Gilberto A. Santiago

Dengue viremia in blood donors identified by RNA and detection of dengue transfusion transmission during the 2007 dengue outbreak in Puerto Rico

BACKGROUND: In 2007, a total of 10,508 suspected dengue cases were reported in Puerto Rico. Blood donations were tested for dengue virus (DENV) RNA and recipients of RNA‐positive donations traced to assess transfusion transmission.

Persistence of Zika Virus in Body Fluids — Preliminary Report

Background To estimate the frequency and duration of detectable Zika virus (ZIKV) RNA in human body fluids, we prospectively assessed a cohort of recently infected participants in Puerto Rico. Methods We evaluated samples obtained from 295 participants (including 94 men who provided semen specimens) in whom ZIKV RNA was detected on reverse‐transcriptase–polymerase‐chain‐reaction (RT‐PCR) assay in urine or blood at an enhanced arboviral clinical surveillance site. We collected serum, urine, saliva, semen, and vaginal secretions weekly for the first month and at 2, 4, and 6 months. All specimens were tested by means of RT‐PCR, and serum was tested with the use of anti–ZIKV IgM enzyme‐linked immunosorbent assay. Among the participants with ZIKV RNA in any specimen at week 4, collection continued every 2 weeks thereafter until all specimens tested negative. We used parametric Weibull regression models to estimate the time until the loss of ZIKV RNA detection in each body fluid and reported the findings in medians and 95th percentiles. Results The medians and 95th percentiles for the time until the loss of ZIKV RNA detection were 15 days (95% confidence interval [CI], 14 to 17) and 41 days (95% CI, 37 to 44), respectively, in serum; 11 days (95% CI, 9 to 12) and 34 days (95% CI, 30 to 38) in urine; and 42 days (95% CI, 35 to 50) and 120 days (95% CI, 100 to 139) in semen. Less than 5% of participants had detectable ZIKV RNA in saliva or vaginal secretions. Conclusions The prolonged time until ZIKV RNA clearance in serum in this study may have implications for the diagnosis and prevention of ZIKV infection. In 95% of the men in this study, ZIKV RNA was cleared from semen after approximately 4 months. (Funded by the Centers for Disease Control and Prevention.)BACKGROUND To estimate the frequency and duration of detectable Zika virus (ZIKV) RNA in human body fluids, we prospectively assessed a cohort of newly infected participants in Puerto Rico. METHODS We evaluated samples obtained from 150 participants (including 55 men) in whom ZIKV RNA was detected on reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay in urine or blood in an enhanced arboviral clinical surveillance site. We collected serum, urine, saliva, semen, and vaginal secretions weekly for the first month and then at 2, 4, and 6 months. All specimens were tested by means of RT-PCR, and serum was tested with the use of anti-ZIKV IgM enzyme-linked immunosorbent assay. Among the participants with ZIKV RNA in any specimen at week 4, biweekly collection continued until all specimens tested negative. We used parametric Weibull regression models to estimate the time until the loss of ZIKV RNA detection in each body fluid and reported the findings in medians and 95th percentiles. RESULTS The medians and 95th percentiles for the time until the loss of ZIKV RNA detection were 14 days (95% confidence interval [CI], 11 to 17) and 54 days (95% CI, 43 to 64), respectively, in serum; 8 days (95% CI, 6 to 10) and 39 days (95% CI, 31 to 47) in urine; and 34 days (95% CI, 28 to 41) and 81 days (95% CI, 64 to 98) in semen. Few participants had detectable ZIKV RNA in saliva or vaginal secretions. CONCLUSIONS The prolonged time until ZIKV RNA clearance in serum in this study may have implications for the diagnosis and prevention of ZIKV infection. Current sexual-prevention guidelines recommend that men use condoms or abstain from sex for 6 months after ZIKV exposure; in 95% of the men in this study, ZIKV RNA was cleared from semen after about 3 months. (Funded by the Centers for Disease Control and Prevention.).

Analytical and Clinical Performance of the CDC Real Time RT-PCR Assay for Detection and Typing of Dengue Virus

Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV). There are four genetically distinct DENVs (DENV-1–4) that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1–4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA) for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1–4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1–4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.

Prolonged Detection of Zika Virus RNA in Pregnant Women.

