Direct detection of Leishmania from clinical samples.

The ability to rapidly and accurately diagnose leishmaniasis is a military priority. Testing was conducted to evaluate diagnostic sensitivity and specificity of field-expedient Leishmania genus and visceral Leishmania specific dual-fluorogenic, hydrolysis probe (TaqMan), polymerase chain reaction assays previously established for use in vector surveillance. Blood samples of patients with confirmed visceral leishmaniasis and controls without the disease from Baringo District, Kenya, were tested. Leishmania genus assay sensitivity was 100% (14/14) and specificity was 84% (16/19). Visceral Leishmania assay sensitivity was 93% (13/14) and specificity 80% (4/5). Cutaneous leishmaniasis (CL) skin scrapes of patients from Honduras were also evaluated. Leishmania genus assay sensitivity was 100% (10/10). Visceral Leishmania assay specificity was 100% (10/10) from cutaneous leishmaniasis samples; no fluorescence above background was reported. These results show promise in a rapid, sensitive, and specific method for Leishmania direct detection from clinical samples.

A Field-Expedient Method for Direct Detection of Enterotoxigenic E Coli and Shigella from Stool.

We describe a field-expedient analytic system that fills a unique and critical public health role and potentially provides a valuable aid in diagnostics. Dual-fluorigenic, hydrolysis probe (TaqMan), PCR assays for detection of causative agents of enterotoxigenic Escherichia coli (ETEC) disease and shigellosis/bacillary dysentery were prepared in a thermal-stable, hydrolytic enzyme resistant format. The assays were packaged as a kit for use with a portable, ruggedized, qRT-PCR thermocycler. The analytical limit of detection of each q RT-PCR: ETEC-STIa, ETEC-STIb, and ETEC-LT assay was 30 colony forming units (CFU) and Shigella/enteroinvasive E coli assay was 3 CFU. During field evaluation, testing was conducted using a blind-panel of 138 stored stool samples previously obtained from enterotoxigenic E coli disease (n=91) and shigellosis (n=47) patients. Sample processing and analyses were completed in 3 days. Test results of the qRT-PCR assays showed promise as aid in pathogen identification when compared to culture, digoxigenin-labeled probe (ETEC), and serotyping (Shigella) the qRT-PCR. The sensitivity of each of the 4 qRT-PCR assays was 100% and specificity was ETEC-STIa (92.4%), ETEC-STIb (92.6%), ETEC-LT (79.6%), and Shigella/enteroinvasive E coli (81.6%). Sequencing of qRT-PCR amplicon indicated that the sensitivity and specificity of each qRT-PCR assay exceeded the comparator methods. The system shows promise as a rapid method for direct detection of ETEC and Shigella from stool and is applicable for use in clinical diagnostics and biosurveillance as an extension of temporary field laboratories or as a part of fixed reference laboratory facilities.

Detection of Dengue Virus in Mosquito Extracts and Human Clinical Samples Using a Field Expedient Molecular Platform.

Dengue fever occurs in localized outbreaks and can significantly erode troop strength and mission readiness. Timely identification of dengue virus (DENV) provides for rapid and appropriate patient management decisions, such as medical evacuation and supportive therapies, as well as help to promote Force Health Protection through vector control and personal protective measures. The "Ruggedized" Advanced Pathogen Identification Device is a field-friendly PCR (Polymerase Chain Reaction) platform that can be used to facilitate early identification of DENV. We developed a dry-format PCR assay on this platform. The assay demonstrated 100% analytical specificity for detecting dengue using a cross-reactivity panel. We used a panel of 102 acute, DENV isolation positive serum samples and 25 DENV negative samples; the assay demonstrated a clinical sensitivity of 97.1% (95% C.I. 91.6-99.4%) and specificity of 96.0% (95% C.I. 79.7-99.9%) in identifying patients with dengue infection. We also used the assay to test mosquito homogenates from 28 adult female Aedes aegypti. A single DENV infected mosquito was identified using the PCR assay and confirmed using immunofluorescence as a reference method. Much of the testing was performed under austere field conditions. Together, our results demonstrate the utility of this assay for detecting DENV in vector and human samples in field environments.

Leveraging arthropod-borne disease surveillance assays for clinical diagnostic use.

