive. Platelet hyperactivity may play a role in the development of hemostatic disorders (eg, disseminated intravascular coagulation) or in tumor metastasis. Further investigation is needed to determine if modification of platelet function in patients with lymphoma affects disease progression or outcome.     

Intracranial haemorrhage associated with Angiostrongylus vasorum infection in three dogs

This report describes three dogs with intracranial haemorrhage secondary to severe coagulation defects associated with Angiostrongylus vasorum infection. The initial case was diagnosed at necropsy, with two subsequent cases diagnosed antemortem and successfully treated. The dogs ranged in age from 14 months to four years and were presented for evaluation of a severe, subacute onset of suspected cerebral disease. Magnetic resonance imaging performed on all three dogs was suggestive of multiple areas of intraparenchymal brain haemorrhage. Coagulation assays showed a consumptive coagulopathy resembling chronic disseminated intravascular coagulation. Postmortem examination of the initial case confirmed the presence of multiple intracranial and extracranial haemorrhages. An unexpected finding was that of a marked multifocal nematode infection of the lungs with an associated vasculopathy. The parasites were confirmed to be A vasorum. In the two other dogs, faecal examination by Baermann technique confirmed A vasorum infection. Both dogs were treated with fenbendazole and one was additionally given a plasma transfusion. Repeated coagulation assays were normal within one week. Neurological examinations were normal for both dogs within six weeks. This case series indicates that A vasorum infection should be considered as a possible aetiology of intracranial haemorrhage in dogs.     

Serum lipase determination in the dog: a comparison of a titrimetric method with an automated kinetic method

An enzymatic, kinetic method for determining serum lipase activity was evaluated and compared to a standard manual method for use in dogs. The kinetic method was a commercial kit adapted for use on a tandem access clinical chemistry analyzer and utilized a series of coupled enzymatic reactions based on the hydrolysis of 1,2-diglyceride by lipase. The manual method was the Cherry-Crandall technique based on the titration of base against the acid formed by hydrolysis of an olive oil substrate by lipase. The correlation between the two methods was very good (r = 0.94). The reference range for 56 clinically healthy dogs assayed by the kinetic method was 90 to 527 U/L. Diseases associated with a greater than twofold elevation in serum lipase activity as determined by the kinetic method included pancreatitis, gastritis with liver disease, and oliguric renal failure with metabolic acidosis. In some cases, pancreatitis was seen with other clinical problems, such as gastroenteritis, diabetic ketoacidosis, duodenal mass, disseminated intravascular coagulation, and septic peritonitis. Diseases associated with serum lipase activity within the reference range or elevated less than twofold included gastritis, gastric ulcer, cholestasis, phenobarbital-induced hepatopathy, colitis, copper hepatopathy, abdominal hematoma, apocrine gland adenocarcinoma, and thrombocytopenia with pneumonia.     

Plasma D-dimer for the diagnosis of thromboembolic disorders in dogs.

D-dimer is formed during thrombus formation when factor XIIIa crosslinks the terminal D-domains of fibrin. The D-dimer epitope is exposed when the thrombus is lysed by plasmin. Thus, D-dimer represents both thrombin and plasmin activation and is specific for fibrinolysis. D-dimer concentrations are increased in dogs with DIC or other thromboembolic disorders, but because D-dimer is an indicator of physiologic or pathologic fibrinolysis, values are elevated in other conditions associated with fibrinolysis, including orthopedic surgery, neoplasia, and internal hemorrhage. It can be used as an ancillary test for the diagnosis of DIC but is not recommended as a sole test for this purpose. D-dimer has the potential to be a useful laboratory test for the detection of pulmonary thromboembolism in dogs. Further studies are needed to determine the appropriate applications for this test in veterinary patients to aid in clinical decision making, treatment, and patient care.     

Escherichia coli associated endotoxemia in dogs with parvovirus infection

Escherichia coli bacteremia and endotoxemia were observed in 3 adult mongrel dogs which had been prediagnosed as canine parvoviral disease. The endotoxin level was 46.5 pg/ml in the plasma of clinical cases, while 2.3 pg/ml in healthy controls. The microflora of the feces was confused in the clinical cases. The percentage of E. coli was major in the feces. Serologically similar strains were isolated from the blood. These strains did not produce enterotoxins such as heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT). Histopathologically, the lesions in the small intestine consisted of epithelial degeneration and necrosis. Viral inclusion bodies were frequently observed in the epithelial cells. Disseminated intravascular coagulation was observed in various tissues including the liver and small intestinal submucosa. After experimental infection with CPV, all dogs showed various clinical signs. CPV was positive in the feces. Endotoxin level in the plasma gradually increased and high level continued for long period from 10 to 30 days. Mean maximum level of endotoxin in the experimental dogs was 73.6 pg/ml. These results indicate that intestinal flora plays a important role in the pathogenesis of CPV infection and that endotoxin is one of the factors which predispose to severe disease after the infection.