Table of Contents:

• Taxonomy Information
• Lifecycle Information
• Genome Information
• Biosafety Information
• Culturing Information
• Epidemiology Information
• Diagnostic Tests Information
• Infected Hosts Information
• Phinet: Pathogen-Host Interaction Network
• Lab Animal Pathobiology & Management
• References
• Data Provenance and Curators

1. Species:
   1. Francisella tularensis (Website 9):
      1. GenBank Taxonomy No.: 263
      2. Description: Francisella tularensis is a small, aerobic, pleomorphic, gram-negative, intracellular and extracellular coccobacillus. It is the causative agent of the disease tularemia(Parola and Raoult, 2001).
      3. Variant(s):
         • Francisella tularensis biogroup tularensis. (Website 10):
           • Common Name: biovar type A.
           • GenBank Taxonomy No.: 119856
           • Parents: Francisella tularensis
           • Description: Francisella tularensis biogroup tularensis (also known as nearctica, type A),(Website 10). is predominantly found in mammalian hosts and arthropod vectors of North America(Parola and Raoult, 2001). It is the most virulent of the F. tularensis subspecies and accounts for approximately 90% of tularemia cases in North America,(Choi, 2002). where rabbits (Sylvilagus) are important reservoirs(Parola and Raoult, 2001). The virulent Schu 4 strain of F. tularensis is type A(Dennis et al., 2001).
         • Francisella tularensis biogroup holarctica. (Website 11):
           • Common Name: biovar type B.
           • GenBank Taxonomy No.: 119857
           • Parents: Francisella tularensis
           • Description: Three biovars of F. tularensis biogroup holarctica have been suggested; biovar I (erythromycin sensitive), biovar II (erythromycin resistant), and biovar japonica(Ellis et al., 2002). Francisella tularensis biogroup holarctica (also known as palaearctica, type B) is more widely distributed in nature and is found in Europe, Asia, and to a minor extent in North America. This subspecies is linked to waterborne disease of rodents and hares, and it is considered to be less pathogenic for mammals than F. tularensis subsp. tularensis(de la Puente-Redondo et al., 2000). A live vaccine strain of F. tularensis (F. tularensis LVS) was derived from a virulent type B strain(Website 17). Vaccine strains 15 and 155 were transferred to the United States from Moscow in 1956. Cultures grown from reconstituted ampules showed that both strain 15 and strain 155 segregated into two colony types, designated blue colony variant or grey colony variant depending on their appearance when viewed microscopically under oblique light. The blue colony variant was shown to be more virulent and immunogenic in small animals than the grey colony variant. Mice immunized with the blue colony variant were protected when challenged with the fully virulent Schu 4 strain. Lyophilized preparations of the blue colony variant were prepared and a live vaccine strain (LVS) was derived after five passages through mice(Ellis et al., 2002).
         • Francisella tularensis holarctica japonica :
           • Parents: Francisella tularensis biogroup holarctica.
           • Description: Francisella tularensis biogroup palaearctica japonica belongs to F. tularensis biogroup holarctica (palaearctica) and is found primarily in Japan(Choi, 2002).
         • Francisella tularensis biogroup mediaasiatica. (Website 12):
           • GenBank Taxonomy No.: 135248
           • Parents: Francisella tularensis
           • Description: Francisella tularensis subsp. mediaasiatica has been found only in central Asia in a part of the former Soviet Union(de la Puente-Redondo et al., 2000).
         • Francisella tularensis biogroup novicida. (Website 13):
           • GenBank Taxonomy No.: 264
           • Parents: Francisella tularensis
           • Description: F. tularensis biogroup novicida was isolated from a Utah water sample in 1950(Farlow et al., 2001). There are only a few known isolates of this biogroup(Johansson et al., 2001). and it is of low virulence(Parola and Raoult, 2001).