Researchers at the Walter Reed Army Institute of Research have taken a joint service approach to filling an identified diagnostic capability gap by leveraging a vector surveillance assay. Specifically, the Army took a field-stable real-time polymerase chain reaction assay, developed by the Air Force, for dengue virus surveillance in arthropod vectors and collaborated with Navy researchers for utility in human diagnostics. As current Department of Defense diagnostic PCR assays employ the Joint Biological Agent Identification and Diagnostic System, the dengue assay was tested for use on this platform. The low rates of false negative and false positive dengue samples in clinical matrices demonstrate excellent utility as a human diagnostic assay. Overall, converting an arboviral vector surveillance assay to human diagnostic assay and potentially vice versa is both cost effective and labor reducing. Codevelopment with harmonization of vector surveillance and diagnostics offers monetary and resource advantages to the Department of Defense and should be considered as a path forward in times when downsizing threatens assay development and pathogen discovery.

A field-expedient method for detection of leptospirosis causative agents in rodents.

We have developed a thermal-stable, pathogenic Leptospira TaqMan PCR assay intended to support pathogen surveillance in reservoir populations. The assay is packaged specifically for use with a portable, ruggedized, real-time PCR thermocycler. Limit of detection was established at ≤ 100 fg (20 organisms). Sensitivity and specificity were 100% concordant with conventional PCR results using a broad test panel of human pathogenic and nonpathogenic Leptospira, genetic near neighbors, and clinically significant organisms. In blind testing using a panel (n=50) of pathogenic Leptospira infected and noninfected Rattus species samples, assay sensitivity results were 100% concordant with conventional PCR. Tests performed under field conditions using wild-collected rodent kidney extracts demonstrated the mobility of the system. During field evaluation, samples were processed and analyzed in 3 hours. Thermal stabilized reagents allowed transportation, storage, and analyses under ambient temperatures. The system provides a promising aid in leptospirosis control programs.

Leishmania detection in sand flies using a field-deployable real-time analytic system.

We describe here the development and evaluation of advanced vector surveillance analytic technologies for real-time leishmaniasis risk assessment. Leishmania genus and visceral leishmaniasis causative agent--specific dual fluorogenic-probe hydrolysis (TaqMan), thermally stable (freeze-dried) polymerase chain reaction assays were developed using field-durable analytic instrumentation. In laboratory testing with a panel of diverse Leishmania species from culture and infected sand flies, the sensitivity and specificity of both assays were 100% concordant with DNA sequencing. In specificity testing with Leishmania genetic near neighbors, clinically significant organisms, and human genomic DNA, no detectable fluorescence above background was observed. Field evaluation was conducted in southern Iraq using wild sand flies. In field testing, Leishmania genus assay was 100% sensitive and 96% specific with a single false-positive result. The visceral leishmaniasis genotype assay was 100% sensitive and 100% specific compared to DNA sequencing. Thermally stable polymerase chain reaction assays vastly simplified transportation and storage. Assay preparation and analysis required less than 2 hours.

Use of vector diagnostics during military deployments: recent experience in Iraq and Afghanistan.

Vector-borne diseases such as malaria, dengue, and leishmaniasis are a threat to military forces deployed outside of the United States. The availability of specific information on the vector-borne disease threat (e.g., presence or absence of a specific disease agent, temporal and geographic distribution of competent vectors, and vector infection rates) allows for effective implementation of appropriate measures to protect our deployed military forces. Vector diagnostics can provide critical, real-time information crucial to establishing effective vector prevention/control programs. In this article we provide an overview of current vector diagnostic capabilities, evaluate the use of vector diagnostics in Operation Enduring Freedom and Operation Iraqi Freedom, and discuss the concept of operations under which vector diagnostics are employed.

Impact of phlebotomine sand flies on U.S. military operations at Tallil Air Base, Iraq: 4. Detection and identification of leishmania parasites in sand flies.

