Laboratory Diagnosis, Treatment and Prevention of Francisella tularensis

Francisella tularensis is a Gram-negative coccobacillus that causes tularemia, a zoonotic disease that can affect various organs and systems in humans and animals. The laboratory diagnosis of Francisella tularensis infection is challenging because of the low number of bacteria in clinical specimens, the fastidious growth requirements of the organism, and the high risk of infection for laboratory personnel. Therefore, a combination of methods is usually employed to confirm the diagnosis, including direct detection, culture and identification, antibody detection, and molecular methods.

The choice of specimen depends on the type and stage of tularemia infection. The most common specimens are:

• Scrapings from infected ulcers
• Lymph node biopsies
• Sputum
• Whole blood
• Serum

Scrapings from infected ulcers are useful for direct detection and culture of the organism. Lymph node biopsies are also helpful for culture and histopathology. Sputum can be collected from patients with pneumonic tularemia. Whole blood can be used for culture and molecular testing. Serum is generally collected from all patients early in disease and during convalescence for antibody detection.

To minimize the loss of viable organisms, samples should be transported to the laboratory within 24 hours. If specimens are to be held longer than 24 hours, specimens should be refrigerated in Amie’s transport medium. F. tularensis should remain viable for up to 7 days stored at ambient temperature in Amie’s medium.

Specimens for molecular testing should be placed in guanidine isothiocyanate buffer, which preserves the DNA and inactivates the organism. This reduces the risk of infection to laboratory personnel and improves the sensitivity of PCR assays.

Specimens should be handled with care and appropriate biosafety precautions, as F. tularensis is highly infectious and can cause severe disease even with a small inoculum. The laboratory should be notified of the suspicion of tularemia before sending the specimens, so that they can prepare the necessary equipment and reagents.

One of the methods to directly detect Francisella tularensis in clinical specimens is staining and fluorescent antibody (FA) testing. This method involves applying a specific dye or antibody that binds to the bacteria and makes them visible under a microscope.

Staining can be done with Gram stain, which is a common technique to differentiate bacteria based on their cell wall structure. F. tularensis appears as small, Gram-negative coccobacilli (rod-shaped bacteria with rounded ends) that are often difficult to see unless the concentration of bacteria is high. Gram stain is useful for specimens such as wound swabs, tissue biopsies, and respiratory aspirates.

FA testing is more sensitive and specific than Gram stain, as it uses antibodies that are labeled with a fluorescent dye and bind only to F. tularensis antigens. FA testing can be performed on smears or sections of lesions, lymph nodes, or other tissues. Commercial kits are available for FA testing and are typically used in reference laboratories. FA testing can also be used to confirm the identity of F. tularensis isolates from culture.

Both staining and FA testing require proper handling and processing of specimens to avoid false-negative results. Specimens should be fixed with methanol or acetone before staining or FA testing to preserve the bacterial morphology and antigenicity. Specimens should also be examined promptly after staining or FA testing to avoid fading of the fluorescence.

Staining and FA testing are rapid and relatively inexpensive methods to directly detect F. tularensis in clinical specimens. However, they have some limitations, such as low sensitivity for some specimen types, cross-reactivity with other bacteria, and potential exposure of laboratory personnel to infectious aerosols. Therefore, these methods should be used in conjunction with other diagnostic methods, such as culture, serology, and molecular assays.

Francisella tularensis is a strictly aerobic bacterium that requires enriched media containing sulfhydryl compounds (such as cysteine, cystine, thiosulfate or IsoVitaleX) for primary isolation. Two commercial media for cultivation of the organism are available: glucose cystine agar and cystine-heart agar, both require the addition of 5% sheep or rabbit blood. However, F. tularensis can also grow on chocolate agar or buffered charcoal yeast extract (BCYE) agar, media supplemented with cysteine that are used in most laboratories.

The organism is slow-growing and requires 2 to 4 days for maximal colony formation. It produces 1-2 mm gray to grayish-white colonies on chocolate agar after more than 48 hours. It is weakly catalase positive and oxidase negative. Identification is done by growth on chocolate agar but not blood agar (blood agar is not supplemented with cysteine) and by demonstrating the reactivity of the bacteria with specific antiserum (i.e., agglutination of the organism with antibodies against Francisella).

