Detection Of West Nile Virus RNA Research Articles

Efficient Detection of West Nile Virus in Urine Specimens by a Novel In-House RT-qPCR Detection Kit.

West Nile Virus (WNV) infection represents a major global public health challenge. Even though most of the patients are asymptomatic, some cases progress to critical condition which may be fatal. Diagnosis traditionally relies on serological methods, but their limitations, including cross-reactivity, highlight the need for alternative approaches. Here, we present the development and validation of a novel RT-qPCR assay for precise and rapid detection of WNV RNA in urine, emerging as a promising specimen due to its noninvasive collection and high viral load. The assay demonstrates high efficiency and sensitivity, with a detection limit comparable to commercially available kits. This study highlights the importance of in-house kit design as a diagnostic tool in regions affected by emerging tropical infections, such as WNV, exemplified Cyprus. It emphasizes the critical role of low-cost, early detection with high sensitivity and specificity in infection control and surveillance efforts.

Diagnosis of West Nile virus infections: Evaluation of different laboratory methods.

The diagnosis of West Nile virus (WNV) is challenging due to short-term and low-level viremia, flavivirus cross-reactivity, and long immunoglobulin M (IgM) persistence. To evaluate different methods for WNV detection [reverse transcription-polymerase chain reaction (RT-PCR), IgM/IgG antibodies, IgG avidity] in serum, cerebrospinal fluid (CSF), and urine samples of patients with confirmed WNV infection. The study included patients with confirmed WNV neuroinvasive infection (n = 62), asymptomatic WNV seropositive individuals (n = 22), and individuals with false-positive WNV IgM antibodies (n = 30). WNV RNA was detected using RT-PCR. A commercial ELISA was used to detect WNV IgM/IgG antibodies with confirmation of cross-reactive samples using a virus neutralization test (VNT). IgG-positive samples were tested for IgG avidity. The WNV-RNA detection rates were significantly higher in the urine (54.5%)/serum (46.4%) than in CSF (32.2%). According to the sampling time, the WNV-RNA detection rates in urine collected within 7 days/8-14/≥ 15 days were 29.4/66.6/62.5% (P = 0.042). However, these differences were not observed in the CSF. The median RT-PCR cycle threshold values were significantly lower in urine (32.5, IQR = 28-34) than in CSF (34.5, IQR = 33-36). The frequency of positive WNV IgM and IgG significantly differed according to the sampling time in serum but not in CSF. Positive IgM/IgG antibodies were detected in 84.3/9.3% of serum samples collected within 7 days, 100/71.1% of samples collected 8-14, and 100% samples collected after ≥ 15 days. Recent WNV infection was confirmed by low/borderline avidity index (AI) in 13.6% of asymptomatic individuals. A correlation between ELISA and AI was strong negative for IgM and strong positive for IgG. No significant correlation between ELISA IgG and VNT was found. The frequency of WNV RNA and antibody detection depends on the sampling time and type of clinical samples. IgG avidity could differentiate recent WNV infections from long-persisting IgM antibodies.

West Nile virus in adults and larvae of Culiseta longiareolata and Culex hortensis (Diptera: Culicidae) captured in Hamedan, western Iran

West Nile virus (WNV) is an emerging arbovirus transmitted by mosquitoes. Although it is considered the most widespread mosquito-borne arbovirus in Iran, vectors of this zoonotic pathogen remain unknown in many regions. This study aimed to assess the presence of WNV in mosquitoes collected in the western city of Hamedan in 2022. Adult mosquitoes were captured using light traps, and mosquito larvae were collected by dipping technique from 45 diverse habitats, including urban, suburban, and rural sites. Specimens were identified and pooled into 69 batches based on their species for viral RNA extraction and Real-Time PCR. In total, 3243 mosquitoes (2209 larvae and 1034 adults) were captured and identified as Culiseta longiareolata, Culex hortensis, Anopheles maculipennis s.l., Culex theileri, Culex pipiens, Anopheles claviger, and Anopheles superpictus s.l. in decreasing order. Molecular screening revealed seven WNV-positive pools of Culiseta longiareolata and Culex hortensis in rural (n = 5) and urban areas (n = 2). Detection of WNV RNA indicates active circulation in mosquitoes and risk of transmission to humans and animals in Hamadan. These findings identify putative vectors in Hamadan, though vectors likely vary regionally in Iran. Further surveillance is needed to elucidate local WNV epidemiology and transmission dynamics fully. Nonetheless, this study provides important baseline evidence of WNV activity to guide prevention strategies in this area.

