Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma

Hue, Kien Duong Thi; Tuan, Trung Vu; Nguyen, Thi Hanh Tien; Bich, Chau Tran Nguyen; Anh, Huy Huynh Le; Wills, Bridget; Simmons, Cameron P.

doi:10.1016/j.jviromet.2011.08.002

Cited by 103 publications

(97 citation statements)

References 24 publications

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“…Blood samples collected at admission (visit 1) of hospitalized patients were tested for DENV NS1 antigen by SD BIOLINE Dengue Duo rapid diagnostic kit (Standard Diagnostic) according to the manufacturer's recommendations. This test was performed by well-trained nurses at the patient's bedside and the result was confirmed at IPC by DENV RNA detection in the same blood sample using a serotype-specific multiplex qRT-PCR method (31). The qRT-PCR had a limit of detection of 0.5-3 plaque-forming units (PFU) and 5-10 cDNA copies per 20-μL reaction, depending on the serotype (31).…”

Section: Methodsmentioning

confidence: 99%

“…S3) (31). Patients with a confirmed DENV infection by NS1 antigen detection and/or qRT-PCR were considered dengue index cases (DICs) that initiated geographic cluster investigations.…”

Section: Methodsmentioning

confidence: 99%

“…All Ae. aegypti females that were used in human-tomosquito DENV transmission experiments were from laboratory-reared F 2 or F 3 generations derived from F 1 parental females that tested negative for DENV, JEV, and chikungunya virus (CHIKV) by virus-specific RT-PCR and for other potential flaviviruses by pan-flavivirus RT-PCR (31,(35)(36)(37).…”

Section: Methodsmentioning

confidence: 99%

“…All F 1 mosquitoes were sorted by sex in a Petri dish placed on ice, pooled into vials of 10 mosquitoes (male and female) per vial, and homogenized. Extracted RNA was tested for DENV, JEV, CHIKV, and other flaviviruses (31,(35)(36)(37).…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Asymptomatic humans transmit dengue virus to mosquitoes

Duong

Lambrechts

Rácz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

381

356

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Three-quarters of the estimated 390 million dengue virus (DENV) infections each year are clinically inapparent. People with inapparent dengue virus infections are generally considered dead-end hosts for transmission because they do not reach sufficiently high viremia levels to infect mosquitoes. Here, we show that, despite their lower average level of viremia, asymptomatic people can be infectious to mosquitoes. Moreover, at a given level of viremia, DENV-infected people with no detectable symptoms or before the onset of symptoms are significantly more infectious to mosquitoes than people with symptomatic infections. Because DENV viremic people without clinical symptoms may be exposed to more mosquitoes through their undisrupted daily routines than sick people and represent the bulk of DENV infections, our data indicate that they have the potential to contribute significantly more to virus transmission to mosquitoes than previously recognized.mosquito experimental infection | Cambodia | Aedes aegypti | human-to-mosquito transmission | dengue W ith 3.97 billion people living in 128 countries currently at risk for infection, dengue viruses (DENV-1 to -4) cause more human morbidity and mortality worldwide than any other arthropod-borne virus (1, 2). Aedes aegypti mosquitoes are the primary vectors of DENV throughout the tropics (3). Dengue prevention relies on the control of Ae. aegypti populations, which is failing in most parts of the world due to lack of resources, lack of political will, and/or ineffective implementation (4).Virus transmission from infected humans to mosquitoes is a critical step in dengue epidemiology, but due to logistical constraints it has been directly examined only in a handful of studies to date (5). In initial experimental infections of human volunteers during the 1920s (6, 7), the onset of clinical symptoms occurred 4-9 d after virus inoculation by mosquito bite (8). DENV-infected humans were infectious to mosquitoes from 2 d before to 2 d after the onset of symptoms, and Ae. aegypti fed on viremic people were able to transmit virus to another person after at least 11 d of extrinsic incubation (8). Results from later studies indicated that, for naturally infected people with clinically apparent dengue, the duration of detectable viremia was on average 4-5 d after the onset of symptoms, but could range from 2 to 12 d (9, 10). Investigators in Vietnam fed Ae. aegypti directly on 208 symptomatic, hospitalized dengue patients and reported that the probability of successful human-to-mosquito DENV transmission was coincident with the kinetics of viremia (11). Dengue patients were infectious up to 5 d after the onset of symptoms, which generally corresponded with "defervescence" (11).All previous studies on human-to-mosquito DENV transmission were limited to people with overt illness and did not consider subclinical infections. An estimated 300 million of the total 390 million DENV infections per year are clinically inapparent or mildly symptomatic, i.e., no illness that disrupted a person's ...

