Development of a novel serological assay for the detection of mpox infection in vaccinated populations.

In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 104 nonendemic locations worldwide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.

Three Years of Shared Service HIV Nucleic Acid Testing for Public Health Laboratories: Worthwhile for HIV-1 but Not for HIV-2.

BACKGROUND: In 2016, HIV-2 nucleic acid testing (NAT) was added to a shared service program that conducts HIV-1 NAT for public health laboratories performing the recommended algorithm for diagnosing HIV. Here, we evaluate the usefulness of HIV-2 NAT in this program as compared with HIV-1 NAT. METHODS: Specimens eligible for HIV-1 NAT were reactive on an HIV-1/2 antibody or antigen/antibody initial test and nonreactive or indeterminate on a supplemental antibody test or were reactive for HIV-1 antigen-only on an HIV-1/2 antigen/antibody initial test. Specimens eligible for HIV-2 NAT were reactive on an initial test, HIV-2 indeterminate or HIV indeterminate on a supplemental antibody test and had no detectable HIV-1 RNA or were reactive for HIV-2 antibody on an HIV-1/2 antigen/antibody test, and this reactivity was not confirmed with a supplemental antibody assay. All specimens were tested in a reference laboratory using APTIMA HIV-1 qualitative RNA and/or a validated qualitative HIV-2 RNA real-time PCR assay. RESULTS: During 2016 to 2019, HIV-1 RNA was detected in 234 (14%) of 1731 specimens tested. HIV-2 RNA was not detected in 52 specimens tested. Median time from specimen collection to reporting of HIV-1 and HIV-2 NAT results by year ranged from 9 to 10 days and from 22 to 27 days, respectively. Two specimens with HIV-2 indeterminate results on a supplemental antibody test had detectable HIV-1 RNA. CONCLUSIONS: A shared service model for HIV-1 NAT is both feasible and beneficial for public health laboratories. However, because no HIV-2 infections were detected, our data suggest that this program should reconsider the usefulness of HIV-2 NAT testing.

Parameters of Mosquito-Enhanced West Nile Virus Infection.

UNLABELLED: The arthropod-borne West Nile virus (WNV) emerged in New York State in 1999 and quickly spread throughout the United States. Transmission is maintained in an enzootic cycle in which infected mosquitoes transmit the virus to susceptible hosts during probing and feeding. Arthropod-derived components within the viral inoculum are increasingly acknowledged to play a role in infection of vertebrate hosts. We previously showed that Culex tarsalis mosquito saliva and salivary gland extract (SGE) enhance the in vivo replication of WNV. Here, we characterized the effective dose, timing, and proximity of saliva and SGE administration necessary for enhancement of WNV viremia using a mouse model. Mosquito saliva and SGE enhanced viremia in a dose-dependent manner, and a single mosquito bite or as little as 0.01 µg of SGE was effective at enhancing viremia, suggesting a potent active salivary factor. Viremia was enhanced when SGE was injected in the same location as virus inoculation from 24 h before virus inoculation through 12 h after virus inoculation. These results were confirmed with mosquito saliva deposited by uninfected mosquitoes. When salivary treatment and virus inoculation were spatially separated, viremia was not enhanced. In summary, the effects of mosquito saliva and SGE were potent, long lasting, and localized, and these studies have implications for virus transmission in nature, where vertebrate hosts are fed upon by both infected and uninfected mosquitoes over time. Furthermore, our model provides a robust system to identify the salivary factor(s) responsible for enhancement of WNV replication. IMPORTANCE: Mosquito-borne viruses are a significant class of agents causing emerging infectious diseases. WNV has caused over 18,000 cases of neuroinvasive disease in the United States since its emergence. We have shown that Culex tarsalis mosquito saliva and SGE enhance the replication of WNV. We now demonstrate that saliva and SGE have potent, long-lasting, and localized effects. Our model provides a robust system to identify the salivary factor(s) and characterize the mechanism responsible for enhancement of WNV replication. These studies could lead to the identification of novel prophylactic or treatment options useful in limiting the spread of WNV, other mosquito-borne viruses, and the diseases that they cause.

