Laboratory diagnosis, treatment and prevention of Rickettsia rickettsii

Rickettsia rickettsii is the causative agent of Rocky Mountain spotted fever (RMSF), a potentially fatal tick-borne disease. Laboratory diagnosis of R. rickettsii infection is important for early and appropriate treatment, as well as for epidemiological surveillance and prevention. However, laboratory diagnosis of R. rickettsii can be challenging due to the following reasons:

• The clinical manifestations of RMSF are nonspecific and can mimic other febrile illnesses.
• The rash, which is a characteristic sign of RMSF, may be absent or delayed in some patients.
• The organism is difficult to culture and requires biosafety level 3 facilities and precautions.
• The serological tests may have low sensitivity in the early phase of infection or cross-reactivity with other rickettsial species.

Therefore, a combination of clinical, epidemiological, and laboratory criteria is recommended for the diagnosis of RMSF. The laboratory methods that can be used for the detection of R. rickettsii include:

• Direct detection methods: These methods aim to identify the presence of R. rickettsii antigen or DNA in clinical specimens, such as skin biopsies or serum. The advantages of these methods are that they can provide rapid and specific results, and that they do not require prior exposure or antibody production by the host. The disadvantages are that they may have low sensitivity due to the low number of organisms in the specimens, and that they may require specialized equipment and expertise. The direct detection methods include:

  • Giemsa or Gimenez stains: These are microscopic techniques that use specific dyes to stain the rickettsiae in tissue sections or smears. The stained organisms appear as small purple dots (coccobacilli) within or outside the host cells. However, these stains are not specific for R. rickettsii and may also stain other bacteria or cellular debris.
  • Direct fluorescent antibody (DFA) test: This is an immunofluorescence technique that uses fluorescent-labeled antibodies that bind to R. rickettsii antigen in tissue sections or smears. The bound antibodies emit light when viewed under a fluorescence microscope, allowing the visualization of the rickettsiae as bright spots. This test is more specific than the stains, but it requires fresh or frozen specimens and a fluorescence microscope.
• Culture: This method involves the isolation and growth of R. rickettsii in artificial media, such as embryonated egg or tissue cultures. The advantages of this method are that it can provide definitive identification and characterization of the isolate, and that it can be used for further studies, such as typing or susceptibility testing. The disadvantages are that it is time-consuming, labor-intensive, and hazardous, as it requires biosafety level 3 facilities and precautions. Moreover, culture is often unsuccessful due to the fastidious nature of R. rickettsii and the presence of inhibitory substances in the specimens.

• Serology: This method detects the presence of R. rickettsii-specific antibodies in the serum of infected patients. The advantages of this method are that it is widely available, relatively inexpensive, and easy to perform. The disadvantages are that it may have low sensitivity in the early phase of infection, as antibodies usually appear after 7 to 10 days of illness onset, and that it may have low specificity due to cross-reactivity with other rickettsial species or non-rickettsial antigens. The serological tests include:

  • Weil-Felix test: This is an agglutination test that uses certain strains of Proteus vulgaris bacteria as antigens. These bacteria share some cross-reactive antigens with R. rickettsii and other rickettsiae, and thus can agglutinate with the serum antibodies of infected patients. However, this test is nonspecific and outdated, and is rarely used in modern laboratories.
  • Microimmunofluorescence (MIF) test: This is an immunofluorescence technique that uses R. rickettsii-specific antigens (outer membrane proteins or lipopolysaccharide) coated on slides. The serum antibodies bind to these antigens and are detected by fluorescent-labeled secondary antibodies under a fluorescence microscope. This test is considered the reference method for serology, as it has high sensitivity and specificity, and can differentiate between different rickettsial species and antibody classes (IgM or IgG).
  • Indirect immunofluorescent antibody (IFA) test: This is a similar technique to MIF, but uses whole-cell antigens instead of specific antigens. This test has lower specificity than MIF, but may have higher sensitivity in some cases.
  • Enzyme-linked immunosorbent assay (ELISA): This is an enzyme-based technique that uses R. rickettsii-specific antigens coated on plates. The serum antibodies bind to these antigens and are detected by enzyme-labeled secondary antibodies that produce a color change when exposed to a substrate. This test has similar sensitivity and specificity to MIF, but may be more convenient and automated.

The serological diagnosis of RMSF is usually based on the demonstration of a fourfold rise in antibody titers between acute and convalescent sera (collected at least two weeks apart), or a single serum titer of 1:64 or higher by MIF or IFA.

