Weil Felix Test

Etiology and Epidemiology

The etiologic agents of rickettsial diseases have diverse transmission mechanisms, wide geographic distributions, and a range of disease manifestations. Rickettsiae are most commonly transmitted through fleas, ticks, mites, or mouse vectors, with humans acting as incidental hosts.[4] The Rickettsiaceae that cause life-threatening infections are R. rickettsii (rocky mountain spotted fever), R. prowazekii (louse-borne typhus), Orientia tsutsugamushi (scrub typhus), R. conorii (Mediterranean spotted fever), R. typhi (murine typhus); and, in rare cases, other organisms of the spotted fever group. Most rickettsial species are limited to a particular region due to climatic conditions and natural host constraints; however, some rickettsia species can be encountered globally, such as R. felis and R. typhi.[5]

Epidemiologic clues can be used to narrow the possible bacterial etiologies of a suspected rickettsial infection.[6] These clues include:

• Environmental exposure to ticks, fleas, or mites in regions and seasons where vector-borne diseases are prevalent, such as spotted fever and typhus rickettsioses, scrub typhus, ehrlichioses, and anaplasmosis
• Travel to or residence in an endemic region
• Exposure to pregnant ruminants, cats, or dogs, indicating possible Q fever
• Exposure to flying squirrels, associated with R. prowazekii typhus
• History of previous louse-borne typhus, which may lead to recrudescent typhus [7]

Pathophysiology

Rickettsiae are obligate intracellular bacteria with tropism for vascular endothelial cells. The clinical manifestations of rickettsial infections arise from the direct injury of these cells and the release of prostaglandins from the cells that further increase vascular permeability. Due to the resulting hypovolemia, the antidiuretic hormone is released, leading to increased sodium excretion and hyponatremia.[8] The clinical manifestations of rickettsioses are similar during the first 5 days, including fever, headache, myalgias, nausea, vomiting, and cough. As the disease progresses, it can diverge into variable disease states, depending on the host response. Possible manifestations include the occurrence of a macular, maculopapular, or vesicular rash; eschar formation; pneumonitis; meningoencephalitis; progressive hypotension; and organ failure consistent with sepsis.[9]

The Weil-Felix test is a nonspecific agglutination test that uses antigenic cross-reactivity between rickettsiae and certain non-motile Proteus serotypes to detect anti-rickettsial antibodies, so-called Weil-Felix antibodies, in a patient's serum. Both rickettsial antigen and Proteus OX (O-specific polysaccharide chain of outer membrane lipopolysaccharide) antigens are recognized by anti-rickettsial antibodies in a patient's serum; upon mixing serum that contains anti-rickettsial antibodies with OX antigen, the resulting reaction can indicate previous or current rickettsial infection.[8]

The rickettsial target of these antibodies was determined to be the lipopolysaccharide (LPS) O-antigen, which is highly conserved across the rickettsiae groups.[2] Amano et al demonstrated chemical and structural similarities between the LPS O-polysaccharides in P. vulgaris OX19 and those in the typhus group, suggesting a shared O-antigen as the mechanism of cross-reactivity. Cross-reactivity occurs between P. vulgaris OX19 and the typhus group, P. mirabilis OXK and the scrub typhus group, and P. vulgaris OX2 and OX19 and the spotted fever group.[10]

The test relies on the cross-reactive immunoglobulin M (IgM) antibodies produced in response to the infection. Unfortunately, these antibodies are not produced at sufficient levels to cause a positive test until 5 to 10 days after the onset of the disease. The serum concentration of the antibodies continues to rise, and a repeated titer 7 to 14 days after the original that shows a 4-fold or greater increase in concentration may be used to confirm the diagnosis.[11]

Specimen Requirements and Procedure

To perform the Weil-Felix test, a blood sample is obtained through venipuncture and allowed to coagulate. It is then centrifuged to allow the collection of a serum sample. An undiluted portion of the serum is combined with a suspension of proteus antigen on a white tile for an initial screening test. If agglutination occurs within 1 minute, the tester can expect a titer of at least 1:20 in the confirmatory tube agglutination test. This confirmatory test consists of serial dilutions of the patient's serum combined with the Proteus antigen. The final dilutions should range from 1:20 to 1:1280, along with a known negative control sample for quality assurance. After mixing and incubating for 4 hours, the tubes are assessed for a reaction. Tubes without agglutination appear unchanged, whereas those with agglutination display a granular appearance. The final result is the most dilute of the titered samples to exhibit agglutination at the end of the test.[12]

Interpreting the Weil-Felix test requires knowledge of the disease course and corresponding immune response. The level of IgM necessary for a positive result is not observed until 5 to 10 days after the onset of illness, and a single negative test cannot exclude disease. A threshold for agglutinins that is considered normal is up to 1:40; however, many factors can contribute to a titer above this threshold in those without a rickettsial infection. Notably, with Proteus OXK suspensions, titers up to 1:160 have been observed in noninfected persons. A positive titer of 1:320 has been observed in 54% of healthy individuals and 62% of individuals with non-rickettsial infections, indicating a low sensitivity at this threshold.[13]

Testing Procedures

The procedure involves collecting a blood sample from the patient in a tube or vial and sending it for analysis. There are two methods for conducting the analysis of the blood sample in the laboratory.[14]

Slide Method 

A small amount of the serum sample from the patient is placed on the slide, after which the preferred antigen drop is added to it. The next step is to rotate the consequential postponement for a minute. If visible agglutination occurs, a positive result is indicated.

Tube Method

In this method, a 2-fold dilution of the serum sample and diluents such as 0.25% phenol saline are added to the tubes. The tube's final volume has to be made up to 1 mL. Antigen deferment is added and incubated for 4 to 6 hours at 50 to 55 °C. Positive results are indicated if there is granulation or visible flocculation.

