Clinical Significance And Detection Of DENV-RNA In Urine From Dengue Fever Patients

Dengue fever is an acute infectious disease caused by dengue virus (DENV), andit is also a major public health problem in the tropical and subtropical regions, whichhas a serious threat to human health and public health security in the world. Denguefever needs laboratory diagnosis to be confirmed. By using RT-PCR method fordetecting DENV nucleic acid, is a rapid, sensitive and specific laboratory diagnosticmethod. Although Blood specimens is commonly used, it still have some deficits,such as short viremia period,invasive procedures and inconvenient use. However,Urine specimens are economic, convenient, safe and noninvasive. Can DENVnucleic acid detection in urine be used for diagnosis of dengue fever? There has rarestudy abroad, and there has no research in China up to now. Thus, to make sure theeffects of DENV nucleic acid testing in urine for diagnosis of dengue patients, whichhas an important significance to improve the diagnosis and control of dengue fever.ObjectivesThis study intends to establish a laboratory method through the detection ofDENV-RNA in urine from dengue fever patients, and to explore its clinicalsignificance.Objects 1.The experimental group: It included57cases of dengue fever patients who werehospitalized in the eighth people’s hospital in Guangzhou from August to Novemberin2013. Cases of dengue fever(DF)and severe dengue fever (SDF) patients were42and15, respectively. We totally collected73plasma specimens and158series ofurines of early-middle-late disease course to carry out experiment.1.1. The median age of dengue fever patients was37.5years with a range of7to74years.Cases of male and female were35and22,respectively.1.2. Totally,73plasma samples were derived from51patients (1-2portions perperson). There were35,33, and5copies of plasma, respectively in the early courseof disease (1-5disease days), middle course (6-9disease days) and late course(10-14disease days). Six patients had no plasma collected.1.3.57cases of dengue fever patients had158copies of urine (2-3portions perperson), in the early, middle and late course of disease, respectively had56,76,26copies of urine.2.The control group: It Included10cases for non-dengue patients and healthypeople,respectively,and each had one copy of urine, a total of20. The non-denguepatients included5cases of HIV/AIDS patients and5cases of hepatitis B patients.Methods1. Confirm dengue patientsWe summarized clinical manifestation and etiology results of57cases of denguefever patients by the review of medical records. According to WHO“The IssuedGuide of Dengue Diagnosis, Treatment, Prevention and Control” in2009, we makesure that57cases of dengue fever patients all reach the laboratory confirmedstandard.2. The specimen collection and preservation2.1. Collect5-10ml urine,within2hours centrifuge10minutes at a speed of2000r/min, take supernatant to1.5ml microcentrifuge tube, then place-80℃in therefrigerator to save,and avoid repeated freezing and thawing;2.2. Collect5ml anticoagulant fresh blood at the same time,then rapidly separate to get plasma to1.5ml microcentrifuge tube, and save in-80℃.3. Anti-DENV-IgM and IgG detection in plasma using ELISAUsing PANBIO DENGUE IgM CAPTURE ELISA and PANBIO CAPTUREDENGUE IgG ELISA(PANBIO company in Australia) kits tested anti-DENV IgMand IgG antibody in plasma collected in fever period.4. The judgment of the primary infection and secondary infection.If anti-DENV-IgM and anti-DENV-IgG were negative at the same time, it can bejudged as the primary infection; however, If anti-DENV-IgG was positive andspecimen collection time was in fever period, can be judged as secondary infectionregardless of the anti-DENV-IgM outcome.5. Detection of DENV-RNA in plasma and urine using RT-PCRTake out a tube of urine specimen from-80degrees fridge, dissolve and blend atroom temperature. According to steps of Daan nucleic acid extracting reagent kitand Daan RT-PCR kit, prepare for RT-PCR reaction system, and then use7500FastReal-time PCR system (American ABI) to amplify specific fragments of denguevirus nucleic acid. Based on the amplification curve and CT value, identify thenegative or positive as a result. Steps of DENV-RNA detection in plasma was thesame with urine.6. The evaluation of renal damageBy using the domestic recognized indicators combined of sex, age, albumin,blood urea nitrogen and creatinine with patients, and according to the Chinese eatingcorrection proprietary software, endogenous creatinine clearance can be calculated.When the endogenous creatinine clearance was less than or equal to70ml perminute, it was able to judge renal damage.7. Statistical methodsSPSS13.0software. Pearson chi-square test. Continuity correction method.Fisher’s exact probability method. P values less than0.05are statistically significant.