Persistence of a Skewed Repertoire of NK Cells in People with HIV-1 on Long-Term Antiretroviral Therapy.

HIV-1 infection greatly alters the NK cell phenotypic and functional repertoire. This is highlighted by the expansion of a rare population of FcRγ- NK cells exhibiting characteristics of traditional immunologic memory in people with HIV (PWH). Although current antiretroviral therapy (ART) effectively controls HIV-1 viremia and disease progression, its impact on HIV-1-associated NK cell abnormalities remains unclear. To address this, we performed a longitudinal analysis detailing conventional and memory-like NK cell characteristics in n = 60 PWH during the first 4 y of ART. Throughout this regimen, a skewed repertoire of cytokine unresponsive FcRγ- memory-like NK cells persisted and accompanied an overall increase in NK surface expression of CD57 and KLRG1, suggestive of progression toward immune senescence. These traits were linked to elevated serum inflammatory biomarkers and increasing Ab titers to human CMV, with human CMV viremia detected in approximately one-third of PWH at years 1-4 of ART. Interestingly, 40% of PWH displayed atypical NK cell subsets, representing intermediate stages of NK-poiesis based on single-cell multiomic trajectory analysis. Our findings indicate that NK cell irregularities persist in PWH despite long-term ART, underscoring the need to better understand the causative mechanisms that prevent full restoration of immune health in PWH.

Subclinical herpesvirus shedding among HIV-1-infected men on antiretroviral therapy.

OBJECTIVE: We evaluated the subclinical shedding of six different herpesviruses in antiretroviral drug-treated HIV-positive [HIV(+)] MSM, and determined how this is associated with markers of inflammation and immune activation. METHODS: We obtained blood, semen, throat washing, urine, and stool from 15 antiretroviral-treated HIV-1-infected MSM with CD4 T-cell reconstitution, and 12 age-matched HIV-negative [HIV (-)] MSM from the Multicenter AIDS Cohort Study at four timepoints over 24 weeks to measure DNA levels of cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1 and 2, human herpesvirus 6 (HHV6), and HHV8. T-cell activation and plasma levels of soluble markers of inflammation and activation were also measured at the corresponding timepoints. RESULTS: HIV(+) participants had a trend for higher total herpesvirus shedding rate. HIV(+) participants also had a significantly higher rate of shedding EBV and CMV compared with the HIV(-) group. Herpesvirus shedding was mostly seen in throat washings. In the HIV(+) group, herpesvirus shedding rate inversely correlated with plasma levels of interferon γ-induced protein 10 and soluble CD163. CMV DNA levels negatively correlated with levels of T-cell activation. There was a trend for a positive correlation between EBV shedding rate and plasma soluble CD14. HHV6 shedding rate negatively correlated with plasma levels of interleukin-6, soluble CD163, and interferon gamma-induced protein 10. Correlations were not observed among HIV(-) individuals. CONCLUSION: Among treated HIV-infected MSM, there are higher subclinical shedding rates of some herpesviruses that occur in different body compartments and negatively correlate with levels of inflammation and immune activation.

Factors associated with real-time RT-PCR cycle threshold values among medically attended influenza episodes.

We evaluated the cycle threshold (CT) values of 1,160 influenza A positive and 806 influenza B positive specimens from two seasons of the US Flu VE Network to identify factors associated with CT values. Low CT values (high genomic load) were associated with shorter intervals between illness onset and specimen collection, young age (ages 3-8 years old), and self-rated illness severity for both influenza A and B. Low CT values were also associated with reported fever/feverishness and age ≥65 years for influenza A.

Viral infections in outpatients with medically attended acute respiratory illness during the 2012-2013 influenza season.

BACKGROUND: While it is known that acute respiratory illness (ARI) is caused by an array of viruses, less is known about co-detections and the resultant comparative symptoms and illness burden. This study examined the co-detections, the distribution of viruses, symptoms, and illness burden associated with ARI between December 2012 and March 2013. METHODS: Outpatients with ARI were assayed for presence of 18 viruses using multiplex reverse transcriptase polymerase chain reaction (MRT-PCR) to simultaneously detect multiple viruses. RESULTS: Among 935 patients, 60% tested positive for a single virus, 9% tested positive for ≥1 virus and 287 (31%) tested negative. Among children (<18 years), the respective distributions were 63%, 14%, and 23%; whereas for younger adults (18-49 years), the distributions were 58%, 8%, and 34% and for older adults (≥50 years) the distributions were 61%, 5%, and 32% (P < 0.001). Co-detections were more common in children than older adults (P = 0.01), and less frequent in households without children (P = 0.003). Most frequently co-detected viruses were coronavirus, respiratory syncytial virus, and influenza A virus. Compared with single viral infections, those with co-detections less frequently reported sore throat (P = 0.01), missed fewer days of school (1.1 vs. 2 days; P = 0.04), or work (2 vs. 3 days; P = 0.03); other measures of illness severity did not vary. CONCLUSIONS: Among outpatients with ARI, 69% of visits were associated with a viral etiology. Co-detections of specific clusters of viruses were observed in 9% of ARI cases particularly in children, were less frequent in households without children, and were less symptomatic (e.g., lower fever) than single infections.