Interfering Factors

The same cross-reactive IgM antibodies used to perform the Weil-Felix test are cross-reactive with other antigens, causing false positives. There have been cases of individuals testing seropositive for anti-R. rickettsii IgM without any supporting evidence to indicate a recent rickettsial infection.[15] The United States Centers for Disease Control and Prevention (CDC) recommends that serological tests should always be paired and appropriately timed to give the best evidence of recent infection. This concept is also crucial for accurately interpreting the Weil-Felix test when used as a diagnostic study. The false-negative result may occur due to excess antibodies in the patient's serum (prozone phenomena). This issue can be obviated by testing with serial dilutions of the patient's serum.[16]

Results, Reporting, and Critical Findings

Interpreting the Weil-Felix test requires knowledge of the disease course and corresponding immune response. The level of IgM necessary for a positive result is not observed until 5 to 10 days after the onset of illness, and a single negative test cannot exclude disease.[15] One way to improve the diagnostic accuracy of the Weil-Felix test is to repeat the test 7 to 14 days after the original positive test and compare the titer levels. A significant increase in the titer level upon repeat testing strongly indicates a recent infection.[17]

Some confirmed rickettsial infections require CDC notification, including anaplasmosis, ehrlichiosis, Q fever, and spotted fever rickettsioses. The Weil-Felix test is not definitively diagnostic but can be included in the report as supporting information. The gold standard for diagnosis of rickettsial infections is indirect immunofluorescent IgG antibody assays of paired serum samples—one taken during the acute phase of the disease and one taken 2 to 4 weeks later in the convalescent phase—which has a much higher sensitivity and specificity.[16] Other options for laboratory evaluation include polymerase chain reaction and other nucleic acid amplification techniques, species-specific enzyme-linked immunosorbent assays, or direct visualization of the bacteria in tissue samples with labeled antibodies. Culturing and isolating the obligate-intracellular rickettsiae is possible but requires specialized methods that are not practical for routine diagnosis.[18]

Clinical Significance

Since its inception a century ago, the Weil-Felix test has mostly been replaced by newer diagnostic studies. However, it is important to remember the test is still in common use among areas without access to modern methods. It is inexpensive, requires little training to perform, and can provide meaningful data supporting the diagnosis of a rickettsial infection.[19] 

Quality Control and Lab Safety

Qualitative and semi-quantitative examinations provide non-numerical results. Qualitative examinations measure the presence or absence of a substance or evaluate cellular characteristics such as morphology. Semi-quantitative examinations offer an estimate of the amount of the substance present.[20] Quality control processes must monitor qualitative and semi-quantitative testing. These processes should use controls that mimic patient samples as much as possible.[21]

Positive and negative controls are recommended for many qualitative and semi-quantitative tests, including procedures that use special stains or reagents and tests with endpoints such as agglutination or color change. These controls should generally be used with each test run. The use of controls also helps to validate a new lot number of test kits or reagents, to check on temperatures of storage and testing areas, and to evaluate the process when new testing personnel is carrying out the testing.[22]

Laboratories must establish a quality control program for their qualitative and semi-quantitative tests.[23] In establishing this program, policies should be set, staff trained, responsibilities assigned, and resources ensured to be available. Ensure that the recording of all quality control data is complete and that the quality manager and the laboratory director appropriately review the information. If QC results are unexpected, patient results should not be reported.[24]

When using traditional controls for qualitative or semi-quantitative tests, the following points should be kept in mind—test control materials in the same manner as testing patient samples; use positive and negative controls, ideally daily or as often as recommended by the manufacturer; choose positive controls that are close to the cut-off value of the test to ensure the detection of weak positive reactions; and for agglutination procedures, include a weak positive control and a negative control and stronger positive control.[21]

The laboratory must participate in the external quality control or proficiency testing program because it is a regulatory requirement published by the Centers for Medicare and Medicaid Services (CMS) in the Clinical Laboratory Improvement Amendments regulations. These programs are helpful to ensure the accuracy and reliability of the laboratory compared to other laboratories performing the same or comparable assays. Required participation and scored results are monitored by CMS and voluntary accreditation organizations.[25] The proficiency testing plan should be included as an aspect of the quality assessment plan and the overall quality program of the laboratory.[26]

All specimens, control materials, and calibrator materials should be considered potentially infectious. Normal precautions required for handling all laboratory reagents should be exercised. Disposal of all waste material should be in accordance with local guidelines. Gloves, a lab coat, and safety glasses should be worn when handling human blood specimens. Plastic tips, sample cups, and gloves that come into contact with blood must be placed in a biohazard waste container.[27] Disposable glassware should be discarded into sharps waste containers. Work surfaces must be protected with disposable absorbent bench top paper, which is discarded into biohazard waste containers weekly or whenever blood contamination occurs. Work surfaces should be wiped weekly.[28]

Enhancing Healthcare Team Outcomes

In regions with limited access to advanced diagnostic tools, the Weil-Felix test remains a valuable resource for diagnosing infections such as endemic typhus. Despite its replacement by more modern methods in many areas, this test can provide crucial information in resource-poor settings. Adequate training on the test's techniques and their clinical significance allows using this test effectively, ensuring timely diagnosis and improving patient outcomes in underserved populations.

Interprofessional team members, including clinicians, nurses, and laboratory technicians, should be familiar with the Weil-Felix test and its role in diagnosing rickettsial infections. A shared understanding of the test's value and limitations enables better collaboration, enhancing diagnostic accuracy and case management. This collaborative approach can help ensure that even in resource-limited environments, patients receive appropriate care based on the available diagnostic tools.