Patients and setting

This research was conducted at Hospital Sultanah Aminah Johor Bahru (HSAJB), a 989-bedded hospital that serves as the main tertiary referral centre of Southern Malaysia, with its patient population reflecting the larger community in Malaysia. The period of study was from January to April 2014, coinciding with one of the peaks of DENV outbreaks. During the study period, all hospitalized patients with a positive non-structural protein 1 (NS1) antigen were identified from the microbiology laboratory database, HSAJB. The initial NS1 testing was done at the microbiology laboratory of HSAJB, using a commercially available rapid dengue diagnostic kit; SD BIOLINE Dengue Duo combo device (Standard Diagnostic Inc., Korea). Secondary DENV infections were detected using Panbio Dengue IgG Capture ELISA, which has incorporated a cut-off value of > 22 Panbio Units, equivalent to HAI level of 1:2560, indicative of secondary infections [18].

Clinical data was retrospectively collected by reviewing the medical case notes, microbiology, haematology and biochemical laboratory results. The clinical data retrieved on admission included demography, vital signs, underlying comorbidities, signs and symptoms, haematological, liver and renal function parameters. Warning signs and severe dengue manifestations were recorded throughout the hospital stay. In addition, nadir platelet counts and results of dengue serology were also noted.

Approval was obtained from the Medical Research Ethics Committee, Ministry of Health Malaysia (NMRR-14-617-21061). Informed consent was not obtained from the patients, as this was a retrospective study and data was analyzed anonymously.

In total 290 patients (non-duplicate) with NS1 antigen positive were identified. Their serum samples were stored at -80 °C for further testing. These tests were conducted at an infectious diseases research laboratory at Monash University Malaysia.

Patients’ serum samples and extraction of viral DNA

Viral RNA was extracted from 200 μl of the original serum using QIAamp viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Extracted RNA was stored either at -80 °C or used for RT-PCR immediately. Complementary DNA (cDNA) from viral RNA was synthesized by reverse transcription using AccessQuick RT-PCR System kit (Promega, USA). The RT mixture consisted of 10 μl (20–50 ng) of extracted RNA, 1 unit of reverse transcriptase enzyme, 12.5 μl of AccessQuick mastermix (2x), 1 μl of random primer and 20 U of RNase inhibitor (RNaseOUT, Invitrogen) in a final volume of 20 μl. The RT mixture was incubated at 65 °C for 5 min (min) followed by 37 °C for 1 h (h) and 72 °C for 5 min. The prepared cDNA was used for multiplex PCR.

Multiplex PCR

DENV serotypes were determined using multiplex PCR [19], which amplified specific target regions using a forward conserved 5’UTR primer and four reverse primers targeting specific regions of the M and C genes of respective DENV-1, -2, -3 and -4 serotypes. To ensure the specificity of the primers to DENV and the absence of cross-reactivity with related flaviviruses, the primers were blasted through the National Centre for Biotechnology Information database [19]. Amplifications were performed as described and the expected size of each of the amplicons was as follows: DENV-1:342 bps, DENV-2: 251 bps, DENV-3: 538 bps and DENV-4: 754 bps. To perform PCR, a primer mix was prepared by mixing 400 nM of forward conserved primer and 200 nM of each reversed primer with appropriate volume of DEPC-treated distilled water. The premix was added to PCR buffer containing 1.5 mM MgCl_2, 0.2 mM of each of the dNTPs, 5U of Taq polymerase and 2 μl of viral cDNA. The thermal cycling profile of this assay consisted of 35 cycles of PCR at 95 °C denaturation for 30 s (s), 60 °C of annealing for 30 s and 72 °C extension for 1 min [19]. PCR contamination was avoided by spatially separating the RNA extraction, cDNA preparation and amplification steps. In order to detect possible contamination, a no template negative control was incorporated in all the PCR reactions.

All samples were also subjected to RT-PCR for Chikungunya virus based on its non-structural protein 1 (nsP1) and glycoprotein E1 (E1) genes [20]. Chikungunya virus infections are relatively common in Malaysia and can mimic DENV infections in clinical presentations. PCR was performed in a Mastercycler gradient machine (Eppendorf, Hamburg, Germany).