Whole-Blood PCR Preferred for Timely Diagnosis of Neuroinvasive West Nile Virus Infections: Lessons From the 2021 Arizona Outbreak.

In 2021, the state of Arizona experienced the largest focal outbreak of West Nile virus (WNV) in US history. Timely and accurate diagnostic testing remains a challenge for WNV due to transient viremia and limited immunoassay specificity. Recent studies have identified whole blood (WB) and urine as more sensitive specimen types for the detection of WNV RNA. We evaluated ordering practices, test performance, and patient characteristics of probable and confirmed cases. In total, we identified 190 probable and proven cases, including 127 patients (66.8%) with neuroinvasive disease. Among all cases, only 29.5% had WNV polymerase chain reaction (PCR) testing ordered on WB, of which 80.3% resulted as positive, including 7 cases in which WNV serologic testing was negative and 5 cases for which serologic testing was not ordered. In comparison, only 23.7% of cases that had cerebrospinal fluid (CSF) PCR ordered had a positive result, including 3 cases that were negative by PCR on WB. In contrast, WNV PCR on WB detected 12 neuroinvasive cases that were CSF PCR negative. WNV PCR testing in urine was only ordered on 2 patients, both of whom were positive. Crossing cycle threshold (Ct) values were not significantly different between WB and CSF specimen types, nor was there a correlation between Ct value and days from symptom onset at the time of sample collection; all specimen types and time points had Ct values, with 98% above 30. WB was positive by WNV PCR in several patients for >7 days (range, 7-25 days) after symptom onset, as was the CSF PCR. Taken together, these findings indicate that WNV PCR testing on WB may be the best initial test for timely diagnosis of WNV infection, irrespective of clinical manifestation; however, if negative in patients with suspected neuroinvasive disease, WNV PCR testing on CSF should be ordered.

The results of entomological monitoring West Nile fever on the territory of the Crimean peninsula in the 2022 season

Objective: to evaluate the role of blood-sucking mosquitoes of various species living in the northern and central regions of the Crimean Peninsula in the transmission of West Nile virus (WNV).Materials and methods: the collection of bloodsucking mosquitoes was carried out in 7 administrative units of the Republic of Crimea with automatic traps and a vertical handheld vacuum cleaner from July 1 to August 5, 2022. The species composition of mosquitoes was determined visually using an MSP-1 stereomicroscope (option 22) using standard keys. Detection of WNV RNA in the samples was performed by RT-PCR. Mosquito infection levels and dominance index were calculated.Results: a common eudominant species for urban and rural biotopes of the surveyed areas was determined Ae. caspius, whose dominance index was 72.4% and 31.3%. Mosquitoes of the species Cx. modestus were the most numerous in rural biotopes (dominance index — 48.4%). The invasive species Ae. albopictus in the central part of the Crimean peninsula, which indicates the expansion of its habitat. Of 18.6 thousand mosquitoes of 12 species, merged in 693 samples, WNV RNA was detected in 14 samples (2.02%), 3 isolates of the pathogen were isolated. A high level of WNV infection of mosquitoes in urban biotopes was established, which amounted to 4.12%. WNV circulation was first confirmed in the northern part of Crimea (Krasnoperekopsky district).Conclusion: the epidemiological significance of mosquitoes of various species on the territory of the Crimean Peninsula has been determined. The data on the spread of WNV in the Republic of Crimea were supplemented, taking into account which the estimated zoning of the territory was carried out according to the degree of risk of infection with WNV. Recommendations are given to strengthen epidemiological surveillance and preventive measures.