Two human immunodeficiency virus Type 2 cases in US blood donors including serologic, molecular, and genomic characterization of an epidemiologically unusual case.

BACKGROUND: Blood donation screening for human immunodeficiency virus Type 2 (HIV-2) has been in place in the United States since 1992. However, only three HIV-2 antibody-positive donors have been reported to date, all detected via HIV-1 cross-reactivity. STUDY DESIGN AND METHODS: Here we identify two additional HIV-2-positive donors by routine anti-HIV-1 and anti-HIV-2 screening, including a first-time male donor living in Georgia having recently immigrated to the United States from West Africa (from a 1998 donation) and a Taiwanese female repeat donor (nurse) living in California with no travel outside of Taiwan or apparent connections to West Africa (from a 2015 donation). Neither donor acknowledged any risk factors, and both remained asymptomatic through follow-up. The second donor was further investigated by serologic, molecular, and genomic assays because of her unusual demographics. She was documented to harbor HIV-2 RNA, albeit sporadically by HIV-2-specific nucleic acid tests (35%-100% of replicates) and at very low levels (<9.6 IU/mL). Metagenomic next-generation sequencing (mNGS) confirmed the identification of a Group B HIV-2 strain, with recovered reads covering 46.9% of the predicted genome. CONCLUSIONS: The estimated frequency of an HIV-2-positive blood donor in the United States is one in 57 million donations. Due to the low frequency and low pathogenicity of HIV-2, public health and blood donation screening efforts must focus on HIV-1 detection and prevention. However, detection of HIV-2 infection in a donor with no apparent link to West Africa suggests that the United States must remain vigilant for HIV-2 virus infections. Ultradeep mNGS may be useful in the future for comprehensive identification of rare transfusion-transmissible agents.

Quantitating SARS-CoV-2 Neutralizing Antibodies from Human Dried Blood Spots.

Background: In the earliest days of COVID-19 pandemic, the collection of dried blood spots (DBS) enabled public health laboratories to undertake population-scale seroprevalence studies to estimate rates of SARS-CoV-2 exposure. With SARS-CoV-2 seropositivity levels now estimated to exceed 94% in the United States, attention has turned to using DBS to assess functional (neutralizing) antibodies within cohorts of interest. Methods: Contrived DBS eluates from convalescent, fully vaccinated and pre-COVID-19 serum samples were evaluated in SARS-CoV-2 plaque reduction neutralization titer (PRNT) assays, a SARS-CoV-2 specific 8-plex microsphere immunoassay, a cell-based pseudovirus assay, and two different spike-ACE2 inhibition assays, an in-house Luminex-based RBD-ACE2 inhibition assay and a commercial real-time PCR-based inhibition assay (NAB-Sure™). Results: DBS eluates from convalescent individuals were compatible with the spike-ACE2 inhibition assays, but not cell-based pseudovirus assays or PRNT. However, the insensitivity of cell-based pseudovirus assays was overcome with DBS eluates from vaccinated individuals with high SARS-CoV-2 antibody titers. Conclusion: SARS-CoV-2 neutralizing titers can be derived with confidence from DBS eluates, thereby opening the door to the use of these biospecimens for the analysis of vulnerable populations and normally hard to reach communities.

Demographic and Clinical Factors Associated With SARS-CoV-2 Spike 1 Antibody Response Among Vaccinated US Adults: the C4R Study.

This study investigates correlates of anti-S1 antibody response following COVID-19 vaccination in a U.S. population-based meta-cohort of adults participating in longstanding NIH-funded cohort studies. Anti-S1 antibodies were measured from dried blood spots collected between February 2021-August 2022 using Luminex-based microsphere immunoassays. Of 6245 participants, mean age was 73 years (range, 21-100), 58% were female, and 76% were non-Hispanic White. Nearly 52% of participants received the BNT162b2 vaccine and 48% received the mRNA-1273 vaccine. Lower anti-S1 antibody levels are associated with age of 65 years or older, male sex, higher body mass index, smoking, diabetes, COPD and receipt of BNT16b2 vaccine (vs mRNA-1273). Participants with a prior infection, particularly those with a history of hospitalized illness, have higher anti-S1 antibody levels. These results suggest that adults with certain socio-demographic and clinical characteristics may have less robust antibody responses to COVID-19 vaccination and could be prioritized for more frequent re-vaccination.