OBJECTIVE: Zika virus infection during pregnancy is a cause of microcephaly and other fetal brain abnormalities. Reports indicate that the duration of detectable viral RNA in serum after symptom onset is brief. In a recent case report involving a severely affected fetus, Zika virus RNA was detected in maternal serum 10 weeks after symptom onset, longer than the duration of RNA detection in serum previously reported. This report summarizes the clinical and laboratory characteristics of pregnant women with prolonged detection of Zika virus RNA in serum that were reported to the U.S. Zika Pregnancy Registry. METHODS: Data were obtained from the U.S. Zika Pregnancy Registry, an enhanced surveillance system of pregnant women with laboratory evidence of confirmed or possible Zika virus infection. For this case series, we defined prolonged detection of Zika virus RNA as Zika virus RNA detection in serum by real-time reverse transcription-polymerase chain reaction (RT-PCR) 14 or more days after symptom onset or, for women not reporting signs or symptoms consistent with Zika virus disease (asymptomatic), 21 or more days after last possible exposure to Zika virus. RESULTS: Prolonged Zika virus RNA detection in serum was identified in four symptomatic pregnant women up to 46 days after symptom onset and in one asymptomatic pregnant woman 53 days postexposure. Among the five pregnancies, one pregnancy had evidence of fetal Zika virus infection confirmed by histopathologic examination of fetal tissue, three pregnancies resulted in live births of apparently healthy neonates with no reported abnormalities, and one pregnancy is ongoing. CONCLUSION: Zika virus RNA was detected in the serum of five pregnant women beyond the previously estimated timeframe. Additional real-time RT-PCR testing of pregnant women might provide more data about prolonged detection of Zika virus RNA and the possible diagnostic, epidemiologic, and clinical implications for pregnant women.

Virus-Specific Differences in Rates of Disease during the 2010 Dengue Epidemic in Puerto Rico

Background Dengue is a potentially fatal acute febrile illness (AFI) caused by four mosquito-transmitted dengue viruses (DENV-1–4) that are endemic in Puerto Rico. In January 2010, the number of suspected dengue cases reported to the passive dengue surveillance system exceeded the epidemic threshold and an epidemic was declared soon after. Methodology/Principal Findings To characterize the epidemic, surveillance and laboratory diagnostic data were compiled. A suspected case was a dengue-like AFI in a person reported by a health care provider with or without a specimen submitted for diagnostic testing. Laboratory-positive cases had: (i) DENV nucleic acid detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in an acute serum specimen; (ii) anti-DENV IgM antibody detected by ELISA in any serum specimen; or (iii) DENV antigen or nucleic acid detected in an autopsy-tissue specimen. In 2010, a total of 26,766 suspected dengue cases (7.2 per 1,000 residents) were identified, of which 46.6% were laboratory-positive. Of 7,426 RT-PCR-positive specimens, DENV-1 (69.0%) and DENV-4 (23.6%) were detected more frequently than DENV-2 (7.3%) and DENV-3 (<0.1%). Nearly half (47.1%) of all laboratory-positive cases were adults, 49.7% had dengue with warning signs, 11.1% had severe dengue, and 40 died. Approximately 21% of cases were primary DENV infections, and 1–4 year olds were the only age group for which primary infection was more common than secondary. Individuals infected with DENV-1 were 4.2 (95% confidence interval [CI]: 1.7–9.8) and 4.0 (95% CI: 2.4–6.5) times more likely to have primary infection than those infected with DENV-2 or -4, respectively. Conclusions/Significance This epidemic was long in duration and yielded the highest incidence of reported dengue cases and deaths since surveillance began in Puerto Rico in the late 1960s. This epidemic re-emphasizes the need for more effective primary prevention interventions to reduce the morbidity and mortality of dengue.

Highly Sensitive Detection of Dengue Virus Nucleic Acid in Samples from Clinically Ill Patients

ABSTRACT Dengue virus (DENV) is a major cause of febrile illness and hemorrhagic fever in tropical and subtropical regions. Typically, patients presenting with acute dengue disease are viremic but may not have yet developed detectable titers of antibody. Therefore, early diagnosis depends mostly on detection of viral components, such as the RNA. To define the potential use of transcription-mediated amplification (TMA) DENV RNA as a diagnostic tool, we first compared its analytic sensitivity using a routine real-time reverse transcription (RT)-PCR and found that TMA is approximately 10 to 100 times more sensitive. In addition, we tested acute-phase serum samples (<5 days post-symptom onset) submitted as part of laboratory-based surveillance in Puerto Rico and determined that among patients with serologically confirmed dengue infection, TMA detected DENV RNA in almost 80% of serum specimens that were negative by the RT-PCR test used for diagnosis and in all specimens with positive RT-PCR results. We conclude that TMA is a highly sensitive method which can detect DENV RNA in approximately 89% of clinical, acute-phase serum specimens.

