Introduction

First described in 1916, the Weil-Felix reaction is a test used to diagnose rickettsial infections. While it has largely been replaced with new serological techniques, the Weil-Felix test continues to hold great importance in resource-limited areas where more advanced methods are unavailable. The known pathogenic rickettsia species are gram-negative, obligate intracellular bacteria that include an increasing number of identified organisms belonging to seven genera (Rickettsia, Orientia, Ehrlichia, Anaplasma, Neorickettsia, Candidatus, Neoehrlichia, and Coxiella). They are closely related and are traditionally separated into three groups: the epidemic and endemic typhus group, the scrub typhus group, and the spotted fever group.[1]

The test was developed upon the observation that certain serotypes of Proteus bacteria display antigenic cross-reactivity with Rickettsia species. Through the isolation of these Proteus antigens, a heterophile agglutination reaction was developed to identify antibodies against the Rickettsia disease groups. P. vulgaris OX19 antigen reacts with antibodies to the typhus group (TG), P. mirabilis OXK antigen reacts with antibodies to the scrub typhus group (STG), and both P. vulgaris OX2 and OX19 antigens react with antibodies to the spotted fever group (SFG).[2]

Due to its low sensitivity and specificity, the Weil-Felix test has fallen out of favor in most clinical settings, and its use is no longer recommended in routine practice. The current gold standard in diagnosing rickettsial infections is indirect immunofluorescence, which is available through most state health departments in areas where infections are common.[3]

Etiology and Epidemiology

The etiologic agents of rickettsial diseases have varied mechanisms of transmission, wide geographic distributions, and an array of disease manifestations. Rickettsiae are most commonly transmitted through flea, tick, mite, 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 SFG organisms. 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 include environmental exposure to ticks, fleas, or mites, at a time and in a region the vector is known to transmit the disease (spotted fever and typhus rickettsioses, scrub typhus, ehrlichioses, anaplasmosis), travel to or residence in an endemic region, exposure to pregnant ruminants, cats, or dogs (Q fever), exposure to flying squirrels (R. prowazekii typhus), and history of previous louse-borne typhus (recrudescent typhus).[7]

Pathophysiology

Rickettsiae are obligate intracellular bacteria with tropism for vascular endothelial cells. The disease manifestations seen with rickettsial infections are owed to direct injury of these cells, as well as the release of prostaglandins from the cells that further increase vascular permeability. Due to the resulting hypovolemia, the antidiuretic hormone is released, resulting in increased sodium excretion and hyponatremia.[8] The clinical manifestations of rickettsioses are similar during the first five days and include fever, headache, myalgias, nausea, vomiting, and cough. As the course 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 utilizes 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 TG rickettsiae, suggesting a shared O-antigen as the mechanism of cross-reactivity. Cross-reactivity is seen between P. vulgaris OX19 and TG rickettsiae, between P. mirabilis OXK and STG rickettsiae, and between P. vulgaris OX2 and OX19 and SFG rickettsiae.[10]

The test relies on the cross-reactive 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 7-14 days after the original that shows a four-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 by 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 there is agglutination within one 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, while a granular appearance is seen in those that exhibit agglutination. 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 seen until 5-10 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. It is especially seen with Proteus OXK suspensions, in which titers up to 1:160 have been observed in non-infected persons. A positive titer of 1:320 has been observed in 54% of healthy persons and 62% of persons with non-rickettsial infections, giving a low sensitivity with this threshold.[13]

Testing Procedures

The procedure involves the collection of the blood sample from the patient in the tube or the vial and sending it for analysis. There are two methods of conducting the analysis in the lab.[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 this results in visible agglutination, then a positive result is indicated.

Tube Method: In this method, a twofold dilution of the serum sample is added to the tubes along with diluents; 0.25% phenol saline. The tube’s final volume has to be made up to 1 ml. Antigen deferment is then added and incubated for four to six hours at 50 to 55 degrees C. Positive results are indicated if there is granulation or visible flocculation.

Interfering Factors

The same cross-reactive IgM antibodies utilized to perform the Weil-Felix test are cross-reactive with other antigens, causing false positives. There are reports of persons seropositive for anti-R. rickettsii IgM with no supporting evidence to indicate a recent rickettsial infection.[15] The United States Centers for Disease Control and Prevention (CDC) suggests serological tests should always be paired and appropriately timed to give the best evidence of recent infection. This concept is also essential in accurately interpreting the Weil-Felix test if it is used as a diagnostic study. The false-negative result may occur due to excess antibodies in the patient's serum (prozone phenomena). This 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 seen until 5-10 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 7-14 days after the original positive test and compare the titer levels. A significant increase in the titer level upon repeat testing is a strong indication of a recent infection.[17]

Some confirmed rickettsial infections require CDC notification. These include 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 two to four weeks later in the convalescent phase), which has a much higher sensitivity and specificity.[16] Other options for laboratory evaluation include polymerase chain reaction (PCR) and other nucleic acid amplification techniques, species-specific ELISA 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 are those that give non-numerical results. Qualitative examinations measure the presence or absence of a substance or evaluate cellular characteristics such as morphology. Semi-quantitative examinations provide an estimate of how much of the measured substance is 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 will also help 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]

The laboratory must establish a quality control program for its qualitative and semi-quantitative tests.[23] In establishing this program, set policies, train staff, assign responsibilities and ensure all resources are available. Make sure that recording of all quality control data is complete and that an appropriate review of the information is carried out by the quality manager and the laboratory director. If QC results are not what is expected, do not report patient results.[24]

When utilizing traditional controls for qualitative or semi-quantitative tests, take in mind the following: Test control materials in the same manner as testing patient samples; use a positive and negative control, preferably once each day of testing, or at least as often as recommended by the manufacturer; choose positive controls that are close to the cut-off value of the test, to be sure the test can detect weak positive reactions; and for agglutination procedures, include a weak positive control as well as a negative control and stronger positive control.[21]

The laboratory must participate in the external quality control or proficiency testing (PT) program because it is a regulatory requirement published by the Centers for Medicare and Medicaid Services (CMS) in the Clinical Laboratory Improvement Amendments (CLIA) regulations. It is 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 PT plan should be included as an aspect of the quality assessment (QA) plan and the overall quality program of the laboratory.[26]

Consider all specimens, control materials, and calibrator materials as potentially infectious. Exercise the normal precautions required for handling all laboratory reagents. Disposal of all waste material should be in accordance with local guidelines. Wear gloves, a lab coat, and safety glasses when handling human blood specimens. Place all plastic tips, sample cups, and gloves that come into contact with blood in a biohazard waste container.[27] Discard all disposable glassware into sharps waste containers. Protect all work surfaces with disposable absorbent bench top paper, which is discarded into biohazard waste containers weekly, or whenever blood contamination occurs. Wipe all work surfaces weekly.[28]

Enhancing Healthcare Team Outcomes

In areas with poor access to advanced testing, this test serves as a valuable diagnostic tool to diagnose infections such as endemic typhus. Adequate training on testing techniques and clinical significance will allow the use of this test in patients and healthcare facilities with limited resources, thereby improving patient outcomes. The interprofessional team members should at least be familiar with this test and understand its value and results, leading to improved diagnosis and enhanced case management.