Routine HIV Test Results in 6 US Clinical Laboratories Using the Recommended Laboratory HIV Testing Algorithm With Geenius HIV 1/2 Supplemental Assay.

BACKGROUND: Geenius HIV 1/2 Supplemental Assay (Geenius; Bio-Rad Laboratories) is the only Food and Drug Administration-approved HIV-1/HIV-2 antibody differentiation test for the second step in the HIV laboratory testing algorithm. We characterized the occurrence of true HIV-1 and HIV-2 infections as well as false results in 6 US clinical laboratories using Geenius. METHODS: We examined routine HIV testing outcome data from the time the laboratories began using the algorithm with Geenius until September 30, 2017. We calculated the positive predictive value for Geenius HIV-1 and HIV-2 reactivity separately. RESULTS: Of 5,046,684 specimens tested, 41,791 had reactive antigen/antibody test results. Most specimens with reactive antigen/antibody results were HIV-1 antibody-positive established infections (n = 32,421), 1,865 of which also had indeterminate HIV-2 bands present. Ninety-three specimens were HIV-2 antibody positive or untypable for HIV-1/HIV-2 antibody. Acute HIV-1 infections were found in 528 specimens; 881 specimens lacked the nucleic acid test to determine the possibility of acute HIV-1 infection. False-positive antigen/antibody test results were present in 7505 specimens. Few specimens (n = 363) had false-positive antigen/antibody results with indeterminate Geenius and negative HIV-1 nucleic acid test results. The positive predictive values of Geenius reactivity were 99.4% for HIV-1 and 4.3% for HIV-2. CONCLUSIONS: Routine testing using the laboratory testing algorithm with Geenius resulted in most specimens resolving as HIV negative or HIV-1 positive. The occurrence of indeterminate HIV-2 bands with a Geenius final assay interpretation of HIV-1 positive was more common than true HIV-2 infections. Reporting indeterminate HIV-2 results in this situation may cause confusion with interpreting HIV infection status.

Multicenter Evaluation of Clinical Diagnostic Methods for Detection and Isolation of Campylobacter spp. from Stool.

The use of culture-independent diagnostic tests (CIDTs), such as stool antigen tests, as standalone tests for the detection of Campylobacter in stool is increasing. We conducted a prospective, multicenter study to evaluate the performance of stool antigen CIDTs compared to culture and PCR for Campylobacter detection. Between July and October 2010, we tested 2,767 stool specimens from patients with gastrointestinal illness with the following methods: four types of Campylobacter selective media, four commercial stool antigen assays, and a commercial PCR assay. Illnesses from which specimens were positive by one or more culture media or at least one CIDT and PCR were designated "cases." A total of 95 specimens (3.4%) met the case definition. The stool antigen CIDTs ranged from 79.6% to 87.6% in sensitivity, 95.9 to 99.5% in specificity, and 41.3 to 84.3% in positive predictive value. Culture alone detected 80/89 (89.9% sensitivity) Campylobacter jejuni/Campylobacter coli-positive cases. Of the 209 noncases that were positive by at least one CIDT, only one (0.48%) was positive by all four stool antigen tests, and 73% were positive by just one stool antigen test. The questionable relevance of unconfirmed positive stool antigen CIDT results was supported by the finding that noncases were less likely than cases to have gastrointestinal symptoms. Thus, while the tests were convenient to use, the sensitivity, specificity, and positive predictive value of Campylobacter stool antigen tests were highly variable. Given the relatively low incidence of Campylobacter disease and the generally poor diagnostic test characteristics, this study calls into question the use of commercially available stool antigen CIDTs as standalone tests for direct detection of Campylobacter in stool.

The Public Health Framework of Legalized Marijuana in Colorado.

On January 1, 2014, Colorado became the first state in the nation to sell legal recreational marijuana for adult use. As a result, Colorado has had to carefully examine potential population health and safety impacts as well as the role of public health in response to legalization. We have discussed an emerging public health framework for legalized recreational marijuana. We have outlined this framework according to the core public health functions of assessment, policy development, and assurance. In addition, we have discussed challenges to implementing this framework that other states considering legalization may face.