Use of Self-Collected Dried Blood Spots and a Multiplex Microsphere Immunoassay to Measure IgG Antibody Response to COVID-19 Vaccines.

Serosurveys can determine the extent and spread of a pathogen in populations. However, collection of venous blood requires trained medical staff. Dried blood spots (DBS) are a suitable alternative because they can be self-collected and stored/shipped at room temperature. As COVID-19 vaccine deployment began in early 2021, we rapidly enrolled laboratory employees in a study to evaluate IgG antibody levels following vaccination. Participants received a DBS collection kit, self-collection instructions, and a brief questionnaire. Three DBS were collected by each of 168 participants pre- and/or postvaccination and tested with a multiplex microsphere immunoassay (MIA) that separately measures IgG antibodies to SARS-CoV-2 spike-S1 and nucleocapsid antigens. Most DBS (99.6%, 507/509) were suitable for testing. Participants with prior SARS-CoV-2 infection (n = 7) generated high S antibody levels after the first vaccine dose. Naïve individuals (n = 161) attained high S antibody levels after the second dose. Similar antibody levels were seen among those vaccinated with Moderna (n = 29) and Pfizer-BioNTech (n = 137). For those receiving either mRNA vaccine, local side effects were more common after the first vaccine dose, whereas systemic side effects were more common after the second dose. Individuals with the highest antibody levels in the week prior to the second vaccine dose experienced more side effects from the second dose. Our study demonstrated that combining self-collected DBS and a multiplex MIA is a convenient and effective way to assess antibody levels to vaccination and could easily be used for population serosurveys of SARS-CoV-2 or other emerging pathogens. IMPORTANCE Serosurveys are an essential tool for assessing immunity in a population (1, 2). However, common barriers to effective serosurveys, particularly during a pandemic, include high-costs, resources required to collect venous blood samples, lack of trained laboratory technicians, and time required to perform the assay. By utilizing self-collected dried blood spots (DBS) and our previously developed high-throughput microsphere immunoassay, we were able to significantly reduce many of these common challenges. Participants were asked to self-collect three DBS before and/or after they received their COVID-19 vaccines to measure antibody levels following vaccination. Participants successfully collected 507 DBS that were tested for IgG antibodies to the spike and nucleocapsid proteins of SARS-CoV-2. When used with self-collected DBS, our relatively low-cost assay significantly reduced common barriers to collecting serological data from a population and was able to effectively assess antibody response to vaccination.

Development of a Novel Serological Assay for the Detection of Mpox Infection in Vaccinated Populations.

In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 103 non-endemic locations world-wide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay (MIA) using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important diagnostic tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.

Mosquito saliva causes enhancement of West Nile virus infection in mice.

West Nile virus (WNV) is transmitted to vertebrate hosts primarily by infected Culex mosquitoes. Transmission of arboviruses by the bite of infected mosquitoes can potentiate infection in hosts compared to viral infection by needle inoculation. Here we examined the effect of mosquito transmission on WNV infection and systematically investigated multiple factors that differ between mosquito infection and needle inoculation of WNV. We found that mice infected with WNV through the bite of a single infected Culex tarsalis mosquito exhibited 5- to 10-fold-higher viremia and tissue titers at 24 and 48 h postinoculation and faster neuroinvasion than mice given a median mosquito-inoculated dose of WNV (10(5) PFU) by needle. Mosquito-induced enhancement was not due to differences in inoculation location, because additional intravenous inoculation of WNV did not enhance viremia or tissue titers. Inoculation of WNV into a location where uninfected mosquitoes had fed resulted in enhanced viremia and tissue titers in mice similar to those in mice infected by a single infected mosquito bite, suggesting that differences in where virus is deposited in the skin and in the virus particle itself were not responsible for the enhanced early infection in mosquito-infected mice. In addition, inoculation of mice with WNV mixed with salivary gland extract (SGE) led to higher viremia, demonstrating that mosquito saliva is the major cause of mosquito-induced enhancement. Enhanced viremia was not observed when SGE was inoculated at a distal site, suggesting that SGE enhances WNV replication by exerting a local effect. Furthermore, enhancement of WNV infection still occurred in mice with antibodies against mosquito saliva. In conclusion, saliva from C. tarsalis is responsible for enhancement of early WNV infection in vertebrate hosts.