Detection and Analysis of West Nile Virus Structural Protein Genes in Animal or Bird Samples.

West Nile virus (WNV) is an important zoonotic pathogen, which is detected mainly by identification of its RNA using PCR. Genetic differentiation between WNV lineages is usually performed by complete genome sequencing, which is not available in many research and diagnostic laboratories. In this chapter, we describe a protocol for detection and analysis of WNV samples by sequencing the entire region of their structural genes capsid (C), preM/membrane, and envelope. The primary step is the detection of WNV RNA by quantitative PCR of the NS2A gene or the C gene regions. Next, the entire region containing the structural protein genes is amplified by PCR. The primary PCR product is then amplified again in parallel reactions, and these secondary PCR products are sequenced. Finally, bioinformatic analysis enables detection of mutations and classification of the samples of interest. This protocol is designed to be used by any laboratory equipped for endpoint and quantitative PCR. The sequencing can be performed either in-house or outsourced to a third-party service provider. This protocol may therefore be useful for rapid and affordable classification of WNV samples, obviating the need for complete genome sequencing.

The value of West Nile virus RNA detection by real-time RT-PCR in urine samples from patients with neuroinvasive forms.

Routine laboratory screening is based on the detection of WNV specific IgM and IgG in blood and cerebrospinal fluid. Confirmation is then classically applied by real timeRT-PCR (rRT-PCR)in Cerebrospinal fluid (CSF), which often gives negative results due to too short virorachia and late sampling.rRT-PCR was applied-for the first time for routine diagnosis purpose-on urine samples. During 2018 outbreak in Tunisia, 107 patients presented WNV neurologic symptoms and were positive for WNV serology. Of them, 95 patients were sampled for urine and 35 were sampled for CSF. Qualitative rRT-PCR was performed on both type of samples. WNV RNA was detected in 50.5% of urine samples (48/95) and in 2.8% of CSF samples (1/35). WNV RNA was detectable from day1 to day41from symptom onset, however, positive urine rate was 53.1% during the first 10days from symptom onset. The proportions of urine-positive and urine-negative samples, based on day of collection, showed no statistical difference (p > 0.005). Cycle threshold (Ct)values ranged from 12 to 39, with no correlation with the day of collection. The lowest Ctvalue was detected for urine sampled on day 5 after symptom onset. A statistically significant difference was found between age groups of confirmed and non confirmed cases (p < 0.001). Our study reported the use of rRT-PCR on urine samples as a confirmatory diagnostic tool for WNV "probable cases" during an outbreak. Our findings underlined the reliability and therapidity of this confirmatory tool, even late, and showed its superiority on CSF investigation.

First neuroinvasive human case of West Nile Disease in Southern Italy: Results of the ‘One Health’ approach

