Guide question 3: Enumerate the most useful important laboratory tests that will aid clinicians in the diagnosis

Guide question 3: Enumerate the most useful important laboratory tests that will aid clinicians in the diagnosisBautista, Carmela VeronicaBautista, Hannah Vernadine

Laboratory studies are used for two purposes: to confirm the diagnosis and to determine the extent of organ involvement and severity of complications. Laboratory confirmation of leptospirosis can be accomplished through isolation of the pathogen or by serologic testing.1LeptospirosisBiochemical, hematologic and urinalysis findings in leptospirosis are not specific, but certain patterns suggest the diagnosis:Elevated BUN and serum creatinine in conjunction with mixed conjugated and unconjugated hyperbilirubinemia with aminotransferase elevation to less than 5 times the upper limit of normalLeptospirosisUrinalysis may show abnormalities of the sediment (leukocytes, erythrocytes, hyaline and granular casts)

Elevation of the non-cardiac isoform of creatine kinaseLeptospirosisHematologic abnormalitiesLeukocytosis (typical in severe disease)LeukopeniaHemolytic anemiaMild to moderate anemiaThrombocytopeniaLeptospirosisChest radiography: Alveolar infiltrates predominate (associated with hemoptysis but not with purulent sputum)Diffuse interstitial infiltrate patterns Small nodular infiltrates and pleura based densities

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.5LeptospirosisSerologic assays are the diagnostic mainstay in leptospirosisMicroscopic Agglutination Test (MAT) gold standardsingle titer exceeding 1:200 or serial titers exceeding 1:100

The confirmation of leptospirosis requires laboratory testing. Definitive diagnosis rests on demonstrating the presence of the organism by culture isolation, detection of nucleic acids or antigen in body fluids, or immunohistochemical visualization in tissue

Microscopic agglutination testing (MAT) uses a battery of antigens taken from common (frequently locally endemic) leptospire serovars.The MAT entails growth of a battery of serovars representing the 26 leptospiral serogroups, incubation of a standard quantity of leptospires with the patient's serum on a microtiter plate, and detection of agglutination by dark-field microscopy.

The highest dilution of serum that yields significant (50%) agglutination is reported as the titer. Although antibody titers are reported by serovar, a positive MAT result reflects the presence only of Leptospira-specific antibodies and cannot be used to precisely identify the infecting serovar because one serovar may induce antibodies that cross-react with other serovars.

In a patient with clinical findings consistent with the disease, a single titer exceeding 1:200 or serial titers exceeding 1:100 suggest leptospirosis; however, neither is diagnostic. A 4-fold rise in titer between acute and convalescent specimens is considered a positive result. The antibody response does not reach detectable levels until the second week of illness, and it can be affected by treatment.

False-negative: specimen obtained before the immune phase of diseaseFalse-positive MAT results may occur with cases ofLegionellainfection, Lyme disease, and syphilis.6

Darkfield microscopy of leptospiral microscopic agglutination test.For the reading of MAT titers it is highly recommended to use a darkfield microscope with a high contrast between the leptospires and the dark background.

The MAT is read by dark-field microscopy. The end point is the highest dilution of serum at which 50% agglutination occurs. Because of the difficulty in detecting when 50% of the leptospires are agglutinated, the end point is determined by the presence of approximately 50% free, unagglutinated leptospires compared to the control suspension

A general recommended standard Endemic area, a serological diagnosis of leptospirosis is confirmed when MAT in sera taken 5-10 days apart rises from less than the starting dilution of the test (usually 1:50, or 1:100), to more than 100, or 4-fold or more, or is initially more than 400.

non-endemic area, a single titre of 50 or more, with a clinically compatible illness, indicates likely leptospirosis the titre will usually rise 4-fold or more in a second specimen a few days later. However, a single titre of 400 or more can lead to only presumptive diagnosis, indicating recent contact with leptospires which are not necessarily the cause of the current illness. 7LeptospirosisOther serologic tests ELISA, indirect hemagglutination, dot blot, and lateral flowBased on solid phase assays

Leptospires can be cultured from blood and CSF (1st 7-10 days of illness) and from urine (beginning the 2nd wk)Blood cultures may be negative if drawn too early or too late. Leptospires may not be detected in the blood until 4 days after the onset of symptoms (7-14 d after exposure). Once the immune system is activated, blood cultures may again become negative. Leptospires may be isolated from the cerebrospinal fluid (CSF) within the first 10 days.