Utility of Newborn Dried Blood Spots to Ascertain Seroprevalence of SARS-CoV-2 Antibodies Among Individuals Giving Birth in New York State, November 2019 to November 2021.

Importance: Serosurveys can be used to monitor population-level dynamics of COVID-19 and vaccination. Dried blood spots (DBSs) collected from infants contain maternal IgG antibodies and are useful for serosurveys of individuals recently giving birth. Objectives: To examine SARS-CoV-2 antibody prevalence in pregnant individuals in New York State, identify associations between SARS-CoV-2 antibody status and maternal and infant characteristics, and detect COVID-19 vaccination among this population. Design, Setting, and Participants: A population-based, repeated cross-sectional study was conducted to detect SARS-CoV-2 nucleocapsid (N) and spike (S) IgG antibodies. Deidentified DBS samples and data submitted to the New York State Newborn Screening Program between November 1, 2019, and November 30, 2021, were analyzed. Exposures: Prenatal exposure to SARS-CoV-2 antibodies. Main Outcomes and Measures: The presence of IgG antibodies to SARS-CoV-2 N and S antigens was measured using a microsphere immunoassay. Data were analyzed by geographic region and compared with reported COVID-19 cases and vaccinations among reproductive-aged females (15-44 years of age). Data were stratified by infant birth weight, gestational age, maternal age, and multiple birth status. Results: Dried blood spot samples from 415 293 infants (median [IQR] age, 1.04 [1.00-1.20] days; 210 805 [51.1%] male) were analyzed for SARS-CoV-2 antibodies. The first known antibody-positive infant in New York State was born on March 29, 2020. SARS-CoV-2 seroprevalence reflected statewide and regional COVID-19 cases among reproductive-aged females in the prevaccine period. From February through November 2021, S seroprevalence was strongly correlated with cumulative vaccinations in each New York State region and in the state overall (rs = 0.92-1.00, P ≤ .001). S and N seroprevalences were significantly lower in newborns with very low birth weight (720 [14.8%] for S and 138 [2.8%] for N, P < .001) and low birth weight (5160 [19.3%] for S and 1233 [4.6%] for N, P = .009) compared with newborns with normal birth weight (77 116 [20.1%] for S and 19 872 [5.2%] for N). Lower N and higher S seroprevalences were observed in multiple births (odds ratio [OR], 0.84; 95% CI, 0.75-0.94; P = .002 for N and OR, 1.24; 95% CI, 1.18-1.31; P < .001 for S) vs single births and for maternal age older than 30 years (OR, 0.87; 95% CI, 0.80-0.94; P < .001 for N and OR, 1.17; 95% CI, 1.11-1.23; P < .001 for S) vs younger than 20 years. Conclusions and Relevance: In this study, seroprevalence in newborn DBS samples reflected COVID-19 case fluctuations and vaccinations among reproductive-aged women during the study period. These results demonstrate the utility of using newborn DBS testing to estimate SARS-CoV-2 seroprevalence in pregnant individuals.

The costs of infection and resistance as determinants of West Nile virus susceptibility in Culex mosquitoes.