Human herpesvirus 8 induces polyfunctional B lymphocytes that drive Kaposi's sarcoma.

UNLABELLED: Kaposi's sarcoma (KS) is an unusual neoplasia wherein the tumor consists primarily of endothelial cells infected with human herpesvirus 8 (HHV-8; Kaposi's sarcoma-associated herpesvirus) that are not fully transformed but are instead driven to excess proliferation by inflammatory and angiogenic factors. This oncogenic process has been postulated but unproven to depend on a paracrine effect of an abnormal excess of host cytokines and chemokines produced by HHV-8-infected B lymphocytes. Using newly developed measures for intracellular detection of lytic cycle proteins and expression of cytokines and chemokines, we show that HHV-8 targets a range of naive B cell, IgM memory B cell, and plasma cell-like populations for infection and induction of interleukin-6, tumor necrosis factor alpha, macrophage inhibitory protein 1α, macrophage inhibitory protein 1ß, and interleukin-8 in vitro and in the blood of HHV-8/HIV-1-coinfected subjects with KS. These B cell lineage subsets that support HHV-8 infection are highly polyfunctional, producing combinations of 2 to 5 of these cytokines and chemokines, with greater numbers in the blood of subjects with KS than in those without KS. Our study provides a new paradigm of B cell polyfunctionality and supports a key role for B cell-derived cytokines and chemokines produced during HHV-8 infection in the development of KS. IMPORTANCE: Kaposi's sarcoma (KS) is the most common cancer in HIV-1-infected persons and is caused by one of only 7 human cancer viruses, i.e., human herpesvirus 8 (HHV-8). It is unclear how this virus causes neoplastic transformation. Development and outgrowth of endothelial cell lesions characteristic of KS are hypothesized to be dependent on virus replication and multiple immune mediators produced by the KS cells and inflammatory cells, yet the roles of these viral and cell factors have not been defined. The present study advances our understanding of KS in that it supports a central role for HHV-8 infection of B cells inducing multiple cytokines and chemokines that can drive development of the cancer. Notably, HIV-1-infected individuals who developed KS had greater numbers of such HHV-8-infected, polyfunctional B cells across a range of B cell phenotypic lineages than did HHV-8-infected persons without KS. This intriguing production of polyfunctional immune mediators by B cells serves as a new paradigm for B cell function and classification.

Influenza and other respiratory virus infections in outpatients with medically attended acute respiratory infection during the 2011-12 influenza season.

BACKGROUND: Respiratory tract infections are a major cause of outpatient visits, yet only a portion is tested to determine the etiologic organism. Multiplex reverse transcriptase polymerase chain reaction (MRT-PCR) assays for detection of multiple viruses are being used increasingly in clinical settings. METHODS: During January-April 2012, outpatients with acute respiratory illness (≤ 7 days) were tested for influenza using singleplex RT-PCR (SRT-PCR). A subset was assayed for 18 viruses using MRT-PCR to compare detection of influenza and examine the distribution of viruses and characteristics of patients using multinomial logistic regression. RESULTS: Among 662 participants (6 months-82 years), detection of influenza was similar between the MRT-PCR and SRT-PCR (κ = 0.83). No virus was identified in 267 (40.3%) samples. Commonly detected viruses were human rhinovirus (HRV, 15.4%), coronavirus (CoV, 10.4%), respiratory syncytial virus (RSV, 8.4%), human metapneumovirus (hMPV, 8.3%), and influenza (6%). Co-detections were infrequent (6.9%) and most commonly occurred among those <18 years old. In regression analyses, compared with non-viral illnesses, RSV and hMPV were significantly more frequent in children and less frequent in 18- to 49-year-olds than in those ≥ 50 years (P = 0.01), fever was more common in hMPV and influenza infections (P = 0.008), nasal congestion was more frequent in CoV, HRV, hMPV, influenza and RSV infections (P = 0.001), and body mass index was higher among those with influenza (P = 0.036). CONCLUSIONS: Using MRT-PCR, a viral etiology was found in three-fifths of patients with medically attended outpatient visits for acute respiratory illness during the influenza season; co-detected viruses were infrequent. Symptoms varied by viral etiology.

Detection of influenza virus infection using two PCR methods.

