Immunocapture of cell surface proteins embedded in HIV envelopes uncovers considerable virion genetic diversity associated with different source cell types.

HIV particles in the blood largely originate from activated lymphocytes and can overshadow variants which may be expressed from other cell types. Investigations of virus persistence must be able to distinguish cells refractory to viral clearance that serve as reservoirs. To investigate additional cell types that may be associated with in vivo HIV expression we developed a virus particle immunomagnetic capture method targeting several markers of cellular origin that become embedded within virion envelopes during budding. We evaluated the ability of markers to better distinguish cell lineage source subpopulations by assessing combinations of different antibodies with cell-sorted in vitro culture and clinical specimens. Various deductive algorithms were designed to discriminate source cell lineages and subsets. From the particle capture algorithms, we identified distinct variants expressed within individuals that were associated with disparate cellular markers. Among the variants uncovered were minority-level viruses with drug resistance mutations undetected by sequencing and often were associated with markers indicative of myeloid lineage (CD3-/CD10-/CD16+ or /CD14+, and CD3-/CD16-/CD14-/CD11c+ or /HLA-DR+) cell sources. The diverse HIV genetic sequences expressed from different cell types within individuals, further supported by the appearance of distinct drug-resistant variants, highlights the complexity of HIV reservoirs in vivo which must be considered for HIV cure strategies. This approach could also be helpful in examining in vivo host cell origins and genetic diversity in infections involving other families of budding viruses.

HIV Immunocapture Reveals Particles Expressed in Semen under INSTI-based Therapy are Largely Myeloid Cell-Derived and Disparate from Circulating Provirus.

As use of HIV integrase strand transfer inhibitors (INSTI) increases and formulations are being developed for maintenance therapies and chemoprophylaxis, assessing virus suppression under INSTI-based regimens in prevention-relevant biologic compartments, such as the male genital tract, is timely. We used cell-source marker virion immunocapture to examine amplification of particle RNA then assessed the phylogenetic relatedness of seminal and blood viral sequences from men with HIV who were prescribed INSTI-based regimens. Seminal plasma immunocaptures yielded amplifiable virion RNA from 13/24 (54%) men, and the sequences were primarily associated with markers indicative of macrophage and resident dendritic cell sources. Genetic distances were greatest (>2%) between seminal virions and circulating proviruses, pointing to ongoing low-level expression from tissue-resident cells. While the low levels in semen predict an improbable likelihood of transmission, viruses with large genetic distances are expressed under potent INSTI therapy and have implications for determining epidemiologic linkages if adherence is suboptimal.

The Effect of Kangaroo Mother Care After Duodenal Obstruction in Neonates.

Objective: The present study aimed to explore the effectiveness of clinical application of kangaroo mother care (KMC) in neonates after surgery for duodenal obstruction in achieving total enteral nutrition (TEN) and shortening the length of hospital stay. Methods: A prospective study of 60 cases of surgery for duodenal obstruction in pediatric patients in the neonatal intensive care unit of Kunming Children's Hospital between January 2018 and December 2019 was conducted. The study subjects included 15 cases with intestinal malrotation, 18 cases with circular pancreas, 10 cases with a duodenal septum, and 17 cases with duodenal atresia or duodenal stenosis. According to the single and double numbers of the operation date, the subjects were randomly divided into the control group and observation group, with 30 cases in each group. The conventional care of enhanced recovery after surgery (ERAS) was carried out in the control group, and KMC based on ERAS conventional care was implemented in the observation group. The difference in the duration to achieve TEN and the length of hospital stay between the two groups of patients after care was compared and analyzed. Results: The average duration to achieve TEN for neonates with duodenal obstruction in the control group was 14.23 ± 3.17 days, while that in the observation group was 12.27 ± 1.15 days. The average length of hospital stay in the control group was 17.22 ± 4.71 days, while that in the observation group was 13.34 ± 2.70 days. There was a significant difference in the duration to achieve TEN and the average length of hospital stay between the two groups (P < 0.05). The duration to achieve TEN and the length of hospital stay in pediatric patients were significantly shorter in the observation group than in the control group. Conclusion: Kangaroo mother care has important clinical significance and application value in shortening the duration to achieve TEN and the length of hospital stay in neonates after surgery for duodenal obstruction.

Phylodynamic Analysis Complements Partner Services by Identifying Acute and Unreported HIV Transmission.

