Diagnostic Tests Hepatitis B

Laboratory studies will be discussed according to the stage of disease.

Acute hepatitis B disease

High levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), at a range of 1000-2000 IU/mL, is the hallmark of this disease, although values 100 times more than the upper limit of normal (ULN) can be identified. Higher values are found in patients with icteric hepatitis. ALT levels are usually higher than AST levels. Alkaline phosphatase (ALP) levels may be elevated, but they are usually not more than 3 times the upper limit of normal. Albumin levels can be slightly low, and serum iron levels may be elevated. In the preicteric period (ie, before the appearance of jaundice), leukopenia (ie, granulocytopenia) and lymphocytosis are the most common hematologic abnormalities and are accompanied by an increase in the erythrocyte sedimentation rate (ESR). Anemia due to a shortened red blood cell survival period is an infrequent finding, although hemolysis may be noted. Thrombocytopenia is a rare finding. Patients with severe hepatitis experience a prolongation of the prothrombin time (PT). Several viral markers can be identified in the serum and the liver. Hepatitis B surface antigen (HBsAg) (Australian antigen) and hepatitis B e antigen (HBeAg) (marker of infectivity) are the first markers that can be identified in the serum. Hepatitis B core antibody (HBcAb) (immunoglobulin M [IgM]) follows. For patients who recover, seroconversion to hepatitis B surface antibody (HBsAb) and hepatitis B e antibody (HBeAb) is observed. The HBcAb is of the IgG class. Patients with persistent HBsAg for longer than 6 months develop chronic hepatitis.

Chronic inactive hepatitis B disease

Healthy carriers have normal AST and ALT levels, and the markers of infectivity (ie, HBeAg, HBV DNA) may be negative. HBsAg, HBcAb of IgG type, and HBeAb are also present in the serum.

Chronic active hepatitis B disease

Patients have mild to moderate elevation of the aminotransferases (5 times the ULN). The ALT levels are usually higher than the AST levels. Extremely high levels of ALT can be observed during exacerbation or reactivation of the disease, and they can be accompanied by impaired synthetic function of the liver (ie, decreased albumin levels, increased bilirubin levels, and prolonged PT).

Hepatitis B virus (HBV) DNA levels are high during this phase. HBsAg and HBcAb of IgG or IgM type (in case of reactivation) are identified in the serum. If the AST levels are higher than the ALT levels, the diagnosis of cirrhosis must be excluded. Hyperglobulinemia is another finding, predominantly with an elevation of the IgG globulins. Tissue-nonspecific antibodies, such as antismooth muscle antibodies (ASMAs) (20-25%) or antinuclear antibodies (ANAs) (10-20%), can be identified. Tissue-specific antibodies, such as antibodies against the thyroid gland (10-20%), can also be found. Mildly elevated levels of rheumatoid factor (RF) are usually present.

Cirrhosis
In early stages of cirrhosis, findings of chronic viral hepatitis can be found. Later on, as the disease progresses, low albumin levels, hyperbilirubinemia, prolonged PT, low platelet count and white blood cell count, and AST levels higher than ALT levels can be identified. ALP levels and gamma-glutamyl transpeptidase (GGT) can be slightly elevated.

DHF

Complete Blood Cell Count

Leukopenia, often with lymphopenia, is observed near the end of the febrile phase of illness. Lymphocytosis, with atypical lymphocytes, commonly develops before defervescence or shock. A systematic review found that patients with dengue had significantly lower total WBC, neutrophil, and platelet counts than patients with other febrile illnesses in dengue-endemic populations.[45] A hematocrit level increase greater than 20% is a sign of hemoconcentration and precedes shock. The hematocrit level should be monitored at least every 24 hours to facilitate early recognition of dengue hemorrhagic fever and every 3-4 hours in severe cases of dengue hemorrhagic fever or dengue shock syndrome. Thrombocytopenia has been demonstrated in up to 50% of dengue fever cases. Platelet counts less than 100,000 cells/ L are seen in dengue hemorrhagic fever or dengue shock syndrome and occur before defervescence and the onset of shock. The platelet count should be monitored at least every 24 hours to facilitate early recognition of dengue hemorrhagic fever.

Metabolic Panel and Liver Enzymes

Hyponatremia is the most common electrolyte abnormality in patients with dengue hemorrhagic fever or dengue shock syndrome. Metabolic acidosis is observed in those with shock and must be corrected rapidly. Elevated blood urea nitrogen (BUN) levels are observed in those with shock. Acute kidney injury is uncommon.[46, 47]

Transaminase levels may be mildly elevated into the several thousands in patients with dengue hemorrhagic fever who have acute hepatitis. Low albumin levels are a sign of hemoconcentration.

