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In this chapter we will discuss the diagnosis and monitoring of individuals with HIV infection. The application and interpretation of these tests does not change dramatically when used in the immunocompromised host. The principal approach to the diagnosis of HIV infection involves serologic testing, although nucleic acid amplification tests play an important role in the diagnosis of acute HIV infection. The algorithm for diagnosis of HIV continues to evolve with newer assays that are able to detect infection within an earlier timeframe after HIV transmission. Viral load testing for HIV-1 is the cornerstone for monitoring patients on antiretroviral therapy. Genotypic and phenotypic resistance tests are employed when antiretroviral resistance is suspected and results help guide therapy. The tropism assay must be performed to determine the efficacy of CCR5 chemokine receptor antagonists. Next-generation sequencing methods are an innovative approach to assessing archived antiretroviral resistance in patients with virologic suppression. The success of antiretroviral therapy with improved long-term outcomes has made transplantation in HIV-infected patients a reality.

Quantitative measurement of viral load is an important parameter in the management of filovirus disease outbreaks because viral load correlates with severity of disease, survival, and infectivity. During the ongoing Ebola virus disease outbreak in parts of Western Africa, most assays used in the detection of Ebola virus disease by more than 44 diagnostic laboratories yielded qualitative results. Regulatory hurdles involved in validating quantitative assays and the urgent need for a rapid Ebola virus disease diagnosis precluded development of validated quantitative assays during the outbreak. Because of sparse quantitative data obtained from these outbreaks, opportunities for study of correlations between patient outcome, changes in viral load during the course of an outbreak, disease course in asymptomatic individuals, and the potential for virus transmission between infected patients and contacts have been limited. We strongly urge the continued development of quantitative viral load assays to carefully evaluate these parameters in future outbreaks of filovirus disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cytomegalovirus (CMV) is an important cause of morbidity and mortality in transplant patients, and is typically monitored using laboratory-developed quantitative molecular assays. Clinicians who use quantitative CMV DNA testing should be aware of a number of aspects of testing that will aid in decision making while managing CMV disease in their patients. These include (1) the specimen type used (whole blood or plasma), (2) the limit of detection and limit of quantification chosen by the clinical laboratory, (3) the linear range of the assay, (4) the reproducibility of the assay within the institution, and (5) the wide variability of viral load values among different assays. The biologic properties of CMV, including its variability within the host and of its half-life, are also important factors in the clinical testing for this virus.

Background: Cytomegalovirus (CMV) DNA viral load testing is routinely performed in centers that serve patients that are immunosuppressed from organ or hematopoietic stem cell transplantation. Clinical laboratories that offer this testing often face practical concerns about the storage of these specimens to ensure accurate measurement for patient care. The published studies that assess CMV DNA stability at 4°C have done so only up to 72 h., Objective: Our objective was to determine the stability of CMV DNA in whole blood and plasma for clinical viral load testing over a 14 day period., Study Design: Twenty-one plasma samples that were CMV-positive and twenty whole blood samples (including eleven CMV-negative whole blood samples spiked with CMV-positive plasma) were stored at 4°C and underwent extraction and amplification at 3 time points: Day 0, Day 7, and Day 14., Results: Log(10) values were calculated and t-test was performed on the values comparing Day 0 to Day 14 for plasma and whole blood. There was no statistically significant difference between Day 0 and Day 14 for both specimen types, including the CMV-negative whole blood specimens that were spiked with CMV-positive plasma., Conclusions: CMV DNA in plasma and whole blood is stable for 14 days at a temperature of 4°C., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background Previous research has shown that rooms of patients with coronavirus disease 2019 (COVID-19) present the potential for healthcare-associated transmission through aerosols containing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, data on the presence of these aerosols outside of patient rooms are limited. We investigated whether virus-containing aerosols were present in nursing stations and patient room hallways in a referral center with critically ill COVID-19 patients. Methods Eight National Institute for Occupational Safety and Health BC 251 2-stage cyclone samplers were set up throughout 6 units, including nursing stations and visitor corridors in intensive care units and general medical units, for 6 h each sampling period. Samplers were placed on tripods which held 2 samplers positioned 102 cm and 152 cm above the floor. Units were sampled for 3 days. Extracted samples underwent reverse transcription polymerase chain reaction for selected gene regions of the SARS-CoV-2 virus nucleocapsid and the housekeeping gene human RNase P as an internal control. Results The units sampled varied in the number of laboratory-confirmed COVID-19 patients present on the days of sampling. Some of the units included patient rooms under negative pressure, while most were maintained at a neutral pressure. Of 528 aerosol samples collected, none were positive for SARS-CoV-2 RNA by the estimated limit of detection of 8 viral copies/m3 of air. Conclusions Aerosolized SARS-CoV-2 outside of patient rooms was undetectable. While healthcare personnel should avoid unmasked close contact with each other, these findings may provide reassurance for the use of alternatives to tight-fitting respirators in areas outside of patient rooms during the current pandemic. [ABSTRACT FROM AUTHOR]

