A field-expedient method for detection of leptospirosis causative agents in rodents.
Article Type: Report
Subject: Rodents (Physiological aspects)
Leptospirosis (Diagnosis)
Leptospirosis (Care and treatment)
Microbiological assay (Methods)
Polymerase chain reaction (Methods)
Authors: McAvin, James C.
Kengluecha, Ampornpan
Takhampunya, Ratree
Richardson, Jason H.
Pub Date: 07/01/2012
Publication: Name: U.S. Army Medical Department Journal Publisher: U.S. Army Medical Department Center & School Audience: Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 U.S. Army Medical Department Center & School ISSN: 1524-0436
Issue: Date: July-Sept, 2012
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 301479554
Full Text: Routine biosurveillance and outbreak response systems are important public health tools which can facilitate prevention of infectious diseases through early detection and identification of pathogen emergence and mitigation of outbreaks through focused and timely response efforts. Rodent-borne zoonoses are a significant cause of morbidity and mortality worldwide and rapid recognition is critical to minimizing disease transmission at the local level and the spread of pathogens globally.

Leptospirosis is one of the most widespread zoonotic diseases in the world. (1-3) Genus Leptospira bacteria are classified into 17 species and over 200 serovars comprised of pathogenic, opportunistic, and nonpathogenic organisms. (4) Leptospira are transmitted by infected wild and domestic animals with rodents recognized as the most significant reservoir. Transmission to humans is by contact with infected urine in water, soil, and surfaces and through direct contact with infected animals.

The absence of a licensed vaccine against Leptospira and limitations in leptospirosis diagnostics and treatment drive the need for efficacious prevention and control. Surveillance of potential sources of Leptospira transmission serves a valuable role in leptospirosis risk assessment. Leptospirosis prevention is dependent on control of infected animals and awareness and elimination of contaminated environmental sources. To most efficiently make use of finite surveillance resources risk assessment activities must be focused on likely transmission foci and the associated environment. Analyses and risk assessment conducted in a timely manner is critical to effectively implementing prevention and control resources in an outbreak or potential outbreak situation.

Disease outbreaks often occur in developing regions and coincide with natural disasters or in war-torn areas. It is under these conditions that rapid disease surveillance, efficacious risk assessment, and appropriate and efficient use of control resources are most critical. However, Leptospira reference methodology by microscopic agglutination test requires up to 3 weeks for culture incubation. (5-8) As such, real-time PCR can serve as a valuable aid in surveillance and provides promise in diagnostics. Rapid and highly sensitive and specific molecular-based detection tests have been developed, however, these technologies are designed for use in a fixed laboratory infrastructure and as such are not suitable for use under austere and extreme field conditions. (7-16) In situations of an underdeveloped, damaged, or totally absent infrastructure, disease surveillance must be conducted without access to laboratory facilities, electricity, or cold-chain resources. Disruption of transportation systems and power grid are some of the obstacles that drive the need for mobile and independently operating disease surveillance systems.

We have developed a highly sensitive and specific, thermal-stable, pathogenic Leptospira species (LPS) PCR detection assay formatted for use with mobile, autonomously operating, field-proven, real-time PCR instrumentation. (17-20) We describe a field-expedient method for sensitive and specific detection of leptospirosis causative agents in rodents.

MATERIALS AND METHODS

Study Site. Field-evaluation was conducted at Buri Ram Province (14[degree] 33' 30" N, 102[degree] 55 ' 30" E), Thailand, 16 to 20 August 2010. The LPS PCR assay, nucleic acid preparation reagents, and PCR instrument were transported, stored, and sample preparation and analyses conducted under ambient temperature (25[degree]C to 33[degree]C) and humidity (80%-100%) conditions. Staff and equipment and supplies were transported by a van to the Field site. The field laboratory was set up and system operations confirmed within 2 hours. The laboratory was established in a single room of a building without environmental control using 2 tabletops (each approximately 1 [m.sup.2]). Sample preparation and analyses were conducted without provisions for spatial separation or containment.