BackgroundWest Nile Disease (WND) is a zoonotic mosquito‐borne infection involving viral pathogens, human and animal hosts, vectors and environment. Cooperation among medical, veterinary and entomological fields has been promoted by the Italian Public Health Authorities, and an integrated West Nile Virus (WNV) Surveillance Plan has been in force in Italy since 2016 to prevent the transmission risk of WND to humans through an early detection of viral circulation by animal and entomological surveillance. This managing model is unique in Europe.ObjectivesThis survey aimed at presenting the ‘One Health’ approach applied in 2016 to the first autochthonous human case of West Nile Neuroinvasive Disease (WNND) in Sicily (Southern Italy).MethodsSerological (anti‐WNV IgM and IgG ELISA, anti‐WNV neutralizing antibodies) and molecular tests were conducted on blood, liquor and urine of a 38‐year‐old man with encephalitis and meningitis. Overall, 2704 adult culicides from 160 mosquito catches were morphologically identified. Female mosquitoes were analysed in pools for WNV RNA detection. Serological (anti‐WNV IgM and IgG ELISA) and molecular analyses for WNV were carried out in 11 horses, 271 chickens and two dogs sampled in farms around the man's residence.Results and conclusionsWNND was confirmed by serological analysis on patient's liquor and serum. Collected mosquito species included Culex pipiens (93.56%, CI95% 92.64%–94.49%), Aedes albopictus (5.25%, CI95% 4.41%–6.09%), Culex hortensis (0.59%, CI95% 0.30%–0.88%), Culiseta longiareolata (0.55%, CI95% 0.27%–0.83%) and Anopheles maculipennis s.l. (0.04%, CI95% –0.04% to 0.11%). Mosquito pools were negative for WNV RNA. Two dogs (100%) and two horses (18.18%, CI95% –4.61 to 40.97%) resulted positive for anti‐WNV specific antibodies.The ‘One Health’ approach allowed to report the first human neuroinvasive WND in Sicily and to confirm the local circulation of WNV in animals of the same area where the clinical case occurred, defining the autochthonous origin of the infection.

Optimizing PCR Detection of West Nile Virus from Body Fluid Specimens to Delineate Natural History in an Infected Human Cohort.

West Nile virus (WNV), a mosquito-borne arbovirus, remains a major global health concern. In this study, we optimized PCR methods then assessed serially-collected whole blood (WB), urine (UR), saliva, and semen specimens from a large cohort of WNV-positive participants to evaluate the natural history of infection and persistent shedding of WNV RNA. Viral RNA extraction protocols for frozen WB and UR specimens were optimized and validated through spiking experiments to maximize recovery of viral RNA from archived specimens and to assess the degradation of WNV RNA in stored UR specimens. The resultant procedures were used in conjunction with PCR detection to identify WNV-positive specimens and to quantify their viral loads. A total of 59 of 352 WB, 10 of 38 UR, and 2 of 34 saliva specimens tested positive for WNV RNA. Although a single semen specimen was positive 22 days post onset, we could not definitively confirm the presence of WNV RNA in the remaining specimens. WNV RNA-positive UR specimens exhibited profound loss of viral RNA during storage, highlighting the need for optimal preservation pre-storage. This study provides optimized methods for WNV RNA detection among different fluid types and offers alternative options for diagnostic testing during the acute stages of WNV.

West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells.

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus of global importance. Neuroinvasive WNV infection results in encephalitis and can lead to prolonged neurological impairment or death. Type I interferon (IFN-I) is crucial for promoting antiviral defenses through the induction of antiviral effectors, which function to restrict viral replication and spread. However, our understanding of the antiviral response to WNV infection is mostly derived from analysis of bulk cell populations. It is becoming increasingly apparent that substantial heterogeneity in cellular processes exists among individual cells, even within a seemingly homogenous cell population. Here, we present WNV-inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine the transcriptional variation and viral RNA burden across single cells. We observed that only a few cells within the bulk population displayed robust transcription of IFN-β mRNA, and this did not appear to depend on viral RNA abundance within the same cell. Furthermore, we observed considerable transcriptional heterogeneity in the IFN-I response, with genes displaying high unimodal and bimodal expression patterns. Broadly, IFN-stimulated genes negatively correlated with viral RNA abundance, corresponding with a precipitous decline in expression in cells with high viral RNA levels. Altogether, we demonstrated the feasibility and utility of WNV-inclusive scRNA-seq as a high-throughput technique for single-cell transcriptomics and WNV RNA detection. This approach can be implemented in other models to provide insights into the cellular features of protective immunity and identify novel therapeutic targets.IMPORTANCE West Nile virus (WNV) is a clinically relevant pathogen responsible for recurrent epidemics of neuroinvasive disease. Type I interferon is essential for promoting an antiviral response against WNV infection; however, it is unclear how heterogeneity in the antiviral response at the single-cell level impacts viral control. Specifically, conventional approaches lack the ability to distinguish differences across cells with varying viral abundance. The significance of our research is to demonstrate a new technique for studying WNV infection at the single-cell level. We discovered extensive variation in antiviral gene expression and viral abundance across cells. This protocol can be applied to primary cells or in vivo models to better understand the underlying cellular heterogeneity following WNV infection for the development of targeted therapeutic strategies.

