High viral fitness during acute HIV-1 infection

High Viral Fitness during Acute HIV-1Infection

Alicia Arnott1,2¤a,Darren Jardine1¤b,Kim Wilson1,Paul R.Gorry3,4,5,Kate Merlin6,Patricia Grey6, Matthew https://www.wendangku.net/doc/318391071.html,w6,Elizabeth M.Dax1,4,Anthony D.Kelleher6,7,Don E.Smith6¤c,Dale A.McPhee1,2,4*and the Pulse Study Team"

1National Serology Reference Laboratory,St Vincent’s Institute,Melbourne,Victoria,Australia,2Department of Microbiology,Monash University,Melbourne,Victoria, Australia,3Burnet Institute,Melbourne,Victoria,Australia,4Department of Microbiology and Immunology,University of Melbourne,Parkville,Victoria,Australia, 5Department of Medicine,Monash University,Melbourne,Victoria,Australia,6National Centre in HIV Epidemiology and Clinical Research,University of New South Wales, Sydney,New South Wales,Australia,7St.Vincent’s Centre for Applied Medical Research,Sydney,New South Wales,Australia

Abstract

Several clinical studies have shown that,relative to disease progression,HIV-1isolates that are less fit are also less pathogenic.The aim of the present study was to investigate the relationship between viral fitness and control of viral load (VL)in acute and early HIV-1infection.Samples were obtained from subjects participating in two clinical studies.In the PULSE study,antiretroviral therapy(ART)was initiated before,or no later than six months following seroconversion.Subjects then underwent multiple structured treatment interruptions(STIs).The PHAEDRA study enrolled and monitored a cohort of individuals with documented evidence of primary infection.The subset chosen were individuals identified no later than12 months following seroconversion to HIV-1,who were not receiving ART.The relative fitness of primary isolates obtained from study participants was investigated ex vivo.Viral DNA production was quantified using a novel real time PCR assay.

Following intermittent ART,the fitness of isolates obtained from5of6PULSE subjects decreased over time.In contrast,in the absence of ART the fitness of paired isolates obtained from7of9PHAEDRA subjects increased over time.However,viral fitness did not correlate with plasma VL.Most unexpected was the high relative fitness of isolates obtained at Baseline from PULSE subjects,before initiating ART.It is widely thought that the fitness of strains present during the acute phase is low relative to strains present during chronic HIV-1infection,due to the bottleneck imposed upon transmission.The results of this study provide evidence that the relative fitness of strains present during acute HIV-1infection may be higher than previously thought.Furthermore,that viral fitness may represent an important clinical parameter to be considered when deciding whether to initiate ART during early HIV-1infection.

Citation:Arnott A,Jardine D,Wilson K,Gorry PR,Merlin K,et al.(2010)High Viral Fitness during Acute HIV-1Infection.PLoS ONE5(9):e12631.doi:10.1371/ journal.pone.0012631

Editor:Esper Georges Kallas,University of Sao Paulo,Brazil

Received May14,2010;Accepted August7,2010;Published September9,2010

Copyright:?2010Arnott et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.

Funding:Alicia Arnott,Monash University Post-graduate Scholarship;Australian Centre for HIV and Hepatitis Research Grant;the Foundation for AIDS Research (amfAR)Grant(106669)and the Australian National Health and Medical Research Council Project Grant(502617).The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.

Competing Interests:The authors have declared that no competing interests exist.

*E-mail:dale@https://www.wendangku.net/doc/318391071.html,.au

¤a Current address:Institut Pasteur,Phnom Penh,Cambodia

¤b Current address:Department of Microbiology,St Vincent’s Hospital,Melbourne,Victoria,Australia

¤c Current address:Head of Research Development,Albion Street Centre,Sydney,New South Wales,Australia

"Members of the Pulse Study Team are listed in the Acknowledgments.

Introduction

HIV-1exists within the host as a swarm of genetically related strains,termed quasispecies[1].The heterogeneity of the quasispecies occurs largely as a result of the highly erroneous reverse transcription process[2].Combined with the rapid rate of virion production(between108and109virions per day)and the large number of infected cells(107to108),the result is a highly diverse HIV-1population[3,4,5].Additionally,recombination between distinct strains within a host can also occur,further increasing diversity within the virus population[1,6].

The inherent genetic diversity of HIV-1facilitates rapid evolution and adaptation to a given or changing environment within the infected host,referred to as viral fitness[6,7]. Adaptation of HIV-1involves migration and dissemination throughout the host,escape from adaptive and innate immune responses,and from antiretroviral drug pressure[6].Fitness therefore is dependent upon viral and host factors,and has been associated with HIV-1disease progression in individuals with chronic HIV-1infection[6,8,9].It is thought that individuals harbouring virus isolates that are attenuated or replicate poorly are able to control virus replication and delay disease progression compared with individuals infected with rapidly replicating virus isolates.A correlation between poor ex vivo replication and VL suppression was observed following analysis of individuals infected with a nef/LTR attenuated strain[9,10,11,12,13,14].In the findings of Trkola et al.(2003),viral fitness of isolates obtained prior to initiation of ART strongly correlated with the degree of VL rebound following treatment cessation in a group of20 individuals with chronic HIV-1infection[8].A strong correlation between ex vivo viral fitness and disease progression was demonstrated following analysis of virus isolates obtained from three well characterised long term survivors(LTS)of HIV-1 infection,and three individuals with chronic,progressive HIV-1

infection[15].Similarly,Campbell et al.(2003)reported a strong linear relationship between HIV-1replication ex vivo and plasma VL for12individuals with chronic HIV-1infection[16]. Collectively,these observations suggest a correlation between ex vivo viral fitness and clinical outcome in chronic HIV-1disease [17].

Little is known regarding viral fitness during the acute phase of infection.From what is known,the fitness of isolates present during acute HIV-1infection is thought to be low relative to isolates present at later stages of infection,due to the significant genetic bottleneck imposed upon transmission[1,18].Indeed, findings from two studies investigating founder viruses and viral diversification in acute HIV-1infection revealed that in the majority of individuals investigated,infection occurred as a result of transmission or expansion of a single founder virus[19,20].The genetic properties required for efficient transmission may differ from those required for effective establishment and dissemination of HIV-1infection throughout the new host.As a result,the adaptive potential of transmitted strains may be reduced[1].

