Higher severe acute respiratory syndrome coronavirus 2 infection rate in pregnant patients.

BACKGROUND: During the early months of the coronavirus disease 2019 pandemic, risks associated with severe acute respiratory syndrome coronavirus 2 in pregnancy were uncertain. Pregnant patients can serve as a model for the success of clinical and public health responses during public health emergencies as they are typically in frequent contact with the medical system. Population-based estimates of severe acute respiratory syndrome coronavirus 2 infections in pregnancy are unknown because of incomplete ascertainment of pregnancy status or inclusion of only single centers or hospitalized cases. Whether pregnant women were protected by the public health response or through their interactions with obstetrical providers in the early months of pandemic is not clearly understood. OBJECTIVE: This study aimed to estimate the severe acute respiratory syndrome coronavirus 2 infection rate in pregnancy and to examine the disparities by race and ethnicity and English language proficiency in Washington State. STUDY DESIGN: Pregnant patients with a polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 infection diagnosed between March 1, 2020, and June 30, 2020 were identified within 35 hospitals and clinics, capturing 61% of annual deliveries in Washington State. Infection rates in pregnancy were estimated overall and by Washington State Accountable Community of Health region and cross-sectionally compared with severe acute respiratory syndrome coronavirus 2 infection rates in similarly aged adults in Washington State. Race and ethnicity and language used for medical care of pregnant patients were compared with recent data from Washington State. RESULTS: A total of 240 pregnant patients with severe acute respiratory syndrome coronavirus 2 infections were identified during the study period with 70.7% from minority racial and ethnic groups. The principal findings in our study were as follows: (1) the severe acute respiratory syndrome coronavirus 2 infection rate was 13.9 per 1000 deliveries in pregnant patients (95% confidence interval, 8.3-23.2) compared with 7.3 per 1000 (95% confidence interval, 7.2-7.4) in adults aged 20 to 39 years in Washington State (rate ratio, 1.7; 95% confidence interval, 1.3-2.3); (2) the severe acute respiratory syndrome coronavirus 2 infection rate reduced to 11.3 per 1000 deliveries (95% confidence interval, 6.3-20.3) when excluding 45 cases of severe acute respiratory syndrome coronavirus disease 2 detected through asymptomatic screening (rate ratio, 1.3; 95% confidence interval, 0.96-1.9); (3) the proportion of pregnant patients in non-White racial and ethnic groups with severe acute respiratory syndrome coronavirus disease 2 infection was 2- to 4-fold higher than the race and ethnicity distribution of women in Washington State who delivered live births in 2018; and (4) the proportion of pregnant patients with severe acute respiratory syndrome coronavirus 2 infection receiving medical care in a non-English language was higher than estimates of pregnant patients receiving care with limited English proficiency in Washington State (30.4% vs 7.6%). CONCLUSION: The severe acute respiratory syndrome coronavirus 2 infection rate in pregnant people was 70% higher than similarly aged adults in Washington State, which could not be completely explained by universal screening at delivery. Pregnant patients from nearly all racial and ethnic minority groups and patients receiving medical care in a non-English language were overrepresented. Pregnant women were not protected from severe acute respiratory syndrome coronavirus 2 infection in the early months of the pandemic. Moreover, the greatest burden of infections occurred in nearly all racial and ethnic minority groups. These data coupled with a broader recognition that pregnancy is a risk factor for severe illness and maternal mortality strongly suggested that pregnant people should be broadly prioritized for coronavirus disease 2019 vaccine allocation in the United States similar to some states.

Disease severity, pregnancy outcomes, and maternal deaths among pregnant patients with severe acute respiratory syndrome coronavirus 2 infection in Washington State.