• Molecular diagnosis: This method detects the presence of R. rickettsii DNA or genes in clinical specimens by polymerase chain reaction (PCR). The advantages of this method are that it can provide rapid and specific results, and that it can detect low levels of organisms in the specimens. The disadvantages are that it may be affected by inhibitors or contaminants in the specimens, and that it requires specialized equipment and expertise. The molecular targets that are used for PCR include gene sequences for outer membrane proteins (OmpA, OmpB), the 17-kDa lipoprotein, citrate synthase, or other genes.

PCR has been shown to be useful for detecting R. rickettsii DNA in skin biopsy specimens from patients with rash, with good sensitivity and high specificity.

The most common specimens used for the laboratory diagnosis of Rickettsia rickettsii are skin biopsies and serum. Skin biopsies are obtained from the rash lesions that appear on the patient`s body, usually on the wrists, ankles, palms, and soles. Serum is collected from the patient`s blood.

Skin biopsies are useful for direct detection methods, such as staining and immunofluorescence, as well as for molecular methods, such as PCR. Serum is mainly used for serological methods, such as antibody detection and agglutination tests.

Skin biopsies should be taken as early as possible in the course of the disease, preferably within the first week of illness. Serum samples should be collected in two phases: acute phase (within the first week of illness) and convalescent phase (2-4 weeks after the onset of symptoms). A fourfold rise in antibody titers between the two phases is indicative of a recent infection.

Skin biopsies and serum samples should be handled with care and transported to the laboratory under appropriate conditions. Skin biopsies should be placed in sterile saline or transport medium and kept refrigerated or frozen until testing. Serum samples should be separated from blood cells by centrifugation and stored at 4°C or lower until testing. Both types of specimens should be labeled with the patient`s name, date of collection, and clinical information. They should also be accompanied by a request form that specifies the tests to be performed and the suspected diagnosis.

Direct detection of R. rickettsii antigen in skin biopsy specimens or serum samples from infected patients can be performed by using specific anti-rickettsial antibodies. This method is rapid and specific, but requires specialized equipment and expertise.

There are two main types of direct detection methods: Giemsa or Gimenez stains, and direct fluorescent antibody (DFA) test.

• Giemsa or Gimenez stains are histochemical stains that can be used to visualize rickettsiae in tissue sections or smears. These stains bind to the DNA and RNA of the bacteria, producing a purple or pink color. However, these stains are not very sensitive and may not detect low numbers of rickettsiae. Moreover, they are not specific and may cross-react with other bacteria or cellular debris.
• DFA test is an immunohistochemical technique that uses fluorescent-labeled antibodies to detect rickettsial antigens in tissue sections or smears. The antibodies bind to the outer membrane proteins or lipopolysaccharides of the bacteria, producing a bright green or yellow fluorescence under a microscope. This test is more sensitive and specific than the stains, but it requires fresh or frozen specimens and a fluorescence microscope. The antibodies used for DFA test should be specific for R. rickettsii and not cross-react with other rickettsial species.

Both methods require experienced personnel to perform and interpret the results. They are also prone to false negatives if the specimens are not collected properly or if the rickettsiae are degraded by host immune response or antibiotics. Therefore, direct detection methods should be used in conjunction with other diagnostic methods, such as culture, serology, or molecular diagnosis.

• Culture is a method of isolating R. rickettsii from skin biopsies or serum samples of infected patients.
• Culture requires inoculating the specimen into embryonated egg or tissue cultures and observing the cytopathic effects of the rickettsiae on the host cells.
• Culture is a definitive and specific method of diagnosis, but it is rarely attempted in routine laboratories because of the associated risk of infection and the need for biosafety level 3 facilities.
• Culture is also time-consuming and technically demanding, and it may take several weeks to obtain results.

Serology is the most common method for diagnosing R. rickettsii infection, as it detects the presence of antibodies against the bacteria in the patient`s blood. However, serological tests have some limitations, such as cross-reactivity with other rickettsial species, low sensitivity in early stages of infection, and dependence on paired serum samples to demonstrate a fourfold rise in antibody titers.