Tailoring public health responses to growing HIV transmission clusters depends on accurately mapping the risk network through which it spreads and identifying acute infections that represent the leading edge of cluster growth. HIV transmission links, especially those involving persons with acute HIV infection (AHI), can be difficult to uncover, or confirm during partner services investigations. We integrated molecular, epidemiologic, serologic and behavioral data to infer and evaluate transmission linkages between participants of a prospective study of AHI conducted in North Carolina, New York City and San Francisco from 2011-2013. Among the 547 participants with newly diagnosed HIV with polymerase sequences, 465 sex partners were reported, of whom only 35 (7.5%) had HIV sequences. Among these 35 contacts, 23 (65.7%) links were genetically supported and 12 (34.3%) were not. Only five links were reported between participants with AHI but none were genetically supported. In contrast, phylodynamic inference identified 102 unreported transmission links, including 12 between persons with AHI. Importantly, all putative transmission links between persons with AHI were found among large clusters with more than five members. Taken together, the presence of putative links between acute participants who did not name each other as contacts that are found only among large clusters underscores the potential for unobserved or undiagnosed intermediaries. Phylodynamics identified many more links than partner services alone and, if routinely and rapidly integrated, can illuminate transmission patterns not readily captured by partner services investigations.

[Assessment of the Availability of Soil Copper and Related Influencing Factors at a County Scale].

Understanding the spatial distribution of total copper, available copper, and the spatial non-stationary relationships between available copper and relevant environmental factors is important for the delineation of soil risk areas and the development of related control measures. This study was conducted in Zhangjiagang County of Jiangsu Province, China. The risk status for soil copper was assessed based on 357 soil samples in the study area. The effects of soil type and land-use type on the concentration of available soil copper were discussed first. Then, ordinary kriging was adopted to map the spatial distribution patterns of the total soil copper and available soil copper, and the spatial distribution map of the copper availability ratio (i.e., available copper/total copper) was also developed for the study area. The risk areas for soil copper were delineated based on the spatial distribution patterns of available soil copper and the copper availability ratio. Finally, a new spatial local regression technique, geographic weighted regression (GWR), was used to explore the local spatial regression relationships between available copper and its three main impact factors (i.e., total soil copper, soil pH, and SOM). Results showed that both soil type and land-use type had some effect on the concentration of available soil copper. The copper availability ratio had a strong spatial heterogeneity, with the higher values mainly in the northeast, southeast, and northwest of the study area and the lower values mainly in the middle and southwest of the study area. The range of the copper availability ratio is 13.56% to 29.15%. The results of the comparison of the traditional ordinary least squares regression (OLSR) and GWR showed that the GWR model had higher fitting accuracy than the OLSR model[i.e., a larger decision coefficient R2, and smaller corrected Akaike information criteria (AICc) and the sum of squares of residuals] in modeling the relationships between available copper and its three main impact factors. The GWR analysis showed that the effect of soil factors on the concentration of soil available copper was non-stationary. The GWR could effectively reveal the spatial non-stationary influence of the related soil factors on the concentration of available soil copper, and the results could explain the reasons for the accumulation of available soil copper in local areas. Potential risk areas for available soil copper were delineated based on the copper availability ratio and the concentration of available soil copper in the study area. The results should be crucial data for developing specific control measures for soil copper at a regional scale.

Sensitive sentinel mutation screening reveals differential underestimation of transmitted HIV drug resistance among demographic groups.

OBJECTIVE: To examine the association of majority and minority-level transmitted HIV drug resistance (TDR) among diverse demographic populations in the United States and assess what different mutations may infer about TDR risk and engagement in care. DESIGN: Used sensitive assays to screen 1070 deidentified convenience plasma specimens from United States national HIV surveillance conducted in 2009-2011 on newly diagnosed persons with no evidence of antiretroviral drug use. METHODS: We applied validated allele-specific PCR for five HIV reverse transcriptase mutations as sentinel markers of TDR. The total and minority-level prevalence of TDR by demographic characteristics was compared. RESULTS: Sensitive screening identified 72% more TDR than conventional sequencing for the five mutations assessed (13.6 vs. 7.9%, P < 0.0001), with K65R having the greatest increase (0-1.7%). One-third of K65R was in persons who also had at least one of the other mutations screened. The total TDR prevalence among whites (16.4%) and blacks (14.9%) was significantly higher than that among Hispanics/Latinos (6.4%) (P = 0.005 and 0.013, respectively). TDR prevalence was highest (23.1%) in those 13-19 years (85% black). TDR prevalence among women (72% black) was nearly as high as among MSM (47% black) (14.3 vs. 15.1%, respectively). CONCLUSION: A significant proportion of TDR, primarily in older, white MSM, was undetected by conventional testing. The greatest underestimation was for rapid-decaying mutations typically associated with the source virus having recent exposure to antiretroviral therapy. However, total TDR prevalence was highest in the less than 20-year age group who were predominantly black, underscoring the importance of prevention efforts for at-risk youth.