BACKGROUND: Evidence is accumulating that coronavirus disease 2019 increases the risk of hospitalization and mechanical ventilation in pregnant patients and for preterm delivery. However, the impact on maternal mortality and whether morbidity is differentially affected by disease severity at delivery and trimester of infection are unknown. OBJECTIVE: This study aimed to describe disease severity and outcomes of severe acute respiratory syndrome coronavirus 2 infections in pregnancy across the Washington State, including pregnancy complications and outcomes, hospitalization, and case fatality. STUDY DESIGN: Pregnant patients with a polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 infection between March 1, 2020, and June 30, 2020, were identified in a multicenter retrospective cohort study from 35 sites in Washington State. Sites captured 61% of annual state deliveries. Case-fatality rates in pregnancy were compared with coronavirus disease 2019 fatality rates in similarly aged adults in Washington State using rate ratios and rate differences. Maternal and neonatal outcomes were compared by trimester of infection and disease severity at the time of delivery. RESULTS: The principal study findings were as follows: (1) among 240 pregnant patients in Washington State with severe acute respiratory syndrome coronavirus 2 infections, 1 in 11 developed severe or critical disease, 1 in 10 were hospitalized for coronavirus disease 2019, and 1 in 80 died; (2) the coronavirus disease 2019-associated hospitalization rate was 3.5-fold higher than in similarly aged adults in Washington State (10.0% vs 2.8%; rate ratio, 3.5; 95% confidence interval, 2.3-5.3); (3) pregnant patients hospitalized for a respiratory concern were more likely to have a comorbidity or underlying conditions including asthma, hypertension, type 2 diabetes mellitus, autoimmune disease, and class III obesity; (4) 3 maternal deaths (1.3%) were attributed to coronavirus disease 2019 for a maternal mortality rate of 1250 of 100,000 pregnancies (95% confidence interval, 257-3653); (5) the coronavirus disease 2019 case fatality in pregnancy was a significant 13.6-fold (95% confidence interval, 2.7-43.6) higher in pregnant patients than in similarly aged individuals in Washington State with an absolute difference in mortality rate of 1.2% (95% confidence interval, -0.3 to 2.6); and (6) preterm birth was significantly higher among women with severe or critical coronavirus disease 2019 at delivery than for women who had recovered from coronavirus disease 2019 (45.4% severe or critical coronavirus disease 2019 vs 5.2% mild coronavirus disease 2019; P<.001). CONCLUSION: Coronavirus disease 2019 hospitalization and case-fatality rates in pregnant patients were significantly higher than in similarly aged adults in Washington State. These data indicate that pregnant patients are at risk of severe or critical disease and mortality compared to nonpregnant adults, and also at risk for preterm birth.

MicroRNA Signature of Epithelial-Mesenchymal Transition in Group B Streptococcal Infection of the Placental Chorioamniotic Membranes.

BACKGROUND: Infection-induced preterm birth is a major cause of neonatal mortality and morbidity and leads to preterm premature rupture of placental chorioamniotic membranes. The loss of amniotic epithelial cells and tensile strength preceding membrane rupture is poorly understood. We hypothesized that intrauterine bacterial infection induces changes in microRNA (miRNA) expression, leading to amniotic epithelial cell loss and membrane weakening. METHODS: Ten pregnant pigtail macaques received choriodecidual inoculation of either group B Streptococcus (GBS) or saline (n = 5/group). Placental chorioamniotic membranes were studied using RNA microarray and immunohistochemistry. Chorioamniotic membranes from women with preterm premature rupture of membranes (pPROM) and normal term pregnancies were studied using transmission electron microscopy. RESULTS: In our model, an experimental GBS infection was associated with changes in the miRNA profile in the chorioamniotic membranes consistent with epithelial to mesenchymal transition (EMT) with loss of epithelial (E-cadherin) and gain of mesenchymal (vimentin) markers. Similarly, loss of desmosomes (intercellular junctions) was seen in placental tissues from women with pPROM. CONCLUSIONS: We describe EMT as a novel mechanism for infection-associated chorioamniotic membrane weakening, which may be a common pathway for many etiologies of pPROM. Therapy based on anti-miRNA targeting of EMT may prevent pPROM due to perinatal infection.