To examine the relationship between ex vivo viral fitness and control of VL in the acute or early chronic stage of HIV-1 infection in this study,viral strains obtained from participants of two clinical cohorts were investigated[21,22].Relative viral fitness was assessed using a highly sensitive,quantitative real time PCR (QPCR)assay to measure production of total HIV-1DNA.Total HIV-1DNA production can be detected as early as3h post-infection ex vivo,preceding production of integrated and circular forms[23].Hence,total HIV-1DNA production was thought to represent a sensitive,early and reliable marker to assess the relative viral fitness of isolates investigated in this study.We found that ex vivo viral replicative fitness did not correlate with coincident plasma VL from individuals in the acute and early chronic stages of HIV-1infection.Surprisingly,the fitness of isolates obtained from individuals prior to,or immediately following seroconversion to HIV-1was equal to or greater than that of isolates obtained from ART na?¨ve individuals with early,chronic HIV-1infection. The results of this study suggest that despite the genetic bottleneck occurring upon transmission of HIV-1,the replication capacity of transmitted strains is not necessarily reduced.As viral pathoge-nicity has been linked to fitness,the findings of this study also suggest that the pathogenicity of isolates present during acute HIV-1infection may be higher than previously thought,perhaps providing further evidence for the initiation of ART during this phase of HIV-1infection.

Methods

Patients

Plasma samples were obtained from20of60participants of the PULSE study[21](Table S1).The PULSE study was designed to investigate whether individuals with acute HIV-1infection could suppress HIV-1replication following multiple structured inter-ruptions(STIs)to ART.Briefly,the PULSE study consisted of four phases:A,B,C and D.Baseline plasma samples were collected from subjects upon enrolment into the study,prior to initiation of ART(Phase A).Subjects received ART[stavudine,lamivudine, ritonavir-boosted indinavir with randomisation to hydroxyurea (HU)or not]until plasma VL decreased to,50RNA copies/ml for three consecutive months.Patients selected were stratified to ensure a balance of acute or early primary HIV-1infection(PHI) with or without HU[21].Once VL was contained below detection in Phase A,subjects underwent carefully monitored STI in Phase B.Subjects remained off ART if the VL remained below5000 RNA copies/ml.Once the VL increased above5000RNA copies/ml,ART was reinitiated as Phase C.Treatment interrup-tion(Phase B)followed by reinitiation of ART(Phase C),occurred a maximum of three times for each subject,prior to entry into Phase D.Phase D was a follow-up phase,a period of clinical monitoring following the completion of the mandated treatment interruptions study[21].

Seventeen participants of the PHAEDRA study were investi-gated in parallel with PULSE study subjects(Table S2).The PHAEDRA study was a natural history cohort study,patients could elect to be treated or not.It was established to monitor immunological and virological characteristics of individuals with acute and early HIV-1infection.Documentation of acquiring HIV within the past12months was the criteria for entry.This particular substudy was restricted to a cohort of patients who had elected not to receive ART.All these participants had serocon-verted to HIV-1at enrollment.Samples were collected at baseline and24,36and52weeks subsequently.Seroconversion for both cohorts was defined according to stages described by Fiebig and collegues[24](Tables S1and S2).For the subjects from whom virus was successfully isolated and further study performed,at baseline the PULSE subjects had a median Fiebig stage of4with a mean VL and CD4T cell count of1383342RNA copies/mL and533.5cells/m l,respectively.At baseline,the median Fiebig stage was6for the PHAEDRA subjects,with a mean VL and CD4 cell count of159286RNA copies/mL and720.7cells/m l, respectively(Tables S1and S2).

Plasma samples were stored at280u C,and patient PBMCs in liquid nitrogen,until required.Research ethics approval was given by St Vincent’s Hospital,Sydney,St Vincent’s Health,Melbourne and the University of New South Wales Research Ethics Committees.All participants signed an informed consent form before study entry.

Cells

Peripheral blood mononuclear cells(PBMCs)were isolated by density gradient centrifugation from buffy packs collected from healthy,HIV-1seronegative individuals,obtained from the Australian Red Cross Blood Service(ARCBS,Melbourne, Australia),as described[25].Cells were maintained in RF-10 medium(RPMI-1640medium supplemented with10%[v/v] heat-inactivated foetal bovine serum,0.03m g/ml L–glutamine, 100U/ml penicillin and100m g/ml streptomycin),and activated with10m g/ml of phytohemagglutinin(PHA)for3days prior to infection with primary HIV-1strains.

Replication of primary isolates can vary considerably in PBMCs from different donors[26].To minimise the impact of donor variability,all donor PBMCs used for virus isolation and viral fitness experiments were screened against a diverse panel of primary HIV-1isolates to determine permissiveness to infection with HIV-1,prior to use.Cells were selected for use in the fitness assay based on the ability to support replication of a genetically diverse panel of primary HIV-1isolates[9].The level of CD4 expression on the surface of PBMCs capable of supporting replication of genetically diverse primary HIV-1strains was significantly higher than on PBMCs that could not(Pate and McPhee,unpublished).To further minimise the effects of donor variability,pooled preferred PHA-PBMCs from two separate HIV-1negative donors were used for all experiments.

Viruses

The reference isolate HIV-1MBC925was isolated from PBMCs collected from an AIDS patient,and characterised as described [27].This highly pathogenic,clade B,CCR5-using primary isolate was selected as it had been observed to replicate efficiently and

reproducibly in PHA-PBMCs(McPhee,D.A.,unpublished)[27]. The use of HIV-1MBC925also enabled a direct comparison between the fitness of isolates present during acute infection relative with that of an isolate obtained from an individual with advanced disease.Virus isolation was attempted from36and34 plasma samples collected from PULSE and PHAEDRA subjects, respectively,by centrifugation over a20%(w/v)sucrose cushion at 450006g for1h.Plasma was preferred as it best represents the circulating quasispecies.The pelleted virus was resuspended in IL-2medium(RF-10medium containing10U/ml IL-2and12mM HEPES)containing16107PHA-PBMCs and cultured for14days [25].Virus production was analysed by measurement of cell-free reverse transcriptase(RT)activity or p24antigen production. Virus isolation was attempted from plasma collected at Baseline prior to the initiation of ART,from all PULSE subjects,and from any additional,available Phase B(STI)plasma sample with a VL $5000RNA copies/ml.Coincident plasma VL measurements ranged from260to7500000RNA copies/ml(Table S1).Isolates were successfully obtained from19of the36plasma samples:15 from Baseline and4from plasma collected subsequent to Baseline.

A strong correlation between plasma virus isolation from PULSE subjects,and high coincident VL,was observed.Virus isolation was unsuccessful from plasma samples with a VL,153000RNA copies/ml(Table S1).