Wild-caught Rodents. Field-evaluation of the LPS assay in vivo sensitivity was conducted using a test panel of wild-caught rodent kidney tissue extracts (n=36). Sampling was conducted for 2 nights in the rice field and forest around the rural villages in 3 study sites (Chamni (14[degree] 47' 18" N, 102[degree] 50' 30" E), Khu Mueng (15[degree] 16' 18" N, 103[degree] 0' 6" E), Lahan Sai (14[degree] 24' 42" N, 102[degree] 51' 36" E) districts of Buri Ram province). In each site, rodent habitats were identified, and small wire live-traps (14 cm wide, 14 cm high, 30 cm long) specially fitted for rodents were set. A mixture of banana and snail was used for bait. The traps were placed in the evening (between 4 PM and 5 PM) and collected early the following morning (7 AM to 8 AM). Captured rodents were euthanized by carbon dioxide overdose.21 Rodent kidneys and spleens were aseptically removed and extract prepared as described below. Rattus rattus was the most prevalent species (subspecies identification was not made). Sample extracts were transported on dry ice from the field site to the Armed Forces Research Institute of Medical Sciences laboratory for confirmation testing using a well established Leptospira gyrase subunit B conventional PCR assay. (15) All animal activities were approved by the Institutional Animal Care and Use Committee and conducted in an Association for Assessment and Accreditation of Laboratory Animal Care International (Frederick, MD) accredited facility and in compliance with the Animal Welfare Act (7 USC [section][section] 2131-2156) and other federal statutes and regulations involving animals.

Preparation of Nucleic Acid Extract. Total nucleic acid extracts were prepared from bacterial cultures, viral cultures, and rodent kidney and spleen tissues using QIAamp DNA Mini kit, QIAamp viral RNA Mini kit (QIAGEN Inc., Valencia, CA), and DNA preparation kit (Wizard Genomic DNA Purification Kit (Promega Corp, Wisconsin)) respectively. Leptospira DNA was quantified using the Qubit fluorometer (Life Technologies, Grand Island, NY) following the manufacturers' instructions. Extracts were stored at -70[degree]C.

Design of PCR Probe and Primer Oligonucleotides. The LPS TaqMan PCR assay primer and probe oligonucleotide sequences may be requested from the primary author. Oligonucleotides were designed de novo by eye targeting a 132 base pair (bp) sequence of the gene encoding Leptospira interrogans serogroup Australis major outer membrane protein (lipl32); GenBank accession number: AY609325.1. Oligonucleotide sequences of human pathogenic Leptospira species were selected considering the following guidelines:

* amplicon length=75-150 bp

* oligonucleotide length= 18-30 bases

* guanine and cytosine content=30%-80%

* primer melting temperature (Tm)=63[degree]C to 67[degree]C

* probe Tm 8[degree]C to 10[degree]C higher than primer Tm

* probe placement relative to primers (proximal)

* avoidance of runs of identical nucleotides to prevent mismatching and nucleotide complementarities to prevent secondary structure (hairpin-loop) formation and oligonucleotide dimerization.

Melting temperatures were quantiied and the absence of signiicant secondary structure formation and dimerization were conirmed with PrimerExpress software (PE Applied Biosystems, Foster City, California). Primer and probe sequence heterology with genomic sequences of closely related species through diverse genera were validated by BLAST (Basic Local Alignment Search Tool) database search. (23) Primer and probe synthesis and quality control were conducted by a commercial vendor (Idaho Technology, Inc, Salt Lake City, Utah). The TaqMan probe contained 2 fluorigenic labels, a 5' reporter dye (6-carboxyfluorescein (FAM)) and a 3' quencher dye (6-carboxytetramethylrhodamine (TAMRA)) (Roche Molecular Diagnostics, Pleasanton, California). (24,25)