Clinical characteristics of 46 pregnant women with a severe acute respiratory syndrome coronavirus 2 infection in Washington State.

BACKGROUND: The impact of coronavirus disease 2019 on pregnant women is incompletely understood, but early data from case series suggest a variable course of illness from asymptomatic or mild disease to maternal death. It is unclear whether pregnant women manifest enhanced disease similar to influenza viral infection or whether specific risk factors might predispose to severe disease. OBJECTIVE: To describe maternal disease and obstetrical outcomes associated with coronavirus disease 2019 in pregnancy to rapidly inform clinical care. STUDY DESIGN: This is a retrospective study of pregnant patients with a laboratory-confirmed severe acute respiratory syndrome coronavirus 2 infection from 6 hospital systems in Washington State between Jan. 21, 2020, and April 17, 2020. Demographics, medical and obstetrical history, and coronavirus disease 2019 encounter data were abstracted from medical records. RESULTS: A total of 46 pregnant patients with a severe acute respiratory syndrome coronavirus 2 infection were identified from hospital systems capturing 40% of births in Washington State. Nearly all pregnant individuals with a severe acute respiratory syndrome coronavirus 2 infection were symptomatic (93.5%, n=43) and the majority were in their second or third trimester (43.5% [n=20] and 50.0% [n=23], respectively). Symptoms resolved in a median of 24 days (interquartile range, 13-37). Notably, 7 women were hospitalized (16%) including 1 admitted to the intensive care unit. A total of 6 cases (15%) were categorized as severe coronavirus disease 2019 with nearly all patients being either overweight or obese before pregnancy or with asthma or other comorbidities. Of the 8 deliveries that occurred during the study period, there was 1 preterm birth at 33 weeks' gestation to improve pulmonary status in a woman with class III obesity, and 1 stillbirth of unknown etiology. CONCLUSION: Severe coronavirus disease 2019 developed in approximately 15% of pregnant patients and occurred primarily in overweight or obese women with underlying conditions. Obesity and coronavirus disease 2019 may synergistically increase risk for a medically indicated preterm birth to improve maternal pulmonary status in late pregnancy. These findings support categorizing pregnant patients as a higher-risk group, particularly those with chronic comorbidities.

Evaluation of a novel screening method for fetal aneuploidy using cell-free DNA in maternal plasma.

Objective: To evaluate the test performance of a novel sequencing technology using molecular inversion probes applied to cell-free DNA screening for fetal aneuploidy. Methods: Two cohorts were included in the evaluation; a risk-based cohort of women receiving diagnostic testing in the first and second trimesters was combined with stored samples from pregnancies with fetuses known to be aneuploid or euploid. All samples were blinded to testing personnel before being analyzed, and validation occurred after the study closed and results were merged. Results: Using the new sequencing technology, 1414 samples were analyzed. The findings showed sensitivities and specificities for the common trisomies and the sex chromosome aneuploidies at >99% (Trisomy 21 sensitivity 99.2 CI 95.6­99.2; specificity 99.9 CI 99.6­99.9). Positive predictive values among the trisomies varied from 85.2% (Trisomy 18) to 99.0% (Trisomy 21), reflecting their prevalence rates in the study. Comparisons with a meta-analysis of recent cell-free DNA screening publications demonstrated equivalent test performance. Conclusion: This new technology demonstrates equivalent test performance compared with alternative sequencing approaches, and demonstrates that each chromosome can be successfully interrogated using a single probe.

Vasa previa: diagnosis and management.