Diagnosis of west Nile neuroinvasive disease in humans

West Nile virus (WNV) is arbovirus distributed all around the world. In humans, 80% of infection cases are asymptomatic. In 20% of infected people, a febrile self-limiting illness is reported. WNV has the potential for fatal neuroinvasive disease. In 1% of cases, the infection may result in neuroinvasive disease with permanent neurological consequences or death outcome. Neurological forms may vary presenting with encephalitis, meningitis, meningoencephalitis or acute flaccid paralysis. Outbreaks with neurological forms of WNV infection were recorded in different areas of Greece, Italy, Romania, Hungary and Serbia. During the period from 2013 to 2016, 114 samples of cerebrospinal fluid and 107 serum samples were taken from 114 patients suspected of WNV neuroinvasive disease (WNND). The presence of specific anti-WNV IgM and IgG antibodies in cerebrospinal fluid (CSF) and sera samples were tested by WNV IgM and IgG ELISA (Euroimmun, Germany). In addition, 48 samples of CSF or/and serum of people with suspected WNV infection were examined by commercial molecular tests - real time RT-PCR (WNV Real-TM, Sacace biotechnologies, Italy). The IgM antibodies against WNV were present in 25.4% (29/114) of CSF samples, and in 31.8% (34/107) of serum samples tested from 114 patients suspected of WNND. The IgG antibodies against WNV were detected in 3.5% (4/114) of CSF samples, and in 11.2% (12/107) of serum samples. The WNV RNA was detected by real time RT-PCR test in 7 out of 48 (14.6%) CSF or/and serum samples. In this study, detection of IgM antibodies in CSF is more frequent than detection of WNV RNA in CSF or serum samples. WNV RNA detection in CSF is confirmatory diagnostic test but has limited utility in the diagnosis of WNV neuroinvasive disease due to low viremia level at the time of clinical presentation of the disease. The limitations in the use of ELISA IgM test are linked to cross - reactivity among flaviviruses and long persistence of IgM antibodies in the serum and CSF.

Superiority of West Nile Virus RNA Detection in Whole Blood for Diagnosis of Acute Infection

The current diagnosis of West Nile virus (WNV) infection is primarily based on serology, since molecular identification of WNV RNA is unreliable due to the short viremia and absence of detectable virus in cerebrospinal fluid (CSF). Recent studies have shown that WNV RNA can be detected in urine for a longer period and at higher concentrations than in plasma. In this study, we examined the presence of WNV RNA in serum, plasma, whole-blood, CSF, and urine samples obtained from patients diagnosed with acute WNV infection during an outbreak which occurred in Israel in 2015. Our results demonstrate that 33 of 38 WNV patients had detectable WNV RNA in whole blood at the time of diagnosis, a higher rate than in any of the other sample types tested. Overall, whole blood was superior to all other samples, with 86.8% sensitivity, 100% specificity, 100% positive predictive value, and 83.9% negative predictive value. Interestingly, WNV viral load in urine was higher than in whole blood, CSF, serum, and plasma despite the lower sensitivity than that of whole blood. This study establishes the utility of whole blood in the routine diagnosis of acute WNV infection and suggests that it may provide the highest sensitivity for WNV RNA detection in suspected cases.

