Introduction

First described in 1916, the Weil-Felix reaction is a test used in the diagnosis of 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 3 groups: the epidemic and endemic typhus group, 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 that could identify the presence of antibodies against the Rickettsia disease groups. P. vulgaris OX19 antigen reacts with antibodies to 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 the diagnosis of rickettsial infections is indirect immunofluorescence, which is available through most state health departments in areas where the 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. 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 certain region due to climatic conditions and natural host constraints; however, some rickettsia species can be encountered globally, such as R. felis and R. typhi.[4][5]

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

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.[7] The clinical manifestations of rickettsioses are similar during the first 5 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, and progressive hypotension and organ failure consistent with sepsis.[8]

The Weil-Felix test is a nonspecific agglutination test that utilizes antigenic cross-reactivity between rickettsiae and certain non-motile Proteus serotypes for detection of 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.[7] The rickettsial target of these antibodies was determined to be the lipopolysaccharide (LPS) O-antigen, which is highly conserved across the rickettsiae groups. Amano et al. demonstrated chemical and structural similarities between the LPS O-polysaccharides present in P. vulgaris OX19 and those present 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.[2][9]

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 5-10 days after the onset of disease. The serum concentration of the antibodies continue 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.[10]

Specimen Requirements and Procedure

In order 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 that are then 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.[11]

Interpretation of 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.[12]

Interfering Factors

The same cross-reactive IgM antibodies utilized to perform the Weil-Felix test are cross-reactive with other antigens, causing a high level of false positives. There are reports of persons seropositive for anti-R. rickettsii IgM with no supporting evidence to indicate a recent rickettsial infection.[13] 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, a concept that is important in the accurate interpretation of the Weil-Felix test as well if it is being used as a diagnostic study.[14]

Results, Reporting, Critical Findings

Interpretation of 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.[13]

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 recent infection.

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. 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.[14][15]

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 that can support the diagnosis of a rickettsial infection. 

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 technique and clinical significance will allow the use of this test in patients and healthcare facilities with limited resources, thereby improving patient outcomes.