Coagulation Studies
Coagulation studies may help to guide therapy in patients with severe hemorrhagic manifestations. Findings are as follows:
y y y

Prothrombin time is prolonged Activated partial thromboplastin time is prolonged Low fibrinogen and elevated fibrin degradation product levels are signs of disseminated intravascular coagulation

Serum Studies
Serum specimens should be sent to the laboratory for serodiagnosis, PCR, and viral isolation. Because the signs and symptoms of dengue fever are nonspecific, attempting laboratory confirmation of dengue infection is important. Serodiagnosis is made based on a rise in antibody titer in paired specimens obtained during the acute stage and during convalescence. Results vary depending on whether the infection is primary or secondary. The IgM capture enzyme-linked immunosorbent assay (MAC-ELISA) has become the most widely used serologic assay for dengue. Other tests are also used, however, including the following:
y y y y

Complement fixation (CF) Neutralization test (NT) Hemagglutination inhibition (HI) IgG ELISA

Draw serum specimens for diagnosis as soon as possible after the onset of illness or hospitalization and at the time of death or discharge from the hospital. Immediately place specimens on wet ice and send to the laboratory. Obtain a second (ie, convalescent) blood sample for convalescent-phase serologic testing 7-21 days after the acute-phase serum specimen was drawn. Ideally, draw the convalescent-phase serum specimen 10 days after the acute-phase specimen. A European study found that if only a single serum sample is available, a single positive result on enzyme-linked ELISA (PanBio IgM or IgG) has a high rate of false positivity and should be confirmed using a second, more specific diagnostic technique. In the absence of further testing, platelet and white blood cell counts can be diagnostically helpful, because the combination of thrombocytopenia and leukopenia is present in 40.4% of confirmed cases but in only 6.1% of false-positive cases.[48, 49]

Ultrasonography
Ultrasonography is a potentially timely, cost-effective, and easily used modality in the evaluation of potential dengue hemorrhagic fever. Positive and reliable ultrasonographic findings include fluid in the chest and abdominal cavities, pericardial effusion, and a thickened gallbladder wall. Thickening of the gallbladder wall may presage clinically significant vascular permeability.[50, 2] The utility of previous studies was limited because patients underwent only a single scan. However, in a study by Srikiatkhachorn et al, daily serial ultrasonographic examinations of the thorax and abdomen proved useful in the evaluation of patients with suspected dengue hemorrhagic fever.[50] Plasma leakage was detected in some patients within 3 days of fever onset. Pleural effusion was the most common sign. Based on ultrasonographic findings, dengue hemorrhagic fever was predicted in 12 patients before hemoconcentration criteria had been met.

Case Definitions
Cases are classified as suspected dengue if they are compatible with the clinical description. They are classified as probable dengue if they are compatible with the clinical definition and satisfy one or more of the following criteria:
y

Supportive serology (reciprocal hemagglutination-inhibition antibody titer greater than 1280, comparable IgG EIA titers, or positive IgM antibody test in late acute or convalescent-phase serum specimen) Occurrence at the same location and time as other confirmed cases of dengue fever

A confirmed case of dengue is one that is compatible with the clinical definition and is confirmed by the laboratory. Criteria for the diagnosis of dengue hemorrhagic fever include a probable or confirmed case of dengue infection and hemorrhagic tendencies as evidenced by one or more of the following:
y y y y y

A positive result from the tourniquet test Petechiae, ecchymoses, or purpura Bleeding from the mucosa, gastrointestinal tract, injection sites, or other sites Hematemesis or melena and thrombocytopenia (< 100,000 cells/ L) Evidence of plasma leakage due to increased vascular permeability

Plasma leakage may manifest as one or more of the following:

y y y

Greater than 20% rise in average hematocrit level for age and sex Greater than 20% drop in hematocrit level following volume replacement compared with baseline Signs of plasma leakage (eg, pleural effusion, ascites, hypoproteinemia)

Dengue shock syndrome is diagnosed in cases meeting all of the above criteria plus evidence of circulatory failure, such as the following:
y y y y y

Rapid, weak pulse Narrow pulse pressure (< 20 mm Hg), with increased peripheral vascular resistance (PVR) and elevated diastolic pressure Hypotension Cool, clammy skin Altered mental status, although the patient may initially remain alert