Laboratory and Clinical Characteristics of Human West Nile Virus Infections during 2011 Outbreak in Southern Greece

During the summer-autumn of 2011, a human outbreak of West Nile virus (WNV) infection occurred in southern Greece, following the first outbreak during 2010 in northern Greece. An investigation was performed to analyze laboratory diagnosis, geographic distribution, and clinical features of WNV cases in southern Greece. Serum and cerebrospinal fluid (CSF) specimens from all patients seeking laboratory diagnosis for suspected WNV infection were tested for the presence of specific WNV immunoglobulin M (IgM) and IgG antibodies. Detection of WNV RNA in CSF and whole blood samples was accomplished by real-time PCR. During August-October of 2011, 31 confirmed or probable cases of WNV infection were identified. In 25 of them, individuals experienced severe neurological manifestations and were classified as WNV neuroinvasive disease cases. Risk factors such as advanced age, hypertension, and diabetes mellitus were identified in most cases with neurological complications. As many as 25 of the WNV cases occurred in the broader region of Athens; the majority of them (17 cases) were identified in municipalities of Eastern Attica, located almost 40 km from the metropolitan area of Athens and 500 km from Central Macedonia, where the 2010 WNV outbreak occurred. The spread of the virus in a newly affected area of the country suggests that WNV has been established in Greece and disease transmission will be continued in the future.

Excretion of West Nile Virus in Urine During Acute Infection

Detection of West Nile virus (WNV) RNA in urine has been anecdotally described and proposed for the diagnosis of WNV infection. This study reports the routine use of real-time reverse-transcription polymerase chain reaction for the detection of WNV RNA in urine to support diagnosis of WNV infection during the large outbreak that occurred in northeastern Italy in 2012. Fourteen of 32 patients (43.8%) with symptomatic WNV infection, defined as neuroinvasive disease and fever, had detectable WNV RNA in urine at the time of diagnosis, at a higher rate and load and for a longer time than detection of WNV RNA in blood. Detection of WNV RNA in urine was less frequent (2 of 14 patients [14.2%]) in blood donors in whom WNV infection was identified by WNV nucleic acid amplification testing. Infectious virus was isolated from the urine of a patient with neuroinvasive disease and a high WNV RNA load in urine.

Virus and Antibody Dynamics in Acute West Nile Virus Infection

The dynamics of the early stages of West Nile virus (WNV) infection can be assessed by follow-up studies of viremic blood donors. A total of 245 donors with WNV viremia were followed up weekly for 4 weeks and then monthly for up to 6 additional months or until seroconversion. Plasma samples were tested for WNV RNA by transcription-mediated amplification (TMA) and for WNV-specific IgM and IgG antibodies. RNA persistence was investigated by 6 replicate TMA tests; samples that were viremic for >40 days were tested for WNV-neutralizing activity. Follow up of 35 additional viremic donors for up to 404 days was conducted to evaluate persistence of WNV-specific antibody. The median time from RNA detection to IgM seroconversion was 3.9 days; to IgG seroconversion, 7.7 days; to RNA negativity by single-replicate TMA, 13.2 days; and to RNA negativity by 6-replicate TMA, 6.1 additional days after results of single-replicate TMA are negative. For 4 donors in whom RNA persisted for >40 days after the index donation, all samples obtained after this threshold were also positive for WNV IgG and neutralizing activity. The mean times to IgM and IgA negativity were 156 and 220 days, respectively. IgM and IgG develop rapidly after viremia and before RNA levels become undetectable, which occurred a mean of 13.2 days after the index donation among donors in this study. WNV RNA detection by replicate TMA rarely persists for >40 days after the index donation and is accompanied by WNV-specific neutralizing antibody, consistent with an absence of WNV transmission via transfusion of seropositive blood components.