BACKGROUND: Vasa previa is a rare condition that is associated with a high rate of fetal or neonatal death when not diagnosed antenatally. The majority of available studies are either small, do not include antepartum data, limited to single institutions, or are biased by inclusion of patients from registries and online vasa previa support groups. OBJECTIVE: The purpose of this study was to investigate the diagnostic and management strategies for this potentially catastrophic entity and to describe further maternal and placental risk factors that may aid in the establishment of a screening protocol for vasa previa. STUDY DESIGN: This was a retrospective multicenter descriptive study that included all pregnancies that were complicated by vasa previa that delivered between January 1, 2000, and December 31, 2012. Nine maternal fetal medicine practices and the hospitals in which they practice participated in data collection of diagnosis, treatment, and maternal-neonatal outcomes. RESULTS: Sixty-eight pregnancies were identified that included the diagnosis of vasa previa or "possible vasa previa" either in the ultrasound record or in the hospital record at the time of delivery. Four cases (5.8%) appeared to resolve on repeat ultrasound examination. Fifteen of the 64 cases that were suspected of having vasa previa could not be verified or were not documented at delivery. Of the remaining 49 cases, where vasa previa was documented, 47 cases (96%) were diagnosed by ultrasound scanning antenatally. Known risk factors for vasa previa were present in 41 of 47 cases (87%). Of the 49 cases, 41 were delivered by planned cesarean delivery at a mean gestational age of 34.7 weeks, and 8 cases required emergent cesarean delivery at a mean gestational age of 34.6 weeks (range, 32.4-36.0 weeks gestation). Seven of these emergent cesarean deliveries had been diagnosed previously; 1 case had not. All of the emergent cesarean deliveries were for vaginal bleeding; 1 case was also for a concerning fetal heart rate, but only 1 of the known cases had a documented ruptured fetal vessel. None of these cases were found to have cervical shortening before the onset of bleeding. One of the undiagnosed cases resulted in a ruptured fetal vessel and a baby with no heart beat at birth who survived but had periventricular leukomalacia at 1 month of age with mild white-matter atrophy. Of the remaining neonates in this group, there were no deaths and no major complications beyond mild respiratory distress syndrome in 9 cases. There were no other major neonatal complications, which included no cases of periventricular leukomalacia, neonatal sepsis, necrotizing enterocolitis, or any grade of intraventricular hemorrhage in the confirmed cases of vasa previa. CONCLUSION: This study confirms most current recommendations that include risk-based ultrasound screening, early hospitalization at 30-34 weeks gestation, antenatal corticosteroids at 30-32 weeks gestation, and elective delivery at 33-34 weeks gestation. Thus, with these recommendations for current identification and management of vasa previa in this series of geographically diverse mostly private practice maternal fetal medicine practices, we have confirmed recent reports that show a dramatic improvement in neonatal survival and complications compared with earlier reports.

Choriodecidual Group B Streptococcal Infection Induces miR-155-5p in the Fetal Lung in Macaca nemestrina.

The mechanisms underlying fetal lung injury remain poorly defined. MicroRNAs (miRNAs) are small noncoding, endogenous RNAs that regulate gene expression and have been implicated in the pathogenesis of lung disease. Using a nonhuman primate model of choriodecidual infection, we sought to determine if differentially expressed miRNAs were associated with acute fetal lung injury. After inoculating 10 chronically catheterized pregnant monkeys (Macaca nemestrina) with either group B streptococcus (GBS) at 1 × 10(6) CFU (n = 5) or saline (n = 5) in the choriodecidual space, we extracted fetal lung mRNA and miRNA and profiled the changes in expression by microarray analysis. We identified 9 differentially expressed miRNAs in GBS-exposed fetal lungs, but of these, only miR-155-5p was validated by quantitative reverse transcription-PCR (P = 0.02). Significantly elevated miR-155-5p expression was also observed when immortalized human fetal airway epithelial (FeAE) cells were exposed to proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]). Overexpression of miR-155-5p in FeAE cells in turn increased the production of IL-6 and CXCL10/gamma interferon-induced protein 10, which are implicated in leukocyte recruitment but also in protection from lung injury. Interestingly, while miR-155-5p decreased fibroblast growth factor 9 (FGF9) expression in a luciferase reporter assay, FGF9 levels were actually increased in GBS-exposed fetal lungs in vivo. FGF9 overexpression is associated with abnormal lung development. Thus, upregulation of miR-155-5p may serve as a compensatory mechanism to lessen the increase in FGF9 and prevent aberrant lung development. Understanding the complicated networks regulating lung development in the setting of infection is a key step in identifying how to prevent fetal lung injury leading to bronchopulmonary dysplasia.