Polymerase Chain Reaction. Wet reagent LPS PCR assay optimization was conducted on the "Ruggedized" Advanced Pathogen Identification Device (R.A.P.I.D.) (Idaho Technology, Inc (ITI), Salt Lake City, Utah). Primers and probe were optimized with R.A.P.I.D. wet reagents and the optimum condition was 5 mmol/L MgCl2, 400 nmol/L primers, 100 nmol/L probe. The master mix contained LPS 400 nmol/L forward and reverse primers, 100 nmol/L TaqMan probe, 200 umol/L each dNTP, 5 mmol/L Mg[Cl.sub.2], 1 x PCR buffer, 1 x stabilization buffer, and Taq Polymerase:Ab: Enzyme diluent (1:1:10.5).

The optimal LPS PCR master mix formula was used for LPS assay preparation and production conducted by an ITI proprietary process. Freeze-dried LPS PCR master mix reagents only required hydration and addition of sample template prior to analysis. Assays were prepared according to the manufacturer's (ITI) instructions. A positive template control reaction was prepared using L interrogans serovar Bangkok at a total concentration of 1 pg template. Negative template control reactions were prepared using PCR grade water. A R.A.P.I.D. standardized PCR thermal cycling protocol consists of an initial DNA denaturation at 95[degree]C for 3 minutes, and PCR for 45 cycles at 95[degree]C for 0 seconds for template denaturation (sinusoidal temperature cycling) and 60[degree]C for 20 seconds of combined annealing and primer extension.

Linearity and Limit of Detection. The linearity of the LPS freeze-dried assay was assessed in order to determine the amplification efficacy and efficiency of the PCR. These data were used to estimate limit of detection (LOD). The estimated value served as the starting point for further evaluation of LOD by replicate sample test. The correlation coefficient ([R.sup.2]) of standard DNA concentrations was used to establish linearity. The slope was used to calculate amplification efficacy and efficiency using the formulas:

Efficacy = -1+10(-1/slope) Efficiency=10(-1/slope)

The LOD was estimated using a standard curve produced by plotting critical threshold (Ct) values versus the logarithm of serial dilutions of L borgpetersenii serogroup Ballum serovar Ballum at 10 ng to 1.0 fg genomic DNA per reaction volume. The Ct values of each log DNA concentration were measured in 2 replicates. Least-squares regression analysis (performed by the R.A.P.I.D. software) plotted Ct as a function of DNA concentration. The R.A.P.I.D. software automatically calculated "best-it" of the regression and a standard curve was established, the linear relationship between APCR cycle number and ADNA concentration. The R2 value was automatically adjusted near or at unity by the R.A.P.I.D. software.

The LOD was estimated as the template concentration at the lowest Ct value above background. The estimated LOD was used to conduct replicate sample testing (n=20). Replicate sample testing was conducted by 3 operators.

Rodent extracts and Leptospira Reference Strains. A test panel of well characterized rodent kidney extracts from sample archives was prepared consisting of 30 pathogenic Leptospira infected tissue extracts and 20 noninfected extracts. Kidney tissue extracts were previously prepared and conirmed positive for pathogenic Leptospira species by Leptospira gyrase subunit B conventional PCR.8 Extracts were archived at -70[degree]C. Prior to LPS assay sensitivity testing, template quality was conirmed using Leptospira gyrase subunit B conventional PCR.

Validation testing of LPS PCR assay sensitivity and speciicity were conducted using a diverse panel of 24 reference serovars of Leptospira species consisting of 22 pathogenic and 2 nonpathogenic serovars (Table 1). Reference strains were obtained from the Department of Leptospirosis Laboratory, National Institute of Animal Health, Thailand. Cultures were grown in Ellinghausen-McCullough-Johnson-Harris medium (Difco Laboratories, Detroit, Michigan) and maintained by weekly subculture at 30[degree]C following established methodology. (22) Reference sample quality was conirmed using Leptospira gyrase subunit B conventional PCR.