BACKGROUND: Understanding the phenotypic consequences of interactions between arthropod-borne viruses (arboviruses) and their mosquito hosts has direct implications for predicting the evolution of these relationships and the potential for changes in epidemiological patterns. Although arboviruses are generally not highly pathogenic to mosquitoes, pathology has at times been noted. Here, in order to evaluate the potential costs of West Nile virus (WNV) infection and resistance in a primary WNV vector, and to assess the extent to which virus-vector relationships are species-specific, we performed fitness studies with and without WNV exposure using a highly susceptible Culex pipiens mosquito colony. Specifically, we measured and compared survival, fecundity, and feeding rates in bloodfed mosquitoes that were (i) infected following WNV exposure (susceptible), (ii) uninfected following WNV exposure (resistant), or (iii) unexposed. RESULTS: In contrast to our previous findings with a relatively resistant Cx. tarsalis colony, WNV infection did not alter fecundity or blood-feeding behaviour of Cx. pipiens, yet results do indicate that resistance to infection is associated with a fitness cost in terms of mosquito survival. CONCLUSIONS: The identification of species-specific differences provides an evolutionary explanation for variability in vector susceptibility to arboviruses and suggests that understanding the costs of infection and resistance are important factors in determining the potential competence of vector populations for arboviruses.

High-Throughput Multiplex SARS-CoV-2 IgG Microsphere Immunoassay for Dried Blood Spots: A Public Health Strategy for Enhanced Serosurvey Capacity.

Early in the pandemic when diagnostic testing was not widely available, serosurveys played an important role in estimating the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in different populations. Dried blood spots (DBS), which can be collected in nonclinical settings, provide a minimally invasive alternative to serum for serosurveys. We developed a Luminex-based SARS-CoV-2 microsphere immunoassay (MIA) for DBS that detects IgG antibodies to nucleocapsid (N) and spike subunit 1 (S1) antigens. The assay uses a 384-well plate format and automated liquid handlers for high-throughput capacity. Specificity was assessed using a large collection of prepandemic DBS and well-characterized sera. Sensitivity was analyzed using serology data from New York State SARS-CoV-2 serosurvey testing and matched diagnostic test results. For DBS, the specificity was 99.5% for the individual N and S1 antigens. Median fluorescence intensity (MFI) values for DBS and paired sera showed a strong positive correlation for N (R2 = 0.91) and S1 (R2 = 0.93). Sensitivity, assessed from 1,134 DBS with prior laboratory-confirmed SARS-CoV-2 infection, ranged from 83% at 0 to 20 days to 95% at 61 to 90 days after a positive test. When stratified using coronavirus disease 2019 (COVID-19) symptom data, sensitivity ranged from 90 to 96% for symptomatic and 77 to 91% for asymptomatic individuals. For 8,367 health care workers reporting detailed symptom data, MFI values were significantly higher for all symptom categories. Our results indicate that the SARS-CoV-2 IgG DBS MIA is sensitive, specific, and well-suited for large population-based serosurveys. The ability to readily modify and multiplex antigens is important for ongoing assessment of SARS-CoV-2 antibody responses to emerging variants and vaccines. IMPORTANCE Testing for antibodies to SARS-CoV-2 has been used to estimate the prevalence of COVID-19 in different populations. Seroprevalence studies, or serosurveys, were especially useful during the early phase of the pandemic when diagnostic testing was not widely available, and the resulting seroprevalence estimates played an important role in public health decision making. To achieve meaningful results, antibody tests used for serosurveys should be accurate and accessible to diverse populations. We developed a test that detects antibodies to two different SARS-CoV-2 proteins in dried blood spots (DBS). DBS require only a simple fingerstick and can be collected in nonclinical settings. We conducted a robust validation study and have demonstrated that our test is both sensitive and specific. Furthermore, we demonstrated that our test is suitable for large-scale serosurveys by testing over 56,000 DBS collected in a variety of community-based venues in New York State during the spring of 2020.

Cumulative incidence and diagnosis of SARS-CoV-2 infection in New York.