The laboratory should be alerted if F. tularensis is suspected so cultures can be incubated for extended periods, due to the fastidious, slow-growing nature of the bacterium. The laboratory should also follow biosafety level 3 precautions when handling the specimens, as the organism is highly infectious and can cause severe disease.

Other methods for identification of F. tularensis include molecular methods such as polymerase chain reaction (PCR) assays and whole-cell MALDI-TOF mass spectrometry. These methods can detect F. tularensis directly in clinical specimens or culture isolates and can also differentiate between subspecies of the bacterium. However, these methods may not be widely available in all laboratories and may require validation and quality control.

Serological tests are useful for detecting antibodies to F. tularensis in serum samples from patients with suspected tularemia. Antibodies usually appear within one week of onset of symptoms and can persist for months or years after recovery. Serological tests can also help confirm the diagnosis of tularemia when culture or molecular methods are negative or unavailable.

There are different types of serological tests available for F. tularensis, such as:

• Whole-cell agglutination (WCA): This test uses whole bacterial cells as antigens and detects both IgM and IgG antibodies. It is simple, rapid and inexpensive, but it may have low sensitivity and specificity, and it may cross-react with other bacteria.
• Enzyme-linked immunosorbent assay (ELISA): This test uses purified or recombinant antigens and detects IgM and IgG antibodies separately. It is more sensitive and specific than WCA, but it may also cross-react with other bacteria and it requires specialized equipment and reagents.
• Microagglutination (MA): This test uses killed bacterial cells coated with gelatin as antigens and detects IgM and IgG antibodies separately. It is more sensitive and specific than WCA, but it may also cross-react with other bacteria and it requires a microscope and trained personnel.
• Immunofluorescence assay (IFA): This test uses fluorescent-labeled antibodies to detect F. tularensis antigens or antibodies in serum or tissue samples. It is highly sensitive and specific, but it may also cross-react with other bacteria and it requires a fluorescence microscope and trained personnel.

The choice of serological test depends on the availability, cost, turnaround time and performance characteristics of each test. The CDC recommends ELISA as the preferred method for routine diagnosis of tularemia. However, serological testing should be performed alongside other diagnostic methods, such as culture or molecular testing, to increase the accuracy of diagnosis.

Serological testing has some limitations, such as:

• It may not detect early infections or anamnestic responses (rapid antibody production after re-exposure) due to the lag time between infection and antibody production.
• It may not differentiate between current, recent or past infections due to the persistence of antibodies after recovery.
• It may not correlate with the severity or type of infection due to the variability of antibody responses among individuals.
• It may produce false positive results due to cross-reactions with other bacteria or non-specific reactions.

Therefore, serological testing should be interpreted with caution and in conjunction with clinical presentation, exposure history and other laboratory findings.

To perform serological testing for F. tularensis, serum samples should be collected from all patients early in disease and during convalescence. Ideally, the first serum sample should be collected within the first week of onset and the second serum sample should be collected 2–3 weeks later. A fourfold or greater increase in antibody titers between acute and convalescent samples or a single titer of 1:160 or greater is considered diagnostic of tularemia. However, some patients may have low or undetectable antibody titers despite having tularemia, especially if they have received antibiotic treatment before seroconversion.

Serological testing for F. tularensis is often performed at commercial laboratories or reference laboratories such as CDC or state public health laboratories. Physicians who suspect tularemia should alert the laboratory to the possible need for special diagnostic and safety procedures due to the high infectivity and potential bioterrorism threat of F. tularensis.

Molecular methods using polymerase chain reaction (PCR) assays have been developed to detect F. tularensis directly in clinical specimens, such as swabs, tissues, blood, or respiratory samples. PCR assays can provide rapid and sensitive diagnosis of tularemia, especially when culture and serology are negative or inconclusive. PCR assays can also be used for typing and subtyping of F. tularensis strains based on single nucleotide polymorphisms (SNPs) or variable number tandem repeats (VNTRs).