HIV reverse-transcriptase drug resistance mutations during early infection reveal greater transmission diversity than in envelope sequences.

BACKGROUND: Drug resistance mutations (DRMs) can serve as distinct, nonpolymorphic markers for evaluating diversity of expressed HIV-1. We screened for DRMs during early-acute viremia and examined the diversity in reverse transcriptase (RT) relative to envelope (env) in cases of transmitted drug resistance. METHODS: We evaluated 111 longitudinal plasma samples collected every 2-7 days from 15 individuals who seroconverted for HIV-1 infection in 1994-2000. The samples were screened with sensitive polymerase chain reaction assays for the commonly transmitted M41L and K70R mutations and for K65R, which was undetected by bulk sequencing. Mutation-positive samples were further characterized by clonal sequencing of RT and env V1-V3. RESULTS: Drug resistance mutations were detected in 4 of 15 seroconverters at 5-50 days of viral nucleic acid expression; most mutations disappeared about the time of seroconversion. Clonal sequencing verified low-level K65R at frequencies of 0.4%-4.9%. In each case, K65R coexisted unlinked with variants carrying 2-5 thymidine analog mutations at frequencies of 1.6%-23.0%. In one seroconverter, variants with M184V and nonnucleoside RT inhibitor mutations were also identified at first RNA expression. Each seroconverter displayed a homogeneous V1-V3 env population. CONCLUSIONS: Reverse-transcriptase DRMs demonstrate that the breadth of variants in transmission may be greater than what is reflected in envelope sequences.

Sensitive tenofovir resistance screening of HIV-1 from the genital and blood compartments of women with breakthrough infections in the CAPRISA 004 tenofovir gel trial.

The Centre for the AIDS Programme of Research in South Africa 004 (CAPRISA 004) study demonstrated that vaginally applied tenofovir gel is a promising intervention for protecting women from sexually acquiring human immunodeficiency virus (HIV). However, the potential for emergence of tenofovir resistance remains a concern in women who seroconvert while using the gel despite the lack of plasma virus resistance as assessed by population sequencing during the trial. We applied highly sensitive polymerase chain reaction-based assays to screen for tenofovir resistance in plasma and vaginal swab specimens. The absence of mutation detection suggested little immediate risk of tenofovir-resistant HIV-1 emergence and forward transmission in settings in which gel users are closely monitored for HIV seroconversion.

CD4(+) cell count, viral load, and drug resistance patterns among heterosexual breakthrough HIV infections in a study of oral preexposure prophylaxis.

We examined CD4 cell count and plasma viral load patterns among Botswana TDF/FTC Oral HIV Prophylaxis Trial (TDF2 study) participants who seroconverted, comparing participants assigned to receive tenofovir/emtricitabine with participants assigned to receive placebo. We also evaluated for antiretroviral drug resistance among the breakthrough HIV infections. Among nine seroconverters assigned to tenofovir/emtricitabine and 24 to placebo, there were no significant differences in their CD4 cell count or viral load profiles over time. Of the four participants who seroconverted on-study while receiving tenofovir/emtricitabine, none became infected as a result of drug-resistant HIV; moreover, no resistance mutations emerged following seroconversion.