Sand flies collected between April 2003 and November 2004 at Tallil Air Base, Iraq, were evaluated for the presence of Leishmania parasites using a combination of a real-time Leishmania-generic polymerase chain reaction (PCR) assay and sequencing of a 360-bp fragment of the glucose-6-phosphate-isomerase (GPI) gene. A total of 2,505 pools containing 26,574 sand flies were tested using the real-time PCR assay. Leishmania DNA was initially detected in 536 pools; however, after extensive retesting with the real-time PCR assay, a total of 456 pools were considered positive and 80 were considered indeterminate. A total of 532 samples were evaluated for Leishmania GPI by sequencing, to include 439 PCR-positive samples, 80 PCR-indeterminate samples, and 13 PCR-negative samples. Leishmania GPI was detected in 284 samples that were sequenced, to include 281 (64%) of the PCR-positive samples and 3 (4%) of the PCR-indeterminate samples. Of the 284 sequences identified as Leishmania, 261 (91.9%) were L. tarentolae, 18 (6.3%) were L. donovani-complex parasites, 3 (1.1%) were L. tropica, and 2 were similar to both L. major and L. tropica. Minimum field infection rates were 0.09% for L. donovani-complex parasites, 0.02% for L. tropica, and 0.01% for the L. major/tropica-like parasite. Subsequent sequencing of a 600-bp region of the "Hyper" gene of 12 of the L. donovani-complex parasites showed that all 12 parasites were L. infantum. These data suggest that L. infantum was the primary leishmanial threat to U.S. military personnel deployed to Tallil Air Base. The implications of these findings are discussed.

Operational vector-borne disease surveillance and control: closing the capabilities gap through research at overseas military laboratories.

Malaria, dengue fever, chikungunya virus, leishmaniasis, and a myriad of other vector-borne diseases pose significant threats to the warfighter and to the overall combat effectiveness of units. Military preventive medicine (PM) assets must accurately evaluate the vector-borne disease threat and then implement and/or advise the commander on countermeasures to reduce a particular threat. The success of these measures is contingent upon the biology of the disease vector and on the tools or methods used to conduct vector/pathogen surveillance and vector control. There is a significant gap between the tools available and those required for operational PM assets to provide real-time, effective surveillance and control. A network of US Army and US Navy overseas laboratories is focused on closing the current capabilities gap. Their mission is to develop and field test tools and methods to enhance the combatant commander's ability to identify and mitigate the threat posed by these vector-borne diseases.

Deployable, field-sustainable, reverse transcription-polymerase chain reaction assays for rapid screening and serotype identification of dengue virus in mosquitoes.

Dengue virus universal and serotype 1 to 4 fluorogenic probe hydrolysis, reverse transcription (RT)-polymerase chain reaction (PCR) assays and positive-control RNA template were freeze-dried in a thermally stable, hydrolytic enzyme-resistant format and deployed for testing in a dengue fever-endemic region of Thailand. The study site presented austere testing conditions. Field-collected Aedes aegypti mosquitoes spiked with inoculated A. aegypti mosquitoes and individual and pooled, field-collected, A. aegypti, A. albopictus, and Culex tritaeniorhynchus mosquitoes were used for RT-PCR assay evaluations. For dengue virus-inoculated A. aegypti mosquitoes and spiked samples, in vitro sensitivity and specificity results for all five assays were concordant with indirect fluorescent antibody assay results. A single pool of field-collected, female, A. aegypti mosquitoes was identified as dengue virus positive. Cross-reactivity was not observed across heterologous serotypes, mosquito vectors, or human DNA. The limit of detection was >7 to < or =70 genomic equivalents. Sample processing and analysis required <2 hours. These results show promise of field-formatted RT-PCR reagents for rapid, sensitive, specific dengue virus screening and serotype identification in mosquitoes under field-deployed conditions.

Real-time fluorescence polymerase chain reaction (PCR) identification of Streptococcus pneumoniae from pleural fluid and tissue.

A diagnosis of pneumococcal pneumonia and empyema was established by real-time fluorescence polymerase chain reaction (PCR) performed in pleural fluid and tissue samples from a child in whom cultures were negative. This case demonstrates that the use of solid-phase nucleic acid purification technology and real-time fluorescence PCR have an application in the rapid diagnosis of S. pneumoniae pneumonia.

Rapid identification of dengue virus by reverse transcription-polymerase chain reaction using field-deployable instrumentation.

