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    Aligned sequences from the GPI gene of representative Leishmania strains. Numbers correspond to the nucleotide sequence starting from the beginning of the L. major GPI gene (systematic name, LmjF12.0530).

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RAPID IDENTIFICATION OF LEISHMANIA COMPLEXES BY A REAL-TIME PCR ASSAY

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  • 1 Infectious Disease Service, Walter Reed Army Medical Center, Washington, DC; Department of Entomology, Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Viral Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland; Infectious Disease Division, Uniformed Services University of the Health Sciences, Bethesda, Maryland; Department of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland

A real-time PCR assay for the detection of four Leishmania complexes (L. Viannia, L. mexicana, L. donovani/infantum, and L. major) was developed and evaluated. The assay was developed to detect the glucosephosphate isomerase gene and capitalizes on DNA sequence variability within that gene for Leishmania complex identification. Primer/probe sets were created and tested against a panel of 21 known negative controls and on DNA extracted from cultured promastigotes or from tissue biopsies from patients with cutaneous leishmaniasis. The assay was highly specific, as no amplification products were detected in the negative control samples while simultaneously retaining a high degree of complex-specific diagnostic accuracy for cultured organisms and patient clinical samples. Real-time PCR offers rapid (within hours) identification of Leishmania to the complex level and provides a useful molecular tool to assist both epidemiologists and clinicians.

INTRODUCTION

Leishmaniasis is a protozoan infection that is endemic throughout the tropical and subtropical regions.1 The World Health Organization estimates that 1.5 million to 2 million new cases occur each year.2 Twenty-two species of Leishmania have been reported to cause human infections.3 In the Old World, cutaneous leishmaniasis is predominantly due to L. major, L. tropica, L. aethiopica, and L. infantum, whereas L. donovani and L. infantum are responsible for visceral disease. In the New World, members of the L. Viannia subgenus (including L. V. braziliensis and L. V. panamensis) and the L. Leishmania mexicana complex cause the majority of cutaneous disease, whereas L. L. chagasi is associated with visceral disease.

Identification of the infecting parasite to the complex or species level is important for prognostic, epidemiologic, and therapeutic reasons.4 The classical method used for this task is isoenzyme analysis, a procedure that is slow, laborious, and requires the growth of cultured promastigote parasites.5 This method of identification is impractical for real-time patient treatment decisions, as the turn-around period from biopsy to successful growth of culture to isoenzyme identification can take weeks. Newer methods of identification that could potentially classify Leishmania directly from patient specimens (abrogating the requirement for culture) include monoclonal antibodies and numerous DNA-based assays including multiplex polymerase chain reaction (PCR), PCR plus sequencing, and restriction fragment length polymorphism (RFLP) analysis.611 Most recently, real-time PCR, a platform that can process a sample in less than an hour, has been reported to rapidly differentiate single nucleotide mutations within a target DNA sequence.12,13 We have previously developed and field tested a real-time PCR assay to diagnose cutaneous and visceral leishmaniasis to the genus level and have used this assay to assess the absence or presence of infection in both vertebrate (human hosts) and invertebrate sand fly vectors.14,15 We now describe the development and testing of a second-generation real-time PCR assay to classify samples to the level of Leishmania complexes and species. This assay has the potential to provide the treating clinician with a rapid identification of the infecting Leishmania species, which subsequently offers the potential for targeted treatment strategies.

MATERIALS AND METHODS

Parasites.

Cultured Leishmania reference strains were obtained from the strain collections of the Walter Reed Army Institute of Research (Silver Spring, MD). The identification of all strains had been accomplished by isoenzyme analysis. The strains were cultured at 24°C in Schneider’s Drosophila Medium (Gibco, Grand Island, NY) containing 20% fetal calf serum.16 Parasites were harvested at a density of 2 × 107 parasites/mL, pelleted at 500 × g for 8 minutes at 4°C, washed three times in phosphate-buffered balanced salt solution (PBSS) pH 7.4, and then resuspended in 500 μL PBSS.

Clinical samples.

Skin biopsy samples were obtained from patients with suspected cutaneous leishmaniasis who presented to the Walter Reed Army Medical Center (Washington, DC). Samples were placed in Schneider’s Drosophila Medium for culture and 70–100% ethanol for PCR processing. The maximum time from sampling to PCR processing was 7 days. All cultures were identified by isoenzyme analysis of cultured promastigotes.