1. Francisella tularensis Information
   1. Stage Information:
      1. Vegetative cell:
         • Size: Francisella tularensis is 0.2-0.5 x 0.7-1.0 microns.
         • Shape: Francisella tularensis is a pleomorphic coccobacilli.
         • Picture(s):
           • Gram Stain Smears of Bacillus anthracis, Yersinia pestis, and Francisella tularensis (Dennis et al., 2001)
             
             
             
             Description: Gram Stain Smears of the Agents of Anthrax (Bacillus anthracis), Plague (Yersinia pestis), and Tularemia (Francisella tularensis), Demonstrating Comparative Morphology, Size, and Staining Characteristics. A, B. anthracis is a large (0.5-1.2 x 2.5-10.0 micrometer), chain-forming, gram-positive rod that sporulates under certain conditions (Gram stain of organism from culture; original magnification x250); B, Y. pestis is a gram-negative, plump, nonspore-forming, bipolar-staining bacillus that is approximately 0.5-0.8 x 1-3 micrometer (Gram stain of smear from infected tissue; original magnification x250); C, F. tularensis is a small (0.2 x 0.2-0.7 micrometer), pleomorphic, poorly staining, gram-negative coccobacillus (Gram stain of organism from culture; original magnification x500) (inset, direct immunofluorescence of smear of F. tularensis; original magnification x400. Sources: A and B, Sherif Zaki, Centers for Disease Control and Prevention; C, Armed Forces Institute of Pathology. Used with permission(Dennis et al., 2001).

1. Genome of Francisella tularensis
   1. Description: The genome of the Schu 4 strain of Francisella tularensis (a virulent type A strain) is being sequenced through a project funded by the United States Army Medical Research and Material Command, UK Ministry of Defence, Swedish Ministry of Defence, and the US Defence Advanced Research Projects Agency. Information regarding that project can be found in Website 16(Website 16). Preliminary results from this project suggest a total genome size of less than 2 Mbp, making this one of the smaller bacterial genomes(Ellis et al., 2002, Prior et al., 2001). The genome of F. tularensis LVS (a type B strain) is being sequenced through the Biology and Biotechnology Research Program at Lawrence Livermore National Laboratory(Website 17). Information regarding that project can be found in Website 17.
2. Genome of Francisella tularensis biogroup novicida.
   1. Plasmid pFNL10(Website 15)
      1. GenBank Accession Number: AF121418
      2. Size: 3990 bp(Pomerantsev et al., 2001, Website 15).
      3. Gene Count: Plasmid pFNL10 has 6 ORFs(Pomerantsev et al., 2001).
      4. Description: Plasmid pFNL10 is found in Francisella tularensis biogroup novicida. F. tularensis biogroup novicida is the only member of the genus that carries a native plasmid(Pomerantsev et al., 2001).

1. Biosafety information for Francisella tularensis
   1. Level: Biosafety level 2 practices and containment should be used for routine diagnostic activities with clinical materials. Biosafety level 3 practices, containment, and facilities should be used for all manipulations of cultures and for experimental studies involving infectious materials with a potential for aerosol and droplet production (centrifuging, grinding, vigorous shaking, growing cultures in volume, and animal studies)(Dennis et al., 2001).
   2. Precautions: Laboratory coat, impervious gloves, and gown (with tight wrists and a tie in the back) should be worn when working with Francisella tularensis. Face masks should be worn when working with infectious material in biosafety cabinet. Bodies of patients who die of tularemia should be handled using standard precautions. Autopsy procedures likely to cause aerosols, such as bone sawing, should be avoided. Clothing or linens contaminated with body fluids of patients infected with F. tularensis should be disinfected per standard precaution protocols such as steam sterilization. Persons handling animals, especially rabbits, should wear impervious gloves. F. tularensis is susceptible to 1% sodium hypochlorite (10% bleach) and standard levels of chlorine in municipal water sources should be protective(Dennis et al., 2001, Website 4).
   3. Disposal: Decontamination before disposal, incineration of animal carcasses, steam sterilization of other laboratory waste(Website 4).

