IMPROVED SENSITIVITY OF DENGUE VIRUS DETECTION BY REVERSE

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					                                                     ∂“∫—π«‘®—¬«‘∑¬“»“ µ√å°“√·æ∑¬å∑À“√                       105
                                                    Armed Forces Research Institute of Medical Sciences


IMPROVED SENSITIVITY OF DENGUE VIRUS DETECTION BY
REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION
(RT-PCR) USING WHOLE BLOOD COMPARED TO SERUM OR
PLASMA
Klungthong C, Mammen MP Jr, Thirawuth V, Narupiti S, Chinnawirotpisan P, Nisalak A,
Kalayanarooj S, Jarman RG

A number of RT-PCR procedures to detect and to identify dengue virus (DENV) serotypes in
clinical specimens have been developed. These techniques are vital to surveillance programs
which require rapid detection of dengue viruses that can signal an impending outbreak and can
serve to trigger community education and vector control measures to minimize transmission
potential. Serum is usually the sample of choice for genomic amplification. We sought to
determine if we could enhance our ability to detect low levels of dengue viremia occurring in
either low viremic states or at the early or late time points of the febrile phase of illness. We
randomly selected 108 patients with suspected acute dengue viral infections whose specimens
were sent to USAMC-AFRIMS for service dengue diagnostics. Specimen processing provided
separate aliquots of whole blood and serum or plasma for RNA extraction. We were able to
detect DENV RNA by RT-PCR in 80 (74%) of the whole blood samples compared with only
56 (52%) of the corresponding serum/plasma samples. All 56 samples that were positive by
DENV RT-PCR were also positive when tested on whole blood. However, an additional 24
samples were positive by DENV RT-PCR when tested on whole blood but not serum/plasma.
Of these, 23 were serologically confirmed with paired sera; 1 could not be serologically confirmed
due to lack of a convalescent specimen. These results indicate that whole blood specimens
provide higher sensitivity of RT-PCR DENV detection than using serum/plasma specimens.
These finding suggests that surveillance strategies that are based on molecular detection can
be strengthened with a simple shift in procedure to extract RNA from whole blood rather than
serum/plasma.

American Society of Tropical Medicine and Hygiene 54th Annual Meeting. Washington,
DC, USA. 11-15 December 2005. (Poster)


IMPROVED SENSITIVITY OF DENGUE VIRUS DETECTION BY THE
REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION
(RT-PCR) USING QIAAMP VIRAL RNA KIT COMPARED TO TRIZOL
Klungthong C, Thirawuth V, Rodpradit P, Narupiti S, Nisalak A, Kalayanarooj S,
Jarman RG, Mammen MP Jr

The dengue virus (DENV) RT-PCR technology is increasingly the modality of choice in the
diagnosis of acute dengue permitting a diagnosis within 24 hours using a single blood collection.
Given that the reliability of this assay is contingent on collecting the sample during the febrile
period when viremia occurs, an increase in its sensitivity lends itself to a wider window of
detection. The sensitivity of this approach largely relies on the RNA extraction step. We evaluated



                                                                                                 √“¬ß“πª√–®”ªï 2548
                                                                                                 Annual Report 2005
               ®—¬«‘∑¬“»“ µ√å°“√·æ∑¬å∑À“√
   106  ∂“∫—π«‘Forces Research Institute of Medical Sciences
       Armed


  the TRIzol and QIAamp Viral RNA isolation methods, two widely used methods for RNA
  extraction, on their comparative ability to extract minute concentrations of viral RNA. The
  sensitivity of RT-PCR with all four serotypes of DENV was assessed following serial viral
  dilution and RNA extraction, in triplicate, using these methods. Both methods were equally and
  reproducibly able to extract DENV RNA from 1 PFU/ml for DENV-1 to -3 and 10 PFU/ml for
  DENV-4. The highest dilutions used with the QIAamp method that resulted in 1 of 3 replicates
  each contributing to a positive RT-PCR result are as follows: 10-2 PFU/ml of DENV-1, 10-6
  PFU/ml of DENV-2, 10-3 PFU/ml of DENV-3, and 10-2 PFU/ml of DENV-4. We were able
  to detect DENV-1 to DENV-4 RNA extracted with TRIzol by RT-PCR from 10-2 PFU/ml in
  1 replicate, 10-3 PFU/ml in 1 replicate, 10-2 PFU/ml in 3 replicates, and 10 PFU/ml in 3
  replicates, respectively. We extended this study to include acute sera from forty suspected
  DENV-infected patients for whom USAMC-AFRIMS has been requested to do service testing.
  These sera were extracted using both QIAamp and TRIzol RNA isolation methods. Twenty-
  eight of 40 acute sera revealed positive RTPCR results after extraction using both QIAamp and
  TRIzol. However, an additional 5 (15%) specimens were only positive when using QIAamp.
  All 33 positive RT-PCR samples were from serologically confirmed DENV-infected cases.
  These indicated that QIAamp Viral RNA Mini kit can improve the sensitivity and increase
  the reproducibility of DENV RT-PCR detection.

  American Society of Tropical Medicine and Hygiene 54th Annual Meeting. Washington,
  DC, USA. 11-15 December 2005. (Poster)


  MOLECULAR CHARACTERIZATION OF DENGUE VIRUS
  SEROTYPE 3 SAMPLED EARLY IN A 2005 OUTBREAK IN TIMOR-
  LASTE
  Chinnawirotpisan P, Mammen MP Jr, Kalayanarooj S, Anjarparidze A, Gibbons RV,
  Nisalak A, Jarman RG

  Dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS),
  caused by dengue virus (DENV) serotypes 1 to 4, remains a re-emerging public health problem
  in Southeast Asia. In early 2005, East Timor experienced a dengue outbreak. Acute sera were
  obtained from 44 hospitalized children, of whom 2 experienced DF and 42 DHF (12 with grade I,
  7 with grade II, 13 with grade III, 8 with grade IV and 2 with unknown grade). Three cases, all
  with DHF grade IV, died. Specimens from these 44 dengue cases were evaluated to determine
  viral serotype, genotype and, furthermore, whether any viral determinants correlated with enhanced
  virulence. Specimens were tested by reverse transcription-polymerase chain reaction (RT-PCR)
  and nested-PCR (using serotype-specific primers) to identify the infecting serotypes. Of the
  44 acute sera evaluated, 25 (53.3%) were positive by nested-PCR (all associated with DHF),
  of which 24 were DENV-3 and 1 was DENV-2. We successfully amplified the envelope (E)
  genes of 14 of 24 DENV-3 strains, al associated with DHF, two of which were associated with
  fatality. Nucleic acid sequences (1,479) nucleotides in length) of the complete E gene sequences
  were aligned with various global sample of DENV-3 E gene sequences from GenBank to generate
  a maximum likelihood (ML) phylogenetic tree using PAUP package. Fourteen isolates were



√“¬ß“πª√–®”ªï 2548
Annual Report 2005