PURPOSE: New York State (NYS) is an epicenter of the SARS-CoV-2 pandemic in the United States. Reliable estimates of cumulative incidence in the population are critical to tracking the extent of transmission and informing policies. METHODS: We conducted a statewide seroprevalence study in a 15,101 patron convenience sample at 99 grocery stores in 26 counties throughout NYS. SARS-CoV-2 cumulative incidence was estimated from antibody reactivity by first poststratification weighting and then adjusting by antibody test characteristics. The percent diagnosed was estimated by dividing the number of diagnoses by the number of estimated infection-experienced adults. RESULTS: Based on 1887 of 15,101 (12.5%) reactive results, estimated cumulative incidence through March 29 was 14.0% (95% confidence interval [CI]: 13.3%-14.7%), corresponding to 2,139,300 (95% CI: 2,035,800-2,242,800) infection-experienced adults. Cumulative incidence was highest in New York City 22.7% (95% CI: 21.5%-24.0%) and higher among Hispanic/Latino (29.2%), non-Hispanic black/African American (20.2%), and non-Hispanic Asian (12.4%) than non-Hispanic white adults (8.1%, P < .0001). An estimated 8.9% (95% CI: 8.4%-9.3%) of infections in NYS were diagnosed, with diagnosis highest among adults aged 55 years or older (11.3%, 95% CI: 10.4%-12.2%). CONCLUSIONS: From the largest U.S. serosurvey to date, we estimated >2 million adult New York residents were infected through late March, with substantial disparities, although cumulative incidence remained less than herd immunity thresholds. Monitoring, testing, and contact tracing remain essential public health strategies.

Mosquitoes inoculate high doses of West Nile virus as they probe and feed on live hosts.

West Nile virus (WNV) is transmitted to vertebrate hosts by mosquitoes as they take a blood meal. The amount of WNV inoculated by mosquitoes as they feed on a live host is not known. Previous estimates of the amount of WNV inoculated by mosquitoes (10(1.2)-10(4.3) PFU) were based on in vitro assays that do not allow mosquitoes to probe or feed naturally. Here, we developed an in vivo assay to determine the amount of WNV inoculated by mosquitoes as they probe and feed on peripheral tissues of a mouse or chick. Using our assay, we recovered approximately one-third of a known amount of virus inoculated into mouse tissues. Accounting for unrecovered virus, mean and median doses of WNV inoculated by four mosquito species were 10(4.3) PFU and 10(5.0) PFU for Culex tarsalis, 10(5.9) PFU and 10(6.1) PFU for Cx. pipiens, 10(4.7) PFU and 10(4.7) PFU for Aedes japonicus, and 10(3.6) PFU and 10(3.4) PFU for Ae. triseriatus. In a direct comparison, in vivo estimates of the viral dose inoculated by Cx. tarsalis were approximately 600 times greater than estimates obtained by an in vitro capillary tube transmission assay. Virus did not disperse rapidly, as >99% of the virus was recovered from the section fed or probed upon by the mosquito. Furthermore, 76% (22/29) of mosquitoes inoculated a small amount of virus ( approximately 10(2) PFU) directly into the blood while feeding. Direct introduction of virus into the blood may alter viral tropism, lead to earlier development of viremia, and cause low rates of infection in co-feeding mosquitoes. Our data demonstrate that mosquitoes inoculate high doses of WNV extravascularly and low doses intravascularly while probing and feeding on a live host. Accurate estimates of the viral dose inoculated by mosquitoes are critical in order to administer appropriate inoculation doses to animals in vaccine, host competence, and pathogenesis studies.

Drug resistance mutations and viral load in human immunodeficiency virus type 2 and dual HIV-1/HIV-2 infected patients in Ghana.

Antiretroviral therapy (ART) and drug resistance studies worldwide have focused almost exclusively on human immunodeficiency virus type 1 (HIV-1). As a result, there is limited information on ART and drug resistance in HIV-2 patients. In Ghana, the HIV epidemic is characterized by the domination of HIV-1, with cocirculating HIV-2. We, therefore, sought to determine viral load and drug resistance mutations in HIV-2 patients to inform the clinical management of such individuals in Ghana.We used purposive sampling to collect blood from 16 consented patients, confirmed as HIV-2 or HIV-1/2 dual infections by serology. A 2-step real-time RT-PCR assay was used to determine plasma HIV-2 RNA viral loads. For drug resistance testing, nucleic acids were extracted from plasma and peripheral blood mononuclear cells. The reverse transcriptase and protease genes of HIV-2 were amplified, sequenced and analyzed for drug resistance mutations and HIV-2 group.HIV-2 viral load was detected in 9 of 16 patients. Six of these had quantifiable viral loads (range: 2.62-5.45 log IU/mL) while 3 had viral loads below the limit of quantification. Sequences were generated from 7 out of 16 samples. Five of these were classified as HIV-2 group B and 2 as HIV-2 group A. HIV-2 drug resistance mutations (M184V, K65R, Y115F) were identified in 1 patient.This study is the first to report HIV-2 viral load and drug resistance mutations in HIV-2 strains from Ghana. The results indicate the need for continuous monitoring of drug resistance among HIV-2- infected patients to improve their clinical management.