1. Cysteine heart agar with sheep blood :
   1. Description: Growth on plates. For culturing on plates of Francisella tularensis, use established inoculation and plating procedures. For tissues, use established laboratory procedure to inoculate media (grind or use a sterile wood stick). Tape plates shut in 2 places to prevent inadvertent opening (alternatives to taping are also acceptable) (Website 1). F. tularensis can be grown from such things as pharyngeal washings, sputum specimens, blood, and fasting gastric aspirates(Dennis et al., 2001). Although growth may be visible as early as 24-48 hours after inoculation, growth may be delayed and cultures should be held for at least 10 days before discarding. Under ideal conditions, bacterial colonies on cysteine-enriched agar are typically 1 mm in diameter after 24-48 hours of incubation, white to gray to bluish-gray, opaque, flat with an entire edge, smooth, and may have a shiny surface. By 96 hours, the colonies are typically 3-5 mm in diameter. On cysteine heart agar, F. tularensis colonies are characteristically opalescent and do not discolor the medium(Dennis et al., 2001). Figure 4 of Dennis et al. (2001), shows F. tularensis colonies with characteristic opalescence on cysteine heart agar with sheep blood (cultured at 37 degrees celcius for 72 hours)(Dennis et al., 2001). Figure A2A, (Website 1), shows F. tularensis Schu 4 strain growing on 6% sheep blood agar at 72 hours. Figure A2b, (Website 1), shows F. tularensis Schu 4 strain on chocolate agar after 72 hours(Website 1).
   2. Medium: F. tularensis grows best in cysteine-enriched broth and thioglycollate broth. It grows best on cysteine heart blood agar, sheep blood agar, and on cysteine-supplemented agar such as buffered charcoal-yeast agar, Thayer-Martin agar, and chocolate agar. Selective agar may be useful when culturing materials from nonsterile sites, such as sputum(Dennis et al., 2001, Website 1).
   3. Optimal Temperature: 35-37 degrees celsius(Dennis et al., 2001, Website 1).
   4. Picture(s):
      • Francisella tularensis Growth at 72 Hours After Inoculation (Dennis et al., 2001)
        
        
        
        Description: These Francisella tularensis colonies show characteristic opalescence on cysteine heart agar with sheep blood (cultured at 37 degrees celsius for 72 hours). Source: Centers for Disease Control and Prevention. Used with permission(Dennis et al., 2001).
2. Axenic culture in TSB-C :
   1. Description: Anthony et al., showed that Francisella tularensis grew in a cell-free medium of tryptic soy broth supplemented with 0.1% cysteine(Anthony et al., 1991). However, growth is slow and requires a large inoculum to obtain visible growth within 24 hours(Ellis et al., 2002).
   2. Medium: Francisella tularensis can be cultured in tryptic soy broth supplemented with 0.1% cysteine (TSB-C)(Anthony et al., 1991).
3. Axenic culture in Mueller-Hinton Broth :
   1. Description: Francisella tularensis can be grown axenically in modified Mueller-Hinton broth(Fortier et al., 1995). The addition of 0.025% ferric pyrophosphate appeared to enhance growth in this medium(Ellis et al., 2002).
   2. Medium: Francisella tularensis can be grown in modified Mueller-Hinton broth(Fortier et al., 1995).
4. Axenic culture in thioglycollate broth :
   1. Description: Francisella tularensis can be grown axenically in thioglycollate broth. In static thioglycollate broth, growth is first seen as a dense band near the top which diffuses throughout as growth progresses(Ellis et al., 2002).
   2. Medium: Thioglycollate broth(Ellis et al., 2002).
5. Intracellular growth on murine macrophages :
   1. Description: Murine macrophages support exponential intracellular growth of Francisella tularensis LVS where it remains in a vacuolar compartment throughout its growth cycle(Fortier et al., 1995). Macrophages (from a peritoneal cell preparation) are adjusted to 1 million cells per ml and incubated as cell pellets in polypropylene tubes in 5% CO2 at 37 degrees celsius before exposure to the bacteria. The macrophages are subsequently exposed to F. tularensis LVS at a multiplicity of infection of 1 for 2 hours at 37 degrees celsius in 5% CO2 in a humid environment(Fortier et al., 1995).
   2. Medium: Murine macrophages and associated F. tularensis bacteria were grown in Dulbecco Modified Eagle Medium (DMEM) with 10% heat-inactivated fetal bovine serum(Fortier et al., 1995).
   3. Optimal Temperature: 37 degrees celsius(Fortier et al., 1995).
   4. Optimal Humidity: Humid environment(Fortier et al., 1995).
   5. Doubling Time: 4 to 6 hours(Fortier et al., 1995).