Rapid, accurate, and cost-effective methods to identify the cause of respiratory tract infections are needed to maximize clinical benefit. Outpatients with acute respiratory illness were tested for influenza using a singleplex reverse transcriptase polymerase chain reaction (SRT-PCR) method. A multiplex RT-PCR (MRT-PCR) method tested for influenza and 17 other viruses and was compared with SRT-PCR using chi-square tests. Among 935 patients, 335 (36%) tested positive for influenza A and influenza B using SRT-PCR. Using MRT-PCR, 320 (34.2%) tested positive for influenza A and influenza B. This study supports MRT-PCR as a comparable method for detecting influenza among patients seeking outpatient care for acute respiratory illnesses.

Clinical evaluation of multiplex real-time PCR panels for rapid detection of respiratory viral infections.

Respiratory viral infections are one of the leading causes of morbidity and mortality, particularly in children, the elderly and immunocompromised persons. Rapid identification of viral etiology is critical in ruling out non-viral infections, initiating antiviral treatment and limiting the spread of the infection. Multiplex assays of more than one viral gene target in a single tube have the advantage of rapid screening of a large number of potential viral pathogens in a short time. A multiplex real-time PCR assay was used in this study for detection of respiratory RNA and DNA viral infections in 728 specimens received from 585 adult and pediatric patients comprised of symptomatic and asymptomatic organ transplant recipients and non-recipients for diagnosis of respiratory illnesses and for routine clinical monitoring. Multiplex PCR was more sensitive than the multiplex immunofluoresence culture assay (R-mix) and also detected additional respiratory viruses that were not covered by the R-mix panel. The number of respiratory viruses detected in symptomatic patients was significantly higher than asymptomatic patients in both adult and pediatric patients. Herpesviral infections were the predominant cause of lower respiratory tract infection in the organ transplant recipients, whereas respiratory syncytial virus was the most common pathogen in non-transplant patients particularly children. Multiplex real-time PCR for detection of respiratory viruses has the potential for rapid identification of viral pathogens. In this era of emerging viral infections, addition of newer viral targets to the multiplex PCR panels will be beneficial in determining both patient management and public health epidemiology.

An optimized sensitive method for quantitation of DNA/RNA viruses in heparinized and cryopreserved plasma.

Sodium heparin, an anticoagulant used widely for blood collection, has been known to inhibit DNA polymerase activity in polymerase chain reaction (PCR) assays. However, all cryopreserved plasma samples collected in the 1980s and early 1990s at the Multicenter AIDS Cohort Study were from heparin-treated blood, which poses a problem in quantifying the target nucleic acids contained in those samples by PCR assay. In this study, a nucleic acid extraction procedure was optimized to remove the heparin from extracted nucleic acids. Using this optimized method, similar human immunodeficiency virus 1 (HIV-1) and cytomegalovirus (CMV) loads of these viruses that were added to normal donor blood from ethylenediaminetetraacetic acid (EDTA), acid citrate dextrose (ACD) or sodium heparin tubes were detected by reverse transcriptase (RT) real-time PCR and real-time PCR. Comparable HIV-1 and CMV loads were also detected in the blood of persons with active HIV-1 and CMV infections collected in EDTA-, ACD- or sodium heparin-treated tubes by RT real-time and real-time PCR. The findings showed that the optimized nucleic acid extraction procedure efficiently removes the heparin inhibition effect on the performance of real-time PCR. This method could be used to extract nucleic acids from archived, heparinized plasma for PCR based quantitation of target molecules.

Relationship of cytomegalovirus load assessed by real-time PCR to pp65 antigenemia in organ transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) infection in immunocompromised patients can lead to viremia associated with morbidity and mortality. Monitoring of viral loads in blood is critical for initiating and monitoring antiviral treatment. OBJECTIVES: Validate quantitative real-time PCR assay targeting the US17 and UL54 regions of the CMV genome for automated DNA and extraction and amplification. STUDY DESIGN: 3422 blood specimens from organ transplant recipients, including longitudinal specimens from 12 organ transplant recipients, were tested by CMV PCR and pp65 antigenemia. RESULTS: CMV PCR for both US17 and UL54, was more sensitive and detected CMV DNA earlier and for longer than the CMV pp65 antigenemia test. Using antigenemia results as a reference standard, an optimal cutoff of 500 normalized copies was calculated for both US17 and UL54 PCR targets based on high sensitivity, specificity, and positive and negative predictive values. CMV DNA levels tracked well with clinical symptoms, response to treatment, and antigenemia. CONCLUSIONS: Detection of persistent increases in CMV DNA levels above 500 normalized copies by this real-time PCR assay is indicative of symptomatic CMV disease in organ transplant recipients. Quantitative real-time PCR for CMV DNA can be used in lieu of antigenemia for monitoring CMV infection and determining when to initiate preemptive treatment.

Diagnosis of human metapneumovirus infection in immunosuppressed lung transplant recipients and children evaluated for pertussis.

Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is known to cause respiratory tract infections in children and immunocompromised individuals. Given the difficulties of identifying hMPV by conventional culture, molecular techniques could improve the detection of this virus in clinical specimens. In this study, we developed a real-time reverse transcription-PCR (RT-PCR) assay designed to detect the four genetic lineages of hMPV. This assay and a commercial real-time nucleic acid sequence-based amplification (NASBA) assay (bioMérieux, Durham, NC) were used to determine the prevalence of hMPV in 114 immunosuppressed asymptomatic and symptomatic lung transplant recipients and 232 pediatric patients who were being evaluated for pertussis. hMPV was detected in 4.3% of the immunosuppressed lung transplant recipients and in 9.9% of children evaluated for pertussis. Both RT-PCR and NASBA assays were efficient in detection of hMPV infection in respiratory specimens. Even though hMPV was detected in a small number of the lung transplant recipients, it was still the most prevalent etiologic agent detected in patients with respiratory symptoms. In both of these diverse patient populations, hMPV infection was the most frequent viral respiratory tract infection identified. Given our findings, infection with hMPV infection should be determined as part of the differential diagnosis of respiratory illnesses.

Clinical utility of CMV early and late transcript detection with NASBA in bronchoalveolar lavages.

BACKGROUND: Cytomegalovirus (CMV) infection can cause severe disease in immunocompromised individuals, with CMV pneumonia, most commonly seen in lung or bone marrow transplant recipients, carrying a particularly high fatality rate. Early and accurate diagnosis of CMV pneumonia is therefore critical. OBJECTIVES: Current diagnostic tests for CMV pneumonia in bronchoalveolar lavage (BAL) specimens are either insensitive or poor prognostic indicators of disease. We therefore examined nucleic acid sequence-based amplification (NASBA) assays for CMV transcripts in BAL for the prediction of CMV pneumonia and associated diseases. STUDY DESIGN: A total of 220 BAL specimens from lung transplant recipients and other patients with suspected viral pneumonia were studied. Ninety-nine samples had previously tested positive for CMV by shell vial (SV) culture, while the other 121 had tested negative. All specimens were assayed for CMV pp67 and immediate early (IE) transcripts by NASBA. Results were correlated with evidence of concurrent or subsequent CMV pneumonia, rejection, and infection with other microbes. RESULTS: From a total of 220 BAL specimens, 27 tested positive for pp67 mRNA, 25 tested positive for IE mRNA, and 17 tested positive for both. Only 10 specimens tested positive for CMV by either or both NASBA assays while testing negative by SV assay. However, 74 specimens were SV positive but negative in both NASBA assays. Detection of CMV by any of the three methods was associated with an increased prevalence of pneumonia (i.e., pulmonary interstitial inflammation with radiographic or clinical evidence of lung injury), but not with pulmonary CMV pathology. Detection of CMV by SV was associated with moderate to severe graft rejection. There was no evidence of increased bacterial or fungal pulmonary infections associated with a positive CMV result by any of the three assays. CONCLUSIONS: Detection of either CMV pp67 or IE mRNA transcripts by NASBA in BAL specimens can occasionally identify CMV infections that are negative by conventional shell vial culture, but does not have sufficient sensitivity or positive predictive value to be employed routinely for pre emptive management of pulmonary CMV disease in transplant recipients.

Distinguishing cytomegalovirus (CMV) infection and disease with CMV nucleic acid assays.

Human cytomegalovirus (CMV) continues to be a significant cause of morbidity and mortality among transplant recipients. Molecular assays have been developed for the detection and quantification of CMV nucleic acid. In evaluating the clinical utility of these assays, correlations with clinical outcome are essential. The Amplicor CMV Monitor and NucliSens CMV pp67 tests were compared to the CMV antigenemia assay for 45 transplant recipients and 1 patient with Wegener's granulomatosis. Twenty-three patients remained antigenemia negative throughout the monitoring period, none of whom developed CMV disease. In this patient group, both the Amplicor and NucliSens assays showed very high specificity; only 1 of the 324 specimens assayed by NucliSens and none of the 303 specimens assayed by Amplicor were positive. Twenty-three patients were antigenemia positive during the monitoring period, 12 of whom developed 13 episodes of symptomatic CMV disease. In this patient group, the NucliSens assay was positive at or before the development of symptoms in 12 of the 13 episodes of CMV disease. All eight patients with symptomatic CMV disease who were tested by the Amplicor assay were positive at or before the development of disease. For the 11 asymptomatic patients, the NucliSens assay was positive less frequently than the antigenemia or Amplicor assays. The NucliSens assay was more likely to be positive at higher antigenemia or viral load levels. Both the NucliSens and Amplicor assays appear to have clinical utility in monitoring patients for CMV disease.