The onset of shock may be subtle, indicated by raised diastolic pressure and increased PVR in an alert patient. WHO classification The accuracy of the World Health Organization (WHO) classification system for dengue has been called into question. A study in Indonesian children found that the WHO classification system was in only modest agreement with the intuitive classification by treating physicians, whereas several modified classification systems were in good agreement.[51] The WHO classification system was found to have a sensitivity of 86% for the detection of dengue shock syndrome.[16] Modified systems that added the above early predictors of compensated shock and considered models using varying combinations of evidence of hemorrhagic tendencies, thrombocytopenia, and hemoconcentration were found to yield higher sensitivities (88-99%). Leptospirosis

Laboratory Studies
Laboratory studies are used for two purposesto confirm the diagnosis and to determine the extent of organ involvement and severity of complications. Laboratory confirmation of leptospirosis can be accomplished in 2 different ways. Isolation of the leptospires from human tissue or body fluids is the criterion standard. Urine is the most reliable body fluid to study because the urine contains leptospires from the onset of clinical symptoms until the third week of infection. Other body fluids (eg, blood, cerebrospinal fluid [CSF]) contain the organism, but the window of opportunity to isolate them is shorter. Tissues (ie, liver, muscle, kidney, skin, eyes) are also sources of identification of the leptospires but are obviously more complicated to acquire. Isolation of leptospires can be difficult and time consuming, involving reference laboratories and often taking several months to complete. More often, paired acute and convalescent serum specimens are used to confirm the diagnosis. Again, this is a delayed means of confirmation because the acute sera are collected 1-2 weeks after onset of symptoms, and the convalescent sera are collected 2 weeks afterward.

Antileptospire antibodies in these samples are detected using the microscopic agglutination test (MAT). The Centers for Disease Control and Prevention (CDC) laboratory in Atlanta, Georgia, performs the MAT using 23 leptospire antigens. A 4-fold rise in MAT titer between acute and convalescent sera with any of these antigens confirms the diagnosis of leptospirosis. See the image below.

Darkfield microscopy of leptospiral microscopic agglutination test. (This image is in the public domain and thus free of any copyright restrictions. Courtesy of the Centers for Disease Control/Mrs. M. Gatton) Faster laboratory methods may strongly suggest the diagnosis of leptospirosis, but they may be no more readily available than the CDC laboratory in Atlanta. A single MAT titer of 1:800 on any sera or identification of spirochetes on dark-field microscopy, when accompanied by the appropriate clinical scenario, is strongly suggestive. In suspected leptospirosis, further laboratory studies should be routinely performed to determine the extent and severity of organ involvement after the acute phase of illness. A CBC count is necessary. See the image below.

A scanning electron micrograph depicting Leptospira atop a 0.1m polycarbonate filter. (This image is in the public domain and thus free of any copyright restrictions. Courtesy of the Centers for Disease Control/Rob Weyant) Significant anemia due to pulmonary and gastrointestinal hemorrhage can occur. The platelet count may be diminished as a component of DIC. Levels of blood urea nitrogen and serum creatinine may be profoundly elevated in the anuric or oliguric phase. Tubulointerstitial nephritis is the main cause of acute renal injury in Weil disease. In addition, renal magnesium and potassium wasting is common in persons with leptospirosis.[11]

Serum bilirubin levels elevate as part of the obstructive disease due to capillaritis in the liver. Levels of hepatocellular transaminases are elevated less often and less significantly (usually < 200 U/L). Jaundice and bilirubinemia disproportional to hepatocellular damage is common in leptospirosis; alkaline phosphatase levels may be elevated 10-fold. Coagulation times may be elevated in patients with hepatic dysfunction and/or DIC. Serum creatine kinase levels (MM fraction) are often elevated in patients with muscular involvement. Analysis of the CSF is useful only in excluding other causes of bacterial meningitis. Leptospires is routinely isolated from the CSF, but this finding does not change management of the disease.

Imaging Studies
Imaging studies are also useful in determining the extent and severity of organ involvement. The most common abnormality on chest radiography is bilateral diffuse airspace disease. Chest radiography may also reveal cardiomegaly and pulmonary edema due to myocarditis. In patients with alveolar hemorrhage due to pulmonary capillaritis, the lung parenchyma may contain multiple patchy infiltrates.

Histologic Findings
Shortly after inoculation and during the incubation period, leptospires are actively replicating in the liver. The leptospires then disseminate throughout the body and infect multiple tissues. Silver staining and immunofluorescence are used to identify leptospires in the liver, spleen, kidney, CNS, muscles, and heart. During this acute phase, histology reveals these organisms without much inflammatory infiltrate. In addition to the finding of leptospires during histologic examination, the pathologic effects of leptospiral toxins are also apparent. See the image below.