Virus isolation was attempted from two plasma samples obtained from each PHAEDRA subject:a sample collected at Baseline and a sample collected at either week24,36or52 subsequent to Baseline.Two sequential isolates were successfully obtained from12of the17PHAEDRA subjects(Table S2).Only one isolate,obtained from plasma collected at Baseline,was obtained from an additional PHAEDRA subject(data not shown). Successful virus isolation from plasma obtained from PHAEDRA subjects did not correlate with plasma VL(Table S2).A total of25 viruses were isolated from PHAEDRA cohort members(Table S2).The relative fitness of18of the25isolates was subsequently investigated in this study.

Virus isolation was attempted from cryopreserved PBMCs available from a subset of PULSE subjects,where plasma was unavailable,or when virus isolation from plasma was unsuccessful, using co-culture with preferred PBMCs as described above[9]. After recovery from storage in liquid nitrogen,the viability of all PBMCs collected from PULSE subjects and subsequently used for co-culture was$70%(data not shown).PBMCs were available from two post-Baseline time-points from six of10PULSE subjects, and one post-Baseline time point from a further four subjects,a total of16samples.Following co-culture,eight additional isolates were successfully obtained from eight PULSE subjects.There was no correlation between VL and successful virus isolation from PBMCs(Table S1).A total of28isolates were obtained from16of 20PULSE subjects;the relative fitness of24of the28isolates was subsequently investigated.

Parallel infection assays

A standardised input of600pg of p24antigen of each primary or reference isolate was incubated with26105PHA-PBMCs for2h,in triplicate.Isolates were minimally passaged in an attempt to ensure isolates reflected the replication competent virus present in vivo [28,29].Where600pg of p24could not be achieved,undiluted infection supernatant was added.Cells were washed in IL-2 medium and transferred to96well plates at26105cells/well in a final volume of200m l,achieved using IL-2medium.Cells were harvested at various time-points between0and158h post-infection.Following harvest,cells were washed and resuspended in200m l of TE buffer.To lyse infected cells,300m l of MagNA Pure lysis buffer(Roche,Castle Hills,NSW,Australia)was added and the cells incubated(15minutes,room temperature).Lysed cells were stored at280u C until required for DNA extraction.Harvested supernatant was stored at220u C and virus production analysed by measurement of cell-free RT activity or p24antigen production. DNA was extracted from HIV-1infected PHA-PBMCs using the Invitrogen Easy DNA kit as per the manufacturer’s instructions, with the exception of the initial cell lysis step.

Measurement of total HIV-1DNA production to estimate relative viral fitness

To evaluate relative viral fitness,a quantitative real time PCR (QPCR)assay was developed to measure production of total HIV-1DNA(extrachromosomal,integrated and2-LTR circular forms) for a period of between96and158h post-infection.Published primer and probe sequences targeting a highly conserved region of the59-LTR and the human Albumin gene,were used(Figure1) [30,31].Prior to use assay sensitivity and intra-and inter-assay variation were extensively tested(Text S1;Tables S3and S4).To detect total HIV-1DNA,the real time PCR reaction mix contained5m l DNA in a final volume of20m l.The mix contained QPCR Probe Mastermix(Integrated Sciences,Australia),100nM dual labelled probe,300nM of HIV-1LTR forward and reverse primers,and nuclease free water(NFW).To detect Albumin DNA, the real time PCR reaction mix contained5m l DNA in a final volume of20m l.The reaction mix contained QPCR Probe Mastermix,100nM dual labelled probe,300nM Albumin forward and reverse primers and NFW.All DNA amplifications were performed using a Stratagene MX3000P real time PCR machine(Integrated Sciences,Australia)with the

following Figure1.Primers and probes used in this study.The original sources and sequences of the primers and probes used in this study are summarised.The fluorophores used were FAM and HEX for the HIV-1LTR and albumin probes,respectively.The quencher used for both probes was TAMRA.

doi:10.1371/journal.pone.0012631.g001

conditions:1cycle at95u C for10mins;40cycles at95u C for30 sec and60u C for1min.

Quantification and calculation of relative viral fitness scores

As each strain was tested in triplicate,the mean Ct for each time point was calculated following QPCR analysis.Copies of target DNA were quantified by converting the mean Ct value generated for each sample to DNA copies using the standard curve generated by MX3000P software from the quantified DNA standards included in each run.Copies of total HIV-1cDNA were calculated per200000cells,the number of PHA-PBMCs in each https://www.wendangku.net/doc/318391071.html,ing the copies of HIV-1DNA measured for each strain, a relative viral fitness score was calculated for each isolate.Total HIV-1DNA production at96h post-infection was measured for all isolates tested;hence calculation of viral fitness scores at this time-point enabled direct comparison between the relative fitness of all PULSE and PHAEDRA isolates tested.A second score at the final time-point tested(either110or158h post-infection)enabled analysis of DNA production for those isolates not detected at96h post-infection.As the viral fitness measured in this study was relative to that of the pathogenic reference strain HIV-1MBC925, fitness scores for test strains were calculated relative to HIV-1 DNA production by HIV-1MBC925from coincident time points. To calculate viral fitness scores,copies of DNA produced by test strains were divided by copies of DNA produced by the reference strain at a coincident time point post-infection[Fitness score= (HIV-1DNA T/HIV-1DNA R)],where HIV-1DNA T and HIV-1 DNA R correspond to copies of HIV-1DNA produced by the test and reference strains,respectively.Fitness scores throughout the text and figures are represented as a fraction of1.Isolates with a relative fitness score of$0.1were classified as fit;isolates with a relative viral fitness score of0.1to0.01were classified as moderately fit;the relative fitness of isolates with a score of,0.01 was classified as low.

Results

Reduced relative fitness of a nef/LTR attenuated virus compared with a primary wild type HIV-1strain,

HIV-1MBC925

Whether primary HIV-1isolates of variable replicative fitness could be distinguished on the basis of total HIV-1DNA production was investigated using HIV-1MBC925and a nef/LTR attenuated isolate,HIV-1D36III.The HIV-1D36III isolate,obtained from a long term non-progressor(LTNP),replicates poorly,as a result of deletions/mutations in the nef/LTR region[9,11,32].