1. Outbreak Locations:
   1. Tularemia is not a disease which is notifiable to the World Health Organization, and the worldwide incidence is not known(Ellis et al., 2002). It is primarily a disease of the northern hemisphere and is most common between 30 degrees and 71 degrees north latitude. Tularemia has been remarkably absent from the United Kingdom, Africa, South America, and Australia(Cross et al., 2000). F. tularensis is found throughout much of North America and Eurasia. In the United States, it has been found in every state excluding Hawaii, with most cases occurring in south-central and western states. In Eurasia, the greatest numbers are reported from northern and central Europe, especially Scandinavian countries and those of the former Soviet Union. F. tularensis is found mostly in rural areas, although urban and suburban exposures occasionally do occur(Dennis et al., 2001).
2. Transmission Information:
   1. From: Invertebrates, Wild vertebrates, Domestic mammals , To: Homo sapiens
      Mechanism: Humans become infected with Francisella tularensis through inoculation of the skin, conjunctival sac or oropharyngeal mucosa with blood or tissue while handling infected animals, or through contact with fluids from infected flies, ticks or other animals(Dennis et al., 2001, Website 4). Although the organism is reported to penetrate intact skin, most investigators believe that penetration occurs through sites of inapparent skin disruption(Cross et al., 2000). The bite of vectors including ticks, deerfly, mosquito, gnats, and bedbugs can also transmit the bacterium to humans(Dennis et al., 2001, Website 4, Choi, 2002). It is rarely spread through bites from animals(Website 4). Transmission from human to human has not been documented(Dennis et al., 2001, Website 4, Choi, 2002, Cross et al., 2000).
   2. From: Food , To: Homo sapiens
      Mechanism: Ingestion of food and drinking water contaminated with Francisella tularensis can be infective for humans(Dennis et al., 2001, Website 4, Choi, 2002).
   3. From: Water , To: Homo sapiens
      Mechanism: Ingestion of food and drinking water contaminated with Francisella tularensis can be infective for humans(Dennis et al., 2001, Website 4, Choi, 2002).
   4. From: Air , To: Homo sapiens
      Mechanism: Humans can become infected with Francisella tularensis by inhaling contaminated aerosols(Dennis et al., 2001, Website 4, Choi, 2002).
3. Environmental Reservoir:
   1. Mud-Infected_Animal_Carcass-Meat-Straw-Water:
      1. Description: Francisella tularensis can survive in: 7 degrees celsius mud for 14 weeks;(Website 4, Feldman, 2003). infected animal carcasses and organs for up to 133 days; non-frozen rabbit meat for 31 days; rabbit meat stored at -15 degrees celsius for at least 3 years; straw for 192 days; water for up to 90 days(Website 4).
   2. Wild vertebrates:
      1. Description: Rabbits, hares, and rodents (including water rats, squirrels, mice, and voles) are considered the be the most important reservoirs for Francisella tularensis(Dennis et al., 2001, Website 3, Parola and Raoult, 2001). Birds may also be infected(Website 4). These animals acquire infection through bites by ticks, flies, and mosquitoes, and by contact with contaminated environments(Dennis et al., 2001).
   3. Domestic mammals:
      1. Description: Domestic mammals including sheep, dogs, and cats can be infected with Francisella tularensis(Website 2, Feldman, 2003).
   4. Invertebrates:
      1. Description: Invertebrates such as ticks are common vectors of Francisella tularensis. In the United States, the important tick vectors are: Amblyoma americanum - the Lone Star tick, Dermacentor andersoni - the wood tick, and Dermacentor variabilis - the dog tick. Other vectors include deerflies, horseflies, and mosquitoes(Website 4, Feldman, 2003, Choi, 2002). Generally, biting flies are the most common vectors in Utah, Nevada, and California, while ticks are the most important vectors east of the Rocky Mountains(Ellis et al., 2002). In central Europe, the ticks Dermacentor reticulatus and Ixodes ricinus are important vectors. In the former Soviet Union, the bacterium is transmitted by both mosquitoes (Aedes, Culex, and Anopheles species) and Ixodes species of tick(Ellis et al., 2002).