Negative control samples.

Negative control DNA was extracted from paraffin-embedded clinical samples representing a wide range of dermatological and tropical diseases. In addition, DNA was extracted from cultures of Trypanosoma rangelli (ATCC 30032) and Crithidia fasiculata (ATCC 11745), two organisms closely related to Leishmania.

DNA extraction.

DNA purification was performed by column chromatography (QIAamp Blood and Blood Products Kit for the cultured promastigotes and QIAamp Tissue Kit for clinical samples; Qiagen, Chatsworth, CA) following the manufacturer’s instructions. For the paraffin-embedded clinical samples, the methods described in the QIAamp Tissue Kit were followed.

Selection of oligonucleotides.

Leishmania complexes produce distinct migration patterns with multilocus enzyme electrophoresis, and glucosephosphate isomerase (GPI) is one of the principal enzymes used to help distinguish various strains.17,18 We hypothesized there might be unique DNA sequences for GPI for each of the major Leishmania complexes but identified only one publication reporting a Leishmania GPI DNA sequence.19 To determine if GPI sequence variability exists among Leishmania, a library of Leishmania GPI DNA sequences was required. Trypanosoma brucei is an organism closely related to Leishmania, and a published sequence of the T. brucei GPI was identified in GenBank. Leishmania mexicana GPI sequence (X78206) was subsequently aligned with the Trypanosoma brucei GPI sequence (X15540) using the CLUSTAL program in the Lasergene software package (DNASTAR, Inc, Madison, WI). Primers forward GPI2 (5′-GAGGCACTGAAGCCGTT-3′) and reverse GPI3 (5′-ATGAAATCACACGGAATGA-3′), complementary to homologous areas and flanking sequences of discordance, were synthesized and used in a hot-start PCR assay.

For this PCR, each 25-μL reaction consisted of 3 μL DNA template, 12.5 pmol primer, 21 μL water, and a PCR bead (PCR Ready to Go Beads, Amersham Pharmacia Biotech, Piscataway, NJ) each of which contains each deoxynucleoside triphosphate, buffer, Taq polymerase and 1.5 mM MgCl2. PCR amplification was performed with a DNA thermocycler (PTC-100, MJ Research, Inc., Watertown, MA) with an initial denaturation step (4 minutes at 94°C), then 40 cycles of 94°C for 30 seconds, 45°C for 30 seconds (annealing), and 72°C for 2 minutes (extension). Ten microliters of the amplified product was run in 2% agarose gel stained with ethidium bromide and visualized under UV light. The remainder of the reaction was purified by column chromatography (QIAquick PCR purification kit, Qiagen, Chatsworth, CA) and sent for nucleotide sequencing using the dideoxy chain-termination method as stipulated by the dichloRhodamine Terminator cycle DNA Sequencing Kit (PE Applied Biosystems, Foster City, CA). Samples were analyzed on a 377 Automated DNA sequencer (PE Applied Biosystems). Further computer analysis was performed using DNA Sequencher 3.1.1 (Gene Codes Corporation, Ann Arbor, MI).

The PCR assay described above generated a 700-bp amplicon from 20 Leishmania isolates representing five complexes: 1) L. V. panamensis (WR 2306, ATCC 50158, WR ER, Guatemala [G#] 011), L. V. braziliensis (ATCC 50135, WR 676), L. V. guyanensis (WR 675); 2) L. L. donovani (WR378), L. L. infantum (ATCC 50134); 3) L. L. mexicana (G#003, G#006, G#007, G#024); 4) L. L. major (WR 1075, WR CSU, WR 2312, WR 1088); 5) L. L. tropica (WR 2179, WR 1091, WR 1063).