Production of viral DNA by both isolates was detected at four h post-infection,however a significant difference in replicative fitness over time was observed(Figure2).The difference between total HIV-1DNA produced by HIV-1MBC925and HIV-1D36III at96h post-infection was38.7-fold(Figure2).It has been observed by studies in our laboratory,and those by Kim and collegues,that a single replication cycle takes between20and24h[33,34].Hence, several rounds of infection were required to demonstrate differences in the kinetics of total HIV-1DNA production.A slow/low replication phenotype was observed for the attenuated virus strain compared with a fast/rapid DNA production profile for the reference virus.Increased DNA production at all time-points tested by HIV-1MBC925relative to HIV-1D36III indicated that the replicative fitness of the reference strain was greater than that of the attenuated isolate.For both virus infections there was an increase followed by a modest decrease between4and12h post infection as observed previously in a study of one step growth kinetics of HIV-1[34].Furthermore,these results indicated that using the QPCR assay,primary HIV-1isolates with variable replicative fitness could be readily distinguished on the basis of viral DNA production over several rounds of replication. Decreased replicative fitness from acute to early chronic HIV-1infection,following treatment with ART(PULSE subjects)

The replicative fitness of isolates obtained from14PULSE subjects was investigated using the QPCR assay.The reference isolate HIV-1MBC925was cultured in parallel with test isolates in each viral fitness experiment,enabling calculation of a relative fitness score and to monitor any potential inter assay variation. Two isolates from different time-points obtained from6PULSE subjects,and8single isolates obtained from8PULSE subjects, were tested(Figure3).From the viral fitness scores calculated

using Figure2.Production of HIV-1DNA by a reference and a known attenuated virus,quantified using the QPCR assay.PHA-PBMCs were infected with6000pg of p24of the reference strain HIV-1MBC925 and known attenuated isolate HIV-1D36III,and cultured for110h. Infected cells were harvested at4,8,12,24,48,72,96and110h post-infection.DNA was extracted and the HIV-1and albumin DNA quantified using QPCR.Copies of HIV-1DNA per200000cells, determined for each isolate,are plotted on a logarithmic scale against time(A).In(B),relative fitness of HIV-1D36III was determined by calculating the amount of HIV-1DNA produced at96h post-infection, expressed as a percentage of HIV-1MBC925DNA production at the same time-point.The results are representative of three experiments.

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total HIV-1DNA production at 158h post-infection,isolates obtained from PULSE subjects were categorised into three groups:high fitness,moderate fitness and low fitness (Figure 3).The seven isolates classified as highly fit were all obtained from plasma collected at Baseline,during acute HIV-1infection,and prior to initiation of ART.For three BL isolates replication was near equivalent to the reference strain used (Figure 3).

Four of the 8isolates classified as moderately fit were obtained from plasma collected at Baseline,four were obtained from plasma collected during STI,subsequent to Baseline.The relative fitness of eight isolates was classified as low,indicating that total HIV-1DNA production by these isolates was less than 1%of total HIV-1DNA production by the reference isolate at a coincident time-point post-infection ex vivo (Figure 3).Six of the 8isolates with low fitness were obtained from plasma collected during STI.Viral DNA was only detected after 96h post-infection for these 8isolates.The rapid kinetics of HIV-1DNA production after 96h post-infection lead to selection of 158h post-infection as the final

timepoint for analysis of relative fitness in subsequent experiments (data not shown).

Over time,following the initiation of ART,the fitness of isolates obtained from 6PULSE subjects decreased (Figure 4).Due to the small number of subjects analysed,the decrease observed was not significant (p =0.14).Furthermore,although decreasing viral fitness coincided with decreasing plasma VL for 4of 6subjects from whom multiple isolates were obtained,overall,viral fitness did not correlate with plasma VL for the 14PULSE subjects investigated (Borderline statistical significance p =0.051;Figure 5).There was no correlation between CD4+T cell counts and relative viral fitness for the PULSE subjects investigated (data not shown).

Increasing replicative fitness during chronic HIV-1infection (PHAEDRA subjects)

Sequential isolates from 9of 12PHAEDRA subjects were analysed using the QPCR assay (Figure 6).Based on relative viral fitness scores calculated using total HIV-1DNA production

at

Figure 3.Viral fitness scores and clinical data for PULSE subjects.Shown are clinical and experimental data obtained for PULSE subjects from which virus was successfully isolated and subsequently tested using the real time PCR assay.Indicated by the column headings are the subject identification code and seroconversion status at Baseline (‘+’indicates subject had seroconverted,‘2’indicates subject was seronegative,‘w +’indicates that a weak antibody response was detected).Also shown are the number of STIs experienced by the subject,whether VL was suppressed below 5000RNA copies/ml upon STI (indicated by ‘controller’or ‘non-controller’),and the phase of the PULSE study during which the relevant sample was collected.The time (in weeks)post Baseline that the sample was collected,coincident VL and CD4+T cell counts and the sample type from which virus was successfully isolated,are also shown.Finally,viral fitness scores calculated using DNA production measured at 96h post-infection ex vivo ,and at the final time-point analysed (158h post-infection),are shown for each isolate.The fitness scores generated for the isolate obtained from subject 3.13were calculated from total HIV-1DNA produced at 60and 72h post-infection.‘ND’indicates that the specified measurement was not done.

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110h post-infection,isolates were classified according to the 3groups used above.Four isolates were categorised as highly fit,3of which were obtained from plasma collected 36weeks subsequent

to Baseline (Figure 6).In contrast,all of the isolates obtained from PULSE subjects that were classified as fit were obtained from plasma collected at Baseline,prior to the initiation of ART (Figure 3).

Of the 5PHAEDRA isolates classified as moderately fit,3isolates were obtained from plasma collected 52weeks subsequent to Baseline,and 2isolates were obtained from plasma collected at Baseline (Figure 6).The relative fitness of 9isolates obtained from PHAEDRA subjects was classified as low.Interestingly,6of the 9isolates with low relative fitness were obtained from plasma collected at Baseline,in contrast to results obtained for the PULSE subjects investigated.

Over time,the relative fitness of isolates obtained from seven PHAEDRA subjects increased significantly (p =0.03;Figure 7).However,viral fitness was not found to correlate with plasma VL following analysis of the 18isolates obtained from PHAEDRA subjects (Figure 8).In addition,relative viral fitness was not found to correlate with CD4+T cell counts for the PHAEDRA subjects investigated (data not shown).

High relative fitness of isolates from acute infection (PULSE)compared with early chronic HIV-1infection (PHAEDRA)

It is widely believed that,due to a genetic bottleneck occurring upon transmission,the fitness of isolates present during acute infection is low relative to isolates obtained later in infection [6,35,36,37,38,39].To investigate this,we compared the viral fitness of isolates obtained from Baseline plasma from PULSE subjects,to those collected 36to 52weeks post-baseline from PHAEDRA subjects.The isolate groups were selected to enable the relative fitness of viruses present during acute HIV-1infection,na?

¨ve to any selection pressures exerted by ART (PULSE),and those found during untreated,early chronic infection (PHAE-DRA),to be compared.