There are different types of PCR assays available for F. tularensis detection, such as conventional PCR, real-time PCR, multiplex PCR, and nested PCR. Real-time PCR is preferred over conventional PCR because it allows simultaneous amplification and detection of the target DNA, reducing the risk of contamination and false positives. Real-time PCR can also quantify the bacterial load and monitor the treatment response. Multiplex PCR can detect multiple targets in a single reaction, such as different genes or species of Francisella. Nested PCR can increase the sensitivity and specificity of the assay by using two sets of primers in two consecutive reactions.

The choice of target genes for PCR assays depends on the specificity and sensitivity required for the diagnosis. Some of the commonly used target genes for F. tularensis detection are:

• 16S rRNA gene: This gene is highly conserved among bacteria and can be used to identify the genus Francisella. However, it cannot discriminate between different species or subspecies of Francisella.
• tul4 gene: This gene encodes a membrane protein that is specific to F. tularensis and can be used to differentiate it from other Francisella species. However, it cannot distinguish between different subspecies of F. tularensis.
• fopA gene: This gene encodes a heat shock protein that is specific to F. tularensis and can be used to differentiate it from other Francisella species. It can also distinguish between the two major subspecies of F. tularensis: type A and type B.
• pdpD gene: This gene encodes a protein involved in intracellular survival and virulence of F. tularensis and can be used to differentiate it from other Francisella species. It can also distinguish between type A and type B subspecies of F. tularensis.
• ISFtu2 element: This is an insertion sequence that is specific to F. tularensis and can be used to differentiate it from other Francisella species. It can also distinguish between type A and type B subspecies of F. tularensis.

PCR assays for F. tularensis detection should be performed in a biosafety level 3 laboratory with appropriate precautions and controls to prevent contamination and ensure accuracy. PCR assays should also be validated with reference strains and clinical specimens before routine use. PCR assays should be interpreted in conjunction with clinical findings and other laboratory tests for a definitive diagnosis of tularemia.

Tularemia is a serious and potentially life-threatening disease that requires prompt and appropriate treatment. The choice of antibiotics depends on the severity and type of infection, as well as the patient`s condition and allergies. The following are some of the commonly used antibiotics for treating tularemia:

• Streptomycin or gentamicin: These are the drugs of choice for severe or complicated cases of tularemia, such as pneumonic, typhoidal, or systemic forms. They are given by injection and have a high efficacy and low relapse rate. However, they may cause side effects such as kidney damage, hearing loss, or allergic reactions.
• Doxycycline or ciprofloxacin: These are oral antibiotics that can be used to treat mild or uncomplicated cases of tularemia, such as ulceroglandular, glandular, oculoglandular, or oropharyngeal forms. They are generally well tolerated and have a lower risk of resistance than other antibiotics. However, they may cause side effects such as nausea, diarrhea, photosensitivity, or tendon rupture.
• Chloramphenicol: This is an oral antibiotic that can also be used to treat mild or uncomplicated cases of tularemia. It has a broad spectrum of activity and can penetrate well into tissues and cells. However, it may cause side effects such as bone marrow suppression, aplastic anemia, or gray baby syndrome.

The duration of treatment usually ranges from 10 to 21 days depending on the antibiotic and the clinical response. Patients should complete the full course of treatment even if they feel better to prevent relapse or resistance. Patients should also monitor their symptoms and seek medical attention if they worsen or do not improve.

In addition to antibiotics, supportive care such as hydration, pain relief, fever control, and wound care may be needed to help the patient recover. Patients should also avoid contact with infected animals or insects and practice good hygiene to prevent spreading the infection to others.

Tularemia is a preventable disease that can be avoided by taking measures such as wearing protective clothing, using insect repellents, avoiding contact with sick or dead animals, cooking meat thoroughly, and wearing masks when handling potentially contaminated materials. There is also a vaccine based on a live-attenuated strain of F. tularensis that can provide some protection for people who are at high risk of exposure. However, the vaccine is not widely available and may cause adverse reactions in some people.

Tularemia is a rare but serious disease that requires early diagnosis and treatment to prevent complications and death. Antibiotics are effective in treating tularemia if given promptly and appropriately. Prevention is the best strategy to avoid getting infected with F. tularensis.

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