Rapid diagnosis and determination of duration of viraemia in dengue fever using a reverse transcriptase polymerase chain reaction

Rapid diagnosis and determination a reverse transcriptase polymerase

of duration of viraemia chain reaction

in dengue fever using

‘Unit of Znfection J. L. Brownl*, R. Wilkinson’, R. N. Davidson’, R. Wall’, G. Lloyd*, J. Howells* and G. Pasvol’ and Tropical Medicine, St May’s Hospital Medical School, Imperial College of Science, Technology and Medicine, Northwick Park Hospital, Harrow, HA I 3 UJ, UK; ‘Diagnosis Group, Centre of Applied Microbiology and Research, Salisbuy, Wiltshire, SP4 OJG, UK Abstract

A rapid, simple diagnostic polymerase chain reaction (PCR) method for the diagnosis of dengue fever was developed using a pair of consensus oligonucleotide primers and validated with laboratory-derived strains of dengue serotypes l-4 and other common flaviviruses. A cluster of 13 patients with clinical dengue fever admitted to a single infectious diseasesunit over a period of 3 months allowed evaluation of this technology. The PCR was positive in all 11 acute dengue casesand negative in 2 convalescent casesand 10 febrile patients recently returned from the tropics in whom an alternative diagnosis was established. In some of the acute cases,viraemia was detected before the development of a dignostic antibody response (indirect immunoglobulin (Ig) G enzyme-linked immunosorbent assay(ELISA) and capture IgM ELBA). In patients from whom sequential sera were taken, defervescenceand recovery from thrombocytopenia coincided with the disappearanceof dengue ribonucleic acid from the blood. Nucleotide sequencing of the PCR products was undertaken in 2 cases(from India and Guyana) and the results showed a close match with previously reported serotype 2 sequencies,suggesting a potential for use of this region of the genome in epidemiological studies. Keywords: denguefever,polymerasechain reaction,diagnosis,persistenceof viraemia Introduction

Dengue fever is usually a mild self-limiting flavivirus infection common throughout the tropics, particularly south-east Asia, the Indian subcontinent and Central and South America. Increased air travel has increased importation of this disease into countries where awareness of the diseaseis low, and where costly and laborious investigations might be embarked upon- in search of a diagnosis. Clinical diagnosis of deneue fever is unreliable and the features may mimic other common diseases such as malaria, typhoid or influenza, and at times may be severeenough to be confusedwith meningoencephalitis. Since confirmation of the diagnosis depends largely on retrospective antibody detection or viral culture, we designed and evaluated a rapid, sensitive laboratory test with which we have determined the duration of viraemia in dengue fever and its relationship to clinical manifestations of the disease. Previously published methods for the detection of dengue ribonucleic acid (RNA) by the reverse transcriptase-polymerase chain reaction (RT-PCR) require prior viral culture (ELDADAH et al., 1991; MORITA et al., 1991), detection by hybridization to type ,specific probes (DEUBEL et al., 1990; HENCHAL et al., 1991; LAILLE et al., 1991; PA0 et al., 1992),a second round of nested PCR to detect serotype-specific RNA (LANCIOTTI et al., 1992), multiple primer sets (DEUBEL et al., 1990; ELDADAH et al:, 1991; LAILLE et al., 1991; PAO et al., 1992) or primers with degeneracy in the ‘wobble’ baseposition of codons necessitating variable nucleotide insertion during synthesis (HENCHAL et al., 1991). Our strategy was to examine the published dengue virus sequencesfor consensus regions shared by all 4 serotypes in order to find a universal pair of oligonucleotide primers suitable for RT-PCR. Materials and Methods Patients and sera

Twenty-three patients recently returned from tropical and sub-tropical areas between October and December 1992 and admitted to the Lister Unit at Northwick Park Hospital, Harrow, UK were studied. Routine tests including blood culture and blood film for malarial parasites were performed and, for those casesin which a diagnosis could not be established, serum was sent to the Centre of Applied Microbiology and Research, Porton Down, UK for further investigation. Sera of all 23 cases, *Author for correspondence.