Endurance, refuge, and reemergence of dengue virus type 2, Puerto Rico, 1986-2007.

To study the evolution of dengue virus (DENV) serotype 2 in Puerto Rico, we examined the genetic composition and diversity of 160 DENV-2 genomes obtained through 22 consecutive years of sampling. A clade replacement took place in 1994–1997 during a period of high incidence of autochthonous DENV-2 and frequent, short-lived reintroductions of foreign DENV-2. This unique clade replacement was complete just before DENV-3 emerged. By temporally and geographically defining DENV-2 lineages, we describe a refuge of this virus through 4 years of low genome diversity. Our analyses may explain the long-term endurance of DENV-2 despite great epidemiologic changes in disease incidence and serotype distribution.

A cluster of dengue cases in American missionaries returning from Haiti, 2010.

Dengue is an acute febrile illness caused by four mosquito-borne dengue viruses (DENV-1 to -4) that are endemic throughout the tropics. After returning from a 1-week missionary trip to Haiti in October of 2010, 5 of 28 (18%) travelers were hospitalized for dengue-like illness. All travelers were invited to submit serum specimens and complete questionnaires on pre-travel preparations, mosquito avoidance practices, and activities during travel. DENV infection was confirmed in seven (25%) travelers, including all travelers that were hospitalized. Viral sequencing revealed closest homology to a 2007 DENV-1 isolate from the Dominican Republic. Although most (88%) travelers had a pre-travel healthcare visit, only one-quarter knew that dengue is a risk in Haiti, and one-quarter regularly used insect repellent. This report confirms recent DENV transmission in Haiti. Travelers to DENV-endemic areas should receive dengue education during pre-travel health consultations, follow mosquito avoidance recommendations, and seek medical care for febrile illness during or after travel.

Broad neutralization of wild-type dengue virus isolates following immunization in monkeys with a tetravalent dengue vaccine based on chimeric Yellow Fever 17D/Dengue viruses

The objective of the study was to evaluate if the antibodies elicited after immunization with a tetravalent dengue vaccine, based on chimeric yellow fever 17D/dengue viruses, can neutralize a large range of dengue viruses (DENV). A panel of 82 DENVs was developed from viruses collected primarily during the last decade in 30 countries and included the four serotypes and the majority of existing genotypes. Viruses were isolated and minimally amplified before evaluation against a tetravalent polyclonal serum generated during vaccine preclinical evaluation in monkey, a model in which protection efficacy of this vaccine has been previously demonstrated (Guirakhoo et al., 2004). Neutralization was observed across all the DENV serotypes, genotypes, geographical origins and isolation years. These data indicate that antibodies elicited after immunization with this dengue vaccine candidate should widely protect against infection with contemporary DENV lineages circulating in endemic countries.

Dengue Virus: Isolation, Propagation, Quantification, and Storage

Dengue is a disease caused by infection with one of the four dengue virus serotypes (DENV‐1, ‐2, ‐3, and ‐4). The virus is transmitted to humans by Aedes sp. mosquitoes. This enveloped virus contains a positive single‐stranded RNA genome. Clinical manifestations of dengue can have a wide range of outcomes varying from a mild febrile illness to a life‐threatening condition. New techniques have largely replaced the use of DENV isolation in disease diagnosis. However, virus isolation still serves as the gold standard for detection and serotyping of DENV and is common practice in research and reference laboratories where clinical isolates of the virus are characterized and sequenced, or used for a variety of research experiments. Isolation of DENV from clinical samples can be achieved in mammalian and mosquito cells or by inoculation of mosquitoes. The experimental methods presented here describe the most common procedures used for the isolation, serotyping, propagation, and quantification of DENV. Curr. Protoc. Microbiol. 27:15D.2.1‐15D.2.24.