Probability of negative mycobacterium tuberculosis complex cultures based on time to detection of positive cultures: a multicenter evaluation of commercial-broth-based culture systems.

We conducted a multicenter study to determine whether Mycobacterium tuberculosis complex (MTBC) cultures in automated broth-based systems could reliably be considered negative sooner than 6 weeks. Laboratory sites used Bactec MGIT or BacT/Alert and tracked results of time to detection of all mycobacteria (TTD-all, n = 1547) and of MTBC (TTD-MTBC, n = 466) over 6-month periods from primarily (93%) respiratory specimens. Cumulative percentages by day detected and median TTD of initial and follow-up specimens were analyzed. The median TTD-MTBC for MGIT (n = 6 sites) was 14 days. For laboratories using standard processing procedures, 100% of MTBC were detected from initial and follow-up specimens in 28 and 35 days, respectively, and no yield of MTBC on solid or MGIT liquid media was observed after 5 weeks. The median TTD-MTBC for BacT/Alert (n = 3 sites) was 18 days, with 95% and 100% detected within 37 and 42 days, respectively. Analysis of TTD of positive MTBC cultures in broth can predict the probability of culture negativity at defined time points. Receipt of interim negative reports earlier than 6 weeks could assist clinicians in considering alternative diagnoses and could alter the timing and prioritization of public health interventions. Laboratories should analyze their own TTD data to inform protocol decisions. Laboratories using MGIT could issue reports of no growth of MTBC on initial specimens as early as 4 weeks and for patients undergoing treatment as early as 5 weeks postinoculation.

Avian Influenza virus glycoproteins restrict virus replication and spread through human airway epithelium at temperatures of the proximal airways.

Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37 degrees C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32 degrees C). These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40 degrees C), rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32 degrees C and 37 degrees C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32 degrees C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2) or A/PR/8/34 (H1N1) genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA) and neuraminidase (NA) from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and suggests that adaptation of avian influenza viruses to efficient infection at 32 degrees C may represent a critical evolutionary step enabling human-to-human transmission.

Evaluation of live attenuated influenza a virus h6 vaccines in mice and ferrets.

Avian influenza A virus A/teal/HK/W312/97 (H6N1) possesses seven gene segments that are highly homologous to those of highly pathogenic human influenza H5N1 viruses, suggesting that a W312-like H6N1 virus might have been involved in the generation of the A/HK/97 H5N1 viruses. The continuous circulation and reassortment of influenza H6 subtype viruses in birds highlight the need to develop an H6 vaccine to prevent potential influenza pandemics caused by the H6 viruses. Based on the serum antibody cross-reactivity data obtained from 14 different H6 viruses from Eurasian and North American lineages, A/duck/HK/182/77, A/teal/HK/W312/97, and A/mallard/Alberta/89/85 were selected to produce live attenuated H6 candidate vaccines. Each of the H6 vaccine strains is a 6:2 reassortant ca virus containing HA and NA gene segments from an H6 virus and the six internal gene segments from cold-adapted A/Ann Arbor/6/60 (AA ca), the master donor virus that is used to make live attenuated influenza virus FluMist (intranasal) vaccine. All three H6 vaccine candidates exhibited phenotypic properties of temperature sensitivity (ts), ca, and attenuation (att) conferred by the internal gene segments from AA ca. Intranasal administration of a single dose of the three H6 ca vaccine viruses induced neutralizing antibodies in mice and ferrets and fully protected mice and ferrets from homologous wild-type (wt) virus challenge. Among the three H6 vaccine candidates, the A/teal/HK/W312/97 ca virus provided the broadest cross-protection against challenge with three antigenically distinct H6 wt viruses. These data support the rationale for further evaluating the A/teal/HK/W312/97 ca vaccine in humans.

A single-amino-acid substitution in a polymerase protein of an H5N1 influenza virus is associated with systemic infection and impaired T-cell activation in mice.