Resistance to tenofovir-based regimens during treatment failure of subtype C HIV-1 in South Africa.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) is increasingly available for patients infected with subtype C HIV-1. This subtype is reported to develop the principal TDF resistance mutation in the HIV reverse transcriptase, K65R, with greater propensity than other subtypes. We sought to describe K65R development during TDF use in a cohort of patients infected with subtype C HIV. METHODS: Using a prospectively followed cohort with 6 monthly HIV RNA assays, we identified virological failure (defined as an HIV RNA > 1,000 copies/ml) during treatment that included TDF. Residual serum, stored at the time of the HIV RNA assay, was used for consensus sequencing and allele-specific PCR. We assessed prevalence of resistance at failure during TDF-containing treatment and associated factors. RESULTS: Among 1,682 patients on a TDF-containing regimen, 270 developed failure of which 40 were assessed for resistance. By sequencing, the K65R was identified in 5 (12%), major non-nucleoside reverse transcriptase inhibitor mutations in 24 (57%) and the M184V/I in 12 (28%) patients. The K65R was associated with lower HIV RNA at failure (HIV RNA 3.3 versus 4.2 log10 copies/ml) and prior stavudine exposure. An additional five patients had minority K65R populations identified by allele-specific PCR. CONCLUSIONS: These data suggest that the K65R prevalence at virological failure is moderately higher in our subtype C population than some non-subtype C HIV cohorts. However, we did not find that the K65R was highly selected in HIV-1 subtype-C-infected patients with up to 6 months of failure of a TDF-containing regimen.

Detection of low-level K65R variants in nucleoside reverse transcriptase inhibitor-naive chronic and acute HIV-1 subtype C infections.

To substantiate reports of greater emergence of the K65R nucleoside reverse transcriptase inhibitor (NRTI) mutation in human immunodeficiency virus type 1 (HIV-1) subtype C, we examined natural low-level K65R expression in subtype C relative to subtypes B and AE. We used allele-specific polymerase chain reaction to screen HIV-1 amplified by reverse-transcription high-fidelity polymerase chain reaction from subtype C-infected South African women and infants and CRF01(subtype AE) from Thailand; all subjects were NRTI naive. We found low-level K65R of unknown clinical significance in NRTI-naive subtype C-infected women and infants at frequencies above the natural occurrence in subtypes B and AE. The frequent appearance of subtype C frameshift deletions at codon 65 supports a propensity for transcription error in this region.

Low-frequency K103N strengthens the impact of transmitted drug resistance on virologic responses to first-line efavirenz or nevirapine-based highly active antiretroviral therapy.

BACKGROUND: There are conflicting data on the impact of low-frequency transmitted drug-resistant mutants on responses to first-line highly active antiretroviral therapy (HAART). METHODS: Patients started nevirapine or efavirenz with two or more nucleoside/nucleotide reverse transcriptase inhibitors in 1998-2007 without a prior resistance test at a median 1.0 (interquartile range, 0.0-3.4) year after diagnosis and with a median 218 (interquartile range, 131-296) CD4 cells/mm3, and had at least 24 weeks of follow up. Pre-HAART plasma samples were tested retrospectively by bulk genotyping and sensitive real-time polymerase chain reaction targeting reverse transcriptase K65R, K103N, Y181C, M184V, and G190A (interpretative cutoff 0.3%-0.9%). RESULTS: Among 93 patients, seven of 18 who experienced virologic failure and zero of 75 who maintained virologic suppression showed pre-HAART resistance, including three with high-frequency mutations detectable by bulk genotyping (two K103N, one G190A) and four with low-frequency K103N detectable only by polymerase chain reaction. Detection of either bulk (P = 0.006) or low-frequency (P = 0.001) resistance was significantly associated with the odds of virologic failure; combining the two markedly increased the strength of the association (P < 0.0001). At failure, the pre-HAART mutations were detected by bulk genotyping in five of seven patients alongside additional reverse transcriptase mutations. CONCLUSIONS: Low-frequency K103N mutants were as prevalent as bulk-detectable variants before starting HAART. Both high- and low-frequency mutants were significantly associated with virologic failure.

Minority HIV-1 drug resistance mutations are present in antiretroviral treatment-naïve populations and associate with reduced treatment efficacy.