To assess the impact of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic on seasonal respiratory viruses, absolute case counts and viral reproductive rates from 2019–2020 were compared against previous seasons. Our findings suggest that the public health measures implemented to reduce SARS-CoV-2 transmission significantly reduced the transmission of other respiratory viruses. [ABSTRACT FROM AUTHOR]

Background: In 2014, passive immunization by transfusion of Ebola convalescent plasma (ECP) was considered for treating patients with acute Ebola virus disease (EVD). Early Ebola virus (EBOV) seroconversion confers a survival advantage in natural infection, hence transfusion of ECP plasma with high levels of neutralizing EBOV antibodies is a potential passive immune therapy. Techniques to reduce the risk of other transfusion-transmitted infections (TTIs) are warranted as recent ECP survivors are ineligible as routine blood donors. As part of an ongoing clinical trial to evaluate the safety and effectiveness of ECP, the impact of amotosalen/UVA pathogen reduction technology (PRT) on EBOV antibody characteristics was examined.Study Design and Methods: Serum and plasma samples were collected from EVD-recovered subjects at multiple timepoints and evaluated by ELISA for antibodies to recombinant EBOV glycoprotein (GP) and irradiated whole EBOV antigen, as well as for EBOV microneutralization, classic plaque reduction neutralization test (PRNT) and EBOV pseudovirion neutralization assay (PsVNA) activity.Results: Six subjects donated 40 individual ECP units. Substantial antibody titers and neutralizing activity results were demonstrated but were generally lower for the ACD plasma samples compared to the serum samples. Anti-EBOV titers by all assays remained essentially unchanged after PRT.Conclusion: Treatment of ECP with PRT to reduce the risk of TTI did not significantly reduce EBOV IgG antibody titers or neutralizing activity. Although ECP was used in the treatment of repatriated patients, no PRT units from this study were transfused to EVD patients. This inventory of PRT-treated ECP is currently available for future clinical evaluation. [ABSTRACT FROM AUTHOR]

Background. Personal protective equipment (PPE) protects healthcare workers (HCWs) caring for patients with Ebola virus disease (EVD), and PPE doffing is a critical point for preventing viral self-contamination. We assessed contamination of skin, gloves, and scrubs after doffing Ebola-level PPE contaminated with surrogate viruses: bacteriophages MS2 and Φ6. Methods. In a medical biocontainment unit, HCWs (n = 10) experienced in EVD care donned and doffed PPE following unit protocols that incorporate trained observer guidance and alcohol-based hand rub (ABHR). A mixture of Φ6 (enveloped), MS2 (nonenveloped), and fluorescent marker was applied to 4 PPE sites, approximating body fluid viral load (Φ6, 105; MS2, 106). They performed a patient care task, then doffed. Inner gloves, face, hands, and scrubs were sampled for virus, as were environmental sites with visible fluorescent marker. Results. Among 10 HCWs there was no Φ6 transfer to inner gloves, hands, or face; 1 participant had Φ6 on scrubs at low levels (1.4 X 10²). MS2 transfer (range, 10¹-106) was observed to scrubs (n = 2), hands (n = 1), and inner gloves (n = 7), where it was highest. Most (n = 8) had only 1 positive site. Environmental samples with visible fluorescent marker (n = 21) were negative. Conclusions. Among experienced HCWs, structured, observed doffing using ABHR protected against hand contamination with enveloped virus. Nonenveloped virus was infrequent on hands and scrubs but common on inner gloves, suggesting that inner gloves, but not necessarily ABHR, protect against hand contamination. Optimizing doffing protocols to protect against all types of viruses may require reinforcing careful handling of scrubs and good glove/hand hygiene with effective agents. [ABSTRACT FROM AUTHOR]