Silver stain, liver, fatal human leptospirosis. (This image is in the public domain and thus free of any copyright restrictions. Courtesy of the Centers for Disease Control/Dr. Martin Hicklin) Leptospirosis may be seen as an infective systemic vasculitis.[12] Leptospiral toxins break down endothelial cell membranes of capillaries. This toxin-mediated process allows for extravasation of blood and leptospires from blood vessels into the supported parenchyma. Secondarily, because

the capillaries are no longer functional, ischemia and cell death can occur. Later in infection, mononuclear cells predominate in the areas of this focal cell necrosis. Leptospires can be identified in immunologically privileged sites, such as renal tubules, CNS, and the anterior chamber of the eyes, for weeks to months after the initial infection. In nonhuman animals, the intended hosts of infection, the leptospires establish residence in these immunologically privileged sites. Provided that the animal survives the initial infection, a chronic carrier state is then established, and histology reveals leptospires at these sites for years after initial infection. Malaria

Blood Smears
A diagnosis of malaria should be supported by the identification of the parasites on a thin or thick blood smear. In rare occasions, P falciparum infection can present without detectable parasitemia. If no alternative diagnosis is found in an at-risk patient with possible cerebral malaria (ie, unrevealing lumbar puncture findings), initiate therapy for presumptive malaria and continue to obtain additional blood smears to confirm the diagnosis. When reading a smear, 200-300 oil-immersion fields should be examined (more if the patient recently has taken prophylactic medication, because this temporarily may decrease parasitemia). One negative smear does not exclude malaria as a diagnosis; several more smears should be examined over a 36-hour period.

Thick smears
Three thick and thin smears 12-24 hours apart should be obtained. The highest yield of peripheral parasites occurs during or soon after a fever spike; however, smears should not be delayed while awaiting fever spikes. Thick smears are 20 times more sensitive than thin smears, but speciation may be more difficult. The parasitemia can be calculated based on the number of infected RBCs. This is a quantitative test.

Thin smears
Thin smears are less sensitive than thick smears, but they allow identification of the different species. This should be considered a qualitative test.

Alternatives to Blood Smear Testing

Alternative diagnostic methods typically are used if the laboratory does not have sufficient expertise in detecting parasites in blood smears.

Rapid diagnostic tests (RDT)

Immunochromatographic tests based on antibody to histidine-rich protein-2 (PfHRP2), parasite LDH (pLDH), or Plasmodium aldolase appear to be very sensitive and specific.[6, 7] Some RDTs may be able to detect P falciparum in parasitemias that are below the threshold of reliable microscopic species identification. Only one RDT (BinaxNOW) has been approved to date for the diagnosis of malaria in the United State.[8] In one study, RDTs were found to perform better than microscopy under routine conditions. RDTs performed by the health facility staff were 91.7% sensitive and 96.7% specific. Microscopy was 52.5% sensitive and 77% specific.[9] In a study from Tanzania, d'Acremont et al reported that antimalarials could be safely withheld from febrile children (< 5 y) who had negative results from an RDT based on PfHRP2.[10] RDTs are less effective when parasite levels are below 100 parasites/mL of blood, and, in rare instances, an RDT test is negative in patients with high parasitemias. For these reasons, confirm RDT test results with a second type of screening test when possible. A false-positive result from an RDT may occur up to 2 weeks or more after treatment due to persistence of circulating antigens.

Other tests
In addition to the RDT listed above, new molecular techniques, such as PCR assay testing and nucleic acid sequence-based amplification (NASBA), are also available for diagnosis. They are more sensitive than thick smears but are expensive and unavailable in most developing countries.[11] The quantitative buffy coat (QBC) is a technique that is as sensitive as thick smears. Because results cannot be used to speciate Plasmodium, a thin smear must be examined. Malaria is a reportable disease. Identification of parasites by any of the above techniques should prompt notification to the local or state health department.

Histologic Findings
The table below compares histologic findings for P falciparum, P vivax, P ovale, and P malariae.

Table 1. Histologic Variations Among Plasmodium Species (Open Table in a new window) Findings Only early forms present in peripheral blood Multiplyinfected RBCs Age of infected RBCs Schffner dots Other features P falciparum Yes P vivax No No P ovale P malariae No

Often RBCs of all ages No Cells have thin cytoplasm, 1 or 2 chromatin dots, and applique forms.

Occasionally Young RBCs Yes Late trophozoites develop pleomorphic cytoplasm.

Rare Young RBCs Yes Infected RBCs become oval, with tufted edges.

Rare Old RBCs No Bandlike trophozoites are distinctive.