The transmission of H5N1 influenza viruses from birds to humans poses a significant public health threat. A substitution of glutamic acid for lysine at position 627 of the PB2 protein of H5N1 viruses has been identified as a virulence determinant. We utilized the BALB/c mouse model of H5N1 infection to examine how this substitution affects virus-host interactions and leads to systemic infection. Mice infected with H5N1 viruses containing lysine at amino acid 627 in the PB2 protein exhibited an increased severity of lesions in the lung parenchyma and the spleen, increased apoptosis in the lungs, and a decrease in oxygen saturation. Gene expression profiling revealed that T-cell receptor activation was impaired at 2 days postinfection (dpi) in the lungs of mice infected with these viruses. The inflammatory response was highly activated in the lungs of mice infected with these viruses and was sustained at 4 dpi. In the spleen, immune-related processes including NK cell cytotoxicity and antigen presentation were highly activated by 2 dpi. These differences are not attributable solely to differences in viral replication in the lungs but to an inefficient immune response early in infection as well. The timing and magnitude of the immune response to highly pathogenic influenza viruses is critical in determining the outcome of infection. The disruption of these factors by a single-amino-acid substitution in a polymerase protein of an influenza virus is associated with severe disease and correlates with the spread of the virus to extrapulmonary sites.

Avian influenza h6 viruses productively infect and cause illness in mice and ferrets.

Influenza pandemic preparedness has focused on influenza virus H5 and H7 subtypes. However, it is not possible to predict with certainty which subtype of avian influenza virus will cause the next pandemic, and it is prudent to include other avian influenza virus subtypes in pandemic preparedness efforts. An H6 influenza virus was identified as a potential progenitor of the H5N1 viruses that emerged in Hong Kong in 1997. This virus continues to circulate in the bird population in Asia, and other H6 viruses are prevalent in birds in North America and Asia. The high rate of reassortment observed in influenza viruses and the prevalence of H6 viruses in birds suggest that this subtype may pose a pandemic risk. Very little is known about the replicative capacity, immunogenicity, and correlates of protective immunity for low-pathogenicity H6 influenza viruses in mammals. We evaluated the antigenic and genetic relatedness of 14 H6 influenza viruses and their abilities to replicate and induce a cross-reactive immune response in two animal models: mice and ferrets. The different H6 viruses replicated to different levels in the respiratory tracts of mice and ferrets, causing varied degrees of morbidity and mortality in these two models. H6 virus infection induced similar patterns of neutralizing antibody responses in mice and ferrets; however, species-specific differences in the cross-reactivity of the antibody responses were observed. Overall, cross-reactivity of neutralizing antibodies in H6 virus-infected mice did not correlate well with protection against heterologous wild-type H6 viruses. However, we have identified an H6 virus that induces protective immunity against viruses in the North American and Eurasian lineages.

HIV-1 extrachromosomal 2-LTR circular DNA is long-lived in human macrophages.

HIV-1 extrachromosomal 2-LTR circles (cc2LTR) are rapidly lost in dividing cell populations and, therefore, might be interpreted as representing new infection and ongoing viral replication. However, recent work demonstrated that cc2LTR persist in infected, growth-arrested T cell lines beyond their predicted half-life as previously determined in dividing cell populations. In this study, the evaluation of the stability of cc2LTR was extended to include primary human macrophages, a natural, non-dividing target of HIV-1. By quantitative real-time PCR, cc2LTR were found to persist out to 21 days post-infection in macrophages infected with both integrase competent and integrase- defective, recombinant HIV-1, whereas in activated CD4(+) T lymphocytes, they rapidly decreased over time. This persistence was associated with persistent, low level expression of the indicator gene, luciferase. These data suggest that the presence of HIV-1 cc2LTR in the PBMC of HIV-1-infected patients on suppressive HAART could be due either to ongoing generation of newly infected dividing cells, or persistence of circles in non-dividing cell populations where they appear to be stable. Furthermore, exrachromosomal circular DNA in this cell population could be a source of persisent viral protein expression.

Using fee-for-service testing to generate revenue for the 21st century public health laboratory.