Non-Leptospira Organisms: Specificity Test Panel. Speciicity testing included a panel of a well characterized nucleic acid extracts consisting of non-Leptospira genetic near neighbors, clinically signiicant organisms, and R. rattus and human DNA (Table 2). Organisms harboring RNA genomes underwent reverse transcription to produce genomic cDNA for testing. The intent of cDNA testing was to conirm exclusion of potential laboratory introduced crossover contaminates.

Data Analysis. Sample identification and specifications were entered electronically in the R.A.P.I.D. operating system run protocol. Analyses and results were automatically archived. The criterion for a positive result was a significant increase in fluorescence over background levels, ie, Ct, defined by an algorithm provided in the R.A.P.I.D. analytical software. The Ct is defined as the first PCR cycle with significant fluorescence when normalized against background fluorescence. Samples with a Ct of [greater than or equal to] 40 were considered negative, while samples with a mean Ct of <40 were considered positive by R.A.P.I.D. analyses.

RESULTS

Linearity. Linear regression analyses of the LPS freeze-dried assay using L borgpetersenii serovar Ballum concentrations ranging from 10 ng to 1 fg of total nucleic acid (2 replicates for each of eight 10-fold dilutions) demonstrated the robustness of the assay. Amplification was linear from 10 ng to 100 fg of template concentration. Slope and best fit of correlation coefficient ([R.sup.2]) and error values were performed automatically by regression analyses software included in the software package of the R.A.P.I.D. operating system. Linearity was quantified at slope=3.378, [R.sup.2]= 1.00, and error=0.0613. Leptospira interrogans serovar Bangkok positive template control (PTC) reaction prepared at 1 pg concentration reported fluorescence at an average Ct value of 31.85 corresponding with L borgpetersenii serovar Ballum 1 pg concentration average Ct value of 32.03.

Limit of Detection. The LOD was estimated at [less than or equal to] 100 fg or [less than or equal to] 20 genome equivalent (ge) based on linear regression analyses results. A total of 60 replicate R.A.P.I.D. runs at 100 fg concentration L borgpetersenii serovar Ballum total nucleic acid template established the LOD at[less than or equal to]100 fg (20 ge). Three operators running 20 replicates reactions each over a 2-day period achieved a replicate test score of 100% (60/60). Operator 1 mean ([mu]) Ct values were 35.02, SD=0.51, and percent coefficient of variation values (CV%)=1.45 where n=20, SE=0.11 and 95% confidence interval (CI)=34.80-35.24. Operator 2 mean Ct values were [mu]=35.38, SE=0.75, and CV%=2.11 where n=20, SE=0.17, and 95% CI=35.05-35.71. Operator 3 mean Ct values were u =35.61, SE=0.63, and CV%=1.76 where n=20, SE=0.14, and 95% CI=35.33-35.89.

Sensitivity and Specificity Testing. In LPS assay sensitivity and specificity testing with Leptospira reference strains, sensitivity and specificity results were 100% concordant with Leptospira gyrase B conventional PCR analyses. (Table 1). Twenty-five Leptospira reference strains consisting of 22 pathogenic serovars were positive by LPS assay analyses and 3 nonpathogenic serovars did not report fluorescence above background. All samples were tested in duplicate at a DNA concentration of 100 fg (1 x LOD). Pathogenic Leptospira sample population Ct values were [mu]=35.16, SD=0.89, and CV%=0.78 where n=22, SE=0.19, and 95% CI=34.79-35.53. Nonpathogenic serovars from the panel tested at 1 pg and 100 pg DNA concentrations (100 x and 1000 x LOD) reported no fluorescence above background. Inhibition of PCR was not observed at 100 pg DNA concentration (1000 x LOD) using 3 pathogenic Leptospira serovars: L interrogans serogroup Australis serovar Bangkok (Ct=15.98), L interrogans serogroup Australis serovar Bratislava (14.34), and L weilii serogroup icterohaemorrhagiae serovar Sarmin (Ct=33.29). A single anomalous result occurred, L weilii serovar Sarmin was detected at 1 pg (Ct=39.62) but did not report fluorescence at the 100 fg LOD level. This result was not included in the statistical analyses because L weilii serovar Sarmin sequence is 100% homologous with primer and probe sequences and as such probable experiment error is under assessment.

