title: Impact of dengue virus (DENV) co-infection on clinical manifestations, disease severity and laboratory parameters.
abstract: The co-circulation of 4 DENV serotypes in geographically expanding area, has resulted in increasing occurrence of DENV co-infections. However, studies assessing the clinical impact of DENV co-infections have been scarce and have involved small number of patients. This study explores the impact of DENV co-infection on clinical manifestations and laboratory parameters.
  
  
  
  This retrospective study involved consecutive hospitalized patients with non-structural protein 1 (NS1) antigen positivity during an outbreak (Jan to April 2014). Multiplex RT-PCR was performed directly on NS1 positive serum samples to detect and determine the DENV serotypes. All PCR-positive serum samples were inoculated onto C6/36 cells. Multiplex PCR was repeated on the supernatant of the first blind passage of the serum-infected cells. Random samples of supernatant from the first passage of C6/36 infected cells were subjected to whole genome sequencing. Clinical and laboratory variables were compared between patients with and without DENV co-infections.
  
  
  
  Of the 290 NS1 positive serum samples, 280 were PCR positive for DENV. Medical notes of 262 patients were available for analysis. All 4 DENV serotypes were identified. Of the 262 patients, forty patients (15.3 %) had DENV co-infections: DENV-1/DENV-2(85 %), DENV-1/DENV-3 (12.5 %) and DENV-2/DENV-3 (2.5 %). Another 222 patients (84.7 %) were infected with single DENV serotype (mono-infection), with DENV- 1 (76.6 %) and DENV- 2 (19.8 %) predominating. Secondary dengue infections occurred in 31.3 % patients. Whole genome sequences of random samples representing DENV-1 and DENV-2 showed heterogeneity amongst the DENVs. Multivariate analysis revealed that pleural effusion and the presence of warning signs were significantly higher in the co-infected group, both in the overall and subgroup analysis. Diarrhoea was negatively associated with co-infection. Additionally, DENV-2 co-infected patients had higher frequency of patients with severe thrombocytopenia (platelet count < 50,000/mm(3)), whereas DENV-2 mono-infections presented more commonly with myalgia. Elevated creatinine levels were more frequent amongst the co-infected patients in univariate analysis. Haemoconcentration and haemorrhagic manifestations were not higher amongst the co-infected patients. Serotypes associated with severe dengue were: DENV-1 (n = 9), DENV-2 (n = 1), DENV-3 (n = 1) in mono-infected patients and DENV-1/DENV-2 (n = 5) and DENV-1/DENV-3 (n = 1) amongst the co-infected patients.
  
  
  
  DENV co-infections are not uncommon in a hyperendemic region and co-infected patients are skewed towards more severe clinical manifestations compared to mono-infected patients.
authors: Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway, 47500, Selangor, Malaysia. amreeta.dhanoa@monash.edu.
authors: Amreeta
authors: 0000-0002-4541-4819
authors: A
authors: Dhanoa
authors: Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway, 47500, Selangor, Malaysia.
authors: Sharifah Syed
authors: SS
authors: Hassan
authors: Clinical School Johor Bahru, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, 80100, Johor Bahru, Johor, Malaysia.
authors: Chin Fang
authors: CF
authors: Ngim
authors: Clinical School Johor Bahru, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, 80100, Johor Bahru, Johor, Malaysia.
authors: Chun Fatt
authors: CF
authors: Lau
authors: Clinical School Johor Bahru, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, 80100, Johor Bahru, Johor, Malaysia.
authors: Teik Seng
authors: TS
authors: Chan
authors: Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway, 47500, Selangor, Malaysia.
authors: Nur Amelia Azreen
authors: NA
authors: Adnan
authors: Monash University Malaysia Genomics Facility, Bandar Sunway, 47500, Selangor, Malaysia.|School of Science, Monash University Malaysia, Bandar Sunway, 47500, Selangor, Malaysia.
authors: Wilhelm Wei Han
authors: WW
authors: Eng
authors: Monash University Malaysia Genomics Facility, Bandar Sunway, 47500, Selangor, Malaysia.|School of Science, Monash University Malaysia, Bandar Sunway, 47500, Selangor, Malaysia.
authors: Han Ming
authors: HM
authors: Gan
authors: Department of Pathology, Hospital Sultanah Aminah Johor Bahru, 80100, Johor Bahru, Johor, Malaysia.
authors: Ganeswrie
authors: G
authors: Rajasekaram
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D002675:Child, Preschool; D060085:Coinfection / Q000453:epidemiology / Q000473:pathology / Q000821:virology; D003715:Dengue / Q000175:diagnosis / Q000453:epidemiology / Q000473:pathology* / Q000821:virology; D003716:Dengue Virus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D004196:Disease Outbreaks; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D015999:Multivariate Analysis; D016017:Odds Ratio; D010802:Phylogeny; D012367:RNA, Viral / Q000302:isolation & purification / Q000378:metabolism; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D065288:Serogroup; D012720:Severity of Illness Index; D017361:Viral Nonstructural Proteins / Q000235:genetics; D055815:Young Adult
chemical_list: C079340:NS1 protein, Dengue virus type 2; C546113:NS1 protein, Dengue virus type 3; C082515:NS1 protein, dengue-1 virus; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins
keywords: Clinical manifestations; Co-infection; DENV; Dengue virus; RT-PCR; Serotype