Relative to isolates obtained from PHAEDRA subjects,replication of PULSE Baseline isolates was considerably slower,with replication of 57%of isolates not detected by 96h post-infection.However,between 96and 110or 158h post-infection,total HIV-1DNA production increased substantially,with replication of 84%of PULSE viruses detected (Figure 3).By comparison,replication of 39%of PHAEDRA post-baseline isolates was not detected by 110h post-infection ex vivo (Figure 6).In addition,the increase in total HIV-1DNA production between 96and 110h post-infection for isolates obtained from PHAEDRA subjects was not substantive relative to wild type or the isolates obtained from PULSE subjects (data not shown).We observed that overall,PULSE Baseline isolates were slower to establish a productive infection relative to the PHAEDRA post-baseline isolates (Figure 9).From this we suggest that the genetic diversity of isolates obtained post-Baseline from the PHAEDRA subjects was greater than that of the PULSE isolates obtained at Baseline,evidenced by greater relative adaptive ability.However,once infection was established,the amount of HIV-1DNA produced by the PULSE Baseline isolates was comparable to,or higher than,the amount of viral DNA produced by PHAEDRA post-Baseline isolates (Figure 9).These findings provide evidence that the relative fitness of isolates present during acute HIV-1infection may be higher than previously thought.When viral fitness scores were plotted relative to the stage of seroconversion the results are even more striking.The most fit viruses were observed during the earliest stage of seroconversion monitored (Figure 10).Conceiv-ably,in vivo viral fitness is compromised as HIV-1infection progresses,in response to selective immunological pressure on replicating

virus.

Figure 4.Decreasing fitness over time observed following analysis of paired isolates obtained from acute HIV-1infection subjects,measured using QPCR.Relative viral fitness scores were calculated for isolates obtained from PULSE subjects and represented on a box-plot.Only subjects from whom a Baseline isolate and at least one additional isolate (Week 27to 106)were obtained were included in the analysis (n =6).Where multiple isolates from additional time-points were obtained,the average of the combined viral fitness scores was used.Shown are viral fitness scores calculated at the final time-point tested (158h;exception was 3.13which was at 72h)ex vivo for paired isolates obtained from 6PULSE subjects.The box represents the middle 50%of values for the data set,the solid line indicates the median value.The vertical ‘whiskers’extending from the box respectively indicate the lowest and highest observed values.The open circle represents an outlier;the asterisk represents an extreme outlier.The significance of the observed changes in viral fitness over time is shown (p =0.14),calculated using a signed rank test.

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Figure 5.Viral fitness did not correlate with VL following analysis of isolates obtained from acute HIV-1infection subjects.Coincident plasma VL measurements (log 10RNA copies/ml)were plotted against relative viral fitness scores (log 10)for 16isolates,obtained from plasma,from PULSE subjects.The Pearson correlation was rho =0.496,p =0.051.

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Discussion

In this study we investigated the relative viral fitness of isolates obtained from individuals with acute and early HIV-1infection.Temporal changes in relative viral fitness were observed for 6and 10subjects participating respectively in the PULSE (acute HIV-1infection)and PHAEDRA (early HIV-1infection)studies (Figures 3,4,6and 7).Consistent with the findings of previous studies investigating viral fitness during untreated HIV-1infection [6,15,36],the relative fitness of paired isolates obtained from 7PHAEDRA subjects increased significantly over time (p =0.03;Figure 7).Viral fitness decreased over time following intermittent ART for 5of the 6PULSE subjects analysed (Figure 4),an observation that might be expected due to the potential bottleneck imposed by suppressive ART.Most unex-pected was the high relative fitness of isolates obtained from PULSE subjects during acute HIV-1infection,prior to the initiation of ART,compared to isolates obtained from individuals with early chronic HIV-1infection.Furthermore,total HIV-1DNA production by several PULSE Baseline isolates was comparable to,or greater than that of the highly pathogenic,primary reference isolate HIV-1MBC925obtained from an individual with AIDS (Figure 3)[27].These findings provide evidence that despite the bottleneck occurring upon transmission,the relative fitness of isolates present during acute HIV-1infection may indeed be high.

To investigate relative viral fitness,a ‘parallel infection assay’was used [17].Parallel infection assays have been successfully used in other studies to examine replication of primary HIV-1isolates in primary cell types [35,40,41].Alternatively,viral fitness can be investigated using a growth competition assay,whereby replication of test and reference strains is compared in the same culture,primarily performed using recombinant viruses [6,15,17,42,43,44,45].The use of recombinant strains,as in recent studies by Miura et al.,[46]and Kong et al.,[47]to investigate the contribution of specific genes to the fitness of viruses during acute infection,does not permit investigation of the fitness of the circulating viral quasispecies.We used a parallel infection assay to enable investigation of the replicative fitness of strains isolated directly from patient plasma,to maximise the clinical relevance of results obtained [8].

It is widely accepted that regardless of the route of HIV-1infection,the virus encounters an extreme genetic bottleneck upon transmission,resulting in a highly homogenous virus population in the recipient [19,37,38,39,48].Decreased genetic diversity is thought to activate Muller’s ratchet [49],therefore,the fitness of strains present during acute infection is thought to be low.As 10of the 20PULSE individuals investigated had not fully seroconverted to HIV-1(Table S1),we anticipated that the fitness of viruses isolated from coincident plasma samples would be low.A virus population with highly constrained genetic diversity would not be expected to readily adapt to

an

Figure 6.Clinical and experimental data obtained for PHAEDRA subjects.Shown are clinical and experimental data obtained for PHAEDRA subjects from which virus was successfully isolated and subsequently tested using the real time PCR assay.Indicated by the column headings are the subject identification code,seroconversion status at Baseline (‘+’indicates subject had seroconverted,‘-’indicates subject was seronegative),and the phase of the PHAEDRA study at which the relevant sample was collected.The time (in weeks)post Baseline that the sample was collected,coincident VL and CD4+T cell counts,and the sample type from which virus was successfully isolated are shown.The viral fitness scores calculated using DNA production measured at 96h post-infection ex vivo ,and at the final time-point analysed (110h post-infection),are shown for each isolate.doi:10.1371/journal.pone.0012631.g006

environment distinct to that found within the host,such as the ex vivo system used in this study to measure relative viral fitness [6].