Mosquitoes do senesce: departure from the paradigm of constant mortality.

Although variation in mortality is considered by virtually all vector-borne disease specialists to be one of the most important determinants of an arthropod's capacity to transmit pathogens, the operational assumption often is that insect vector mortality is independent of age. Acceptance of the non-senescence assumption leads to the erroneous conclusion that mosquito age is unimportant, results in misleading predictions regarding disease reductions after vector control, and represses study of other aspects of mosquito biology that change with age. We brought large-scale laboratory life table techniques (N > 100,000) to bear on the question of age-dependent mortality in the mosquito vector of dengue virus, Aedes aegypti. Mortality was highly age dependent in both sexes. Mortality was low at young ages (< 10 days old), steadily increased at middle ages, and decelerated at older ages. A newly derived age-dependent model of pathogen transmission shows the importance of young mosquitoes and population age structure to transmission dynamics. Departure from the age-independent mortality paradigm encourages research on overlooked complexities in mosquito biology, the need for innovative methods to study mosquito population dynamics, and the need to study age-dependent changes for an accurate understanding of mosquito biology and pathogen transmission.

Mortality and reproductive dynamics of Aedes aegypti (Diptera: Culicidae) fed human blood.

Mortality is a critical factor in determining a mosquito's ability to transmit pathogens. We investigated the effect of human blood feeding and reproduction on mortality of the dengue virus vector, Aedes aegypti, by conducting a life-table study of male and female mosquitoes maintained on one of three diets: 10% sucrose, human blood or human blood plus 10% sucrose. We examined the effect of host availability by offering human blood to mosquitoes every day or every other day. Mortality of females was age-dependent and best fit by a logistic or logistic-Makeham model. The availability of blood increased survival; survival of females fed blood plus sugar was greater than those only fed sugar. There was a peak in mortality of females fed blood alone early in life that coincided with the initiation of oviposition. When females in the blood alone group were offered blood daily, their mortality was significantly lower than when they were offered blood every other day. Unlike some previous studies, females fed blood plus sugar had higher fitness than females fed blood alone. Increased fitness may have been due to differences in housing mosquitoes individually in separate cages versus as a group of many mosquitoes in each cage. It was not due to longer survival of males who had access to sugar as a food source. Our results demonstrate that reproductively active Ae. aegypti exhibit age-dependent mortality, which refutes the assumption of age-independent mosquito mortality and underscores the need to incorporate age-dependent factors into pathogen transmission models and research on mosquito biology in general.

West Nile virus infection decreases fecundity of Culex tarsalis females.

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) persistently infects many mosquito tissues, and it has been associated with cytopathological changes in midgut muscles and salivary glands. However, the effects of WNV infection on mosquito fitness (survival and reproduction) are not known. We conducted a life table study of individually housed female Culex tarsalis Coquillett. After an initial bloodmeal from a WNV-infected or uninfected chicken, mosquitoes were provided sucrose and offered weekly opportunities to feed on a hanging blood drop. WNV transmission status was determined by testing the remaining blood drop for virus after mosquito feeding. Dead mosquitoes and eggs were collected daily. Mosquito legs and bodies were tested for WNV, and eggs were counted and allowed to hatch. Two replicates of this experiment were performed, with a total of 62 mosquitoes that fed on a WNV-infected chicken (of which 21 became infected) and 43 mosquitoes that fed on an uninfected chicken. Fecundity of WNV-infected mosquitoes was significantly lower than that of uninfected mosquitoes, especially during the first oviposition. WNV infection was associated with smaller egg rafts, whereas increasing wing length and WNV titer in the legs had a positive effect on egg raft size. Additionally, infected mosquitoes had lower egg hatch rates than did uninfected mosquitoes. There were no significant differences in survival between infected and uninfected mosquitoes. Blood feeding rates were higher in infected mosquitoes than in uninfected mosquitoes. A small amount of virus (average, 378; range, 5-5000 plaque-forming units) was transmitted to the blood drops fed upon by infected mosquitoes. Although WNV infection negatively impacts mosquito reproduction, facets of mosquito biology that are critical to virus transmission success were either not affected (survival) or changed in such a way as to result in enhanced vectorial capacity (blood feeding).