There are different types of serological tests available for R. rickettsii detection, each with its own advantages and disadvantages:

• The Weil-Felix test is an old and non-specific test that relies on the agglutination of certain strains of Proteus vulgaris bacteria that share antigens with some rickettsiae. It is cheap and easy to perform, but it has low sensitivity and specificity, and it cannot distinguish between different rickettsial species .
• The microimmunofluorescence (MIF) test is considered the reference method for R. rickettsii serology. It uses fluorescent-labeled antibodies that bind to specific outer membrane proteins or lipopolysaccharide antigens of the bacteria on a slide. It has high sensitivity and specificity, and it can differentiate between spotted fever group and typhus group rickettsiae. However, it requires specialized equipment and trained personnel, and it may not detect antibodies until the second week of illness .
• The indirect immunofluorescent antibody (IFA) test is similar to the MIF test, but it uses whole-cell antigen instead of purified antigens. It can detect both IgG and IgM antibodies, which may indicate acute or past infection. It is simpler and cheaper than the MIF test, but it has lower specificity and may cross-react with other rickettsial species .
• The enzyme linked immunosorbent assay (ELISA) is a newer serological test that uses enzyme-labeled antibodies that bind to specific antigens of R. rickettsii on a plate. It can measure the amount of antibodies in the serum quantitatively, and it can be automated and standardized. It has higher sensitivity than the IFA test in early stages of infection, but it may also cross-react with other rickettsial species .

A definitive diagnosis of R. rickettsii infection is made by demonstrating a fourfold or greater rise in antibody titers between acute-phase and convalescent-phase sera, or by demonstrating an antibody titer of 1:64 or more in a single serum sample . These antibodies are usually detected 2–3 weeks after the onset of symptoms, and they may persist for a long time after recovery .

Molecular diagnosis is a fast and accurate method to identify R. rickettsii infection in clinical specimens. It involves the amplification and detection of specific segments of rickettsial DNA or outer membrane protein (Omp) genes by polymerase chain reaction (PCR).

PCR has several advantages over other diagnostic methods, such as:

• It can be performed on various types of specimens, including skin biopsies, blood, cerebrospinal fluid, and tissue samples.
• It can detect R. rickettsii DNA even in low concentrations or in cases of early infection, before antibodies are produced.
• It can differentiate R. rickettsii from other closely related rickettsiae, such as R. parkeri or R. conorii, by targeting species-specific gene sequences.
• It can provide results within hours, compared to days or weeks for culture or serology.

However, PCR also has some limitations, such as:

• It requires specialized equipment and trained personnel to perform and interpret the results.
• It may yield false-negative results if the specimen is contaminated with inhibitors of PCR or if the DNA is degraded by storage or transport conditions.
• It may yield false-positive results if the specimen is contaminated with rickettsial DNA from previous cases or environmental sources.

Therefore, PCR should be used as a complementary tool to support the clinical diagnosis of Rocky Mountain spotted fever, and not as a sole criterion for confirmation.

Several gene targets have been used for PCR detection of R. rickettsii, including:

• The OmpA gene, which encodes a major surface antigen that is specific for spotted fever group rickettsiae.
• The OmpB gene, which encodes another surface antigen that is conserved among all rickettsiae but varies in sequence among different species.
• The 17-kDa lipoprotein gene, which encodes a highly immunogenic protein that is present in all rickettsiae.
• The citrate synthase gene (gltA), which encodes an enzyme involved in the Krebs cycle that is essential for rickettsial survival.

The choice of gene target depends on the availability of primers and probes, the sensitivity and specificity of the assay, and the purpose of the test (e.g., screening or confirmation).

Some examples of PCR assays that have been developed and validated for R. rickettsii detection are:

• A real-time PCR assay that uses TaqMan probes to amplify and detect a 401-bp fragment of the OmpA gene.
• A nested PCR assay that uses two sets of primers to amplify and detect a 532-bp fragment of the OmpB gene.
• A multiplex PCR assay that uses three pairs of primers to amplify and detect fragments of the OmpA, OmpB, and 17-kDa lipoprotein genes simultaneously.
• A conventional PCR assay that uses primers to amplify and detect a 401-bp fragment of the gltA gene.

These PCR assays have shown high sensitivity and specificity for R. rickettsii detection in various clinical specimens, such as skin biopsies from patients with rash , blood samples from patients with fever, and cerebrospinal fluid samples from patients with neurological complications.

Molecular diagnosis of R. rickettsii infection by PCR can provide rapid and reliable results that can help guide the appropriate treatment and management of patients with Rocky Mountain spotted fever.