if not used immediately, were separatedwithin 24 h and stored at -70°C until examined for the oresence of dengue RNA by RT-PCR. All nucleic acid &tractions, complementary deoxyribonucleic acid (cDNA) syntheses and PCR reactions were controlled by known positive and negative controls. Dengne serology

Dengue immunoglobulin (Ig) M antibodies were detected by the method of KUNO et al. (1991). In brief, 96well microtitre plates (Falcon, Probind@) were sensitized with 100 PL per well of an affinity-purified goat anti-human IgM antibody (Nordic Immunology) diluted in 0.05 M carbonate-bicarbonate buffer, pH 9.6, and incubated overnight at 4°C. After washing, 50 PL of test and positive control sera diluted 1:40-1:640 in phosphate-buffered saline containing 0.1% Tween 20@ (PBST) were applied in duplicate to the wells and incubated at 37°C for 1 h. After washing in PBST, a standard amount of tissue culture dengue antigen was added and detected after further washing with a peroxidase-conjugated antiflavivirus monoclonal antibody. For IgG antibody detection, the microtitre plate wells were sensitized with 100 PL of the appropriate dengue serotype antigen diluted in carbonate buffer and incubated overnight. The plates were washed with PBST and 50 PL aliquots of control and test sera, diluted 1:100-I:800 in PBST with 0.05% horse serum (Gibco Life Technologies) were added. Incubation was performed at 37°C for 30 min on a heated shaker (Wellwarm 18, Denley). After further washing, 50 PL aliquots of affinity purified goat anti-human IgG conjugated with horse-radish peroxidase diluted 1:2000 in PBST were added and the plates incubated at 37°C for 30 min. In both tests, antibodv fixation was detected bv the addition of 1OdPL of a substrate solution containing tetramethvlbenzidine dihvdrochloride fTMB. Siama) and 0.014% hydrogen peroxide in 0.05%citrate biffer; pH 5. The colour reaction was stopped by the addition of 25 PL of 2N sulphuric acid and absorbance values at 492nm were measured with an automatic enzyme-linked immunosorbent assayplate reader(Anthos 2001@,Denley). The optical density (OD) values of each test were compared with those of a standard positive serum of known titre and were considered positive if the OD was greater than the mean of the negative controls + 3 standard deviations.

DIAGNOSIS OF DENGUE BY PCR

141

Oligonucleotide primer design

We analysed published nucleic acid sequencedata for dengue 1 (MASON et al., 1987; Fu et al., 1992), dengue 2 (DEUBEL et al., 1986; HAHN et al., 1988; IFUEet al., 1989), dengue 3 (OSATOMI & SUMIYOSHI, 1990), dengue 4 (ZHAO et al., 1986) and other flaviviurses (‘Japaneseencephalitis, yellow fever, tick-borne encephalitis) in order to identify specific consensus regions within the dengue genome suitable for oligonucleotide primer synthesis. Such regions are likely to have essential biological functions and be conserved in all dengue species.Two candidate regions were identified: (i) a short section in the 5 ’ untranslated region unique to dengue (sense primer 5 ‘TAGAGAGCAGATCTCTG3 ‘) and (ii) a section from the nucleocapsid region shared by dengue and Japaneseencephalitis, but absent from yellow fever and tick-borne encephalitis viruses (antisenseprimer 5 ‘CGCGTTTCAGCATATTGA3 ‘). The predicted size of the PCR product was 72 nucleotides (nt) long in the caseof dengue serotypes1,2 and 3, and 74 in dengue4 (Fig. 4, i). In order to confirm the specificity of these oligonucleotide primers, RNA extraction and the PCR were done with laboratory strains of dengue 1 (Hawaiian), dengue 2 (New Guinea), dengue 3 (H87), dengue 4 (H241), Japanese encephalitis (Nakayma), yellow fever (Asbi), St Louis encephalitis, West Nile virus (B956), and tick-borne encephalitis.