   5. Air:
      1. Description: Contaminated aerosols are a source of human infection with Francisella tularensis(Dennis et al., 2001, Choi, 2002).
   6. Food:
      1. Description: Contaminated food (including fresh and frozen rabbit meat) is a source of Francisella tularensis infection(Website 4).
   7. Water:
      1. Description: Contaminated water is a source of Francisella tularensis infection(Choi, 2002). It is postulated that F. tularensis may survive in water in association with amebae(Berdal et al., 1996, Berdal et al., 2000).
   8. Soil:
      1. Description: Francisella tularensis can contaminate soil and vegetation(Dennis et al., 2001).
   9. Vegetation:
      1. Description: Francisella tularensis can contaminate soil and vegetation(Dennis et al., 2001).
4. Intentional Releases:
   1. Intentional Release Information:
      1. Description: Francisella tularensis has long been considered a potential biological weapon. It was one of a number of agents studied at Japanese germ warfare research units operating in Manchuria between 1932 and 1945; it was also examined for military purposes in the West. A former Soviet Union biological weapons scientist, Ken Alibek, has suggested that tularemia outbreaks affecting tens of thousands of Soviet and German soldiers on the eastern European front during World War II may have been the result of intentional use. Following the war, there were continuing military studies of tularemia. In the 1950s and 1960s, the US military developed weapons that would disseminate F. tularensis aerosols; concurrently, it conducted research to better understand the pathophysiology of tularemia and to develop vaccines and antibiotic prophylaxis and treatment regimens. In some studies, volunteers were infected with F. tularensis by direct aerosol delivery systems and by exposures in an aerosol chamber. A live attenuated vaccine was developed that partially protected against respiratory and subcutaneous challenges with the virulent Schu 4 strain of F. tularensis, and various regimens of streptomycin, tetracyclines, and chloramphenicol were found to be effective in prophylaxis and treatment. By the late 1960s, F. tularensis was one of several biological weapons stockpiled by the US military. According to Alibek, a large parallel effort by the Soviet Union continued into the early 1990s and resulted in weapons production of F. tularensis strains engineered to be resistant to antibiotics and vaccines(Dennis et al., 2001). In 1969, a World Health Organization expert committee estimated that an aerosol dispersal of 50 kg of virulent F. tularensis over a metropolitan area with 5 million inhabitants would result in 250,000 incapacitating casualties, including 19,000 deaths. Illness would be expected to persist for several weeks and disease relapses to occur during the ensuing weeks or months. It was assumed that vaccinated individuals would be only partially protected against an aerosol exposure. Referring to this model, the Centers for Disease Control and Prevention (CDC) recently examined the expected economic impact of bioterrorist attacks and estimated the total base costs to society of an F. tularensis aerosol attack to be $5.4 billion for every 100,000 persons exposed(Dennis et al., 2001).
      2. Emergency Contact: After seeking prompt medical attention, local and state health departments should be immediately notified if exposure to Francisella tularensis is suspected. If criminal activity is suspected, these agencies will notify the CDC and the FBI(Website 3).
      3. Delivery Mechanism: In biological warfare it is anticipated that the bacteria would be delivered as a cloud to the target population, making entry through the airways into the lungs the most common route, although ingestion and entry through skin wounds is also possible(Website 7).
      4. Containment: Disinfection of contaminated articles may be accomplished using a 0.05% hypochlorite solution(Website 7).

