Transmission experiments support clade-level differences in the transmission and pathogenicity of Cambodian influenza A/H5N1 viruses

Influenza A/H5N1 has circulated in Asia since 2003 and is now enzootic in many countries in that region. In Cambodia, the virus has circulated since 2004 and has intermittently infected humans. During this period, we have noted differences in the rate of infections in humans, potentially associated with the circulation of different viral clades. In particular, a reassortant clade 1.1.2 virus emerged in early 2013 and was associated with a dramatic increase in infections of humans (34 cases) until it was replaced by a clade 2.3.2.1c virus in early 2014. In contrast, only one infection of a human has been reported in the 6 years since the clade 2.3.2.1c virus became the dominant circulating virus. We selected three viruses to represent the main viral clades that have circulated in Cambodia (clade 1.1.2, clade 1.1.2 reassortant, and clade 2.3.2.1c), and we conducted experiments to assess the virulence and transmissibility of these viruses in avian (chicken, duck) and mammalian (ferret) models. Our results suggest that the clade 2.3.2.1c virus is more “avian-like,” with high virulence in both ducks and chickens, but there is no evidence of aerosol transmission of the virus from ducks to ferrets. In contrast, the two clade 1 viruses were less virulent in experimentally infected and contact ducks. However, evidence of chicken-to-ferret aerosol transmission was observed for both clade 1 viruses. The transmission experiments provide insights into clade-level differences that might explain the variation in A/H5N1 infections of humans observed in Cambodia and other settings

Evaluation of the performances of six commercial kits designed for dengue NS1 and anti-dengue IgM, IgG and IgA detection in urine and saliva clinical specimens

Background: Rapid diagnostic tests (RDTs) have been commercialized in order to help physicians in dengue diagnosis. Until recently, only blood samples were used for those tests but it has been shown in several studies that urine and saliva can also be employed for dengue diagnosis. RDTs for the detection of NS1 antigen and anti-dengue IgG, IgM and IgA in urine and saliva specimens have thus been developed by Standard Diagnostics. The aim of this study was to evaluate the performances these new commercial assays. Methods: Two panels of clinical specimens were used: one for the evaluation of the NS1-detection devices and the second for the evaluation of the antibody-detection kits. Each panel consisted of urine and saliva specimens collected sequentially from 86 patients with a confirmed dengue infection. A total of 291 saliva and 440 urine samples were included in the NS1-evaluation panel and 530 saliva and 528 urine specimens constituted the antibody-evaluation panel. All samples were tested in parallel by in-house ELISAs and by the commercial RDTs. Results: The RDTs demonstrated an overall sensitivity of 15.5 %/27.9 %/10.7 % for NS1/IgG/IgA detection in urine samples and 20.4 %/ 34.8 %/11 %/6.2 % for NS1/IgG/IgM/IgA detection in saliva samples. Compared to the in-house NS1 ELISA, the results obtained with the NS1 RDT demonstrated a good correlation with urine samples (kappa coefficient: 0.88) but not with saliva specimens (kappa coefficient: 0.28). RDTs designed for antibody detection in saliva and urine were extremely specific (100 %), but less sensitive than the in-house ELISAs (i.e., reduction of the overall sensitivity by 12.2 % for the RDT designed for IgG detection in urine and by 23.7 % for the RDT detecting anti-DENV IgM in saliva). IgM were not detected in urine, either by RDT or ELISA. Conclusions: Although the RDTs evaluated here offer an apparently attractive approach for dengue diagnosis, this study suggests that these new commercial kits would require further improvement to increase the sensitivity

Additional file 2: of Evaluation of the performances of six commercial kits designed for dengue NS1 and anti-dengue IgM, IgG and IgA detection in urine and saliva clinical specimens

Concordance between RDTs and IPC ELISAs for NS1, anti-DENV IgG/IgM and anti-DENV IgA detection in saliva and urine. (PDF 29 kb

Additional file 1: of Evaluation of the performances of six commercial kits designed for dengue NS1 and anti-dengue IgM, IgG and IgA detection in urine and saliva clinical specimens

Characteristics of the rapid diagnostic tests for NS1, anti-DENV IgG/IgM and anti-DENV IgA detection in saliva and urine according to the manufacturer’s instructions. (PDF 87 kb

Value of Routine Dengue Diagnostic Tests in Urine and Saliva Specimens.

International audienceBackgroundDengue laboratory diagnosis is essentially based on detection of the virus, its components or antibodies directed against the virus in blood samples. Blood, however, may be difficult to draw in some patients, especially in children, and sampling during outbreak investigations or epidemiological studies may face logistical challenges or limited compliance to invasive procedures from subjects. The aim of this study was to assess the possibility of using saliva and urine samples instead of blood for dengue diagnosis.Methodology/Principal FindingsSerial plasma, urine and saliva samples were collected at several time-points between the day of admission to hospital until three months after the onset of fever in children with confirmed dengue disease. Quantitative RT-PCR, NS1 antigen capture and ELISA serology for anti-DENV antibody (IgG, IgM and IgA) detection were performed in parallel on the three body fluids. RT-PCR and NS1 tests demonstrated an overall sensitivity of 85.4%/63.4%, 41.6%/14.5% and 39%/28.3%, in plasma, urine and saliva specimens, respectively. When urine and saliva samples were collected at the same time-points and tested concurrently, the diagnostic sensitivity of RNA and NS1 detection assays was 69.1% and 34.4%, respectively. IgG/IgA detection assays had an overall sensitivity of 54.4%/37.4%, 38.5%/26.8% and 52.9%/28.6% in plasma, urine and saliva specimens, respectively. IgM were detected in 38.1% and 36% of the plasma and saliva samples but never in urine.ConclusionsAlthough the performances of the different diagnostic methods were not as good in saliva and urine as in plasma specimens, the results obtained by qRT-PCR and by anti-DENV antibody ELISA could well justify the use of these two body fluids to detect dengue infection in situations when the collection of blood specimens is not possible

Partial dependence plots for the most influential variables explaining NS1 antigen detection in plasma, urine and saliva.

<p>Partial dependence plots for the most influential variables explaining NS1 antigen detection in plasma, urine and saliva.</p

Partial dependence plots for the most influential variables explaining anti-DENV antibodies detection in plasma, urine and saliva.

<p>Partial dependence plots for the most influential variables explaining anti-DENV antibodies detection in plasma, urine and saliva.</p

Detection of anti-DENV antibodies in plasma, urine and saliva.

<p>Percentage of samples that tested positive by MAC-ELISA (A), AAC-ELISA (B) and IgG indirect ELISA (C) in urine, saliva and plasma samples according to time after onset of fever (D for day, W for week and M for month).</p

Mean viral load measured by qRT-PCR in plasma, urine and saliva.

<p>Mean viral load measured by qRT-PCR in plasma, urine and saliva.</p

Positivity rates of NS1 protein detection in plasma, urine and saliva.

<p>Percentage of samples that tested positive for NS1 protein by capture ELISA in urine, saliva and plasma, by day of sampling after onset of fever (n = 193, 197 and 217 patients with a confirmed dengue infection included for NS1 detection in urine, saliva and plasma, respectively).</p