10 PL of a mixture containing 10 mM HEPES-Hcl, pH 6.9, O-2 mM EDTA, pH 8, and 50 ng of the synthetic antisense oligonucleotide primer. The solution was heated at 90°C for 2 min and quenched on ice. The volume was increased to 20 ILL to give final concentrations of 50 mM Tris-HCL pH 7.5, 75 mM potassium chloride, 3 mM magnesium chloride, 10 mM dithiothreitol, and 0.5 mM of each deoxynucleotide. Twenty units of ribonuclease inhibitor (Pharmacia) and 15 units of cloned Moloney murine leukaemia virus reverse transcriptase (Gibco BRL) were added and the mixture was incubated at 37°C for 90 min. PCR umpla)icution

PCR amplification was performed in a 50 PL mixture containing 10 mM Tris-HCL pH 8.3, 50 mM potassium chloride, 1.5 mM magnesium chloride, 0.01% gelatin, 0.7

Nucleic acid extraction

Nucleic acid extraction was performed using a guanidinium isothiocyanate chaotropic method for reasons of safety and simplicity (BOOMet uZ.,1990).In brief, 100 yL of serum were added to 900 ILL of lysis solution (120 g guanidium isothiocyanate in 100 mL 0.1 M Tris, pH 6.4, with 22 mL of ethylenediaminetetraacetic acid (EDTA), 0.2 M pH 8.0, and 2.6 g of Triton X-100@) and 40 PL of fine silica prepared by gravity sedimentation. RNA bound to silica was separated by centrifugation and washed twice with wash solution (120 g guan-idinium isothiocyanate/lOO mL 0.1 M Tris, pH 6.4), twice with 70% ethanol, and once with 100% acetone. After drying in air, RNA was eluted from the silica using distilled water containing ribonuclease inhibitor (Pharmacia, 20 units/sample). Reverse transcription

Five PL of sample RNA solution were diluted to give Table. Clinical features and results of enzyme-linked with dengue fever

Case

Age

no.

kears)

Sexa

2 3 4 5 6 7 8 9 10 11 12 13

24 57 57 43 58 29 23 56 22 58 45 22 63

M F M M F M M M M M M F F

Fig. 1. Gel electrophoresis of polymerase chain reaction amplification products from laboratory strains of flavivirus. Lane 1, 100 bp DNA size markers; lane 2, negative control; lane 3, dengue 1; lane 4, dengue 2; lane 5, dengue 3; lane 6, dengue4; lane 7, Japaneseencephalitis; lane 8, yellow fever; lane 9, St Louis encephalitis; lane 10, West Nile; lane 11, tickborne encephalitis.

immunoassays

and polymerase

Region visited _. Clinical features Thailand Fever, headache India Vomiting, fever, thrombocytopenia Guyana Fever, vomiting, confusion, thrombocytopenia India Fever, thrombocytopenia India Diarrhoea, vomiting, fever, thrombocytopenia Thailand Fever, thrombocytopenia, leucopenia India Fever, thrombocytopenia India Headache,fever, thrombocytopenia, leucopenia India Fever, thrombocytopenia, leucopenia India Fever, rash, thrombocytopenia India Fever, thrombocytopenia, headache Thailand Malaise, afebrile, thrombocytopenia India Rash, afebrile, thrombocytopenia

chain reaction in 13 patients

IgM

ELIsAb IgG PCRb

+

+

+

-

-C

+

-C

+

+

-c

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

-

+

-d

-

+

-d

aF= female, M = male. bEnzyme-linked immunosorbent assay(ELISA) and polymerasechain reaction (PCR) results; +, positive and -, negative. CInitially negative but becamepositive during convalescence. dPatients were convalescent when the PCR was done.