The sequences for these amplicons have been deposited in GenBank with the following accession numbers: 1) L. V. panamensis (WR 2306, accession no. AY974210), (ATCC 50158, accession no. AY974211), (WR ER, accession no. AY974212), (Guatemala [G#] 011, accession no. AY974213), L.V. braziliensis (ATCC 50135, accession no. AY974214), (WR 676, accession no. AY974215), L. V. guyanensis (WR 675, accession no. AY974199); 2) L. L. donovani (WR378, accession no. AY974201), L. L. infantum (ATCC 50134, accession no. AY974200); 3) L. L. mexicana (G#003, accession no. AY974208), (G#006, accession no. AY974206), (G#007, accession no. AY974207), (G#024, accession no. AY974209); 4) L. L. major (WR 1075, accession no. AY974204), (WR CSU, accession no. AY974202), (WR 2312, accession no. AY974205), (WR 1088, accession no. AY974203); 5) L. L. tropica (WR 2179, accession no. AY974216), (WR 1091, accession no. AY974217), (WR 1063, accession no. AY974218).

Using the CLUSTAL program in the Lasergene software package (DNASTAR, Inc, Madison, WI), these amplicons were aligned and nucleotide differences among the various Leishmania complexes were identified. Using PRIMER EXPRESS software (PE Biosystems, Foster City, CA), appropriate probes and flanking primers were designed to specifically identify each Leishmania complex. The following primer/probe combination for each complex was designed and synthesized: L. Viannia complex, primers LV-f 5′-CAACAAAATGCTTCGCAACAG-3′ and LV-r 5′-CGCAACGCCTTCATGGA-3′ and LV-probe 5′-CGACGGGATATTGTTTGACTT-3′; L. L. mexicana complex, primers Lm-f 5′-CCAGTCCCAGAACACAAACATG-3′ and Lm-r 5′-CCTATCGACCAACACAGAAAAGG-3′ and probe Lm-probe 5′-ATGCCGAACTCCCGAA-3′; L. infantum/donovani complex, primer Lid-f 5′-CCAGATGCCGACCAAAGC-3′ and Lid-r 5′-CGCGCACGTGATGGATAAC-3′ and probe Lid-probe 5′-ATCGGCAGGTTCT-3′; L. major complex, primer Lmaj-f 5′-TTCTGCTCCGTCGGTGTAGA-3′, primer Lmaj-r 5′-GCTTTCGATTGGCTACGACAA-3′ and probe Lmaj-probe 5′-CCTGTCAGGAATTCCACAAA-3′. The probes contained a 5′-reporter dye (FAM, 6-carboxy-fluorescein) and a downstream 3′-quencher dye (TAMRA, 6-carboxy-tetramethyl-rhodamine).

Real-time PCR assay conditions.

PCR amplification and detection for the L. mexicana, L. Viannia, and L. infantum/donovani assays were performed with the Smart Cycler (Cepheid, Sunnyvale, CA), with preincubation at 95°C for 2 minutes, followed by 40 cycles of 2-step incubations at 95°C for 15 seconds and 60°C for 30 seconds. The reactions were conducted in a 26-μL volume containing 3.0 μL of DNA template, 1 PCR bead (Amersham Biosciences, Piscataway, NJ), an additional 2.35 mM MgCl2 (total 3.83 mM MgCl2), 200 nM of each oligonucleotide primer, and 20 nm of an oligonucleotide fluorogenic probe. PCR amplification and detection for the L. major assay were performed with the Smart Cycler (Cepheid, Sunnyvale, CA) with preincubation at 95°C for 2 minutes followed by 40 cycles of 2-step incubations at 95°C for 15 seconds and 68°C for 20 seconds. Reactions for the L. major assay were conducted in a 26-μL volume containing 3.0 μL DNA template, 1 PCR bead (Amersham Biosciences), an additional 3.25 mM MgCl2 (total 4.75 mM MgCl2), 800 nM of each oligonucleotide primer, and 200 nM of an oligonucleotide fluorogenic probe.

The presence of amplified product (a positive result) was defined as when the fluorescent signal exceeded an automatic noise-based defined threshold, and values were recorded as the second derivative of the primary signal, which is the point of greatest change along the amplification curve. Stringent measures to control for contamination included performing sham DNA extractions (using water instead of a biopsy specimen for the DNA extraction step) and inclusion of nontemplate negative controls with each PCR assay. For the testing of negative control clinical samples (human tissue), all samples that were negative with the Leishmania PCR assays were subsequently tested with a PCR assay containing primers for the β-actin gene to determine if amplifiable DNA was extracted from the sample. (TaqMan β actin control kit, Applied Biosystems, Foster City, CA).