Gel elution and sequencing of amplicons

The detection and identification of DENV direct from serum samples by RT-PCR was accomplished based on the product size of the amplified-serotype specific amplicons by electrophoreses in a 1.5-2 % agarose gel stained with ethidium bromide. PCR products were cut from the gel, extracted using the QIAquick Gel Extraction kit (Qiagen, Germany) and were directly sequenced in both forward and reverse directions using the specific primers by a commercial sequencing services (First base, Singapore). Random amplicons of DENVs (DENV-1: 30, DENV-2: 30, DENV-3: 13 and DENV-4:1) were selected from the PCR reactions that showed both single and dual DENV infections. The identities of the sequences were confirmed by Basic Local Alignment Search Tool (BLAST). The sequences obtained in the present study and other sequences retrieved from the GenBank were aligned in ClustalW (2.1).

Virus propagation in C6/36 cells and total viral RNA extraction for next generation sequencing (NGS)

Confluent Aedes albopictus C6/36 monolayer cells were grown and maintained in minimum essential medium (MEM) supplemented with 2 % fetal bovine serum (FBS), HEPES buffer and 1 % penicillin/streptomycin (100 U/mL penicillin, 100 μg/mL streptomycin; Gibco®; USA). Virus isolation was performed by inoculating 50 μl of original serum onto C6/36 monolayer cells in Leighton tubes which were incubated at 30 °C for 7 to 10 days for growth of viruses. Viral RNA was extracted from 200 μl of the first blind passage of the serum-infected C6/36 culture supernatant and cDNA was synthesized using the method described above. The cDNA was used for multiplex PCR [19] and NGS. The amplicons derived from the multiplex PCR of supernatant of the first passage of the C6/36 infected cells were compared with those derived directly from serum. To confirm the DENV serotypes and to determine the heterogeneity of these viruses, random samples of five DENV-1 and six DENV-2 from mono-infected samples were subjected to NGS.

Whole genome sequencing of DENV

Synthesized cDNA was converted into double stranded DNA using NEBNext® mRNA Second Strand Synthesis Module (New England Biolabs, Ipwich, MA) according to the manufacturer’s instructions. The reaction was purified using Ampure bead XP (0.8× vol. ratio), normalized to 0.2 ng/uL based on Qubit quantification (Invitrogen, Carlsbad, CA) and tagmented with Nextera XT (Illumina, San Diego, CA) according to the manufacturer’s instructions for small insert size library. The constructed libraries were quantified, normalized and sequenced on the MiSeq sequencer located at the Monash University Malaysia Genomics Facility (run configuration of 2 × 150 bps paired-end read). Reference mapping to the complete genome of DENV was performed using MITObim version 1.8 (default setting) [21]. The assembled genomes of 6 DENV-2 and 5 DENV-1 (DENV-2: TM26, TM78, TM181, TM198, TM213, TM296; DENV-1: TM24, TM50, TM99, TM100, TM242) along with additional closely related genomes of DENV isolated from the South East Asia and Oceania regions were used to infer evolutionary relationship. Nucleotide alignment based whole genome sequence was performed using MAFFT v7.127b (default alignment setting) and a maximum likelihood phylogenetic tree was constructed using FastTree version 2.1.8 with the Jukes-Cantor + CAT model [22, 23]. Tree visualization and editing was performed using FigTree v1.4.1 (http://tree.bio.ed.ac.uk/software/figtree/). Further classification of genotypes in each serotype was determined using the Genotype Determination and Recombination Detection tool on Virus Pathogen Resource (http://www.viprbrc.org/brc/genotypeRecombination.spg?method=ShowCleanInputPage&decorator=flavi_dengue).

Definition

Based on population background study conducted in Malaysia, the haematocrit parameters used to evaluate haemoconcentration were >40 % in female adults, > 46 % in male ≤ 60 years, > 42 % in male > 60 years and > 38 % in children [28]. Leukopenia was defined as leukocyte count < 4,000/mm³ and thrombocytopenia as platelet count <150,000/mm³. Severe thrombocytopenia was referred to as platelet count < 50,000/mm³, a value shown to be associated with additional severe manifestations [7]. Paediatric patients were defined as patients aged less than 18 years. Secondary DENV infections categorization was based on the results of Panbio dengue IgG capture ELISA [18]. Pleural effusion or ascites was diagnosed based on conventional x-rays or ultrasound of the thorax and abdominal region. Diarrhoea was defined as the passage of three or more loose stools per day [28]. The simultaneous detection of more than one DENV serotypes was classified as co-infection, in contrast to mono-infection where only one DENV serotype was identified.

Data was analyzed using the Statistical Package for Social Sciences (SPSS version 20.0); comparing patients with and without DENV co-infections. To further pinpoint the variances in clinical and laboratory findings attributable to a particular serotype and its co-infection, subgroup analysis (DENV-1 and DENV-2 with its respective co-infection) was performed. Similar analysis was not conducted for DENV-3 and DENV-4 as the numbers were too small for valid statistical comparison.