Uterine rupture risk after periviable cesarean delivery.

OBJECTIVE: To investigate the risk of uterine rupture in women with prior periviable cesarean delivery and prior term cesarean delivery independent of initial incision type. METHODS: We conducted a retrospective longitudinal cohort study using Washington state birth certificate data and hospital discharge records, identifying primary cesarean deliveries performed at 20-26 weeks and 37-41 weeks of gestation with subsequent delivery between 1989 and 2008. We compared subsequent uterine rupture risk in the two groups considering both primary incision type and subsequent labor induction and augmentation. RESULTS: We identified 456 women with index periviable cesarean delivery and 10,505 women with index term cesarean delivery. Women with index periviable cesarean delivery were younger, more frequently of nonwhite race, more likely to smoke, and more likely to have hypertension. Women in the periviable group had more index classical incisions (42% compared with 1%, P<.001) and fewer subsequent inductions and augmentations (8% compared with 16%, P<.001). Uterine rupture in the subsequent pregnancy occurred more frequently among women in the index periviable group than those in the index term group (8/456 [1.8%] compared with 38/10,505 [0.4%], odds ratio [OR] 4.9, 95% confidence interval [CI] 2.3-10.6). This relationship persisted among women with a low transverse incision (4/228 [1.8%] compared with 36/9,558 [0.4%], OR 4.7, 95% CI 1.7-13.4). CONCLUSION: Cesarean delivery at periviability compared with term is associated with an increased risk for uterine rupture in a subsequent pregnancy, even after low transverse incision. These data support judicious use of cesarean delivery at periviable gestational ages and inform subsequent counseling. LEVEL OF EVIDENCE: II.

Impact of simulation and team training on postpartum hemorrhage management in non-academic centers.

OBJECTIVE: Prompt recognition and response to postpartum hemorrhage (PPH) are vital in preventing maternal morbidity and mortality. We conducted a multi-center study to evaluate in situ simulation and team training for PPH among experienced clinical teams in non-academic hospitals in urban and rural communities. METHODS: A longitudinal intervention study was performed in six Oregon community hospitals. All teams responded to an in situ simulated delivery and postpartum hemorrhage using trained actors and an obstetric birthing simulator, followed by a debriefing and training session. The simulation scenario was then repeated in 9-12 months. All sessions were digitally video recorded and independently reviewed by two obstetricians using a structured evaluation form. PPH management including clinical response times were compared before and after team training using Student's paired t-test and McNemar's test. RESULTS: Twenty-two teams completed paired case simulations. Team training significantly improved response times in the management of PPH, including the recognition of PPH, time to administer first medication, performance of uterine massage and time to administer second medication. Medical management (use of three indicated medications) improved after training from 27.3% to 63.6%, p = 0.01. CONCLUSIONS: Simulation and team training significantly improved postpartum hemorrhage response times among clinically experienced community labor and delivery teams.

Synergy and interactions among biological pathways leading to preterm premature rupture of membranes.

Preterm premature rupture of membranes (PPROM) occurs in 1% to 2% of births. Impact of PPROM is greatest in low- and middle-income countries where prematurity-related deaths are most common. Recent investigations identify cytokine and matrix metalloproteinase activation, oxidative stress, and apoptosis as primary pathways to PPROM. These biological processes are initiated by heterogeneous etiologies including infection/inflammation, placental bleeding, uterine overdistention, and genetic polymorphisms. We hypothesize that pathways to PPROM overlap and act synergistically to weaken membranes. We focus our discussion on membrane composition and strength, pathways linking risk factors to membrane weakening, and future research directions to reduce the global burden of PPROM.