West Nile Virus Adheres to Human Red Blood Cells in Whole Blood

Background. West Nile virus (WNV) is endemic in the United States. It is transmissible by blood transfusion, and the nation's blood supply is currently screened for WNV. Documented transmission of WNV infection through red blood cell (RBC) units in which the plasma co-component had a low viral load could be explained, in at least 1 instance, by cell-association of WNV; in this case, the RBC unit was released as negative by minipool nucleic acid testing (NAT) performed on plasma but was intermittently NAT-positive when subsequently tested as an individual sample. We hypothesized that a proportion of WNV bound to blood cells and was not measured by NAT performed on plasma samples. We have investigated whether WNV binds to RBCs, leading to reduction of WNV RNA detection by NAT performed on plasma samples.Methods. Equal volumes of leukoreduced RBCs and their corresponding plasma components from 20 blood donors with NAT results that were positive for WNV were tested in 5 replicates by reverse-transcriptase polymerase chain reaction TaqMan for WNV. In addition, aliquots from 8 of the RBC units were tested by infectivity assays using Vero cells.Results. The reverse-transcriptase polymerase chain reaction TaqMan assay showed that the viral load in the RBC components exceeded that in the corresponding plasma units by 1 order of magnitude. In addition, viruses associated with the RBCs were infectious in Vero cell cultures.Conclusions. These observations reinforce the notion that extraction of viral RNA from whole blood could improve assay sensitivity for blood donor screening and further reduce the residual risk of WNV transmission through transfusion.

Collaborative study to evaluate a working reagent for West Nile virus RNA detection by nucleic acid testing

A nucleic acid test (NAT) assay reference reagent for West Nile virus (WNV) RNA, consisting of heat-inactivated WNV grown in tissue culture and diluted in pooled, negative human plasma, was evaluated and quantitated in a collaborative study in which 14 laboratories participated. Participants were requested to assay serial half-log and log dilutions of the reagent to determine the RNA endpoint. A single endpoint for each such dilution series was calculated with the maximum likelihood method, which assumes that the probability of a positive result at a given dilution follows a Poisson distribution. The calculated endpoint was used to give an estimated "NAT-detectable units per mL" (not necessarily equivalent to genome equivalents/mL or copies/mL). The assays used by participants included qualitative and quantitative NAT assays and both the commercial WNV assays (Chiron and Roche). The estimated number of detectable units per mL for the 14 laboratories varied from log 2.0 to 3.0 with the exception of two outliers. The overall mean titer for all the assays was log 2.52 detectable units per mL (330 detectable units/mL). Multiple testing of individual vials by two laboratories indicated that there was no evidence of vial-to-vial variation in WNV content of the reference reagent. A reference reagent for WNV NAT assays has been established. The mean titer of the reagent, with the results from 14 laboratories, was 330 detectable units per mL.

West Nile virus blood transfusion‐related infection despite nucleic acid testing

A case of West Nile virus (WNV) encephalitis associated with transfusion of blood that did not react when tested for WNV by minipool (MP) nucleic acid testing (NAT) is described. A Nebraska man developed clinical encephalitis 13 days after surgery and transfusion of 26 blood components. Antibody testing confirmed WNV infection. An investigation was initiated to determine the source of this infection. The patient's family members were interviewed to identify risk factors for WNV infection. Residual samples were retested for WNV RNA using transcription-mediated amplification (TMA) assay and two polymerase chain reaction (PCR) assays. Blood donors' follow-up serum samples were collected. All samples were tested for WNV-specific immunoglobulin M antibodies. The patient's family denied recent mosquito exposure. The 20 blood components collected after July 2003 did not react when tested for WNV in a six-member MP-NAT at the time of donation. Retrospective individual testing identified one sample as WNV-reactive by the TMA assay and one of the PCR assays. Seroconversion was demonstrated in the donor associated with this sample. WNV RNA detection by individual donation NAT demonstrates viremic blood escaping MP-NAT and supports transfusion-related WNV transmission. MP-NAT may not detect all WNV-infected blood donors, allowing WNV transmission to continue at low levels. WNV NAT assays might vary in sensitivity and pooling donations could further impact test performance. Understanding MP NAT limitations can improve strategies to maintain safety of the blood supply in the United States.