·Results：1. Result of anti-DENV-IgM and Anti-DENV-IgG in plasmaThere were51cases of IgM positive, and8cases of IgG positive. 2. Result of the judgment of the primary infection and secondary infectionThe results suggested the primary infection patients with43cases, secondaryinfection patients with8cases. Meanwhile,6patients without the plasma collectionwere unable to determine the primary and secondary infection.3. Detection of DENV-RNA in urine3.1. Detection rate of DENV-RNA in urineDENV-RNA in the urine with57Dengue fever patients,40patients werepositive, the detection rate was70.2%.DENV-RNA detection in the urine with20cases of control group was all negative.3.2. Results of DENV-RNA in urine on different day of onsetThe detection rate of DENV-RNA in urine was0（0/0）on1day of onset,0（0/0）on2day of onset,60%（3/5）on3day of onset,56%（10/18）on4day of onset,61%（19/31）on5day of onset,50%（12/24）on6day of onset,80%（16/20）on7dayof onset,67%（14/21）on8day of onset,45%（5/11）on9day of onset,67%（6/9）on10day of onset,29%（2/7）on11day of onset,67%（4/6）on12day of onset,0（0/2）on13day of onset,50%（1/2）on14day of onset. The results showed, theearliest can detect on the onset of3disease day, and the longest on the onset of14day. Except for9,11and13disease days, detection rate of DENV-RNA test in theurine was higher than50%.7to8day of onset had the highest positive rate, reachingto73.2%（N=30/41）.3.3. Results of DENV-RNA detection in the urine at different disease courseIn disease course of the early, middle and late, detection rate of DENV-RNA inurine were57.1%,61.8%, and50%, respectively. The results showed in the middleof disease course, DENV-RNA detection in urine was higher.4. The comparison of detection results of DENV-RNA in urine and plasma4.1. The comparison of detection rate of DENV-RNA in urine and plasmaThe detection rate of DENV-RNA in plasma was96.1%. The detection rate ofDENV-RNA in urine was70.2%,with P value equal to0.000,which had a significantdifference in statistics of the rates. The results showed the detection rate of DENV nucleic acid in urine was lower than that of plasma. More than that, six patients whohad no plasma samples collected had a positive result in DENV-RNA detection inurine,so it showed the detection of DENV-RNA in urine can be a laboratorydiagnosis supplement compared to that of plasma in confirming Dengue fever.4.2. The comparison of detection rate of DENV-RNA in urine and plasma atdifferent disease course4.2.1. DENV-RNA in plasma can be detected as long as the12thdisease day, andwhen it comes to urine, the longest time can be14thday.4.2.2. Early in the course of the disease, the detection rate of DENV-RNA in theplasma (100%)was higher than that of the urine(57.1%),with P value equal to0.000,which had a significant difference in statistics of the rates. However, thedetection rate of DENV-RNA in urine was higher than that of plasma at the seventhday in the mid-time of disease. At late in the course of the disease, when plasmasamples were not collected, DENV-RNA detection in urine could be used asdiagnosis method.4.3Plasma negatie but DENV-RNA can be detected in urineOne patient and another patient on the twelfth day after being attacked, theirDENV-RNA detection in plasma were negative, but had a positive result ofDENV-RNA detection in urine.5. The analysis of related factors about DENV-RNA detection rate in urine.5.1The relationship between severity of dengue illness and the detection rate ofDENV-RNA in urine.DF and SDF patients, urine DENV-RNA detection rate were76.2%and53.3%respectively, with P value equal to0.183, which meant there was no significantdifference of the rates. Results showed that DF or SDF had nothing to do with thedetection rate of DENV-RNA in urine.5.2. The relationship between primary infection or secondary infection and thedetection rate of DENV-RNA in urine. The detection rate of DENV-RNA in urine of primary infection and secondaryinfection respectively were67.4%and75%. When we compared them, P value wasequal to0.994and had no significant difference in statistics. So, primary infection orsecondary infection had nothing to do with the detection rate of DENV-RNA inurine.5.3. The relationship between renal damage and the detection rate ofDENV-RNA in urine.Only42.9%of patients whose kidney damaged had positive results ofDENV-RNA detection in urine, and only22.2%of patients whose results ofDENV-RNA detection in urine was positive had renal damage. Thus, renal damagehad nothing to do with the detection rate of DENV-RNA in urine.Conclusions1. DENV-RNA can be detected in urine from dengue patients, and DENV-RNAdetection in urine was higher in the middle of disease course.2. RT-PCR is a kind of convenient, economical, safe laboratory diagnosis method todetect DENV-RNA in urine from dengue fever patients.