Evaluation of dried blood spot protocols with the Bio-Rad GS HIV Combo Ag/Ab EIA and Geenius™ HIV 1/2 Supplemental Assay.

OBJECTIVE: FDA-approved antigen/antibody combo and HIV-1/2 differentiation supplemental tests do not have claims for dried blood spot (DBS) use. We compared two DBS-modified protocols, the Bio-Rad GS HIV Combo Ag/Ab (BRC) EIA and Geenius™ HIV-1/2 (Geenius) Supplemental Assay, to plasma protocols and evaluated them in the CDC/APHL HIV diagnostic algorithm. METHODS: BRC-DBS p24 analytical sensitivity was calculated from serial dilutions of p24. DBS specimens included 11 HIV-1 seroconverters, 151 HIV-1-positive individuals, including 20 on antiretroviral therapy, 31 HIV-2-positive and one HIV-1/HIV-2-positive individuals. BRC-reactive specimens were tested with Geenius using the same DBS eluate. Matched plasma specimens were tested with BRC, an IgG/IgM immunoassay and Geenius. DBS and plasma results were compared using the McNemar's test. A DBS-algorithm applied to 348 DBS from high-risk individuals who participated in surveillance was compared to HIV status based on local testing algorithms. RESULTS: BRC-DBS detects p24 at a concentration 18 times higher than in plasma. In seroconverters, BRC-DBS detected more infections than the IgG/IgM immunoassay in plasma (p=0.0133), but fewer infections than BRC-plasma (p=0.0133). In addition, the BRC/Geenius-plasma algorithm identified more HIV-1 infections than the BRC/Geenius-DBS algorithm (p=0.0455). The DBS protocols correctly identified HIV status for established HIV-1 infections, including those on therapy, HIV-2 infections, and surveillance specimens. CONCLUSIONS: The DBS protocols exhibited promising performance and allowed rapid supplemental testing. Although the DBS algorithm missed some early infections, it showed similar results when applied to specimens from a high-risk population. Implementation of a DBS algorithm would benefit testing programs without capacity for venipuncture.

Enhanced early West Nile virus infection in young chickens infected by mosquito bite: effect of viral dose.

Mosquito transmission of arboviruses potentially affects the course of viral infection in the vertebrate host. Studies were performed to determine if viral infection differed in chickens infected with West Nile virus (WNV) by mosquito bite or needle inoculation. Mosquito-infected chickens exhibited levels of viremia and viral shedding that were up to 1,000 times higher at 6, 12, and 24 hours post-feeding (PF) compared with those inoculated with 10(3) PFU by needle. Follow-up studies were conducted to determine if enhanced early infection was due to a higher viral dose inoculated by mosquitoes. Needle inoculation with successively higher doses of WNV led to higher early viremia and viral shedding; a dose >or= 10(4) PFU by needle was required to attain the high early viremia observed in mosquito-infected chickens. Mosquitoes inoculated WNV at this level as estimated by feeding on a hanging drop of blood (mean: 10(2.5), range: 10(0.7)-10(4.6) PFU). These results indicate that enhanced early infection in mosquito-infected chickens may be explained by higher viral dose delivered by mosquitoes. On the other hand, chickens infected by multiple mosquitoes (N = 3-11) had viremic titers that were 25-50 times higher at 6 and 12 hours PF than in chickens infected by a single mosquito, suggesting that viral dose is not the only factor involved in enhanced early infection. The likelihood that enhanced early infection in mosquito-infected chickens is due to a higher viral dose inoculated by mosquitoes and/or other factors (saliva, inoculation location, or viral source) is discussed.