Besides the laboratory diagnosis methods mentioned above, some other tests can provide useful information about the patient`s condition and the severity of the disease. These tests include:

• Blood cell count: Leukopenia (low white blood cell count) is present initially, then mild leukocytosis (high white blood cell count); patients usually have a normal WBC count. Thrombocytopenia (low platelet count) is also common and can indicate a risk of bleeding complications.
• Sodium levels: Hyponatremia (low sodium levels in the blood) may be decreased in more severe cases of Rocky Mountain spotted fever, and can be associated with neurological symptoms, such as confusion, seizures, or coma.
• Liver enzymes: Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) may be slightly elevated in some patients, indicating liver inflammation or damage.
• Cerebrospinal fluid analysis: Mild cerebrospinal fluid pleocytosis (increased number of white blood cells in the fluid that surrounds the brain and spinal cord) with monocyte predominance (higher proportion of a type of white blood cell) may be seen in patients with meningitis or encephalitis caused by R. rickettsii infection.

These tests are not specific for R. rickettsii infection, but they can help to monitor the patient`s clinical status and to rule out other possible causes of the symptoms. They can also guide the appropriate management and treatment of the patient. Therefore, they should be performed as part of the routine evaluation of suspected cases of Rocky Mountain spotted fever.

The treatment of Rocky Mountain spotted fever (RMSF), the most severe and common rickettsial disease in the United States, is based on the early administration of antibiotics. The mortality rate of untreated RMSF can be as high as 30%, but with appropriate therapy, it can be reduced to less than 1%.

The drug of choice for RMSF is tetracycline (doxycycline), which inhibits the protein synthesis of rickettsiae and prevents their replication and growth. Doxycycline is effective against all stages of the disease and can also prevent complications such as gangrene, renal failure, and neurological damage. Doxycycline is recommended for all patients with suspected or confirmed RMSF, regardless of their age or pregnancy status. Although tetracyclines are generally contraindicated for pregnant women and young children because of their potential adverse effects on teeth and bones, these risks are outweighed by the benefits of treating a life-threatening infection. Doxycycline should be given orally or intravenously at a dose of 100 mg twice daily for adults and 2.2 mg/kg twice daily for children under 45 kg. The duration of treatment is usually 5 to 7 days, or until the patient has been afebrile for at least 3 days.

An alternative drug for RMSF is chloramphenicol, which also inhibits the protein synthesis of rickettsiae. Chloramphenicol may be used in patients who are allergic to tetracyclines or who cannot tolerate them due to gastrointestinal side effects. However, chloramphenicol has its own drawbacks, such as bone marrow suppression, aplastic anemia, and gray baby syndrome. Chloramphenicol should be given orally or intravenously at a dose of 50 to 75 mg/kg per day in four divided doses for adults and children. The duration of treatment is similar to doxycycline.

Another class of antibiotics that has shown activity against R. rickettsii is fluoroquinolones, such as ciprofloxacin. Fluoroquinolones interfere with the DNA replication and transcription of rickettsiae by inhibiting their topoisomerases. Ciprofloxacin may be used in patients who are allergic or intolerant to both tetracyclines and chloramphenicol, or in cases of severe or complicated RMSF that require combination therapy. Ciprofloxacin should be given orally or intravenously at a dose of 500 mg twice daily for adults and 15 mg/kg twice daily for children. The duration of treatment is similar to doxycycline.

The use of other antibiotics, such as penicillins, cephalosporins, macrolides, sulfonamides, and aminoglycosides, is not recommended for RMSF because they have no or minimal activity against R. rickettsii and may delay the initiation of effective therapy.

The treatment of RMSF should be started as soon as possible after the onset of symptoms, preferably within the first five days of illness. Delayed treatment may increase the risk of severe complications and death. The diagnosis of RMSF should be based on clinical signs and epidemiological factors, rather than waiting for laboratory confirmation, which may take several weeks. Empirical treatment should be given to any patient who presents with fever, rash, headache, and history of tick exposure in an endemic area.

In addition to antibiotic therapy, supportive care is also important for the management of RMSF. Patients should be monitored for signs of shock, organ failure, coagulopathy, and secondary infections. Fluid and electrolyte balance should be maintained and blood transfusions may be required in cases of severe anemia or bleeding. Corticosteroids may be considered in patients with refractory shock or cerebral edema, but their role is controversial and may increase the risk of secondary infections.

Prevention of RMSF relies on avoiding tick bites and removing attached ticks promptly. People who live or travel in areas where RMSF is endemic should wear protective clothing, use insect repellents containing DEET or permethrin, and check their bodies and pets for ticks daily. Ticks should be removed carefully with tweezers or a tick removal device, without squeezing or crushing them. Prophylactic use of doxycycline after a tick bite is not recommended because it may mask the early symptoms of RMSF and delay diagnosis and treatment.

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