Abstract: Background: Since switching to the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, v. 1.0 from the Amplicor HIV-1 Monitor Test, v. 1.5, an increase in detectable viral load results was noted. We were concerned that this was due to the use of Plasma Preparation Tubes (PPT) in this test. Objective: To assess the impact of different pre-analytical processing conditions on HIV-1 viral load results on the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test. Study design: Sixty-three HIV-infected patients were consented and had 3 PPTs and 1 K2EDTA drawn for HIV-1 viral load testing. Three methods of PPT processing were compared against the referent K2EDTA tube which was spun at 1100× g for 20min, poured off and frozen; PPT1 was refrigerated with an additional centrifugation prior to testing, PPT2 was processed similarly to EDTA, and PPT3 was centrifuged, frozen and centrifuged again prior to testing. Results: PPT1 and PPT3 yielded results that were most similar to the referent EDTA processing, with a concordance correlation coefficient (CCC) of 0.80 and 0.85, compared to PPT2 with CCC of 0.37. Both PPT1 and PPT3 involved additional centrifugation prior to testing. In 26 patients with residual samples from the PPT2 processing, 9 (34.6%) were found to have the presence of proviral DNA, which likely contributed to the elevated HIV-1 RNA viral loads in these individuals. Conclusion: PPTs can be used in the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test with an additional centrifugation in order to avoid misleading elevated HIV-1 RNA viral loads that may change patient management. [Copyright &y& Elsevier]

Background Human cytomegalovirus (CMV) is the leading cause of intrauterine and perinatal viral infection. The most common route of CMV transmission in newborns is through breastmilk and this can lead to infant morbidity and mortality. Breast milk that has been frozen for an extended period may need to be tested for CMV DNA to determine the source of infection. It has been a challenge for clinical laboratories to ensure the stability of CMV DNA in frozen breast milk for accurate viral load measurement. Objectives To evaluate the stability of CMV DNA in breast milk by testing quantitative viral loads over a 28-day period for breast milk stored at 4 °C and a 90-day period for breast milk stored at −20 °C. Study design Baseline viral loads were determined on day 0 and the samples stored at 4 °C underwent extraction and amplification at four time points, up to 28 days. The samples stored at −20 °C underwent extraction and amplification at five time points up to 90 days. Log 10 values were calculated and t -test, Pearson’s coefficient, and concordance correlation coefficient were calculated. Results There was no statistically significant difference between the time points by t -test, and correlation coefficients showed greater than 90% concordance for days 0 and 28 as well as days 0 and 90 at both storage temperatures tested. Conclusions The concentration of CMV DNA in breast milk was stable for 28 days at 4 °C and 90 days at −20 °C as the concentrations did not differ significantly from the baseline viral loads. [ABSTRACT FROM AUTHOR]

• A modified Aptima HIV-1 Quant protocol can quantify RNA using 100 μL of plasma. • The standard and modified protocols had high agreement above 50 copies/mL. • Using microtainers for blood collection could expand access to treatment and care. Currently, FDA-approved HIV-1 viral load (VL) assays use venipuncture-derived plasma. The Hologic Panther system uses 0.7 mL total volume for the Aptima HIV-1 Quant Assay standard (APT-Quant-std) and dilution (APT-Quant-dil) protocols. However, smaller plasma volumes from fingerstick whole blood (FSB) collected in EDTA-microtainer tubes (MCT) could provide an easier sample collection method for HIV-1 VL testing. To evaluate the performance of the APT-Quant-std compared to the Roche CAP/CTM and Abbott m2000RT VL assays and an alternative APTQuant 1:7 dilution protocol, the latter using 100 μL of MCT-derived plasma from FSB. Linearity was determined using commercial HIV-1 RNA plasma controls. Dilutions ranging 1.56–2.95 log10 copies/mL were prepared to determine the APT-Quant-dil Limit of Quantitation (LOQ) using Probit analysis. Specificity of APT-Quant-std was calculated using 326 HIVnegative samples. To evaluate agreement, 329 plasma specimens were tested with APT-Quant-std, CAP/CTM, and m2000RT. Forty-seven matched venipuncture and MCT-derived plasma specimens were tested with APT-Quant-std and APT-Quant-dil. Among the RNA controls, specificity was 99.69 % for APT-Quant-std. The R2 values were 0.988 (APT-Quant-std/CAP/CTM), 0.980 (APT-Quant-std/ m2000RT), and 0.997 (APT-Quant-std/APT-Quant-dil). The APT-Quant-dil LOQ was estimated at 2.7 log10 copies/mL (500 copies/mL) (95 %CI 2.62–2.87). At 2.3 log10 copies/mL (200 copies/mL), the overall agreement was 91.0 % for APT-Quant-std/CAP/CTM, 85.7 % for APT-Quant-std/m2000RT, and 82.9 % for APT-Quant-std/APT-Quant-dil. Quantified APT-Quant-std results were on average 0.2 log10 copies/mL higher than CAP/CTM and m2000RT and 0.14 log10 copies/mL higher than APT-Quant-dil. APT-Quant showed similar performance compared to the CAP/CTM and m2000RT assays and remains sensitive and accurate using the dilution protocol. [ABSTRACT FROM AUTHOR]