However,7of the 13isolates obtained from plasma collected at Baseline from PULSE subjects were classified as highly fit (Figures 3and 9).Indeed,analogous to the findings of this study,rapidly replicating variants have been identified in similar,smaller studies investigating the fitness of isolates present during acute and early HIV-1infection [6,36,40].In the findings by Ferbas et al.(1996)for one individual,high viral fitness was observed following analysis of the ex vivo fitness of isolates obtained at the time of peak viremia,but prior to seroconversion [36].Kong et al.(2008)recently reported that strains with

higher

Figure 7.Increasing viral fitness over time observed following analysis of paired isolates obtained from early chronic HIV-1infection subjects,measured using QPCR.Relative viral fitness scores were calculated for isolates obtained from PHAEDRA subjects and represented on a box-plot.Only subjects from whom a Baseline isolate and at least one additional isolate (Week 36to 52)were obtained were included in the analysis (n =8).Shown are viral fitness scores calculated at the final time-point tested (110h)ex vivo for paired isolates obtained from 8PHAEDRA subjects.The box represents the middle 50%of values for the data set,the solid line indicates the median value.The vertical ‘whiskers’extending from the box respectively indicate the lowest and highest observed values.The asterisk represents an extreme outlier.The significance of the observed changes in viral fitness over time is shown (p =0.03),calculated using a signed rank test.

doi:10.1371/journal.pone.0012631.g007

Figure 8.Viral fitness did not correlate with VL following analysis of isolates obtained from early chronic HIV-1infection subjects.Coincident plasma VL measurements (log 10RNA copies/ml)were plotted against relative viral fitness scores (log 10)for 16isolates,obtained from plasma,from PHAEDRA subjects.The Pearson correlation was rho =0.133,p =0.697.

doi:10.1371/journal.pone.0012631.g008

Figure 9.Increased fitness of Baseline isolates obtained from acute HIV-1infection subjects relative to isolates obtained subsequent to Baseline from early chronic HIV-1infection subjects.Relative viral fitness scores were calculated for isolates obtained from PULSE and PHAEDRA subjects.Shown on a box-plot are the viral fitness scores generated for the Baseline isolates obtained from 13PULSE subjects compared with the viral fitness scores of the ‘Late’isolates obtained from eight PHAEDRA subjects,at 96h PI (A)and at the final time-point tested (158h PI for PULSE and 110h PI for PHAEDRA isolates;B).The box represents the middle 50%of values for the data set;the solid line indicates the median value.The vertical ‘whiskers’extending from the box respectively indicate the lowest and highest observed values.Outliers are represented by an open circle;extreme outliers are represented by an asterisk.The significance of difference in viral fitness between the two groups at 96h PI (A;p =0.12)and the final time-point tested (B;p =0.45)is shown.doi:10.1371/journal.pone.0012631.g009

replicative fitness with respect to the env gene were vertically transmitted by mothers with chronic HIV-1infection [47].Combined with the observation of highly fit strains present during acute HIV-1infection in this study,these results suggest the bottleneck that occurs upon initial transmission of HIV-1does not necessarily result in loss of fitness.

The level of relative viral fitness has been linked to the genetic diversity of the viral quasispecies.Kong et al.(2008)reported transmission of multiple virus strains;Borderia et al.(2010)recently demonstrated a direct correlation between increasing genetic diversity and increasing in vivo viral fitness of clonal populations [47,50].Troyer et al.(2005)reported strong correlation between genetic diversity of the viral quasispecies,and ex vivo viral fitness [6].In our study,with subjects that were therapy naive,viral fitness increased over time for 7of the 9PHAEDRA subjects investigated.Observations that genetic diversity correlates with viral fitness are certainly not novel;fitness of an RNA virus population increasing with genetic diversity is described by the Red Queen hypothesis [51].This has been applied extensively in the field of HIV-1research [7,40],and is highly relevant given the level of genetic diversity of the viral quasispecies present in infected individuals.Cloning of the env sequences of isolates obtained from PULSE subjects is currently underway,to investigate whether the observed high level of fitness correlated with genetic diversity of the quasispecies present at baseline,during acute infection.

Following commencement and subsequent interruption of suppressive ART,viral fitness decreased for 5of 6PULSE subjects investigated (Figure 3).Analogous to the findings of this study,reduced viral fitness was also observed for individuals experiencing STI following initiation of ART during acute infection by Wang et al.(2007)[52].Suppressive antiretroviral therapy can result in the development of drug resistant mutations in the viral quasispecies to evade inhibition,which has been shown to reduce viral fitness [6,21].Development of drug resistance mutations in this study was not suspected as VL

suppression was observed upon resumption of ART in all PULSE subjects investigated [21].Instead,analogous to the findings of Wang et al.,(2007)[52]and Borderia et al.(2010)[50],decreasing relative viral fitness over time was thought to be a direct result of a genetic bottleneck created by suppressive ART,activating Muller’s ratchet [6].Muller proposed that when genetically diverse populations are randomly reduced,such as during treatment with ART,or the development of potent immune responses,the overall fitness of the population also decreases [6,40].The fitness of Baseline isolates obtained from 6of the 9PHAEDRA subjects was also classified as low (Figure 6).At Baseline,all PHAEDRA subjects had clearly seroconverted to HIV-1(Table S2).The observed low relative fitness may have resulted from mutation of the viral quasispecies as a direct result of the development of potent immune responses following seroconversion.Indeed,escape from targeted immune responses has been observed in similar studies investigating anti-HIV-1immune responses during early HIV-1infection [17,53].

The accumulation of escape mutations can incur a high fitness cost to the virus,depending on the genomic location of the mutation [54,55,56].Indeed,Goonetilleke and colleagues (2009)reported that selection of viral escape mutants,following development of adaptive T-cell responses,occurred rapidly following containment of peak viremia in 4individuals with acute HIV-1infection confirming earlier studies [57,58,59].However,there was no obvious fitness cost to the viruses studied [59].Similarly,as relative viral fitness increased subsequent to Baseline for 7of 9PHAEDRA subjects investigated in this study,accumulation of deleterious mutations seems unlikely.Not as restrictive as suppressive ART,development of potent immune responses upon seroconversion may have created a ‘‘wider’’bottleneck,limiting but not preventing the expansion and diversification of the viral quasispecies [6].Consequently,we propose that increasing fitness subsequent to seroconversion observed for 7of 9PHAEDRA subjects occurred as a result of virus evolution and diversification within the host to

evade

Figure 10.Viral fitness of baseline isolates obtained from acute HIV-1infection subjects (PULSE)and from early chronic HIV-1infection subjects (PHAEDRA)relative to the stage of seroconversion as detailed by Fiebig and collegues (24).Baseline viral fitness scores for both PULSE (orange)and PHAEDRA (mauve)subjects from final timepoints (110h or 158h)have been grouped according to the stage of seroconversion (Fiebig stages IV,V or VI;reference 24)for direct comparison.doi:10.1371/journal.pone.0012631.g010

adaptive immune responses[6,7,40,51].Although contribution of cellular immune responses to containment of virus replication has not been investigated we are currently assessing neutralising antibody responses for both the PULSE and the PHAEDRA subjects.