RESULTS

Construction of a Leishmania GPI sequence library.

DNA sequencing and alignment of 20 Leishmania amplicons was performed to construct a library of Leishmania GPI sequences. Figure 1 demonstrates the alignment of the five Leishmania complexes sequenced. The primers (underlined) and internal probes (bolded) used to specifically identify four of the Leishmania complexes are listed. Due to space constraints, only a representative strain from each complex is presented, and only the regions of the amplicons demonstrating the areas of primer and probe sequences are shown (not the entire 700-bp sequence). The numbers on Figure 1 correspond to the nucleotide sequence starting from the beginning of the L. major GPI gene (systematic name, LmjF12.0530).

Specificity of each real-time PCR assay using cultured parasites.

DNA extracted from a panel of 26 Leishmania promastigote culture isolates (6 L. L. mexicana, 2 L. V. braziliensis, 5 L. V. panamensis, one L. V. guyanensis, 5 L. L. major, 3 L. L. tropica, 1 L. L. infantum, and 3 L. L. donovani) was tested with each set of primer/probe oligonucleotides in each real-time PCR assay. As displayed in Table 1, there was concordance in the classification of each complex of Leishmania by the real-time PCR assays and isoenzyme analysis.

Specificity of each real-time PCR assay using negative control samples.

DNA extracted from 21 clinical samples and cultures of two closely related organisms (T. rangelli and C. fasiculata) was tested with each Leishmania PCR assay. As listed in Table 2, there was no amplification of DNA detected with any of the four assays tested, while results of the β-actin control kit confirmed the presence of extracted DNA (for the human clinical samples).

Limit of detection of each assay.

Although not statistically rigorous, the limit of detection for these studies was defined as the lowest concentration of DNA at which the assay detected 3 out of 3 replicates. For all assays, this was determined to be 5.6 pg, which is equivalent to approximately 165.4 genome copies.

Results of real-time PCR assay using clinical samples.

Clinical samples were available from 50 patients who had undergone tissue biopsy with a resultant successful culture and isoenzyme identification (37 L. major, 2 L. V. panamensis, 2 L. tropica, and 9 L. mexicana). A portion of the tissue biopsy had also been processed for DNA extraction and had demonstrated the presence of detectable Leishmania DNA by a genus-specific PCR assay.14 An aliquot (3 μL) of the extracted DNA from each sample was then tested with each of the four Leishmania complex real-time PCR assays. The L. major specific assay amplified only DNA from the 37 clinical samples identified by isoenzyme testing as being L. major and not from samples identified as being L. V. panamensis, L. tropica, or L. mexicana. Similarly, the L. Viannia assay amplified DNA only from the L. V. panamensis samples, and the L. mexicana assay only amplified samples from the L. mexicana samples. The L. infantum/donovani assay did not amplify DNA from any of the clinical samples. The samples tested with these assays were obviously highly select, as they were positive by both culture and a genus-level PCR before being subjected to testing with these prototype assays. Given the selected nature of the samples, and the small number of isolates available, no statistical analysis of the clinical sensitivity of each assay (as compared with the traditional diagnostic modalities of microscopy and culture) was performed, and these results are simply “proof-of-principle” that these prototype assays can detect Leishmania DNA in human biopsy samples.

DISCUSSION

Traditional diagnosis of leishmaniasis rests on the direct culture of the organism or visualization of the amastigote stage of the parasite in tissue biopsies. Polymerase chain reaction (PCR) testing to diagnose Leishmania infection has been shown to offer enhanced sensitivity over more traditional diagnostic methods.20,21 Real-time PCR is a relatively new advancement in the diagnostic arena and employs fluorescent labels to enable the continuous monitoring of amplicon (PCR product) formation throughout the reaction. The major advantages of real-time PCR are that it is extremely rapid with results often obtained within 1 hour after DNA processing, it is less labor intensive (there are no gels to run and more samples can be processed at one time), there is less risk of contamination (the PCR tubes remain closed during the entire process), and it is highly specific by using a combination of amplification primers and an internal hybridization probe to detect a target gene. This technique has been widely reported for use in the diagnosis of a variety of diseases, including malaria and adenovirus infection.22,23 A previously described assay,14 designed to identify Leishmania to the genus level (but which cannot discriminate among the various strains) has been used at our institution to diagnose American servicemen afflicted with cutaneous leishmaniasis acquired during deployment to Iraq in support of Operation Iraqi Freedom. A comparison of this PCR assay with standard diagnostic modalities (when using the physician’s decision to offer treatment of leishmaniasis as the “gold standard”) demonstrated the PCR assay to be significantly more sensitive (97%) than culture (78%) and expert microscopy (76%).24

We believe that the most clinically important implication of the assays reported in the current paper is the ability to rapidly discriminate among the various Leishmania species. Traditionally, identification of Leishmania species requires tissue biopsy followed by the successful propagation of the organism in culture. Once an adequate density of parasites is achieved, isoenzyme analysis can be performed. The turn-around time for this process (which can take weeks) makes it impractical for use as a clinical decision tool. By identifying the infecting species within hours, a real-time PCR assay allows the potential for targeted treatment. Given the coexistence of more than one Leishmania species or complex in a given geographic area, and our current inability to rapidly discriminate among them, physicians often treat many cases of cutaneous leishmaniasis with antimony (which requires intramuscular, intravenous, or intralesional administration and is potentially toxic). Published studies suggest, however, that some species of Leishmania may respond well to alternative agents, such as ketoconazole for L. L. mexicana and fluconazole for L. major.25,26 Real-time PCR offers an opportunity to provide clinicians with point-of-care diagnosis down to the species/complex level and permit consideration of alternative treatments to antimony-based therapeutics.

Another potential use of real-time PCR is to rapidly discriminate among Leishmania complexes in archived samples of tissue or dried sandflies. This technique was recently used by U.S. entomologists in Iraq and identified the presence of L. major–infected sandflies well in advance of the first American cases.15 Identification of the prevalent Leishmania species in sandflies can alert clinicians to potential subsequent human cases.

In the future, a potential use for this technique would be as a real-time “test of cure” assay to assess response to therapy. In one such study of human immunodeficiency virus–infected patients with visceral leishmaniasis, clinical relapse was associated with persistence of peripheral blood Leishmania DNA levels.27

Importantly, although the breadth of Leishmania complexes and species archived in our library of live parasites and Leishmania DNA is large, some representative species are absent (i.e., L. V. peruviensis) or are limited to single isolates (i.e., L. V. guyanensis). Due to possible sequence variability in some species of Leishmania that are not well represented in this study set, we cannot exclude that the results of the real-time PCR may result in a falsely negative assay. In our laboratory, we first perform genus-level identification with a rRNA assay using primers/probe conserved among all Leishmania.14 We then perform a complex-level assay based on the geographic source of the sample. At the present time, culture and isoenzyme analysis of clinical samples is performed on all samples, as any isolate that is negative by PCR, but positive by culture, would prompt an investigation of the rRNA and/or GPI sequences to determine if there are nucleotide sequences distinct from our primers and probes.

There are other limitations to real-time PCR, to include the risk of contamination (resulting in falsely positive results), and the need for operator training. In addition, the cost of the assay is prohibitive for many laboratories (the thermocycler and computer used for this study cost approximately $30,000) and not feasible for many remote or poorly equipped endemic areas. Despite these restrictions, real-time PCR is a step toward providing clinicians with an assay that can rapidly distinguish among the various Leishmania complexes. With further improvements, one can foresee a completely automated assay that provides point-of-care results and allows therapy to be targeted against a specific pathogen.

Table 1

Results of four PCR assays when testing cultured promastigote samples

Complex by isoenzyme identificationReference no.L. Viannia assayL. mexicana assayL. major assayL. infantum/donovani assay
PCR, polymerase chain reaction.
L. mexicanaG-007NegativePositiveNegativeNegative
L. mexicanaG-003NegativePositiveNegativeNegative
L. mexicanaG-006NegativePositiveNegativeNegative
L. mexicanaG-024NegativePositiveNegativeNegative
L. mexicanaATCC 50157NegativePositiveNegativeNegative
L. mexicanaATCC 50156NegativePositiveNegativeNegative
L. V. panamensisWR G011PositiveNegativeNegativeNegative
L. V. panamensisWR2304PositiveNegativeNegativeNegative
L. V. panamensisWR2306PositiveNegativeNegativeNegative
L. V. panamensisATCC 50158PositiveNegativeNegativeNegative
L. V. panamensisERPositiveNegativeNegativeNegative
L. V. guyanensisWR675PositiveNegativeNegativeNegative
L. V. braziliensisWR676PositiveNegativeNegativeNegative
L. V. braziliensisATCC 50135PositiveNegativeNegativeNegative
L. majorWR1075NegativeNegativePositiveNegative
L. majorWR CSUNegativeNegativePositiveNegative
L. majorATCC 50122NegativeNegativePositiveNegative
L. majorWR 2312NegativeNegativePositiveNegative
L. majorWR 1088NegativeNegativePositiveNegative
L. tropicaWR1063NegativeNegativeNegativeNegative
L. tropicaATCC 50130NegativeNegativeNegativeNegative
L. tropicaWR 2179NegativeNegativeNegativeNegative
L. donovaniWR 520NegativeNegativeNegativePositive
L. infantumATCC 50134NegativeNegativeNegativePositive
L. donovaniWR 378NegativeNegativeNegativePositive
L. donovaniWR130ENegativeNegativeNegativePositive
Table 2

Results of testing negative control samples with each PCR assay

DiagnosisSource of sampleLeishmania PCR assays resultsB-actin assay results
PCR, polymerase chain reaction.
Compound nevusSkinNegativePositive
Intradermal nevusSkinNegativePositive
Basal cell carcinomaSkinNegativePositive
Condyloma acuminatumSkinNegativePositive
Marked solar elastosisSkinNegativePositive
Seborrheic keratosisSkinNegativePositive
Chronic perivascular inflammationSkinNegativePositive
NeurofibromaSkinNegativePositive
Chagas diseaseHeartNegativePositive
ToxoplasmosisBrainNegativePositive
Histoplasma capsulatumLiverNegativePositive
MycetomaSkinNegativePositive
Buruli ulcerSkinNegativePositive
MyiasisSkinNegativePositive
ChromomycosisSkinNegativePositive
Granuloma inguinaleSkinNegativePositive
Granuloma annulareSkinNegativePositive
ScabiesSkinNegativePositive
SporotrichosisSkinNegativePositive
Trypanosoma rangelliCultureNegativeNot performed
Crithidia fasiculataCultureNegativeNot performed
Figure 1.
Figure 1.

Aligned sequences from the GPI gene of representative Leishmania strains. Numbers correspond to the nucleotide sequence starting from the beginning of the L. major GPI gene (systematic name, LmjF12.0530).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 6; 10.4269/ajtmh.2005.73.999

*

Address correspondence to Glenn Wortmann, 6900 Georgia Ave., NW, Walter Reed Army Medical Center, Washington, DC 20307-5001. E-mail: glenn.wortmann@na.amedd.army.mil

Authors’ addresses: Lisa Hochberg, Huo-hsu Houng, Colleen Sweeney, Peter Weina, and Christian F. Ockenhouse, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910. Glenn Wortmann, Michael Zapor, and Naomi Aronson, Walter Reed Army Medical Center, 6900 Georgia Ave., NW, Washington, DC 20307-5001, E-mail: glenn.wortmann@na.amedd.army.mil.

Acknowledgments: Byran A. Arana, Niddia R. Rizzo, Flora Arana, Medical Entomology Research and Training Unit, Universidad del Valle de Guatemala, Guatemala City, Guatemala, for providing the Guatemalan clinical samples. Ron Neafie, Armed Forces Institute of Pathology, Washington, DC, for providing the negative control samples. Jeff Ryan, Col (ret), U.S. Army, Department of Entomology, WRAIR, for support and guidance.

Disclaimer: The views expressed here are those of the authors and should not be construed to reflect the views of the United States government.

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Author Notes

Reprint requests: Glenn Wortmann, Walter Reed Army Medical Center, 6900 Georgia Ave., NW, Washington, DC 20307-5001, Telephone: 202-782-6740, E-mail: glenn.wortmann@na.amedd.army.mil.
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