142

units of recombinant DNA Taq polymerase (Perkin Elmer, Cetus), 200 mM of each deoxynucleotide, 40 ng of each of the oligonucleotide primers and 5 PL of the cDNA as a template. After 3 min denaturation at 93“C, 40 cycles of amplification were conducted at 94*C for 1 min, 51°C for 1 min, and 72°C for 1.5 min. PCR product analysis

Reaction products were discriminated by electrophoresis through Visigel ‘” separation matrix (Strategene, Cambridge, UK) containing ethidium bromide (0.4 yg/mL) and visualized by transillumination with ultraviolet light. A HaeIII digest of $X174 provided convenient molecular weight markers as the smallest fragment of 72 nt was the same size as the expected dengue RTPCR product. Nucleic acid sequencing

Sequencing of the PCR product from 2 cases(from India and Guyana) was performed to verify the specificity of the test. Single stranded DNA was prepared by streptavidin capture (DynabeadsTM,Dynal Ltd, Wirral, UK) of RT-PCR products synthesised with biotinylated oligonucleotide primers. Sense and antisense oligonucleotides end-labelled with 33p were used for thermocycle sequencing (TaquenceTM cycle sequencing kit, United StatesBiochemical Corporation). Results R T-PCR and serology

The results of the RT-PCR test with the reference flaviviruses are shown in Fig. 1. The oligonucleotide primers, as predicted, gave amplification products with all 4 dengue serotypes and the test was negative with the other viruses. There was, however, some weak amplification with Japaneseencephalitis virus but this generated a higher molecular weight product (lane 7), which could be distinguished from that of dengue. Eleven of the 13 patients with clinically suspected dengue fever on the basis of recent relevant travel, thrombocytopenia (platelets less than 200~ 109/L), negative blood cultures and 3 daily negative malarial films were positive for dengue RNA by RT-PCR (Table 1). These patients had a history of fever ranging from 1 to 4 days before admission. Two afebrile patients (casesno. 12 and 13), negative for dengue RNA but with dengue IgG antibody only, were considered to be convalescent on the basis of their prolonged clinical history. One of these had been travelling for 3 months and gave a history of a febrile illness in the first week of travel. Three patients (casesno. 2, 3 and 4), who were RT-PCR positive but dengue IgG antibody negative on admission, seroconverted in convalescence and only 2 of these had IgM antibody on presentation. None of the sera from the 10 patients with diseasesother than dengue fever such as campylobacter enteritis, coxsackie B virus infection, malaria, measles, tuberculosis, typhoid fever and urinary tract infection were positive by PCR. In one patient (case no. 3) with a lymphocytic cerebrospinal fluid (CSF) and pyuria, dengue serology and serum RT-PCR were positive, but viral nucleic acid could not be detected in the urine or CSF taken at the time of the acute illness. None of the patients exhibited features of dengue haemorrhagic fever/dengue shock syndrome and there was no death.

Fig. 2. Gel electrophoresis of polymerase chain reaction amplification products. (a) Lane 1, negative control; lane 2, HaeIII @X174size markers (lowest band 72 nucleotides); lanes 3-4, case 1 acute sera; lanes 5-6, case 1 convalescent sera; lanes 7-12, case 2 sequential sera. (b) Lane 1, negative control; lanes 2-7, case 3 sequential sera; lane 8, Hat-III @X174size markers; lanes 9-l 1, case3 sequential urine samples; lane 12, case3 cerebrospinal fluid.