Dengue virus universal and dengue serotype 1 to 4, fluorogenic probe hydrolysis (TaqMan), reverse transcription-polymerase chain reaction assays were developed for screening and serotype identification of infected mosquito vectors and human sera using a field-deployable, fluorometric thermocycler. Dengue universal and dengue 1 to 4 serotype assay in vitro sensitivity and specificity results were 100% concordant when tested with total nucleic acid extracts of multiple strains of dengue serotype 1 to 4, yellow fever, Japanese encephalitis, West Nile, and St. Louis encephalitis viruses. The in vitro sensitivity and specificity results for all five assays were concordant when tested with a blind panel of 27 dengue virus-infected mosquitoes, 21 non-dengue (yellow fever, West Nile, or St. Louis encephalitis) flavivirus-infected mosquitoes, and 11 uninfected mosquitoes and with clinical specimens consisting of a human serum panel of eight dengue viremic and 31 non-dengue-infected febrile patient serum samples. No cross-reaction occurred with vector species or human genomic DNA. Sample processing and polymerase chain reaction required <2 hours.

Identification of Aedes aegypti and its respective life stages by real-time polymerase chain reaction.

An Aedes aegypti-specific, fluorogenic probe hydrolysis (Taq-Man), polymerase chain reaction assay was developed for real-time screening using a field-deployable thermocycler. Laboratory-based testing of A. aegypti, A. aegypti (Trinidad strain), Culex pipiens, Culex quinquefasciatus, Anopheles stephensi, and Ochlerotatus taeniorhynchus individual adult mosquitoes and mixed pools (n = 10) demonstrated 100% concordance in both in vitro sensitivity (six of six samples) and specificity (10 of 10 samples). A single adult A. aegypti was identified in a pool of 100 non-A. aegypti mosquitoes. The limit of detection of A. aegypti egg pools was five individual eggs. Field testing was conducted in central Honduras. An A. aegypti and Culex spp. panel of individual and mixed pools (n = 30) of adult mosquitoes, pupae, and larvae demonstrated 100% concordance in sensitivity (22 of 22 samples) and 97% concordance in specificity (29 of 30 samples), with one false-positive result. Field testing of an A. aegypti and Culex spp. blind panel (n = 16) consisting of individual and mixed pools of adult mosquitoes, pupae, and larvae demonstrated 90% concordance in sensitivity (nine of 10 samples) and 88% concordance in specificity (14 of 16 samples).

Identification of Francisella tularensis using real-time fluorescence polymerase chain reaction.

A Francisella tularensis-specific, TaqMan probe-based, real-time fluorescence polymerase chain reaction (PCR) assay required approximately 60 minutes and consistently achieved a sensitivity of < or = 10 fg of F. tularensis genomic DNA (five genome equivalents). Specificity testing against a genomic DNA cross-reaction panel comprised of 22 bacterial organisms representing closely related species, diverse genera, and human genomic DNA resulted in no false positives of significance. The assay was conducted on a field-deployable thermocycler, the R.A.P.I.D. ("Ruggedized" Advanced Pathogen Identification Device), a microbial identification system that can provide rapid and accurate identification F. tularensis.

A real-time fluorescence polymerase chain reaction assay for the identification of Yersinia pestis using a field-deployable thermocycler.

Real-time fluorescence polymerase chain reaction is a microbial identification method that can provide rapid and accurate results using a field-deployable thermocycler, the RAPID ("ruggedized" advanced pathogen identification device). A Yersinia pestis-specific TaqMan assay required approximately 75 minutes and achieved a sensitivity of 100 fg of Y. pestis genomic DNA (20 genome equivalents). Specificity testing against a genomic DNA cross-reaction panel comprised of 22 bacterial species encountered in the respiratory tract resulted in no false positives. No cross-reaction occurred with human genomic DNA.

Real-time PCR detection of salmonella in suspect foods from a gastroenteritis outbreak in kerr county, Texas.

In June 2001, an outbreak of acute gastroenteritis among 109 attendees of a church picnic in Kerr County, Texas, was reported. A 5'-nuclease PCR assay was used to screen for Salmonella in nine food items from the buffet line. Barbeque chicken B tested positive for Salmonella, and no amplification was detected in the remaining food items. These PCR findings were consistent with culture results and were confirmed by direct nucleotide sequencing. Salmonella enterica serotype Panama was cultured from both food and patient stool samples.