Archived and wild-captured rodent kidney tissue extracts tested by the LPS assay demonstrated 100% sensitivity compared to the Leptospira gyrase subunit B conventional PCR assay (Table 3). Using a test panel of 50 archived rodent tissue extracts, 30 Leptospira infected extracts were positive by LPS assay analyses and 20 noninfected extracts did not report fluorescence above background (Table 3). Sample preparation and blind testing were conducted under controlled laboratory conditions. Leptospira infected rodent extract Ct values were [micro]=29.50, SD=3.31, and CV%= 10.98 where n=30, SE=0.60, and 95% CI=28.32-30.68.

In field evaluation using a test panel of 36 wild-captured rodent tissue extracts, 4 Leptospira infected extracts were positive by LPS assay analyses and 32 noninfected extracts did not report fluorescence above background (Table 3). Sample preparation and testing were conducted under field conditions. Wild-captured rodent extract Ct values were u=34.34, SD=4.83, and CV%=23.36 where n=4, SE=2.42, and 95% CI=29.61-39.07.

Specificity Testing Using Negative Control Organisms. Specificity of the LPS assay was 100% concordant with a diverse panel of well characterized non-Leptospira organisms (Table 2). No cross-reaction occurred with human or Rattus species undiluted extracts from blood or kidney tissue, respectively. Ten common infectious disease agents and Total nucleic acid extract from 10 infectious disease agents and cDNA prepared from 7 viruses were tested at a concentration of 1000 x LOD. No fluorescence above background was observed for all non-Leptospira organisms tested.

Throughout laboratory validation testing and field evaluation, PTC reactions reported fluorescence at the expected Ct value ([approximately equal to]32) and negative template control reactions did not report fluorescence above background.

COMMENT

Our results clearly show that the LPS assay is a robust, portable, highly sensitive, and specific test for the detection of pathogenic Leptospira species In evaluation with Leptospira infected rodent kidney extracts, the assay proved to be sensitive with no false negative or false positive results. The stability of the assay was evidenced by the reproducibility of PTC results. Use of the assay with the R.A.P.I.D. provided a highly mobile, standalone, real-time PCR analytic system for field-deployed rodent surveillance. During field evaluation, the system was configured and normal operations confirmed in less than 2 hours. Sample processing and analyses were completed in less than 3 hours. The system is unique in its ability to fill an important public heath role as it provides rapid pathogenic Leptospira detection capability under austere and extreme operating conditions.

Targeting transmission risk areas and identifying preventable conditions help focus control resources. Correctly collected and interpreted data on rodent infection rate and prevalence of contaminated environment integrated with other key transmission indicators such as confirmed leptospirosis cases (where epidemiological data is available), virulence of the circulating Leptospira serovar, rodent infestations and population densities, reproduction rate, terrain and climatic conditions, provide for efficacious transmission risk assessment. These data collected in a spatially focused and expedient manner, augment the predictive power of field surveillance allowing decision makers to dedicate control resources for focused application of animal abatement measures, treatment of habitat, and increased public awareness. The value of animal and environmental surveillance is enhanced by field-expedient detection capability.