Categorical variables were expressed as numbers and percentages and comparison amongst variables was determined by the Fisher’s exact test or Chi-squared test. Continuous variables were expressed as median ± interquartile range (IQR) and comparison was made using the non-parametric Mann–Whitney test. The odds ratio (OR) and its 95 % confidence intervals (CI) were calculated. The p-value < 0.05 (two-tailed) was taken as the level of significance. We then performed a multivariate logistic regression analysis by including clinical manifestations and laboratory parameters which were significant in univariate analysis (P < 0.05), to evaluate the factors independently associated with co-infections. To obtain more reliable results, variables with more than 5 % of missing data were excluded from the final model.

Serotype distribution and phylogenetic analysis of DENV

In total 290 non-duplicate NS1 antigen positive serum samples were identified during the study period. DENV serotypes were determined by multiplex RT-PCR directly from original serum samples and from the supernatant of the first passage of C6/36 serum infected cells. The results showed the amplicons generated from both the multiplex RT-PCR were consistent with each other. Ten of the 290 samples were PCR negative for DENV. Based on the primer designs [19] these 10 samples were also negative for other flaviviruses. All the 290 samples were negative for Chikungunya virus. Of the remaining 280 samples, single DENV serotypes indicating mono-infection were detected in 238 (85 %) samples, while dual serotypes indicating co-infection were found in 42 (15 %) samples.

Sequencing of representative amplicons from the mono and co-infected samples confirmed the serotype of each of the DENV. The consensus sequence of DENV-1 amplicon of size 342 bps, DENV-2 of 251 bps and DENV-3 of 538 bps were 95 % to 100 % similar for all the randomly selected 30 amplicons of DENV-1, 30 amplicons of DENV-2 and 13 amplicons of DENV-3 respectively.

Phylogenetic analysis based on whole genome sequence indicates some degrees of genomic heterogeneity among the DENV strains as evidenced by the terminal branch length in the maximum likelihood tree. The constructed phylogenomic tree exhibited the expected clustering of viral sequences based on their serotypes and genotypes (Fig. 1). All six DENV-2 isolates reported in this study fall within the same Cosmopolitan genotype and also share a common ancestry with strong bootstrap support (100 %) to DENV-2 virus isolated from regions located south of Peninsular Malaysia e.g. Singapore and Indonesia. This pattern of monophyletic clustering is also observed with the newly isolated DENV-1 virus but with a lower bootstrap support (60 %) in the DENV-1 clade with the exception of strain TM242, which is basal to the rest of the DENV-1 strains included in the phylogenomic analysis. Additionally, only TM242 has a different genotype-V while the other four reported DENV-1 strains are genotype-I. In both DENV-1 and DENV-2 lineages, the viral isolates from northern Southeast Asia countries such as Thailand, Cambodia and Vietnam are sister taxa to all or most viral isolates from Malaysia, Singapore and Indonesia, suggesting correlation with biogeography.

Demography and comorbidities

Baseline demographic and comorbidity data is shown in Table 1. The patients’ age ranged from 3 to 75 years (median 27.75 years). There were 38 (14.5 %) paediatric patients. Majority of the patients were Malays (59.9 %). Excluding 14 pregnancies, one-fifth of patients had at least one pre-existing comorbidity; diabetes mellitus being the commonest.

The impact of DENV co-infection on clinical and laboratory parameters on admission

Overall, the most commonly reported symptoms on admission were fever (99.2 %), vomiting (62.6 %), myalgia (59.5 %) and arthralgia (56.9 %). Particularly noteworthy was the presence of diarrhoea in almost half the patients (45.8 %). The median (IQR) duration of symptoms before hospitalization was 4 (1 to 10) days. Raised haematocrit, severe thrombocytopenia (platelets < 50,000/mm³) and leukopenia on admission were detected in 54.2 %, 15 % and 66.8 % of patients respectively. A substantial proportion of patients had elevated liver enzymes; 78.2 % and 61 % for AST and ALT respectively, with levels more than 10 times the normal upper limit noted in 5.5 % of patients. Elevated creatinine levels were observed in 13.5 % of patients. However, except for two patients, one with pre-existing renal failure, the creatinine levels were within 50 % of the upper limits of normal.

