The emergence and spread of Zika virus (ZIKV) presented a challenge to the diagnosis of ZIKV infections in areas with transmission of dengue (DENV) and chikungunya (CHIKV) viruses. To facilitate ...detection of ZIKV infections, and differentiate these infections from DENV and CHIKV, we developed the Trioplex real-time RT-PCR assay (Trioplex assay). Here, we describe the optimization of multiplex and singleplex formats of the assay for a variety of chemistries and instruments to facilitate global standardization and implementation. We evaluated the analytical performance of all Trioplex modalities for detection of these three pathogens in serum and whole blood, and for ZIKV in urine. The limit of detection for the three viruses and in different RNA-extraction modalities is near 10
genome copy equivalents per milliliter (GCE/mL). Simultaneous testing of more than one specimen type from each patient provides a 6.4% additional diagnostic sensitivity. Overall, the high sensitivity of the Trioplex assay demonstrates the utility of this assay ascertaining Zika cases.
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.
During the 2013 dengue epidemic in Luanda, Angola, 811 dengue rapid diagnostic test-positive cases were reported to the Ministry of Health. To better understand the magnitude of the epidemic and ...identify risk factors for dengue virus (DENV) infection, we conducted cluster surveys around households of case-patients and randomly selected households 6 weeks after the peak of the epidemic. Of 173 case cluster participants, 16 (9%) exhibited evidence of recent DENV infection. Of 247 random cluster participants, 25 (10%) had evidence of recent DENV infection. Of 13 recently infected participants who had a recent febrile illness, 7 (54%) had sought medical care, and 1 (14%) was hospitalized with symptoms consistent with severe dengue; however, none received a diagnosis of dengue. Behavior associated with protection from DENV infection included recent use of mosquito repellent or a bed net. These findings suggest that the 2013 dengue epidemic was larger than indicated by passive surveillance data.
We evaluated nucleic acid amplification testing (NAAT) for Zika virus on whole-blood specimens compared with NAAT on serum and urine specimens among asymptomatic pregnant women during the 2015-2016 ...Puerto Rico Zika outbreak. Using NAAT, more infections were detected in serum and urine than in whole blood specimens.
West Nile virus (WNV) was isolated from a human blood donor, a dead falcon, and mosquitoes in Puerto Rico in 2007. Phylogenetic analysis of the 4 isolates suggests a recent introduction of lineage I ...WNV that is closely related to WNV currently circulating in North America.
Background. Anti-dengue virus (DENV) immunoglobulin M (IgM) seroconversion has been the reference standard for dengue diagnosis. However, paired specimens are rarely obtained, and the interval for ...this testing negates its usefulness in guiding clinical case management. The presence of DENV viremia and appearance of IgM during the febrile phase of dengue provides the framework for dengue laboratory diagnosis by using a single serum specimen. Methods. Archived paired serum specimens (n = 1234) from patients with laboratory-confirmed dengue from 2005 through 2011 were used to determine the diagnostic performance of real-time reverse transcription polymerase chain reaction (RT-PCR), for detection of DENV serotypes 1–4, and enzyme-linked immunosorbent assays (ELISAs), for detection of DENV nonstructural protein 1 (NS1) antigen and anti-DENV IgM. Results. During 1–3 days after illness onset, real-time RT-PCR and NS1 antigen testing detected 82%–69% and 90%–84% of cases, respectively, as viremia levels declined, while anti-DENV IgM ELISA detected 5%–41% of cases as antibody appeared. Over the 10-day period of the febrile phase of dengue, the cumulative effect of using these 3 types of tests in a diagnostic algorithm confirmed ≥90% of dengue cases. Conclusions. The use of molecular or NS1 antigen tests to detect DENV and one to detect anti-DENV IgM in a single serum specimen collected during the first 10 days of illness accurately identified ≥90% of dengue primary and secondary cases.
We reconstructed the SARS-CoV-2 epidemic caused by Omicron variant in Puerto Rico by sampling genomes collected during October 2021-May 2022. Our study revealed that Omicron BA.1 emerged and replaced ...Delta as the predominant variant in December 2021. Increased transmission rates and a dynamic landscape of Omicron sublineage infections followed.
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.
Guillain-Barré syndrome (GBS) is an uncommon autoimmune disorder that follows infection or vaccination, and increased incidence has been reported during Zika virus (ZIKV) transmission. During the ...2016 ZIKV epidemic, the Puerto Rico Department of Health (PRDH) implemented the Enhanced GBS Surveillance System (EGBSSS). Here, we describe EGBSSS implementation and evaluate completeness, validity, and timeliness.
GBS cases were identified using passive surveillance and discharge diagnostic code for GBS. Completeness was evaluated by capture-recapture methods. Sensitivity and positive predictive value (PPV) for confirmed GBS cases were calculated for both case identification methods. Median time to completion of key time steps were compared by quarter (Q1-4) and hospital size.
A total of 122 confirmed GBS cases with onset of neurologic illness in 2016 were identified. Capture-recapture methodology estimated that four confirmed GBS cases were missed by both identification methods. Identification of cases by diagnostic code had a higher sensitivity than passive surveillance (89% vs. 80%), but a lower PPV (60% vs. 72%). There was a significant decrease from Q1 to Q3 in median time from hospital admission to case reporting (11 days vs. 2 days, p = 0.032) and from Q2 to Q3 in median time from specimen receipt to arbovirus laboratory test reporting (35 days vs. 26 days, p = 0.004).
EGBSSS provided complete, valid, and increasingly timely surveillance data, which guided public health action and supported healthcare providers during the ZIKV epidemic. This evaluation provides programmatic lessons for GBS surveillance and emergency response surveillance.
Dengue is a potentially fatal acute febrile illness caused by four mosquito-transmitted dengue viruses (DENV-1-4). Although dengue outbreaks regularly occur in many regions of the Pacific, little is ...known about dengue in the Republic of the Marshall Islands (RMI). To better understand dengue in RMI, we investigated an explosive outbreak that began in October 2011. Suspected cases were reported to the Ministry of Health, serum specimens were tested with a dengue rapid diagnostic test (RDT), and confirmatory testing was performed using RT-PCR and IgM ELISA. Laboratory-positive cases were defined by detection of DENV nonstructural protein 1 by RDT, DENV nucleic acid by RT-PCR, or anti-DENV IgM antibody by RDT or ELISA. Secondary infection was defined by detection of anti-DENV IgG antibody by ELISA in a laboratory-positive acute specimen. During the four months of the outbreak, 1,603 suspected dengue cases (3% of the RMI population) were reported. Of 867 (54%) laboratory-positive cases, 209 (24%) had dengue with warning signs, six (0.7%) had severe dengue, and none died. Dengue incidence was highest in residents of Majuro and individuals aged 10-29 years, and ∼95% of dengue cases were experiencing secondary infection. Only DENV-4 was detected by RT-PCR, which phylogenetic analysis demonstrated was most closely related to a virus previously identified in Southeast Asia. Cases of vertical DENV transmission, and DENV/Salmonella Typhi and DENV/Mycobacterium leprae co-infection were identified. Entomological surveys implicated water storage containers and discarded tires as the most important development sites for Aedes aegypti and Ae. albopictus, respectively. Although this is the first documented dengue outbreak in RMI, the age groups of cases and high prevalence of secondary infection demonstrate prior DENV circulation. Dengue surveillance should continue to be strengthened in RMI and throughout the Pacific to identify and rapidly respond to future outbreaks.
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