Group B streptococcal infection of the choriodecidua induces dysfunction of the cytokeratin network in amniotic epithelium: a pathway to membrane weakening.

Early events leading to intrauterine infection remain poorly defined, but may hold the key to preventing preterm delivery. To determine molecular pathways within fetal membranes (chorioamnion) associated with early choriodecidual infection that may progress to preterm premature rupture of membranes (PPROM), we examined the effects of a Group B Streptococcus (GBS) choriodecidual infection on chorioamnion in a nonhuman primate model. Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118-125 days gestation (term = 172 days) received choriodecidual inoculation of either GBS (n = 5) or saline (n = 5). Cesarean section was performed in the first week after GBS or saline inoculation. RNA extracted from chorioamnion (inoculation site) was profiled by microarray. Single gene, Gene Set, and Ingenuity Pathway Analysis results were validated using qRT-PCR (chorioamnion), Luminex (amniotic fluid, AF), immunohistochemistry, and transmission electron microscopy (TEM). Despite uterine quiescence in most cases, significant elevations of AF cytokines (TNF-α, IL-8, IL-1ß, IL-6) were detected in GBS versus controls (p<0.05). Choriodecidual infection resolved by the time of cesarean section in 3 of 5 cases and GBS was undetectable by culture and PCR in the AF. A total of 331 genes were differentially expressed (>2-fold change, p<0.05). Remarkably, GBS exposure was associated with significantly downregulated expression of multiple cytokeratin (CK) and other cytoskeletal genes critical for maintenance of tissue tensile strength. Immunofluorescence revealed highly significant changes in the CK network within amniocytes with dense CK aggregates and retraction from the cell periphery (all p = 0.006). In human pregnancies affected by PPROM, there was further evidence of CK network retraction with significantly shorter amniocyte foot processes (p = 0.002). These results suggest early choriodecidual infection results in decreased cellular membrane integrity and tensile strength via dysfunction of CK networks. Downregulation of CK expression and perturbations in the amniotic epithelial cell intermediate filament network occur after GBS choriodecidual infection, which may contribute to PPROM.

Choriodecidual infection downregulates angiogenesis and morphogenesis pathways in fetal lungs from Macaca nemestrina.

BACKGROUND: Intrauterine exposure to amniotic fluid (AF) cytokines is thought to predispose to bronchopulmonary dysplasia (BPD). We evaluated the effects of GBS exposure on RNA expression in fetal lung tissue to determine early molecular pathways associated with fetal lung injury that may progress to BPD. METHODS: Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118-125 days gestation (term = 172 days) received choriodecidual inoculation of either: 1) Group B Streptococcus (n = 5) or 2) saline (n = 5). Cesarean section and fetal necropsy was performed in the first week after GBS or saline inoculation regardless of labor. RNA was extracted from fetal lungs and profiled by microarray. Results were analyzed using single gene, Gene Set, and Ingenuity Pathway Analysis. Validation was by RT-PCR and immunohistochemistry. RESULTS: Despite uterine quiescence in most cases, fetal lung injury occurred in four GBS cases (intra-alveolar neutrophils, interstitial thickening) and one control (peri-mortem hemorrhage). Significant elevations of AF cytokines (TNF-α, IL-8, IL-1ß, IL-6) were detected in GBS versus controls (p<0.05). Lung injury was not directly caused by GBS, because GBS was undetectable by culture and PCR in the AF and fetal lungs. A total of 335 genes were differentially expressed greater than 1.5 fold (p<0.05) with GBS exposure associated with a striking upregulation of genes in innate and adaptive immunity and downregulation of pathways for angiogenesis, morphogenesis, and cellular growth and development. CONCLUSIONS: A transient choriodecidual infection may induce fetal lung injury with profound alterations in the genetic program of the fetal lung before signs of preterm labor. Our results provide a window for the first time into early molecular pathways disrupting fetal lung angiogenesis and morphogenesis before preterm labor occurs, which may set the stage for BPD. A strategy to prevent BPD should target the fetus in utero to attenuate alterations in the fetal lung genetic program.