Fig. 3. Temporal relationship between viraemia (determined by reverse transcriptase polymerase chain reaction and graded semi-quantitatively: f, a distinct 72 nucleotide band in the gel; +, faint but discernible 72 nucleotide band; -, no band). thrombocvtaemia (0. olateletsx log/L) and fever (--, “C) in4 casesof denguefever: ~ ”

Duration of viraemia

With 4 patients we were able to follow the daily evolution of viraemia by RT-PCR and relate this to clinical state (Fig. 2). In all cases,the disappearanceof viral nucleic acid from the serum preceded the resolution of thrombocytopenia, defervescenceand clinical recovery. Nucleic acid and amino acid sequences

The nucleic acid sequences of the reference strains and the RT-PCR products from patients no. 2 and 3 are

Fig. 4. Nucleic acid(i) and amino acid (ii) sequencesof reverse transcriptase polymerase chain reaction products in dengue fever patients 2 and 3 compared with published sequences.‘New Guinea isolate (1st~ et al., 1989); ‘Jamaican isolate (DEUBELet al., 1986); ‘candidate vaccine strain (multiple passages)(H.~N et al., 1988); dSingapore isolate (Fu et al., 1992);?rlauru island (Western Pacific) isolate (MASONer al., 1987).

DIAGNOSIS OF DENGUE BY PCR

143

shown in Fig. 4, i. The PCR product from patient no. 2 (from Delhi) differed by only one nucleotide from the dengue 2 sequence reported from New Guinea (IRIE et al., 1989), and the sequence from patient no. 3 (from Guyana) was identical to the Jamaican dengue 2 isolate (DEUBEL et al., 1986). The translated sequences(Fig. 4, ii) showed that the amino acids of case no. 2 and the New Guinea isolate were identical. and differed from those of patient no. 3 and the Jamaican isolate by the substitution of asparagine at position 10 with another uncharged polar amino acid, serine.

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Discussion

Eldadah, Z., Asher, D., Godec, M., Pomeroy, K., Goldfard, L., Feinstone, S., Levitan, H., Gibbs, C., jr & Gajdusek, D. (1991). Detection of flaviviruses by reverse transcriptase polymerase chain reaction. Journal of Medical Virology, 33,

This study has considerably raised our awareness of the diagnosis of dengue fever in febrile travellers from tropical and subtropical areas. Our sensitive, specific and simple assay for the laboratory diagnosis of acute dengue fever could be of value to units which deal with such patients. In addition, the amplification of a slightly higher molecular weight product with Japanese encephalitis could also be of potential diagnostic value, although this would need to be confirmed with clinical studies. The clinical presentations of the 2 diseasesare rather different, but in difficult casesa dengue and Japanese encephalitis positive control should be run in order to make the necessarysize distinction. Our data on the duration of dengue viraemia as determined by RT-PCR accord with tissue culture studies (GUBLER et al., 1981).The timing of the disappearanceof viral nucleic acid from serum and defervescenceand reversal of thrombocytopenia was striking, and explains the negative PCR result in the 2 afebrile patients with serological evidence of convalescent infection. During the period of study, October to December 1992. we documented 11 cases of malaria and onlv 2 casesof typhoid in our unit. Many of the dengue fever patients did not have classical features of dengue such as headache, myalgia and bone pain, whilst a number had gastrointestinal symptoms on presentation. One particular patient (case no. 3) presented with a meningoencephalopathic picture necessitating a computerized tomography brain scan, lumbar puncture and empirical intravenous antibiotics. In conclusion, we have defined and validated a set of oligonucleotide primers which form the basis of a rapid laboratory diagnostic test for all 4 serotypes of dengue virus, and have shown that viral RNA disappears from the blood at the time of defervescence.Dengue fever was more common than expected and a number of caseshad unusual clinical presentations. Medical practitioners should consider dengue fever in the differential diagnosis of all febrile travellers from tropical and sub-tropical areas, particularly when thrombocytopenia is present and blood films for malaria are negative. Acknowledgements We thank David Howells of the Human ResourcesCentre in the former Clinical Research Centre at Northwick Park for valuable assistance in determining the strategy for sequencing the short 72nt RT-PCR product and for synthesizing the biotinylated oligonucleotide primers. This work was funded in part by a grant from the Mason Medical Research Foundation.

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