Epidemiological survey and screening strategy for dengue virus in blood donors from Yunnan Province

The rapid expansion of dengue fever is an evolving worldwide public health threat. The World Health Organization (WHO) estimates the overall prevalence of dengue at about 3.9 billion infections in mosquito-prone regions in 128 countries world-wide [1]. Roughly 96 million (25%) of these are identified clinically, with an annual incidence of about 390 million new cases [1]. In Africa, dengue is mostly unquantified, but recent outbreaks suggest that large parts of the continent are at increased risk [2].

Dengue virus enters the human body through the bite of Aedes mosquitoes, replicates in capillary endothelial cells and the mononuclear macrophage system, then enters the bloodstream as viremia. After infection, DENV replicates in the human body for 3 to 14 days before symptoms appear [3]. Since more than 75% of people infected with DENV are asymptomatic, the prevalence of people actively infected with dengue fever during outbreaks increases the risk of DENV infection among adult blood donors. At present, the dengue virus (DENV) is listed as a transfusion- transmitted disease (TTD) by the international transfusion association, the AABB [4]. To reduce the risk of transfusion-transmission of DENV, routine nucleic acid testing (NAT) of blood donor samples is recommended by several high prevalence countries and regions, such as Honduras [5].

China has recorded three outbreaks of dengue hemorrhagic fever. Most recently, this has included the large outbreak that spread to Guangdong Province in 2014, with 6024 confirmed cases and 6 deaths [6, 7]. This outbreak has sparked increasing concerns about DENV infection throughout the country, particularly with regard to blood safety. In China, DENV has not been included in routine screening of blood donors. However, with the rapidly increased population flow due to tourism, DENV has been spreading quickly, and serologic evidence of dengue infection among blood donors has been documented in Guangdong and Guangxi provinces [3, 8].

Given that no effective antiviral agents to treat dengue infection are available and that transmission of DENV by blood donors in mainland China is rising, investigation of the prevalence of DENV in high-risk areas is imperative. This includes analyzing DENV-positive blood donors’ demographic characteristics and evaluating whether wide-range DENV antibody screening is needed.

Xishuangbanna Dai Autonomous Prefecture in Yunnan Province is a region close to Laos and other Southeast Asian countries where dengue is prevalent and outbreaks occur every other year. Potentially infected blood donors in this area pose a threat to blood safety nationwide. However, prior to the study reported here, no data on dengue virus infection among blood donors in Yunnan Province has been collected or reported. In this study, we cooperated with Yunnan Xishuangbanna Blood Center to investigate the prevalence of dengue virus among blood donors, and, based on our findings, to develop a DENV screening strategy for high prevalence seasons and in high prevalence areas.

In China, blood donation samples are routinely tested for HBV, HCV, HIV, and syphilis, but DENV has not been included in the routine donor testing. In the laboratory, testing for dengue is done using ELISA to screen for IgG/IgM, then confirmation using a colloidal gold method to test for NS1, and RT-PCR for specific RNA. Sensitivity of the NS1 test in the febrile phase of dengue can exceed 90% for primary infections, that is, those without prior infection, and antigenemia may persist for several days after the resolution of fever [10, 11].

In this study, we tested for evidence of DENV infection in blood samples from Xishuangbanna, a high-risk area of southwestern mainland China. Total serological prevalence was high at 26.53% (598 out of 2254) using anti-DENV-IgG and/or IgM ELISA. This prevalence may represent the highest serological prevalence ever reported [3, 7, 8]. Prevalence was significantly different between first-time and repeat donors, consistent with other pathogens such as HBV’s serological prevalence among blood donor categories [12, 13].

We also saw significant differences in the serological prevalence among occupational, educational, and ethnic groups. The serological prevalence for farmers and lower education-level donors was higher. We speculate that this occupation-related phenotype has something to do with the donors’ living environment where the risk of DENV transmission was high. In fact, DENV is mainly transmitted by the Aedes aegyptis, often found in residential areas, containers (such as tanks, basins, discarded tires, etc.), plant containers (such as bamboo tubes, tree holes, etc.), and stone pools. It is most likely that residential areas where farmers live are more suitable for Aedes aegypti.

A few donors were RNA and/or NS1 positive among IgG and/or IgM reactive donors. This may be due to the overlapping of various markers’ time of appearance in plasma after DENV infection [14]. RNA and NS1 of DENV are the earliest markers appearing in plasma—either can appear within the first 24 h in which patients become symptomatic—and lasting 5 to 7 days [14]. In contrast, IgG (secondary infection) and IgM appear in plasma after day 4 and last 6 days [14]. This result shows that NS1 can be detected at the same time as viral RNA. However, the detection of NS1 might be limited during secondary infection because of pre-induced adaptive immunity. Generally, the window period of nucleic acid detection is shorter than that of antibody detection. However, for dengue virus, the risk of missing the true positive case is high if there is only a nucleic acid test to screen for dengue virus prevalence because the nucleic acid disappears quickly from plasma: 590 out of 598 (98.66%) ELISA reactive samples were PCR negative. Our findings, together with others [15], indicate that to increase the likelihood of DENV detection and reduce the risk of blood transfusion-transmitted DENV, it may be essential to perform a joint test for of DENV RNA, NS1, and IgG/IgM antibodies.

Table 2 and Table 3 show that the donors who were RNA and/or NS1 positive were mostly farmers or other occupations with higher exposure to standing water. Efforts at mosquito control in the surrounding environment will help reduce DENV infection, but multilayer testing will be necessary to decrease blood transfusion-transmitted DENV.

The prevalence rate of DENV in Xishuangbanna Blood Center is higher than most other blood centers that have implemented DENV donor screening. In order to reduce the risk of transfusion-transmitted DENV, it may be necessary to propose a DENV screening strategy based on these findings in high prevalence seasons and high prevalence areas.