Intensification of antiretroviral therapy accelerates the decay of the HIV-1 latent reservoir and decreases, but does not eliminate, ongoing virus replication.

This study evaluated whether intensification of standard antiretroviral therapy with abacavir, with or without efavirenz, leads to better viral suppression and acceleration of the rate of HIV-1 decay. Ten HIV-1-infected individuals were enrolled in a prospective, open-label study and received standard, combination antiretroviral therapy with either 3 or 4 agents. The rate of decay of the HIV-1 latent reservoir and the frequency of intermittent viremia were compared between 5 patients who underwent treatment intensification and 5 control subjects with comparable baseline characteristics. When compared with control patients, the median half-life (t1/2) of the latent reservoir decreased from 31 to 10 months (P = 0.016) in subjects who had treatment intensification. The frequency of intermittent viremia/year also decreased in 4 of 5 individuals following intensification (2.4/y vs. 0.8/y). These data suggest that ongoing virus replication during standard antiretroviral therapy is due, in part, to the inadequate antiviral potency of current regimens. Despite better viral suppression, treatment intensification did not completely block viral replication, as evidenced by continuing intermittent viremia in some individuals. Additional studies are needed to understand the host- and pathogen-related determinants of incomplete pharmacologic control of HIV-1 replication.

Infectivity and replication capacity of drug-resistant human immunodeficiency virus type 1 variants isolated during primary infection.

It is believed that replication capacity is an important determinant of human immunodeficiency virus type 1 (HIV-1) pathogenicity and transmissibility. To explore this, we conducted a comprehensive analysis of the replication properties of nine drug-resistant and nine drug-susceptible viral isolates derived from patients with primary HIV-1 infection. Viral isolates were tested for single-cycle infectivity in the GHOST cell line. The infectivity of isolates carrying resistance-associated mutations was significantly higher than that of drug-susceptible isolates. Additionally, the growth kinetics of these isolates were determined in CD4+ T lymphocytes. Drug-resistant isolates replicated as well as drug-susceptible viruses. Insertion of the resistance-conferring regions into an NL4-3-based molecular background resulted in chimeras that displayed a modest but significant reduction in replication capacity compared to the drug-susceptible chimeric viruses. Of note, two multidrug-resistant isolates and one protease inhibitor-resistant isolate displayed higher rates of infectivity and growth kinetics than the other drug-resistant or drug-susceptible isolates. These distinct replicative features, however, were not seen in the corresponding chimeras, indicating that changes within the C-terminal region of Gag as well as within the protease and reverse transcriptase genes contribute to but are not sufficient for the level of compensatory adaptation observed. These findings suggest that some drug-resistant viruses isolated during primary infection possess unique adaptive changes that allow for both high viral replication capacity and resistance to one or more classes of antiretroviral drugs. Further studies are needed to elucidate the precise regions that are essential for these characteristics.

Discontinuation of antiretroviral therapy commenced early during the course of human immunodeficiency virus type 1 infection, with or without adjunctive vaccination.

Sixteen subjects were treated with highly active antiretroviral therapy within 120 days of the onset of symptoms of newly acquired human immunodeficiency virus type 1 (HIV-1) infection. Eleven of the 16 participated in an adjunctive therapeutic vaccine trial. After a mean of 3.2 years of treatment, they elected to discontinue therapy. Virus rebound occurred in all subjects and was followed by a spontaneous, transient although significant reduction in log plasma HIV-1 RNA level, ranging from 0.3 to 3.1 log(10) copies/mL. Despite evidence of the induction of HIV-1-specific cell-mediated immune responses, plasma viremia was not persistently suppressed to <500 copies/mL in any subject. The magnitude and dynamics of virus rebound were similar in both vaccinated and unvaccinated subjects. Nevertheless, given the transient suppression of viremia observed in nearly all subjects after treatment has been discontinued, further investigations of adjunctive vaccination with optimized antiretroviral therapy in treating HIV-1 infection are warranted.