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Section: Methodsmentioning

confidence: 99%

“…S3) (31). Patients with a confirmed DENV infection by NS1 antigen detection and/or qRT-PCR were considered dengue index cases (DICs) that initiated geographic cluster investigations.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Asymptomatic humans transmit dengue virus to mosquitoes

Duong

Lambrechts

Rácz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

381

356

View full text Add to dashboard Cite

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“…IgM/IgG ratios <1.8 but >1.2 were called indeterminate. DENV plasma viremia levels were measured by a validated, quantitative RT-PCR assay that has been described previously (28) and used in recent clinical trials (29,30). In the validation process the RT-PCR assay was calibrated against infectious virus (grown in insect cells), and the ratio between genome copies per mL and plaque-forming units per mL was 214:1 for DENV-1, 73:1 for DENV-2, 436:1 for DENV-3, and 101:1 for DENV-4.…”

Section: Methodsmentioning

confidence: 99%

Host and viral features of human dengue cases shape the population of infected and infectious Aedes aegypti mosquitoes

Nguyen

Kien

Tuan

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Dengue is the most prevalent arboviral disease of humans. The host and virus variables associated with dengue virus (DENV) transmission from symptomatic dengue cases (n = 208) to Aedes aegypti mosquitoes during 407 independent exposure events was defined. The 50% mosquito infectious dose for each of DENV-1-4 ranged from 6.29 to 7.52 log10 RNA copies/mL of plasma. Increasing day of illness, declining viremia, and rising antibody titers were independently associated with reduced risk of DENV transmission. High early DENV plasma viremia levels in patients were a marker of the duration of human infectiousness, and blood meals containing high concentrations of DENV were positively associated with the prevalence of infectious mosquitoes 14 d after blood feeding. Ambulatory dengue cases had lower viremia levels compared with hospitalized dengue cases but nonetheless at levels predicted to be infectious to mosquitoes. These data define serotype-specific viremia levels that vaccines or drugs must inhibit to prevent DENV transmission.engue is globally the most important mosquito-borne viral disease of humans, with a global burden of ∼100 million cases per annum (1, 2). Aedes aegypti mosquitoes are the primary mosquito vectors of dengue viruses (DENV), of which there are four virus types (DENV-1-4). Multiple factors influence the likelihood of infection and dissemination of DENV in Ae. aegypti and include the amplitude of daily temperature fluctuations (3), mean temperature (4), and the genotype of mosquito and virus (5), among others (6). The extrinsic incubation period (EIP), a critical determinant of vector competence (7,8), is widely accepted to be 7-14 d for DENV in Ae. aegypti, although a recent modeling analysis of historical DENV transmission data has suggested a wider range of 2-15 d at 30°C (9). A major caveat to many of these observations is that they stem from laboratory experiments with artificially generated virus-spiked blood meals and often in-bred colony mosquitoes.The temporal and virological variables associated with the transmission of DENV from a naturally infected human to a biting Ae. aegypti mosquito are poorly understood. Natural history studies of experimental DENV infection of small cohorts of human volunteers in the 1920s by Siler et al. (10,11), likely using DENV-4 (12), and subsequent studies by Simmons et al. (13), likely using DENV-1 (12), suggested that the window of time before the onset of clinical symptoms that DENV-1 or DENV-4 could be transmitted to Ae. aegypti mosquitoes was 6-18 h or 2 d, respectively (14). After fever onset, the duration of infectiousness was 4-5 d for DENV-1 and up to 2 d for DENV-4, with an EIP in the mosquito of 10 d or more. Consistent with this, mosquitobiting studies by Gubler et al. in the 1960s (15-18) collectively estimated that dengue cases were infectious for 4-5 d after illness onset (range, 2-12 d). The human viremia level required to infect Ae. aegypti mosquitoes is unknown, and therefore it is uncertain what percentage of symptomatic (or asymptomatic...

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