Multivariate analysis showed that the following clinical findings and laboratory results were independently associated with co-infections. Overall, diarrhoea was negatively associated with co-infections (OR: 0.326; 95 % CI: 0.149-0.711). Likewise, diarrhoea was less common in DENV-1 (OR: 0.339; 95 % CI: 0.151-0.764) and DENV-2 co-infections (OR: 0.24; 95 % CI: 0.077-0.753), compared to the mono-infected group. In addition, DENV-2 co-infected group had a higher frequency of patients with severe thrombocytopenia on admission (OR: 12.561; 95 % CI: 1.297-121.647), whereas the DENV-2 mono-infected group had a higher number of patients manifesting with myalgia (OR: 0.306; 95 % CI: 0.102-0.919). Although elevated creatinine levels were significantly higher in the co-infected group in univariate analysis, it was not subjected to multivariate analysis, as missing values were > 5 %.

Comorbidities and pregnancies were not associated with co-infection, both in overall and sub-group analysis. Likewise, no significant association with co-infection was elicited when each comorbidity (Table 1) was analyzed separately. Co-infection was not related to gender, ethnicity, age and secondary DENV infections. Similarly, there was no significant difference in the frequency of haemorrhagic manifestations and raised haematocrit between the two groups.

The impact of DENV co-infection on disease severity

In univariate analysis (Table 4), the presence of at least one warning sign was significantly higher amongst the co-infected group (OR: 2.89; 95 % C1: 0.99-8.50). Amongst the warning signs, co-infected patients were 12 times at increased risk of developing pleural effusion (OR: 12.22; 95 % C1: 2.16-69.19). A higher proportion of co-infected (15 %) patients had at least one severe dengue manifestation compared to mono-infected (5 %) patients (OR: 3.39; 95 % C1: 1.174-9.76). Except for severe bleeding, all the other entities of severe dengue appeared at higher frequency in the co-infected group. However, the difference was statistically significant only for fluid accumulation with respiratory distress (OR: 8.11; 95 % C1: 1.74-37.76). Compared to the mono-infected group, DENV-1 co-infected patients had a significantly higher numbers of pleural effusion (OR: 9.6; 95 % C1: 1.69-54.48), severe dengue manifestations (OR: 3.25; 95 % C1: 1.08-9.76) and fluid accumulation with respiratory distress (OR: 6.36; 95 % C1: 1.36-29.70). Warning signs, pleural effusion and fluid accumulation with respiratory distress were also significantly higher in the DENV-2 co-infected group.

Multivariate analysis revealed that pleural effusion (OR: 12.227; 95 % CI: 1.998 -74.817) and the presence of warning signs (OR: 3.143; 95 % CI: 1.047-9.429) were positively associated with co-infections. Subgroup analysis comparing DENV-2 mono and co-infections, revealed similar characteristics as above. Subgroup analysis comparing DENV-1 mono and co-infected patients, revealed that pleural effusion (OR: 11.824; 95 % CI: 1.936-72.203) was positively associated with co-infections. The associations in the multivariate analysis were maintained when adjusted for comorbidity and patients with secondary dengue infections.

We found no statistical differences between the two groups and its sub-groups in other manifestations of disease severity, such as hypotension, shock, admission to an intensive care unit, the need for mechanical ventilation or supplemental oxygen and hospitalization duration of > 3 days. The median (IQR) length of hospital stay was 3 (1–16) days for the mono-infected and 3 (1–18) days for the co-infected groups.

Strengths and limitations

The strength of this study is that it involved a large number of co-infected patients, allowing more reliable data interpretation. Moreover, since blood samples were collected during the acute phase of disease, the serum samples were NS1 antigen and RT-PCR positive, which permitted serotyping and viral isolation.

However, there are several limitations to this current study. All retrospective studies depend on the obtainability, accuracy and completeness of medical data. Therefore, the influence of certain clinical findings such as petechial rash and Hess’s test were difficult to determine as these tests/findings may not have been performed or recorded in medical charts. Moreover, this study had a relatively small number of patients with pleural effusion which may result in a low statistical power to detect true association. Chest x-rays were performed at the discretion of the attending clinicians, according to clinical findings. However, chest x-rays can only detect significant pleural effusions. The use of serial chest ultrasound can improve detection of small pleural effusions [39], although this may not be practical because of budgetary constraints and service restraints. However, despite this, all patients were managed with a standardized dengue clinical care management algorithm and laboratory and clinical parameters essential for patient monitoring were carefully recorded in the medical charts, which helped improve reliability of data collected. While a prospective study would be more reliable and accurate, the practicality of such a study is questionable because of the low numbers of co-infections in most reports. Finally, our study was conducted in a tertiary referral hospital and involved only hospitalized patients. Thus, these findings may not be generalized to patients with less severe manifestations, not necessitating hospitalization.