Evolving patterns of HIV-1 resistance to antiretroviral agents in newly infected individuals.

OBJECTIVE: To assess temporal changes in prevalence of transmitted HIV-1 drug resistance in a homogeneous cohort of newly infected individuals. METHODS: Pretreatment genotypic and phenotypic drug resistance was tested in 154 subjects with primary HIV-1 infection identified between 1995 and 2001 (group A; n = 76) and 1999 and 2001 (group B; n = 78). Sequence analysis was assessed by population-based sequencing. Virus susceptibility to antiretroviral agents was determined by the PhenoSense assay (ViroLogic). RESULTS: The frequency of resistance-associated mutations in protease (PR) and reverse transcriptase (RT) genes increased from 13.2% (1995-1998) to 19.7% (1999-2001). Although the overall prevalence of viruses with phenotypic resistance did not vary (1995-1998, 10.0%; 1999-2001, 10.8%), the distribution of drug classes changed [nucleoside RT inhibitor (NRTI): 8.3% to 2.7%; non-NRTI: 5.0% to 8.1%; protease inhibitors (PI): 1.7% to 5.4%]. The decrease of phenotypic resistance to NRTI in 1999-2001 was caused by the absence of transmitted lamivudine-resistant variants. Phenotypically susceptible variants with aspartic acid or serine residues at position 215 of RT (5.3%; P = 0.04) instead emerged. Hypersusceptibility to PI decreased from 18.3% to 5.4% (P = 0.02) while the amino acid substitutions in PR increased over time: M36I (6.6% to 19.7%) and A71V/T (3.9% to 15.8%). CONCLUSIONS: There was an increase in the number of HIV-1 variants with both genotypic and phenotypic resistance to non-NRTI and PI over time. Furthermore, viruses with altered genotypes compatible with thymidine analogue or PI exposure but susceptible phenotypes were seen in 1999-2001. The latter findings suggest transmission of viruses from subjects who have either changed or discontinued therapy.

Origin of human immunodeficiency virus type 1 quasispecies emerging after antiretroviral treatment interruption in patients with therapeutic failure.

The emergence of antiretroviral (ARV) drug-resistant human immunodeficiency virus type 1 (HIV-1) quasispecies is a major cause of treatment failure. These variants are usually replaced by drug-sensitive ones when the selective pressure of the drugs is removed, as the former have reduced fitness in a drug-free environment. This was the rationale for the design of structured ARV treatment interruption (STI) studies for the management of HIV-1 patients with treatment failure. We have studied the origin of drug-sensitive HIV-1 quasispecies emerging after STI in patients with treatment failure due to ARV drug resistance. Plasma and peripheral blood mononuclear cell samples were obtained the day of treatment interruption (day 0) and 30 and 60 days afterwards. HIV-1 pol and env were partially amplified, cloned, and sequenced. At day 60 drug-resistant variants were replaced by completely or partially sensitive quasispecies. Phylogenetic analyses of pol revealed that drug-sensitive variants emerging after STI were not related to their immediate temporal ancestors but formed a separate cluster, demonstrating that STI leads to the recrudescence and reemergence of a sequestrated viral population rather than leading to the back mutation of drug-resistant forms. No evidence for concomitant changes in viral tropism was seen, as deduced from env sequences. This study demonstrates the important role that the reemergence of quasispecies plays in HIV-1 population dynamics and points out the difficulties that may be found when recycling ARV therapies with patients with treatment failure.