There were several limitations to the present study.The use of an ex vivo system,such as that used in this and other studies,does not reflect the sensitivity of the virus to antiretroviral drugs, chemokines or additional inhibitory agents that may affect fitness in vivo.Furthermore,for6of the14PULSE subjects from whom plasma virus could not be isolated,virus was isolated from PBMC (Figure3).In addition to PBMC-derived isolates,for5of these6 subjects,virus was obtained from plasma collected at distinct time-points throughout the study.The fitness of both PBMC and plasma derived viruses was subsequently investigated(Figure3).It has long been understood that HIV-1can evolve separately in distinct physiological compartments[60,61].In addition,it is a widely held belief that the current,circulating viral quasispecies are present in the plasma and that cellular reservoirs of HIV-1 contain archived strains.However,the findings of recent studies suggest otherwise[62,63].Indeed,we observed that the kinetics of HIV-1DNA production by the PBMC-derived isolates tested in this study were distinct relative to plasma derived isolates obtained at different time-points from the same PULSE subject(data not shown).

Combined,observations of the relative fitness of PULSE and PHAEDRA isolates suggest selection of the fittest virus,or viruses, upon transmission which progressively become less fit upon development of adaptive immune pressure and/or commence-ment of antiviral therapy.Further studies to investigate the long-term impact of viral fitness on disease progression are warranted. Muira and collegues recently reported the attenuated replication capacity of isolates obtained from individuals who became HIV-1 controllers during early infection[46].In this study,none of the PULSE subjects from whom Baseline isolates with high replicative fitness were obtained controlled HIV-1replication in the absence of therapy(data not shown).Although the role of viral fitness in disease progression remains unclear,what is clear from the findings of this study is that the fitness of strains present during acute/early HIV-1infection can be high.

In conclusion,the findings of this study suggest that despite the bottleneck transmission of a strain or strains with high relative fitness does occur.Furthermore,these results suggest that viral fitness decreases subsequent to the development of adaptive immune pressure and/or commencement of antiviral therapy. The findings of this study make a substantial contribution towards understanding that the selection process during transmission of HIV-1from donor to recipient can be for a very fit virus. Supporting Information

Text S1Detail of viral fitness QPCR assay validation.

Found at:doi:10.1371/journal.pone.0012631.s001(0.04MB DOC)

Table S1Clinical and virus isolation data for PULSE subjects. Shown are the clinical results and the results of attempted virus isolation from plasma or PBMCs obtained from PULSE subjects;‘‘triangle’’indicates that virus isolation was attempted from the sample indicated.A single asterisk indicates the sample used for virus isolation was plasma;a double asterisk indicates that virus isolation was attempted from PBMCs when either plasma was not available or virus isolation from plasma was unsuccessful.Shown is the subject identification number followed by the phase of the PULSE study during which the sample was collected.‘‘A’’,‘‘B’’and‘‘C’’indicate PULSE study Phases A,B and C.The subsequent number indicates during which of up to three B or C phases sample collection occurred;prefaced by‘‘W’’(weeks),the following number indicates duration of the specified phase at sample collection.Seroconversion status according to the Fiebig et al[24]stages,coincident CD4+T cell counts and plasma VL at the time of sample collection,are shown:‘‘.log105.88’’indicates VL was above the upper limit of detection,and was not quantified. Whether subjects received HU in addition to ART is indicated. Reverse transcriptase and p24antigen EIA assay results, performed following virus isolation,are also shown:‘‘ND’’indicates culture supernatant was not tested using the RT assay;‘‘NQ’’indicates that the relevant result for the isolate was above or below the limit of detection for the assay and was not quantified;‘‘2’’indicates that virus isolation was attempted but RT activity or p24antigen were not detected.

Found at:doi:10.1371/journal.pone.0012631.s002(0.32MB DOC)

Table S2Clinical and virus isolation data for PHAEDRA subjects.Shown are the clinical results and the results of attempted virus isolation from plasma obtained from PHAEDRA subjects.A single asterisk indicates the plasma sample from which virus isolation was attempted.Indicated by the column headings are the subject identification code,the phase of the PHAEDRA study at which the relevant sample was collected,and seroconversion status according to Fiebig et al[24].Coincident CD4+T cell counts and plasma VL at the time of sample collection are shown:‘‘.log10 5.88’’indicates VL was above the upper limit of detection,and was not quantified.Reverse transcriptase and p24antigen EIA assay results,performed following virus isolation,are also shown:‘‘ND’’indicates culture supernatant was not tested using the RT assay;‘‘NQ’’indicates that the relevant result for the isolate was above or below the limit of detection for the assay and was not quantified;‘‘2’’indicates that virus isolation was attempted but RT activity or production of p24antigen was not detected subsequently.

Found at:doi:10.1371/journal.pone.0012631.s003(0.17MB DOC)

Table S3Intra-assay variation analysis for the QPCR assay.To examine intra-assay variation,20replicates of each HIV-1(A)and albumin(B)DNA standard were tested in the same run.Data represent the mean Ct value(Mean),standard deviation(SD)and coefficient of variation(COV,expressed as a percentage)for each standard.‘‘N’’indicates the number of replicates detected for each standard.

Found at:doi:10.1371/journal.pone.0012631.s004(0.04MB DOC)

Table S4Inter-assay variation analysis.To examine inter-assay variation,five consecutive runs with the HIV-1(A)and albumin (B)DNA standards were performed.Standards were tested in triplicate within each run.Shown are the mean Ct values obtained for each standard following each of the five independent runs. Data represent the total number of replicates detected(N),mean Ct value(Mean),standard deviation(SD)and coefficient of variation(COV,expressed as a percentage)for each standard. Mean,SD and COV values were calculated using Ct values obtained for each replicate detected of the specified standard.A HIV-1negative non-amplification control(NAC)was included, consisting of cellular DNA.ND indicates that the specified sample was not detected.

Found at:doi:10.1371/journal.pone.0012631.s005(0.06MB DOC)

Acknowledgments

Members of the Pulse Study Team comprised:

National Centre in HIV Epidemiology and Clinical Research,UNSW: D Smith,K Petoumenous,K Irvine,P Grey,R Munro,M Law,J Kaldor,and D Cooper.

St Vincent’s Hospital,Darlinghurst,Sydney:

A Carr,R Feilden,M Lacey,S Pett and DA Cooper.

407Doctors,Darlinghurst,Sydney:

R Macfarlane,D Baker,W Genn,H McLeod,R Vale.

Holdsworth House General Practice,Darlinghurst,Sydney:

M Bloch,D Quan,D Austin,S Miller.

Taylor Square Private Clinic,Darlinghurst,Sydney:

R Finlayson,R Richardson,J Price.