Limitations in leptospirosis diagnostics must be addressed. Achieving a definitive diagnosis across both the acute and immune phases of leptospirosis is challenging because clinical symptoms are easily confused with those of other common diseases. (5,26) The treatment of leptospirosis can be enhanced by rapid and highly sensitive diagnostics. (5) Antibiotics are most effective when started by day 5 of disease onset and as such early diagnosis would be beneficial in the treatment of leptospirosis. (4) However, while rats may shed up to 108 spirochetes per ml of urine, leptospirosis patient sample concentrations present challenges in detection limit. The presence of Leptospira organism/DNA can vary from very low to high levels during the acute (2-7 days) and immune (0-30 days) phases of the disease depending on the seriousness of the infection. (27) Patient urine sample concentration of Leptospira ranges from [10.sup.2] to [10.sup.4] spirochetes per ml and the asymptomatic urinary range is [10.sup.1] to [10.sup.3] spirochetes per ml. (28) Blood sample concentration of Leptospira ranges from [10.sup.1] to [10.sup.5] spirochetes per ml. (29) Diagnosis of leptospirosis is usually retrospective because of the length of the time required for diagnosis by microscopic agglutination test (MAT) reference methodology. (5-8) The MAT and other agglutination-based tests have been developed for more rapid and convenient diagnostics, however, these methods have limitations in specificity. (30) An approved molecular-based human diagnostic test does not currently exist that does not require confirmation testing by Leptospira isolation and culture. Molecularbased methodologies describing direct detection from clinical samples are not currently well represented in the literature. It is our intent to transition the LPS assay to human diagnostic use. We will address challenges in achieving efficacious PCR-based leptospirosis diagnostics by enhancing the high sensitivity and specificity of the LPS assay procedurally, adapting specialized protocols to concentrate patient samples, and the development of extraction and PCR internal positive controls.

Our results show that the LPS TaqMan assay is a field-expedient method for sensitive and specific detection of leptospirosis causative agents in rodents.

ACKNOWLEDGEMENTS

Thanks to Stuart Tyner and Panita Gosi, Department of Immunology, Armed Forces Research Institute of Medical Sciences (Bangkok) for providing samples and support in testing.

This work was funded by the Military Infectious Diseases Research Program, US Army Medical and Materiel Research Command, Fort Detrick, Maryland. The joint efforts of the Departments of the Army and Air

Force were conducted through the Walter Reed Army Institute of Research and the USAF 59th Medical Wing Memorandum of Agreement.

DISCLAIMER

Reference to trade name, vendor, proprietary product or specific equipment is not an endorsement, a guarantee or a warranty by the Department of the Defense or US Armed Forces, and does not imply an approval to the exclusion of other products or vendors that also may be suitable.

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James C. McAvin, MS

Ampornpan Kengluecha, MS

Ratree Takhampunya, PhD

LTC Jason H. Richardson, MS, USA

AUTHORS

Mr McAvin is a Molecular Biologist with the 59th Medical Wing, Lackland Air Force Base, Texas, temporarily assigned to the Armed Forces Research Institute of

Medical Sciences (AFRIMS), Bangkok, Thailand.

Ms Ampornpan is a Medical Research Technologist in the Diagnostic and Reemerging Diseases Section, Entomology Department, AFRIMS, Bangkok, Thailand.

Dr Ratree is Chief of the Molecular Biology Section, Entomology Department, AFRIMS, Bangkok, Thailand.

LTC Richardson is Director, Entomology Branch, Center for Infectious Disease Research, Walter Reed Army Institute of Research, Silver Spring, Maryland.
Table 1. Reference strains tested by Leptospira pathogenic
spp (LPS) PCR

Serogroup                   Serovar               Strain

Pathogenic L. interrogans

Australis             Bratislava            Jez Bratislava
Autumnalis            Autumnalis            Akiyami A
Australis             Bangkok               Bangkok-D92
Bataviae              Bataviae              Swart
Canicola              Canicola              Hond Utrecht IV
Djasiman              Djasiman              Djasiman
Hebdomadis            Hebdomadis            Hebdomadis
Icterohaemorrhagiae   Icterohaemorrhagiae   RGA
Pomona                Pomona                Pomona
Pyrogenes             Pyrogenes             Salinem