Leptospires may be isolated from the urine for several weeks after the initial infection. In some patients, urine cultures may remain positive for months or years after the onset of illness. Positive urine cultures may take as long as 8 weeks to grow.

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The laboratory exams for leptospirosis is time or phase dependent as you can see in the row for culture and serology. Blood specimens may be taken early on in the course of the ilness whereas CSF and Urine samples can be taken after a certain period of time.

Specimens 1 and 2 for serology are acute-phase serum samples; specimen 3 is a convalescent-phase serum sample that may facilitate detection of a delayed immune response; and specimens 4 and 5 are follow-up serum samples that can provide epidemiologic information, such as the presumptive infecting serogroup.9Malaria

Typhoid FeverGold standard: CultureIsolation from blood, marrow, other sterile sites, rose spots, stools, intestinal secretionsBlood culture: SN = 40-80%Marrow culture: SN = 55-90%Stool culture: Negative in 60-70% (1st week) ; can be positive in 3rd week if untreatedLeukopenia, neutropenia (15-25%)Leukocytosis in children (1st 10 days)Moderately elevated liver function tests and muscle enzyme levelsWidal test febrile agglutininsPCR, DNA probe assays not yet been developed for clinical useOther than a positive culture, no specific laboratory test is diagnostic for typhoid fever

11DengueCBCLeukopeniaThrombocytopenia (< 100 x 109/L)High Hct (>20% above baseline)Serum aminotransferase elevationRecovery phase: IgM ELISA or paired serology during recovery Acute Phase: antigen-detection ELISA or RT-PCR Virus- readily isolated from blood if mosquito inoculation or mosquito cell culture is used

Serological diagnosis is complicated by cross-reactivity of IgG antibodies to heterologous flavivirus antigens. A variety of methods are available; the most commonly used methods are E/M viral protein-specific capture IgM or IgG ELISA and the hemagglutination inhibition test. IgM antibodies develop within a few days of illness. Neutralizing and hemagglutination-inhibiting antibodies appear within a week after onset of dengue fever.

Analysis of paired acute and convalescent sera to show a significant rise in antibody titer is the most reliable evidence of an active dengue infection.

Isolation of the virus is difficult. The current favored approach is inoculation of a mosquito cell line with patient serum, coupled with nucleic acid assays to identify a recovered virus.

NS-1 Antigen- may be + during first 3-4 days of illness

12InfluenzaVirus detected in throat swabs, nasopharyngeal swabs, washes, sputumTissue culture (within 4872 h after inoculation)Rapid tests:Immunologic or enzymatic techniquesRt-PCR reaction most Sn and Sp

The most sensitive and specific in vitro test for influenza virus is reverse-transcriptase polymerase chain reaction; this test proved particularly important in detecting the 20092010 pandemic A/H1N1 viruses, for which some rapid antigen detection tests were poorly sensitive.13InfluenzaSerologyObtained in 10-14 days after onset of illnessUseful only in retrospect> 4-fold rise of titer HI or CF

Serologic methods for diagnosis require comparison of antibody titers in sera obtained during the acute illness with those in sera obtained 1014 days after the onset of illness and are useful primarily in retrospect. Serum antibody responses, which can be detected by the second week after primary infection, are measured by a variety of techniques: hemagglutination inhibition (HI), complement fixation (CF), neutralization, enzyme-linked immunosorbent assay (ELISA), and antineuraminidase antibody assay

Fourfold or greater titer rises as detected by HI or CF or significant rises as measured by ELISA are diagnostic of acute infection. Other laboratory tests generally are not helpful in the specific diagnosis of influenza virus infection. Leukocyte counts are variable, frequently being low early in illness and normal or slightly elevated later. Severe leukopenia has been described in overwhelming viral or bacterial infection, while leukocytosis with >15,000 cells/L raises the suspicion of secondary bacterial infection.

Thrombocytopenia may be present.14