OBJECTIVES: The decrease in appropriations for state public health laboratories (SPHLs) has become a major concern as tax revenues and, subsequently, state and federal funding, have decreased. These reductions have forced SPHLs to pursue revenue-generating opportunities to support their work. We describe the current state of funding in a sampling of SPHLs and the challenges these laboratories face as they implement or expand fee-for-service testing. METHODS: We conducted surveys of SPHLs to collect data concerning laboratory funding sources, test menus, fee-for-service testing, and challenges to implementing fee-for-service testing. RESULTS: Most SPHLS receive funding through three revenue sources: state appropriation, federal funding, and fee-for-service testing (cash funds). Among SPHLs, state appropriations ranged from $0 to more than $6 per capita, federal funding ranged from $0.10 to $5 per capita, and revenue from fee-for-service testing ranged from $0 to $4 per capita. The tests commonly performed on a fee-for-service basis included assays for sexually transmitted diseases, mycobacterial cultures, newborn screening, and water testing. We found that restrictive legislation, staffing shortages, inadequate software for billing fee-for-service testing, and regulations on how SPHLs use their generated revenue are impediments to implementing fee-for-service testing. CONCLUSIONS: Some SPHLs are considering implementing or expanding fee-for-service testing as a way to recapture funds lost as a result of state and federal budget cuts. This analysis revealed many of the obstacles to implementing fee-for-service testing in SPHLs and the potential impact on SPHLs of continued decreases in funding.

Outbreak of shiga toxin-producing Escherichia coli serotype O26: H11 infection at a child care center in Colorado.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) O26:H11 is an emerging cause of disease with serious potential consequences in children. The epidemiology and clinical spectrum of O26:H11 are incompletely understood. We investigated an outbreak of O26:H11 infection among children younger than 48 months of age and employees at a child care center. METHODS: Every employee at the center (n = 20) and every child <48 months (n = 55) were tested for STEC and administered a questionnaire. Thirty environmental health inspections and site visits were conducted. A cohorting strategy for disease control was implemented. RESULTS: Eighteen confirmed and 27 suspect cases were detected. There were no hospitalizations. The illness rate was 60% for children and employees. The risk of being a case in children <36 months was twice the risk among children of 36 to 47 months (risk ratio: 2.10; 95% confidence interval: 1.00, 4.42). The median duration of shedding among symptomatic confirmed cases was 30.5 days (range: 14-52 days). Four (22%) confirmed cases were asymptomatic and 3 (17%) shed intermittently. Nearly half (49%) of the household contacts of confirmed cases developed a diarrheal illness. The outbreak was propagated by person-to-person transmission; cohorting was an effective disease control strategy. CONCLUSIONS: This was the largest reported outbreak of O26:H11 infection in the United States and the largest reported non-O157 STEC outbreak in a US child care center. Non-O157 STEC infection is a differential diagnosis for outbreaks of diarrhea in child care settings. Aggressive disease control measures were effective but should be evaluated for outbreaks in other settings.

Emerging respiratory viruses: challenges and vaccine strategies.

The current threat of avian influenza to the human population, the potential for the reemergence of severe acute respiratory syndrome (SARS)-associated coronavirus, and the identification of multiple novel respiratory viruses underline the necessity for the development of therapeutic and preventive strategies to combat viral infection. Vaccine development is a key component in the prevention of widespread viral infection and in the reduction of morbidity and mortality associated with many viral infections. In this review we describe the different approaches currently being evaluated in the development of vaccines against SARS-associated coronavirus and avian influenza viruses and also highlight the many obstacles encountered in the development of these vaccines. Lessons learned from current vaccine studies, coupled with our increasing knowledge of the host and viral factors involved in viral pathogenesis, will help to increase the speed with which efficacious vaccines targeting newly emerging viral pathogens can be developed.

Nef expressed from human immunodeficiency virus type 1 extrachromosomal DNA downregulates CD4 on primary CD4+ T lymphocytes: implications for integrase inhibitors.