BACKGROUND: Transmitted HIV-1 drug resistance can compromise initial antiretroviral therapy (ART); therefore, its detection is important for patient management. The absence of drug-associated selection pressure in treatment-naïve persons can cause drug-resistant viruses to decline to levels undetectable by conventional bulk sequencing (minority drug-resistant variants). We used sensitive and simple tests to investigate evidence of transmitted drug resistance in antiretroviral drug-naïve persons and assess the clinical implications of minority drug-resistant variants. METHODS AND FINDINGS: We performed a cross-sectional analysis of transmitted HIV-1 drug resistance and a case-control study of the impact of minority drug resistance on treatment response. For the cross-sectional analysis, we examined viral RNA from newly diagnosed ART-naïve persons in the US and Canada who had no detectable (wild type, n = 205) or one or more resistance-related mutations (n = 303) by conventional sequencing. Eight validated real-time PCR-based assays were used to test for minority drug resistance mutations (protease L90M and reverse transcriptase M41L, K70R, K103N, Y181C, M184V, and T215F/Y) above naturally occurring frequencies. The sensitive real-time PCR testing identified one to three minority drug resistance mutation(s) in 34/205 (17%) newly diagnosed persons who had wild-type virus by conventional genotyping; four (2%) individuals had mutations associated with resistance to two drug classes. Among 30/303 (10%) samples with bulk genotype resistance mutations we found at least one minority variant with a different drug resistance mutation. For the case-control study, we assessed the impact of three treatment-relevant drug resistance mutations at baseline from a separate group of 316 previously ART-naïve persons with no evidence of drug resistance on bulk genotype testing who were placed on efavirenz-based regimens. We found that 7/95 (7%) persons who experienced virologic failure had minority drug resistance mutations at baseline; however, minority resistance was found in only 2/221 (0.9%) treatment successes (Fisher exact test, p = 0.0038). CONCLUSIONS: These data suggest that a considerable proportion of transmitted HIV-1 drug resistance is undetected by conventional genotyping and that minority mutations can have clinical consequences. With no treatment history to help guide therapies for drug-naïve persons, the findings suggest an important role for sensitive baseline drug resistance testing.

[Association between particulate air pollution and daily respiratory and cardiovascular hospital admissions].

OBJECTIVE: To establish concentration-response relationship model for high particulate air pollution and daily hospital admissions for early warning system. METHODS: The Poisson generalized additive model was used with natural cubic spline smoothing for air pollutants of PM10, SO2, NO2, CO to determine the shape of concentration-response relationship. And piecewise linear regression was used for risk analysis. RESULTS: Age-specific analysis suggested the non-linear association between particulate air pollution and hospital admissions in all age groups. In respiratory and cardiovascular admissions, the percentage increase was 0.716%-2.145% and 0.65% for 10 microg/m3 increase in PM10, respectively. Cause-specific analysis suggested the non-linear association between particulate air pollution and hospital admissions for all diseases. The significant association was found with COPD and ischaemic heart disease, and the related percentage increase was 2.94% and 1.94%, respectively. Heating and noheating period analysis suggested the effect of PM10 was higher in noheating period than that in heating period. CONCLUSION: Compared with mortality, particulate air pollution should affect the hospital admissions more greatly, the percentage increase should be higher for hospital admissions than that of mortality (0.25%).

Simple PCR assays improve the sensitivity of HIV-1 subtype B drug resistance testing and allow linking of resistance mutations.

BACKGROUND: The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing. METHODOLOGY: We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing. SIGNIFICANCE: Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.

Emergence of drug-resistant HIV-1 after intrapartum administration of single-dose nevirapine is substantially underestimated.

Conventional sequence analysis detects human immunodeficiency virus (HIV)-1 drug resistance mutations in approximately 40% of women shortly after they receive intrapartum single-dose nevirapine (SD-NVP). Using sensitive real-time polymerase chain reaction assays for the K103N and Y181C resistance mutations, we tested genotyped virus before and after SD-NVP in 50 South African women infected with HIV-1 subtype C. By sequence analysis, 40 women had no detectable resistance mutations, and an additional 6 women were negative for Y181C after SD-NVP. We found K103N in 16 (40%) of 40 women and Y181C in 5 (11%) of 46 women at 6-36 weeks postpartum. Clonal sequencing confirmed K103N in 5 of 5 representative samples and Y181C in 4 of 4 samples. Four of the 5 women with newly identified Y181C also had K103N. These findings indicate that resistance mutations emerged in at least 65% of the women after SD-NVP and emphasize the importance of further research to determine the clinical implications.

Human caliciviruses as a cause of severe gastroenteritis in Peruvian children.

To define the role of human caliciviruses (HuCVs) in severe childhood gastroenteritis, fecal and paired serum samples from 233 Peruvian children hospitalized with gastroenteritis (case patients) and fecal samples from 248 control subjects were evaluated. Overall, 128 case patients (55%) demonstrated HuCV infection by either fecal (n=81 [35%]) or serological (n=96 [41%]) testing. HuCVs were more prevalent in fecal samples from case patients than those from control subjects (35% vs. 13%; P<.001). HuCV infection was more prevalent among case patients without another pathogen than in those who had a coinfecting pathogen (77% [40/52] vs. 49% [88/181]; P<.001). HuCVs appear to be an important cause of gastroenteritis in Peruvian children.