L. borgpetersenii

Ballum                Ballum                RATTUS SP 127
Javanica              Javanica              Veldrat Bataviae 46
Mini                  Mini                  Sari
Sejroe                Sejroe                M84
Tarassovi             Tarassovi             Perepelitsin

L. kirschneri

Cynopteri             Cynopteri             3522 C
Grippotyphosa         Grippotyphosa         Moskva V

L. noguchii

Louisiana             Louisiana             LSU 1945
Panama                Panama                CZ 214

L. weilii

Celledoni             Celledoni             Celledoni

L. santarosai

Shermani              Shermani              1342 K

L. inadai

Manhao                Manhao                Li 130

Nonpathogenic L. biflexa

Semaranga             Patoc                 Patoc I
Andamana              Andamana              CH 11

L. meyeri

Ranarum               Ranarum               ICF

                         LPS Results *
Serogroup             (mean Ct) ([dagger])

Pathogenic L. interrogans

Australis                    35.44
Autumnalis                   33.82
Australis                    34.50
Bataviae                     35.36
Canicola                     35.47
Djasiman                     35.31
Hebdomadis                   36.22
Icterohaemorrhagiae          35.18
Pomona                       35.12
Pyrogenes                    35.16

L. borgpetersenii

Ballum                       34.32
Javanica                     34.89
Mini                         34.09
Sejroe                       35.33
Tarassovi                    35.50

L. kirschneri

Cynopteri                    35.06
Grippotyphosa                34.25

L. noguchii

Louisiana                    34.82
Panama                       34.99

L. weilii

Celledoni                    34.77

L. santarosai

Shermani                     35.67

L. inadai

Manhao                       38.23

Nonpathogenic L. biflexa

Semaranga                   Negative
Andamana                    Negative

L. meyeri

Ranarum                     Negative

* Pathogenic Leptospira sample population (n = 22) mean Ct=35.16,
SD = 0.89, CV% = 0.78.

([dagger]) Leptospira strain mean Ct value represents duplicate
testing at the LOD concentration (100 fg).

Table 2. Results of negative control testing.

Species                              LPS Results

Human blood                           Negative
Rodent blood (Rattus rattus)          Negative
Escherichia coli                      Negative
Shigella flexneri                     Negative
Shigella sonnei                       Negative
Pseudomonas aeruginosa                Negative
Klebsiella pneumoniae                 Negative
Enterobacter aerogenes                Negative
Staphylococcus aureus                 Negative
Staphylococcus typhimurium            Negative
Streptococcus pyogenes                Negative
Bartonella doshiae                    Negative
Plasmodium falciparum                 Negative
Plasmodium vivax                      Negative
Japanese Encephalitis Virus (cDNA)    Negative
West Nile Virus (cDNA)                Negative
Tembusu Virus (cDNA)                  Negative
Dengue Virus Serotype 1 (cDNA)        Negative
Dengue Virus Serotype 2 (cDNA)        Negative
Dengue Virus Serotype 3 (cDNA)        Negative
Dengue Virus Serotype 4 (cDNA)        Negative

Table 3. Results of rodent kidney tissue testing

Samples              No. Samples   No. True Pos   No. True Neg

Archived rodent          50             30             20
extract *

Wild-captured            36             4              32
rodents ([dagger])

Samples               LPS PCR Sensitivity      gyrB PCR Sensitivity

Archived rodent      100% (30/30 + 0)(100%)   100% (30/30 + 0)(100%)
extract *

Wild-captured         100% (4/4 + 0)(100%)     100% (4/4 + 0)(100%)
rodents ([dagger])

* Archived rodent extract (n = 30) mean Ct=29.5, SD = 3.31, CV% = 10.98.

([dagger]) Wild-captured rodent extract (n = 4) mean Ct=34.34,
SD = 4.83, CV% = 23.36.
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