Recently developed integrase inhibitors targeting the HIV-1 integrase (IN) protein block integration of HIV DNA in the target cell, preventing subsequent virus replication. In the absence of integration, viral DNA is shunted towards the formation of extrachromosomal DNA (E-DNA). Although HIV-1 E-DNA does not support productive replication, it is transcriptionally active and produces viral proteins. However, the significance of E-DNA in virus replication and pathogenesis is poorly understood. In this study, the functional activity of the HIV-1 Nef protein expressed in the absence of viral integration was analysed. Using both a recombinant HIV-1 IN defective virus and a diketo acid IN inhibitor, evidence was provided showing that Nef expressed from E-DNA downregulates CD4 surface expression on primary CD4(+) T lymphocytes. These results suggest that proteins expressed in the absence of integration may have potential clinical consequences, an issue that should be further explored with the introduction of IN inhibitors.

Mice susceptible to SARS coronavirus.

Murine models of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) will greatly advance research on this emerging virus. When BALB/c mice were simultaneously inoculated intranasally and orally, replication of SARS-CoV was found in both lung and intestinal tissue.

Discovery of novel human and animal cells infected by the severe acute respiratory syndrome coronavirus by replication-specific multiplex reverse transcription-PCR.

The severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of the recent outbreak of severe acute respiratory syndrome. VeroE6 cells, fetal rhesus monkey kidney cells, and human peripheral blood mononuclear cells were the only cells known to be susceptible to SARS-CoV. We developed a multiplex reverse transcription-PCR assay to analyze the susceptibility of cells derived from a variety of tissues and species to SARS-CoV. Additionally, productive infection was determined by titration of cellular supernatants. Cells derived from three species of monkey were susceptible to SARS-CoV. However, the levels of SARS-CoV produced differed by 4 log(10). Mink lung epithelial cells (Mv1Lu) and R-Mix, a mixed monolayer of human lung-derived cells (A549) and mink lung-derived cells (Mv1Lu), are used by diagnostic laboratories to detect respiratory viruses (e.g., influenza virus); they were also infected with SARS-CoV, indicating that the practices of diagnostic laboratories should be examined to ensure appropriate biosafety precautions. Mv1Lu cells produce little SARS-CoV compared to that produced by VeroE6 cells, which indicates that they are a safer alternative for SARS-CoV diagnostics. Evaluation of cells permissive to other coronaviruses indicated that these cell types are not infected by SARS-CoV, providing additional evidence that SARS-CoV binds an alternative receptor. Analysis of human cells derived from lung, kidney, liver, and intestine led to the discovery that human cell lines were productively infected by SARS-CoV. This study identifies new cell lines that may be used for SARS-CoV diagnostics and/or basic research. Our data and other in vivo studies indicate that SARS-CoV has a wide host range, suggesting that the cellular receptor(s) utilized by SARS-CoV is highly conserved and is expressed by a variety of tissues.

CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus.

Angiotensin-converting enzyme 2 (ACE2) is a receptor for SARS-CoV, the novel coronavirus that causes severe acute respiratory syndrome [Li, W. Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran, M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C., et al. (2003) Nature 426, 450-454]. We have identified a different human cellular glycoprotein that can serve as an alternative receptor for SARS-CoV. A human lung cDNA library in vesicular stomatitis virus G pseudotyped retrovirus was transduced into Chinese hamster ovary cells, and the cells were sorted for binding of soluble SARS-CoV spike (S) glycoproteins, S(590) and S(1180). Clones of transduced cells that bound SARS-CoV S glycoprotein were inoculated with SARS-CoV, and increases in subgenomic viral RNA from 1-16 h or more were detected by multiplex RT-PCR in four cloned cell lines. Sequencing of the human lung cDNA inserts showed that each of the cloned cell lines contained cDNA that encoded human CD209L, a C-type lectin (also called L-SIGN). When the cDNA encoding CD209L from clone 2.27 was cloned and transfected into Chinese hamster ovary cells, the cells expressed human CD209L glycoprotein and became susceptible to infection with SARS-CoV. Immunohistochemistry showed that CD209L is expressed in human lung in type II alveolar cells and endothelial cells, both potential targets for SARS-CoV. Several other enveloped viruses including Ebola and Sindbis also use CD209L as a portal of entry, and HIV and hepatitis C virus can bind to CD209L on cell membranes but do not use it to mediate virus entry. Our data suggest that the large S glycoprotein of SARS-CoV may use both ACE2 and CD209L in virus infection and pathogenesis.