1. Organism Detection Test:
   1. Gram stain (Website 1):
      1. Time to Perform: 2-to-7-days
      2. Description: Gram staining of Francisella tularensis organisms reveals the presence of tiny (0.2-0.5 microns X 0.7-1.0. microns) pleomorphic, poorly staining, mainly single cell, Gram-negative coccobacilli. The Gram stain interpretation may be difficult because the cells are minute and faintly staining(Website 1).
   2. Growth on chocolate agar (Ellis et al., 2002):
      1. Time to Perform: 2-to-7-days
      2. Description: The Centers for Disease Control and Prevention guidelines recommend the use of cysteine heart agar supplemented with 9% heated sheep red blood cells (CHAB) if growth on general microbiological agars indicates the presence of Francisella tularensis(Ellis et al., 2002). A heavy inoculum will yield visible growth in 18 hours, but the appearance of individual colonies may require 2 to 4 days of incubation. F. tularensis grows slowly at 37 degrees celsius and poorly at 28 degrees celsius, and this can be exploited to distinguish F. tularensis from Yersinia pestis, Francisella philomiragia, and F. tularensis biogroup (subspecies) novicida, all of which grow well at 28 degrees celsius(Ellis et al., 2002). On CHAB, F. tularensis colonies are 2 to 4 mm in size, greenish-white, round, smooth, and slightly mucoid. On media containing whole blood there is usually a small zone of alpha-hemolysis surrounding the colonies(Ellis et al., 2002).
2. Immunoassay Test:
   1. cELISA - capture enzyme-linked immunosorbent assay (Grunow et al., 2000):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: A capture enzyme-linked immunosorbent assay (cELISA) using two monoclonal antibodies (Ft-11 and Ft-27) that are specific for lipopolysaccharide (LPS) of Francisella tularensis subspecies holarctica and Francisella tularensis subspecies tularensis was reported in 2000. The cELISA showed no cross-reactivity with Francisella tularensis subspecies novicida, Francisella philomiragia, Brucella spp., Yersinia spp., Escherichia coli, and Burkholderia spp(Grunow et al., 2000). An important step in the test was the extraction of LPS from the bacteria. The extraction resulted in a 10-fold increase in sensitivity, inactivation of infectious bacteria in subsequent steps, and the lysis of eukaryotic cells (which released intracellular bacteria). The detection limit of the cELISA using LPS-extracted samples was 10 e3 bacteria / milliliter in phosphate buffered saline and 10 e4 bacteria / milliliter in human serum. The assay was also reported to have a similar level of sensitivity in spiked human urine, sputum, and stool(Grunow et al., 2000).
   2. Enzyme-linked immunosorbent assay (Website 5, Ellis et al., 2002, Syrjala et al., 1986, Website 19):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Because of the difficulty and danger of culturing Francisella tularensis, most cases of tularemia are currently diagnosed using the clinical presentation and / or serological evidence(Ellis et al., 2002). A measurable antibody response to Francisella tularensis usually occurs in 50-70% of cases about 2 weeks after the onset of disease(Website 5, Ellis et al., 2002). Titers reach a maximum in 4-8 weeks(Website 5). Enzyme-linked immunosorbent assay (ELISA) has been used for detection of serum antibodies by using a sonicate of the LVS strain and F. tularensis nonviable cells in phenolized saline are available commercially from BD (Becton, Dickinson, and Company; Franklin Lakes, New Jersey)(Website 19). The ELISA detected either IgM, IgA, or IgG in the sera of patients during the second week of tularemia. The antibodies generally persisted and were detectable by ELISA at high titer for six months(Syrjala et al., 1986).
   3. Immunochromatographic Assay (Dipstick) (Grunow et al., 2000):
      1. Time to Perform: minutes-to-1-hour
      2. Description: An immunochromatographic assay using an affinity purified polyclonal antibody and a monoclonal antibody to lipopolysaccharide (LPS) of Francisella tularensis live vaccine strain (LVS, biogroup holarctica) was reported in 2000. The test could detect 10 e6 and 10 e6-e7 bacteria / milliliter of phosphate buffered saline (PBS) or human serum, respectively. The detection limit was increased ten-fold to 10 e5 bacteria / milliliter of PBS and 10 e5-e6 bacteria / milliter of human serum if an LPS extraction method of sample preparation was used. This makes the immunochromatographic test approximately 100 times less sensitive than the capture ELISA that employs monoclonal antibodies Ft-11 and Ft-27. The test was completed in approximately 15 minutes after extraction of LPS, with high bacterial loads showing a positive signal in less than 1 minute. The authors suggest that this assay is a fast and simple method for field diagnosis of tularemia, but the diagnosis should be confirmed by more sensitive techniques such as capture ELISA or PCR(Grunow et al., 2000).
   4. Agglutination (Ellis et al., 2002):
      1. Time to Perform: minutes-to-1-hour
      2. Description: Because of the difficulty and danger of culturing Francisella tularensis, most cases of tularemia are currently diagnosed using the clinical presentation and / or serological evidence(Ellis et al., 2002). A measurable antibody response to Francisella tularensis usually occurs in 50-70% of cases about 2 weeks after the onset of disease(Website 5, Ellis et al., 2002). Titers reach a maximum in 4-8 weeks(Website 5). Agglutination is an accepted method for detection of serum antibodies. F. tularensis nonviable cells in phenolized saline are available commercially from BD (Becton, Dickinson, and Company; Franklin Lakes, New Jersey)(Website 19). A fourfold increase in titer during illness or a single titer of 1:160 or greater is considered diagnostic for infection(Ellis et al., 2002).
3. Nucleic Acid Detection Test: