Optimized Diagnosis of Acute Dengue Fever in Swedish Travelers by a Combination of Reverse Transcription-PCR and Immunoglobulin M Detection
http://www.100md.com
微生物临床杂志 2005年第6期
Swedish Institute for Infectious Disease Control, SE-171 82 Solna
Microbiology and Tumor Biology Center, Karolinska Institutet, SE-171 77, Stockholm, Sweden
ABSTRACT
The dengue viruses (genus Flavivirus, family Flaviviridae) are mosquito borne and cause 100 million cases of dengue fever each year in most tropical and subtropical areas of the world. Increased global travel has been accompanied by an increased import not only of dengue but also of severe fevers of unknown origin to Sweden. Fifty-seven Swedish travelers to dengue epidemic areas, with clinical and serologically diagnosed dengue fever, were included in this study. To find fast and reliable methods to diagnose dengue in the early phase of the disease, patient acute-phase sera were investigated for the presence of dengue-specific immunoglobulin M (IgM) antibodies by enzyme-linked immunosorbent assay (ELISA) and also for dengue serotype (DEN-1 to DEN-4)-specific RNA by different PCR assays. The results showed that 15/20 (75%) of the samples collected 5 days or later post onset of disease, but only 5/37 (14%) of the samples collected on days 0 to 4, contained dengue-specific IgM. Of the samples collected on days 0 to 4 post onset, dengue RNAs of subtypes 1, 2, and 3 were detected by multiplex and/or by TaqMan PCR in 29/37 (78%); of these PCR-positive samples, 93% (27/29) were found IgM negative. By a combination of IgM ELISA and PCR assays, 84% (48/57) of the acute-phase samples were found to be positive. Our results demonstrated that detection of dengue viral RNA by reverse transcription-PCR and Taq-Man PCR is an excellent tool for the early diagnoses of dengue fever and that the IgM assay is a reliable complement for samples collected from day 5 post onset.
INTRODUCTION
More than 2.5 billion people live in dengue fever (DF) epidemic areas in tropical and subtropical areas of Asia, Africa, and South and Central America. Dengue is by far the most common global arboviral infection. Global population growth, unplanned urbanization, lack of effective mosquito control, and increased air travel have all contributed to significantly increased dengue epidemic activity during the past 20 years. It is estimated that 100 million cases of DF, 500,000 cases of dengue hemorrhagic fever (DHF), and approximately 25,000 fatal cases will occur each year at the beginning of the 21st century (4, 13). Currently, in spite of extensive research, there are no vaccines available. Dengue viruses (genus Flavivirus, family Flaviviridae) are mosquito borne, and the principal vector (Aedes aegypti) is a day-biting domestic mosquito that breeds in natural or artificial water (5). Flaviviruses have a positive-strand RNA genome of approximately 11,000 bases which encodes three structural and seven nonstructural proteins.
Dengue virus infections cause a spectrum of symptoms ranging from unapparent, mild febrile illness to fatal hemorrhagic disease (9). Most common is the classical DF, "break bone fever." The major symptoms of DF include a sudden onset with rash, high fever, and head- and backache. Hemorrhagic symptoms are not uncommon and may range from mild to severe. Some of the differential diagnoses during the acute phase of DF include influenza, malaria, measles, and typhoid fever. The more severe DHF is more common in children (13). Plasma leakage from the capillaries may lead to shock and, without therapy, death. Infection with one of the four dengue serotypes (DEN-1 to DEN-4) will provide lifelong immunity to the infecting serotype, but there is no cross-protective immunity against the other serotypes. Reinfection with a second serotype has been associated with a more severe form of disease and is a significant risk factor for DHF (6, 7, 9).
During the acute phase of the illness, which may last for 2 to 10 days, dengue viruses may circulate in the peripheral blood. Reverse transcription (RT)-PCR- and real-time PCR-based methods have recently been developed to detect dengue viral RNA during the viremic phase (1-3, 8, 11). Virus-specific serum immunoglobulin M (IgM) is generally produced within 5 days of disease onset with subsequent production of IgG (14-17).
The risk of infection for the average tourist is low compared to that of the local populations, mainly due to the limited time of exposure, staying in hotels with air conditioning, and the use of repellents. During the past 10 years, an average of approximately 50 dengue diagnoses per year has been made in Swedish travelers using serological methods, i.e., IgG indirect immunofluorescence assay (IFA), and since 2002 this has been complemented with dengue IgM enzyme-linked immunosorbent assay (ELISA) at the Swedish Institute for Infectious Disease Control (12). A seroconversion, significant titer rise, or a significant IFA titer of 320 is generally not seen until 7 to 13 days after the onset of disease (19). Therefore, there has been a need for a more sensitive, fast, and reliable method that can facilitate diagnosis during the early phase of the illness. In this study, we investigated 57 serum samples from patients with a defined dengue infection. The aim was to determine the optimal methods for confirmation of acute dengue virus disease in relation to the sampling dates.
MATERIALS AND METHODS
Serum samples. Paired serum samples from a total of 57 patients (samples from 44 patients collected during 1997 to 2001 and from 13 patients collected in 2002) with seroconversion or a significant (fourfold) rise in antibody titers for dengue virus and clinical symptoms of DF were included in the study. The patients had been diagnosed by IFA performed as previously described (19). The majority of the patients were Swedish travelers returning from Southeast Asia. Serum samples were stored at –20°C.
Dengue IgM ELISA. Serum samples from 1997 to 2001 were retrospectively tested, while the serum samples from 2002 were tested consecutively using the Dengue IgM Capture ELISA (PanBio, Brisbane, Australia) according to the manufacturer's instructions.
Virus propagation. Viral RNA was prepared from dengue virus type 1 (strain West Pac [GenBank accession no. U88535] and strain Hawaii), dengue type 2 (strain New Guinea C, GenBank accession no. M29095), dengue type 3 (strain H-87, GenBank accession no. M93130), and dengue type 4 (strain H-241, GenBank accession no. S66064). Vero cells (ATCC CCL-81) were grown until confluent in 25-cm2 tissue culture flasks at 37°C in minimal essential medium (Gibco BRL, Invitrogen, Life Technologies, Paisley, United Kingdom), supplemented with penicillin-streptomycin solution (50 U/ml and 50 μg/ml, respectively; Gibco) and 5% heat-inactivated fetal calf serum (Gibco). The cells were inoculated with the respective dengue viruses and grown for 1 week in minimal essential medium as described above, except that fetal calf serum was reduced to 2%. The supernatants were collected, clarified by centrifugation for 10 min at 1,000 x g, and subsequently aliquoted and stored at –70°C prior to RNA extraction. All handling of live virus was carried out in a biosafety level 3 laboratory.
RNA extractions. RNA was extracted from the supernatants of the virus-infected Vero cells and acute-phase patient sera. This was done manually using either the TriPure Isolation Reagent (Boehringer Mannheim GmbH) or the QIAamp Viral RNA Mini Kit (QIAGEN, GmbH, Hilden, Germany) or automatically using the MagAttract Viral RNA M48 Kit (QIAGEN) in a BioRobot M48 (GenoVision; QIAGEN) according to the manufacturer's instructions. The extracted RNA was kept in diethyl pyrocarbonate-treated H2O and stored at –20°C prior to cDNA synthesis.
Multiplex dengue RT-PCR. A multiplex two-enzyme RT-PCR was performed as previously described by Harris et al. (8). Briefly, five primers targeting the capsid gene were included in the assay, resulting in products of different sizes (DEN-1, 482 bp; DEN-2, 119 bp; DEN-3, 290 bp; DEN-4, 389 bp). The serum samples from 1997 to 2001 were analyzed retrospectively, while the samples from 2002 were analyzed consecutively. Five microliters of extracted RNA was used as a template in a 25-μl reaction volume.
Synthesis of cDNA. The cycling reactions were performed in a thermocycler (Gene Amp PCR system 2700 or 2400; Applied Biosystems, Foster City, CA [ABI]). In a 20-μl reaction volume, a mixture of 1 μl random nonamers (Sigma-Aldrich, St. Louis, MO), 1 μl 10 mM deoxynucleoside triphosphates (ABI), 5 μl distilled water, and 5 μl viral RNA was incubated at 65°C for 5 min and immediately chilled on ice. A mixture of 4 μl 5x first-strand buffer (Invitrogen), 2 μl 0.1 M dithiothreitol (Invitrogen), and 1 μl (10 U/μl) RNase inhibitor (Invitrogen) was added and incubated at 25°C for 10 min, 37°C for 2 min, and 4°C for the remainder of the time. One microliter (200 U/μl) of Moloney murine leukemia virus reverse transcriptase (Invitrogen) was added, and the mixture was incubated at 37°C for 50 min, 70°C for 2 min, and 4°C for the remainder of the time. The cDNAs were stored at –20°C and used subsequently in the TaqMan PCR.
TaqMan PCR. A TaqMan PCR previously described by Callahan et al. was performed (1).
Modified TaqMan PCR. A modification of the published TaqMan PCR was established (1). The PCR mixtures consisted of TaqMan Universal PCR Master Mix (ABI), primers (Invitrogen), and probes (ABI). Primer and probe optimization was performed, and 900 nM forward primer, 900 nM reverse primer, and 250 nM probe were found to be optimal in all four TaqMan PCRs. In addition, two wobbled DEN-2 primers were constructed (DEN-2-forward, 5' CAY GGC CCT KGT GGC R 3'; DEN-2-reverse, 5' CCC CAT CTY TTY ARW ATC CCT G 3'). In a 25-μl reaction volume, 5 μl of cDNA was used as the template. In the negative template control, 5 μl distilled water was used as a substitute for the cDNA. The cycling reactions were performed in a 7900HT Sequence Detection System (ABI), and the cycling profile was 50°C for 2 min, 95°C for 10 min, and 45 cycles of 95°C for 15 s and 60°C for 1 min.
Construction of dengue TaqMan PCR standards. PCR primers targeting sequences upstream and downstream of the regions of the TaqMan primers were selected using the Primer ExpressSoftware (ABI) (Table 1). The viral cDNA was amplified in 25-μl PCR mixtures containing 5 μl cDNA, 2.5 μl 10x buffer II (ABI), 2.5 μl 25 mM MgCl2 (ABI), 2 μl 10 mM deoxynucleoside triphosphates (ABI), 0.2 to 1.0 μl of 10 μM forward and reverse primers and, 0.2 μl (1 U/μl) AmpliTaq DNA polymerase (ABI). The reaction mixtures were cycled at 94°C 5 min, followed by 30 cycles of 94°C 30 s, 52 to 63°C 1 min, and 72°C 1 min, 72°C 10 min, and 4°C for the remainder of the time. The PCR amplification products were then electrophoresed, and the products of expected sizes were collected. The products were purified with a GFX PCR and Gel Band Purification Kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. The PCR products were subsequently ligated into the linearized plasmid vector pGEM-T (Promega SDS Biosciences, Falkenberg, Sweden) and transformed into JM 109 High Efficiency Competent cells (Promega SDS). Plasmid DNA was purified using the QIAGEN Plasmid Maxi Kit (QIAGEN) according to the manufacturer's instructions. The automated Sanger sequencing method was used to check the inserts of the purified plasmid DNA products for the correct sequence.
Determination of the sensitivity of the dengue TaqMan PCR. The molecular weights and molecules/ml of the double-stranded DNA clones were calculated, and standard curves were constructed. The plasmids were separately diluted 10-fold in Tris-EDTA buffer and tested in triplicate in the TaqMan PCR assays. The obtained cycle threshold (CT) values of the lowest dilutions were dispersed. In order to stabilize the PCRs, carrier DNA was included; i.e., the dengue plasmids were diluted at 103 to 107 molecules/ml and mixed with various amounts of carrier DNA to always obtain the same total amount of DNA.
TaqMan PCR analysis of serum samples. The samples were analyzed in duplicate (2 x 5 μl) in the four specific dengue TaqMan PCR assays. Positive reactions were plotted against the standard curves, and the CT values and the genome content/ml serum were calculated.
RESULTS
Dengue IgM ELISA analysis. IgM antibodies could be detected in 35% (20/57) of the acute-phase patient samples by ELISA, and traces of IgM antibodies could be detected (equivocal test results) in an additional 5% (3/57). In the samples collected between 0 and 4 days post disease onset, only 14% (5/37) were IgM positive, while IgM antibodies were detected in 75% (15/20) of the samples collected from day 5 onward (Fig. 1 and Table 2; see Table 4).
Multiplex dengue RT-PCR analysis. Dengue virus-specific RNA could be detected in 53% (30/57) of the patient samples by multiplex RT-PCR. Thirty-seven samples were collected between days 0 and 4, and of these, 68% (25/37) were found RT-PCR positive (Table 2). In contrast, 82% (9/11) of the samples collected in 2002 (days 0 to 4), which had not been repeatedly freeze-thawed, were found RT-PCR positive. Thirteen samples were of the DEN-1 serotype, nine of the DEN-2 serotype, and eight of the DEN-3 serotype. No samples belonging to the DEN-4 serotype were detected (see Table 4).
Comparison of the published and modified TaqMan PCR methods. Prepared viral RNA from dengue virus serotypes 1 to 4 was tested according to the published method (1). The received CT values were compared to the values received by our modified method (data not shown). We found an increased sensitivity by the separate reversed transcription step with random nonamers compared to the linked RT step.
Construction of dengue TaqMan PCR standards. Specific plasmid standards for each of the four dengue virus serotypes were constructed. PCR primers were selected covering the target sequences for the TaqMan PCR. The dengue viral RNA was reversely transcribed using random nonamers, and PCRs were performed with four different primer pairs (Table 1). Four serotype-specific products with the expected sizes were generated (DEN-1, 401 bp; DEN-2, 301 bp; DEN-3, 353 bp; DEN-4, 451 bp). The fragments of each serotype were pooled and cloned into the pGEM-T vector, and the resulting clones were propagated further. After expansion of the clones, plasmid DNA was purified and subsequently sequenced. The obtained data were confirmed against known sequences in GenBank.
Determination of the sensitivity of the TaqMan PCR. When the plasmid standards were first tested individually, low DNA concentrations gave dispersed results. To create more-stable standards, two standards were mixed together, to generate a higher and equal amount of total DNA in each test tube. This resulted in more-reliable tests, which were also working properly at very low copy numbers. The correlation values (R2) generated by the four serotype-specific TaqMan PCR assays, based on at least three independent tests, were between 0.954 and 0.982, and the slope values were between –3.219 and –3.452. The detection limits were estimated at 500 molecules/ml for all four assays (Table 3).
TaqMan PCR analysis. Dengue-specific RNA was detected in 56% (32/57) of the samples by TaqMan PCR. Seventy-three percent (27/37) of the samples collected between days 0 and 4 were found positive, while only 25% (5/20) of the samples collected at day 5 or later were positive (Table 2). One hundred percent (11/11) of the samples collected in 2002 (days 0 to 4) were found positive. Sixteen samples were of the DEN-1 serotype, seven of the DEN-2 serotype, and nine of the DEN-3 serotype. DEN-4 RNA was not detected in any of the samples. The number of genomes/ml in the acute samples varied between 1 x 103 and 5 x 108 (Table 1). The received mean number of genomes/ml for each sampling day demonstrated a gradual decline over time (Fig. 2).
DEN-2 TaqMan PCR analysis with wobbled primers. Construction of optimized DEN-2 primers was performed by wobbling of the forward and reverse primers at a total of seven positions. Six patient samples were retested with the wobbled DEN-2 primers and the DEN-2 probe. All six samples had previously been found DEN-2 positive by the multiplex RT-PCR, while only two had been found DEN-2 positive by the TaqMan PCR (Table 4). The result revealed that dengue virus RNA could be detected in two additional patient samples.
Comparison and summary of the test results. A total of 57 acute-phase serum samples were tested by dengue virus IgM ELISA, by the dengue virus multiplex RT-PCR, and by the dengue virus serotype-specific (DEN-1 to DEN-4) TaqMan PCRs. In 75% (15/20) of the samples collected 5 days or more following the onset of disease, IgM antibodies were detected by the ELISA. Of 37 patient samples collected at days 0 to 4, only 5 were found positive for dengue virus-specific IgM. In contrast, 68% (25/37) of the serum samples collected 0 to 4 days following the onset of disease were found to be positive by RT-PCR, and 73% (27/37) were positive by the TaqMan PCR (Fig. 1 and Table 4). The serotype could be determined in 78% (29/37) by PCR in the samples collected days 0 to 4 (Table 4). Eight samples yielded discrepant results; i.e., they were positive in only one of the two PCR systems. All samples that were found positive by both PCR assays were identified as the same dengue virus serotype by both assays. Seventeen samples were determined to be of the DEN-1 serotype, nine of the DEN-2 serotype, nine of the DEN-3 serotype, but none of the DEN-4 serotype. Eight samples (8/37) collected between days 0 and 4 were found negative by the PCR assays, but in three of these IgM antibodies could be detected (Fig. 1; Table 4). A dengue diagnosis during the acute phase was obtained in 86% (32/37) of the samples collected on days 0 to 4 and in 80% (16/20) of the samples collected 5 days or more post onset of disease by a combination of all three methods (Table 2).
DISCUSSION
The Swedish Institute for Infectious Disease Control receives serum samples from several hundred Swedish patients with suspected DF after traveling to epidemic countries each year. Increasing travel made it necessary to establish reliable diagnostic tools, not only for rapid identification of acute dengue viral infections but also for the discrimination of severe imported fevers of unknown origin. With knowledge of the sampling date, it is possible to choose optimal methods for dengue diagnosis during the different stages of the disease. Increased awareness among clinicians regarding patient records, including sampling dates, constitutes an important mission (2).
In this study, the acute-phase patient samples had previously been tested in the routine diagnostics for dengue virus-specific IgG by IFA, where they were all found to be negative (<10) or with only low titers (not clinically significant).
To optimize the diagnosis of early patient samples, we modified the recently published assays by a separate cDNA synthesis before performing the TaqMan PCR (1). One of the advantages of a separate RT step, with random primers, is the possibility of using the cDNA for various RNA virus PCRs. Furthermore, the reaction volumes in the TaqMan PCR assays were reduced from 50 μl to 25 μl. While the standards in the publication were generated by spiking normal serum with dengue viruses of known titer, we established serotype-specific plasmid standards. The modified TaqMan PCRs were used to analyze the acute-phase sera for both the presence and the amount of viral RNA. The results were compared to the data generated by a commercial dengue virus IgM ELISA and by a multiplex dengue virus RT-PCR.
The DEN-1 multiplex PCR primers were targeting a different region in the dengue virus genome (C versus NS-5) of another DEN-1 strain (Hawaii versus West Pac) compared to the DEN-1 TaqMan primers. The primers for DEN-2, DEN-3, and DEN-4 multiplex RT-PCR and TaqMan PCR were all located in the same region (C) of the virus genome. While the TaqMan assays were shown to be more sensitive compared to the multiplex RT-PCR in some cases, they also seemed to be more sensitive to sequence variations. Four samples, negative in the multiplex RT-PCR, were found positive by the TaqMan DEN-1 assay with relatively low amounts of viral RNA (3,000 to 20,000 genomes per ml serum), indicating that the TaqMan PCR is more sensitive than the multiplex RT-PCR. On the other hand, the DEN-2 TaqMan assay was not able to detect dengue virus RNA in four samples found positive by the multiplex RT-PCR. The discrepant results may be due to mismatches in primer and/or probe sequences (10). Mismatches were found in both the DEN-1 and DEN-2 probes and the primers when their sequences were compared against known sequences in the GenBank database (data not shown). In an attempt to fine tune the assay, we designed two wobbled DEN-2 primers. Two additional patient samples became positive after retesting with the wobbled primers. An additional explanation for the discrepant results may be the quality of the samples, given that some of the earlier samples had been stored at –20°C for several years and repeatedly freeze-thawed.
Real-time PCR has several advantages over traditional PCR, one of which is the collection of data already during the exponential reaction phase. An increase in the reporter fluorescent signal is thereby directly proportional to the number of generated PCR products, and it is thus possible to detect even a twofold change. There is no post-PCR processing, i.e., no ethidium bromide-gel detection is needed, and there is no risk of contamination in the closed TaqMan PCR plate system. One disadvantage, however, is the need for an almost perfect match between the primer and probe sequences.
The use of plasmid DNA as standards in TaqMan PCR is advantageous, since well-characterized and identical DNA is easy to process in large amounts. However, a disadvantage is that when working with plasmids extreme caution must be exercised to prevent contamination of the PCR. The four dengue virus plasmids that were produced showed almost identical detection limits (molecules/ml) and correlation (R2) in the TaqMan PCRs. Furthermore, the efficiencies (slopes) of the four PCR assays were almost 100%. A correlation of the viral load with clinical data may provide useful information concerning the pathogenesis of dengue virus-associated disease (18).
In this study, we have demonstrated that by a combination of IgM ELISA and PCR assays, a dengue diagnosis can be determined in as many as 84% (48/57) of single acute-phase samples. When the analyses were consecutively performed on samples not repeatedly freeze-thawed, 100% (13/13) of the samples were positive either by PCR or by IgM ELISA.
ACKNOWLEDGMENTS
We thank Kirill Nemirov for valuable advice during the cloning procedure.
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Microbiology and Tumor Biology Center, Karolinska Institutet, SE-171 77, Stockholm, Sweden
ABSTRACT
The dengue viruses (genus Flavivirus, family Flaviviridae) are mosquito borne and cause 100 million cases of dengue fever each year in most tropical and subtropical areas of the world. Increased global travel has been accompanied by an increased import not only of dengue but also of severe fevers of unknown origin to Sweden. Fifty-seven Swedish travelers to dengue epidemic areas, with clinical and serologically diagnosed dengue fever, were included in this study. To find fast and reliable methods to diagnose dengue in the early phase of the disease, patient acute-phase sera were investigated for the presence of dengue-specific immunoglobulin M (IgM) antibodies by enzyme-linked immunosorbent assay (ELISA) and also for dengue serotype (DEN-1 to DEN-4)-specific RNA by different PCR assays. The results showed that 15/20 (75%) of the samples collected 5 days or later post onset of disease, but only 5/37 (14%) of the samples collected on days 0 to 4, contained dengue-specific IgM. Of the samples collected on days 0 to 4 post onset, dengue RNAs of subtypes 1, 2, and 3 were detected by multiplex and/or by TaqMan PCR in 29/37 (78%); of these PCR-positive samples, 93% (27/29) were found IgM negative. By a combination of IgM ELISA and PCR assays, 84% (48/57) of the acute-phase samples were found to be positive. Our results demonstrated that detection of dengue viral RNA by reverse transcription-PCR and Taq-Man PCR is an excellent tool for the early diagnoses of dengue fever and that the IgM assay is a reliable complement for samples collected from day 5 post onset.
INTRODUCTION
More than 2.5 billion people live in dengue fever (DF) epidemic areas in tropical and subtropical areas of Asia, Africa, and South and Central America. Dengue is by far the most common global arboviral infection. Global population growth, unplanned urbanization, lack of effective mosquito control, and increased air travel have all contributed to significantly increased dengue epidemic activity during the past 20 years. It is estimated that 100 million cases of DF, 500,000 cases of dengue hemorrhagic fever (DHF), and approximately 25,000 fatal cases will occur each year at the beginning of the 21st century (4, 13). Currently, in spite of extensive research, there are no vaccines available. Dengue viruses (genus Flavivirus, family Flaviviridae) are mosquito borne, and the principal vector (Aedes aegypti) is a day-biting domestic mosquito that breeds in natural or artificial water (5). Flaviviruses have a positive-strand RNA genome of approximately 11,000 bases which encodes three structural and seven nonstructural proteins.
Dengue virus infections cause a spectrum of symptoms ranging from unapparent, mild febrile illness to fatal hemorrhagic disease (9). Most common is the classical DF, "break bone fever." The major symptoms of DF include a sudden onset with rash, high fever, and head- and backache. Hemorrhagic symptoms are not uncommon and may range from mild to severe. Some of the differential diagnoses during the acute phase of DF include influenza, malaria, measles, and typhoid fever. The more severe DHF is more common in children (13). Plasma leakage from the capillaries may lead to shock and, without therapy, death. Infection with one of the four dengue serotypes (DEN-1 to DEN-4) will provide lifelong immunity to the infecting serotype, but there is no cross-protective immunity against the other serotypes. Reinfection with a second serotype has been associated with a more severe form of disease and is a significant risk factor for DHF (6, 7, 9).
During the acute phase of the illness, which may last for 2 to 10 days, dengue viruses may circulate in the peripheral blood. Reverse transcription (RT)-PCR- and real-time PCR-based methods have recently been developed to detect dengue viral RNA during the viremic phase (1-3, 8, 11). Virus-specific serum immunoglobulin M (IgM) is generally produced within 5 days of disease onset with subsequent production of IgG (14-17).
The risk of infection for the average tourist is low compared to that of the local populations, mainly due to the limited time of exposure, staying in hotels with air conditioning, and the use of repellents. During the past 10 years, an average of approximately 50 dengue diagnoses per year has been made in Swedish travelers using serological methods, i.e., IgG indirect immunofluorescence assay (IFA), and since 2002 this has been complemented with dengue IgM enzyme-linked immunosorbent assay (ELISA) at the Swedish Institute for Infectious Disease Control (12). A seroconversion, significant titer rise, or a significant IFA titer of 320 is generally not seen until 7 to 13 days after the onset of disease (19). Therefore, there has been a need for a more sensitive, fast, and reliable method that can facilitate diagnosis during the early phase of the illness. In this study, we investigated 57 serum samples from patients with a defined dengue infection. The aim was to determine the optimal methods for confirmation of acute dengue virus disease in relation to the sampling dates.
MATERIALS AND METHODS
Serum samples. Paired serum samples from a total of 57 patients (samples from 44 patients collected during 1997 to 2001 and from 13 patients collected in 2002) with seroconversion or a significant (fourfold) rise in antibody titers for dengue virus and clinical symptoms of DF were included in the study. The patients had been diagnosed by IFA performed as previously described (19). The majority of the patients were Swedish travelers returning from Southeast Asia. Serum samples were stored at –20°C.
Dengue IgM ELISA. Serum samples from 1997 to 2001 were retrospectively tested, while the serum samples from 2002 were tested consecutively using the Dengue IgM Capture ELISA (PanBio, Brisbane, Australia) according to the manufacturer's instructions.
Virus propagation. Viral RNA was prepared from dengue virus type 1 (strain West Pac [GenBank accession no. U88535] and strain Hawaii), dengue type 2 (strain New Guinea C, GenBank accession no. M29095), dengue type 3 (strain H-87, GenBank accession no. M93130), and dengue type 4 (strain H-241, GenBank accession no. S66064). Vero cells (ATCC CCL-81) were grown until confluent in 25-cm2 tissue culture flasks at 37°C in minimal essential medium (Gibco BRL, Invitrogen, Life Technologies, Paisley, United Kingdom), supplemented with penicillin-streptomycin solution (50 U/ml and 50 μg/ml, respectively; Gibco) and 5% heat-inactivated fetal calf serum (Gibco). The cells were inoculated with the respective dengue viruses and grown for 1 week in minimal essential medium as described above, except that fetal calf serum was reduced to 2%. The supernatants were collected, clarified by centrifugation for 10 min at 1,000 x g, and subsequently aliquoted and stored at –70°C prior to RNA extraction. All handling of live virus was carried out in a biosafety level 3 laboratory.
RNA extractions. RNA was extracted from the supernatants of the virus-infected Vero cells and acute-phase patient sera. This was done manually using either the TriPure Isolation Reagent (Boehringer Mannheim GmbH) or the QIAamp Viral RNA Mini Kit (QIAGEN, GmbH, Hilden, Germany) or automatically using the MagAttract Viral RNA M48 Kit (QIAGEN) in a BioRobot M48 (GenoVision; QIAGEN) according to the manufacturer's instructions. The extracted RNA was kept in diethyl pyrocarbonate-treated H2O and stored at –20°C prior to cDNA synthesis.
Multiplex dengue RT-PCR. A multiplex two-enzyme RT-PCR was performed as previously described by Harris et al. (8). Briefly, five primers targeting the capsid gene were included in the assay, resulting in products of different sizes (DEN-1, 482 bp; DEN-2, 119 bp; DEN-3, 290 bp; DEN-4, 389 bp). The serum samples from 1997 to 2001 were analyzed retrospectively, while the samples from 2002 were analyzed consecutively. Five microliters of extracted RNA was used as a template in a 25-μl reaction volume.
Synthesis of cDNA. The cycling reactions were performed in a thermocycler (Gene Amp PCR system 2700 or 2400; Applied Biosystems, Foster City, CA [ABI]). In a 20-μl reaction volume, a mixture of 1 μl random nonamers (Sigma-Aldrich, St. Louis, MO), 1 μl 10 mM deoxynucleoside triphosphates (ABI), 5 μl distilled water, and 5 μl viral RNA was incubated at 65°C for 5 min and immediately chilled on ice. A mixture of 4 μl 5x first-strand buffer (Invitrogen), 2 μl 0.1 M dithiothreitol (Invitrogen), and 1 μl (10 U/μl) RNase inhibitor (Invitrogen) was added and incubated at 25°C for 10 min, 37°C for 2 min, and 4°C for the remainder of the time. One microliter (200 U/μl) of Moloney murine leukemia virus reverse transcriptase (Invitrogen) was added, and the mixture was incubated at 37°C for 50 min, 70°C for 2 min, and 4°C for the remainder of the time. The cDNAs were stored at –20°C and used subsequently in the TaqMan PCR.
TaqMan PCR. A TaqMan PCR previously described by Callahan et al. was performed (1).
Modified TaqMan PCR. A modification of the published TaqMan PCR was established (1). The PCR mixtures consisted of TaqMan Universal PCR Master Mix (ABI), primers (Invitrogen), and probes (ABI). Primer and probe optimization was performed, and 900 nM forward primer, 900 nM reverse primer, and 250 nM probe were found to be optimal in all four TaqMan PCRs. In addition, two wobbled DEN-2 primers were constructed (DEN-2-forward, 5' CAY GGC CCT KGT GGC R 3'; DEN-2-reverse, 5' CCC CAT CTY TTY ARW ATC CCT G 3'). In a 25-μl reaction volume, 5 μl of cDNA was used as the template. In the negative template control, 5 μl distilled water was used as a substitute for the cDNA. The cycling reactions were performed in a 7900HT Sequence Detection System (ABI), and the cycling profile was 50°C for 2 min, 95°C for 10 min, and 45 cycles of 95°C for 15 s and 60°C for 1 min.
Construction of dengue TaqMan PCR standards. PCR primers targeting sequences upstream and downstream of the regions of the TaqMan primers were selected using the Primer ExpressSoftware (ABI) (Table 1). The viral cDNA was amplified in 25-μl PCR mixtures containing 5 μl cDNA, 2.5 μl 10x buffer II (ABI), 2.5 μl 25 mM MgCl2 (ABI), 2 μl 10 mM deoxynucleoside triphosphates (ABI), 0.2 to 1.0 μl of 10 μM forward and reverse primers and, 0.2 μl (1 U/μl) AmpliTaq DNA polymerase (ABI). The reaction mixtures were cycled at 94°C 5 min, followed by 30 cycles of 94°C 30 s, 52 to 63°C 1 min, and 72°C 1 min, 72°C 10 min, and 4°C for the remainder of the time. The PCR amplification products were then electrophoresed, and the products of expected sizes were collected. The products were purified with a GFX PCR and Gel Band Purification Kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. The PCR products were subsequently ligated into the linearized plasmid vector pGEM-T (Promega SDS Biosciences, Falkenberg, Sweden) and transformed into JM 109 High Efficiency Competent cells (Promega SDS). Plasmid DNA was purified using the QIAGEN Plasmid Maxi Kit (QIAGEN) according to the manufacturer's instructions. The automated Sanger sequencing method was used to check the inserts of the purified plasmid DNA products for the correct sequence.
Determination of the sensitivity of the dengue TaqMan PCR. The molecular weights and molecules/ml of the double-stranded DNA clones were calculated, and standard curves were constructed. The plasmids were separately diluted 10-fold in Tris-EDTA buffer and tested in triplicate in the TaqMan PCR assays. The obtained cycle threshold (CT) values of the lowest dilutions were dispersed. In order to stabilize the PCRs, carrier DNA was included; i.e., the dengue plasmids were diluted at 103 to 107 molecules/ml and mixed with various amounts of carrier DNA to always obtain the same total amount of DNA.
TaqMan PCR analysis of serum samples. The samples were analyzed in duplicate (2 x 5 μl) in the four specific dengue TaqMan PCR assays. Positive reactions were plotted against the standard curves, and the CT values and the genome content/ml serum were calculated.
RESULTS
Dengue IgM ELISA analysis. IgM antibodies could be detected in 35% (20/57) of the acute-phase patient samples by ELISA, and traces of IgM antibodies could be detected (equivocal test results) in an additional 5% (3/57). In the samples collected between 0 and 4 days post disease onset, only 14% (5/37) were IgM positive, while IgM antibodies were detected in 75% (15/20) of the samples collected from day 5 onward (Fig. 1 and Table 2; see Table 4).
Multiplex dengue RT-PCR analysis. Dengue virus-specific RNA could be detected in 53% (30/57) of the patient samples by multiplex RT-PCR. Thirty-seven samples were collected between days 0 and 4, and of these, 68% (25/37) were found RT-PCR positive (Table 2). In contrast, 82% (9/11) of the samples collected in 2002 (days 0 to 4), which had not been repeatedly freeze-thawed, were found RT-PCR positive. Thirteen samples were of the DEN-1 serotype, nine of the DEN-2 serotype, and eight of the DEN-3 serotype. No samples belonging to the DEN-4 serotype were detected (see Table 4).
Comparison of the published and modified TaqMan PCR methods. Prepared viral RNA from dengue virus serotypes 1 to 4 was tested according to the published method (1). The received CT values were compared to the values received by our modified method (data not shown). We found an increased sensitivity by the separate reversed transcription step with random nonamers compared to the linked RT step.
Construction of dengue TaqMan PCR standards. Specific plasmid standards for each of the four dengue virus serotypes were constructed. PCR primers were selected covering the target sequences for the TaqMan PCR. The dengue viral RNA was reversely transcribed using random nonamers, and PCRs were performed with four different primer pairs (Table 1). Four serotype-specific products with the expected sizes were generated (DEN-1, 401 bp; DEN-2, 301 bp; DEN-3, 353 bp; DEN-4, 451 bp). The fragments of each serotype were pooled and cloned into the pGEM-T vector, and the resulting clones were propagated further. After expansion of the clones, plasmid DNA was purified and subsequently sequenced. The obtained data were confirmed against known sequences in GenBank.
Determination of the sensitivity of the TaqMan PCR. When the plasmid standards were first tested individually, low DNA concentrations gave dispersed results. To create more-stable standards, two standards were mixed together, to generate a higher and equal amount of total DNA in each test tube. This resulted in more-reliable tests, which were also working properly at very low copy numbers. The correlation values (R2) generated by the four serotype-specific TaqMan PCR assays, based on at least three independent tests, were between 0.954 and 0.982, and the slope values were between –3.219 and –3.452. The detection limits were estimated at 500 molecules/ml for all four assays (Table 3).
TaqMan PCR analysis. Dengue-specific RNA was detected in 56% (32/57) of the samples by TaqMan PCR. Seventy-three percent (27/37) of the samples collected between days 0 and 4 were found positive, while only 25% (5/20) of the samples collected at day 5 or later were positive (Table 2). One hundred percent (11/11) of the samples collected in 2002 (days 0 to 4) were found positive. Sixteen samples were of the DEN-1 serotype, seven of the DEN-2 serotype, and nine of the DEN-3 serotype. DEN-4 RNA was not detected in any of the samples. The number of genomes/ml in the acute samples varied between 1 x 103 and 5 x 108 (Table 1). The received mean number of genomes/ml for each sampling day demonstrated a gradual decline over time (Fig. 2).
DEN-2 TaqMan PCR analysis with wobbled primers. Construction of optimized DEN-2 primers was performed by wobbling of the forward and reverse primers at a total of seven positions. Six patient samples were retested with the wobbled DEN-2 primers and the DEN-2 probe. All six samples had previously been found DEN-2 positive by the multiplex RT-PCR, while only two had been found DEN-2 positive by the TaqMan PCR (Table 4). The result revealed that dengue virus RNA could be detected in two additional patient samples.
Comparison and summary of the test results. A total of 57 acute-phase serum samples were tested by dengue virus IgM ELISA, by the dengue virus multiplex RT-PCR, and by the dengue virus serotype-specific (DEN-1 to DEN-4) TaqMan PCRs. In 75% (15/20) of the samples collected 5 days or more following the onset of disease, IgM antibodies were detected by the ELISA. Of 37 patient samples collected at days 0 to 4, only 5 were found positive for dengue virus-specific IgM. In contrast, 68% (25/37) of the serum samples collected 0 to 4 days following the onset of disease were found to be positive by RT-PCR, and 73% (27/37) were positive by the TaqMan PCR (Fig. 1 and Table 4). The serotype could be determined in 78% (29/37) by PCR in the samples collected days 0 to 4 (Table 4). Eight samples yielded discrepant results; i.e., they were positive in only one of the two PCR systems. All samples that were found positive by both PCR assays were identified as the same dengue virus serotype by both assays. Seventeen samples were determined to be of the DEN-1 serotype, nine of the DEN-2 serotype, nine of the DEN-3 serotype, but none of the DEN-4 serotype. Eight samples (8/37) collected between days 0 and 4 were found negative by the PCR assays, but in three of these IgM antibodies could be detected (Fig. 1; Table 4). A dengue diagnosis during the acute phase was obtained in 86% (32/37) of the samples collected on days 0 to 4 and in 80% (16/20) of the samples collected 5 days or more post onset of disease by a combination of all three methods (Table 2).
DISCUSSION
The Swedish Institute for Infectious Disease Control receives serum samples from several hundred Swedish patients with suspected DF after traveling to epidemic countries each year. Increasing travel made it necessary to establish reliable diagnostic tools, not only for rapid identification of acute dengue viral infections but also for the discrimination of severe imported fevers of unknown origin. With knowledge of the sampling date, it is possible to choose optimal methods for dengue diagnosis during the different stages of the disease. Increased awareness among clinicians regarding patient records, including sampling dates, constitutes an important mission (2).
In this study, the acute-phase patient samples had previously been tested in the routine diagnostics for dengue virus-specific IgG by IFA, where they were all found to be negative (<10) or with only low titers (not clinically significant).
To optimize the diagnosis of early patient samples, we modified the recently published assays by a separate cDNA synthesis before performing the TaqMan PCR (1). One of the advantages of a separate RT step, with random primers, is the possibility of using the cDNA for various RNA virus PCRs. Furthermore, the reaction volumes in the TaqMan PCR assays were reduced from 50 μl to 25 μl. While the standards in the publication were generated by spiking normal serum with dengue viruses of known titer, we established serotype-specific plasmid standards. The modified TaqMan PCRs were used to analyze the acute-phase sera for both the presence and the amount of viral RNA. The results were compared to the data generated by a commercial dengue virus IgM ELISA and by a multiplex dengue virus RT-PCR.
The DEN-1 multiplex PCR primers were targeting a different region in the dengue virus genome (C versus NS-5) of another DEN-1 strain (Hawaii versus West Pac) compared to the DEN-1 TaqMan primers. The primers for DEN-2, DEN-3, and DEN-4 multiplex RT-PCR and TaqMan PCR were all located in the same region (C) of the virus genome. While the TaqMan assays were shown to be more sensitive compared to the multiplex RT-PCR in some cases, they also seemed to be more sensitive to sequence variations. Four samples, negative in the multiplex RT-PCR, were found positive by the TaqMan DEN-1 assay with relatively low amounts of viral RNA (3,000 to 20,000 genomes per ml serum), indicating that the TaqMan PCR is more sensitive than the multiplex RT-PCR. On the other hand, the DEN-2 TaqMan assay was not able to detect dengue virus RNA in four samples found positive by the multiplex RT-PCR. The discrepant results may be due to mismatches in primer and/or probe sequences (10). Mismatches were found in both the DEN-1 and DEN-2 probes and the primers when their sequences were compared against known sequences in the GenBank database (data not shown). In an attempt to fine tune the assay, we designed two wobbled DEN-2 primers. Two additional patient samples became positive after retesting with the wobbled primers. An additional explanation for the discrepant results may be the quality of the samples, given that some of the earlier samples had been stored at –20°C for several years and repeatedly freeze-thawed.
Real-time PCR has several advantages over traditional PCR, one of which is the collection of data already during the exponential reaction phase. An increase in the reporter fluorescent signal is thereby directly proportional to the number of generated PCR products, and it is thus possible to detect even a twofold change. There is no post-PCR processing, i.e., no ethidium bromide-gel detection is needed, and there is no risk of contamination in the closed TaqMan PCR plate system. One disadvantage, however, is the need for an almost perfect match between the primer and probe sequences.
The use of plasmid DNA as standards in TaqMan PCR is advantageous, since well-characterized and identical DNA is easy to process in large amounts. However, a disadvantage is that when working with plasmids extreme caution must be exercised to prevent contamination of the PCR. The four dengue virus plasmids that were produced showed almost identical detection limits (molecules/ml) and correlation (R2) in the TaqMan PCRs. Furthermore, the efficiencies (slopes) of the four PCR assays were almost 100%. A correlation of the viral load with clinical data may provide useful information concerning the pathogenesis of dengue virus-associated disease (18).
In this study, we have demonstrated that by a combination of IgM ELISA and PCR assays, a dengue diagnosis can be determined in as many as 84% (48/57) of single acute-phase samples. When the analyses were consecutively performed on samples not repeatedly freeze-thawed, 100% (13/13) of the samples were positive either by PCR or by IgM ELISA.
ACKNOWLEDGMENTS
We thank Kirill Nemirov for valuable advice during the cloning procedure.
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