Burwood Road Clinic,Burwood,Sydney:

N Doong.

Centre for Immunology,St.Vincent’s Hospital:

A Kelleher,J Zaunders,P Cunningham,C Satchell,M-L Munier,K McGhie.

Prahan Market Clinic,Prahan,Melbourne:

N Roth,H Wood.

Victorian Infectious Disease Reference Laboratory,Melbourne:

C Birch,T Middleton.

Miami Sexual Health Clinic,Gold Coast:

J Chuah.

Author Contributions

Conceived and designed the experiments:AA DJ PRG PG ADK DES DAM.Performed the experiments:AA KW.Analyzed the data:AA DJ KW ML EMD ADK DES DAM.Contributed reagents/materials/analysis tools:KW PRG KM PG ML EMD ADK DES.Wrote the paper:AA PRG DAM.

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HIV初筛实验室相关工作制度样本

HIV初筛实验室工作制度 1、本实验操作人员必要经省艾滋病初检技术培训学习合格后方可持证上岗。 2、严格遵守样本采集、保存制度；样品运送解决制度；遵守实验室操作、检 验成果报告解决和保密、实验室污染物废弃物以及工作人员防护消毒隔离制度。 3、积极参加上级实验室业务培训和技术考核。 4、必要参加省临检中心室间质量评价工作。 5、未尽事宜按科室实验室按科室工作制度和有关管理条例执行。

HIV抗体检测程序及其流程 1、血液标本验收合格后，用初筛试剂进行检测，如呈阴性反映，则作HIV抗体阴性报告； 2、初筛检测成果呈阳性反映标本，须进行重复检测。复检时用两种不同初筛检测试剂复测； 3、如两种试剂复检成果均呈阴性反映，则作HIV抗体阴性报告；如均呈阳性反映，或有一份阳性，该标本需送上级实验室加以进一步证明。送检时应将重新采集该受检者血液标本和原有血液标本一并送检。 4、检测成果鉴定和解决 对HIV抗体阳性者应做好征询、保密和报告工作。对HIV抗体阴性者，如近期有高危行为如性乱史、吸毒史、受血史，或有急性流感样症状等状况，为排除窗口期也许，建议每3个月复查一次，持续2次。对HIV抗体可疑对象要做好征询和随访工作。5、、反馈与报告程序 （一）初筛检测中发现HIV抗体阳性反映标本，应尽快(城区普通规定在48小时内，农村规定在96小时内)将血样连同原始实验资料（涉及厂家批号、试剂种类、有效期，如ELISA实验应附上阴性、阳性对照值，Cut off值及样品OD值）和送检化验单送卫生行政部门指定初筛中心实验室，再转送确认实验室，或直接送至HIV抗体确认实验室。送检化验单必要由初筛实验室一名直接实验操作人员和一名中级技术职称以上负责人员签名。初筛实验室不得向受检者宣布初检阳性反映成果； （二）做好标本收集与检测登记工作，每月5日按统一表格向卫生行政部门指定HIV抗体初筛中心实验室报告检测状况，如无HIV抗体中心实验室，则直接向省级HIV抗体确认中心报告。

HIV-1病毒载量试剂1技术参数

HIV-1病毒载量试剂1技术参数 1.名称：人类免疫缺陷病毒（1型）核酸检测试剂盒（PCR-荧光法）。 2.用途：用于定量检测人血浆中人类免疫缺陷病毒（1型）。 3.原理：应用实时荧光定量聚合酶链式反应原理。 4.检测项目：HIV-1RNA定量检测。 5.检测亚型：HIV-1M、O组。 6.检测方法：全自动核酸分离纯化，全自动核酸扩增和实时荧光PCR方法。 7.适用样品种类：非肝素抗凝血浆。 8.定量方式：内部标准品定量。 9.质控品：提供阴性、弱阳性、强阳性对照质控品。 10.技术要求： a.检测灵敏度：≤20copies/ml,置信度≥95%。 b.检测范围：20-1×107copies/ml。 c.检测特异性：100%。 d.重复性：CV值≤0.3log。 e.规格:48T/盒。 f.即开即用型液体试剂。 11.试剂储存条件：2-8℃保存。 12.试剂组份：包括核酸提取、纯化、扩增和检测的全部试剂。 13.抗污染方案：采用UNG酶防止PCR产物污染。 14.样品处理能力：可以同时进行检测1-72个样品 15.备件：含完整实验过程中所需的相关耗材及洗液。 16.适用设备：适用于CobasAmpliprepCobasTaqman全自动病毒载量检测系统。 17.认证文件：中国医疗器械进口注册证等文件。 18.供货计划：按用户要求数量准时分批供货，试剂到货时有效期大于10个 月。

HIV-1病毒载量试剂2技术参数 1．名称：人类免疫缺陷病毒（HIV-1）核酸（RNA）提取及检测试剂盒 2．用途：用于定量检测人血浆中人类免疫缺陷病毒（1型）。3．检测方法：应用RT-PCR和Real-time实时荧光定量检测技术HIV-1型各组病毒载量。 4．检测项目：HIV-1RNA定量检测。 5.检测亚型：HIV-1型M组(A-H)、O组、N组。 6.适用设备：m2000sp自动核酸提取仪，m2000rt实时荧光定量PCR仪; 7．定量方式：外部标准品定量 8．技术要求 8.1检测灵敏度:≤40copies/mL（0.6mL、1.0mL样本体系） 8.2特异性：100%(95%CI99.28-100%) 8.3检测范围:40-107copies/mL 8.4适用样本种类:血浆(ACD-A或EDTA抗凝) 8.5检测样本体积:可选0.2mL、0.5mL、0.6mL和1.0mL 8.6检测方式:96孔PCR反应板 8.7检测批量：一次检测批量可选24，48，72和96 8.8内测标准差(SD):≤0.25log copies/mL 8.9内参要求：与样本一起提取和扩增，质控整个实验过程; 9.认证文件：中国医疗器械进口注册证等文件。 10.供货计划：按用户要求数量准时分批供货，到货时试剂有效期大于10个月。

HIV的实验室诊断

HIV实验室诊断 一、HIV病毒是带有包膜的RNA逆转录病毒，在我国流行的是HIV-1。 二、HIV实验室诊断 人体感染HIV后，血液中最先出现HIV抗原，然后很快消失直到疾病后期才重新出现。几周后出现IgM抗体并很快消失，此后,IgG抗体出现并一直存在。因此，HIV感染的实验室诊断以抗体检测为主，病毒及相关抗原的检测为辅。 抗体检测分为初筛试验和确证试验两种，初筛试验为阳性的血清必须进一步确证，确证实验为阳性的方可报告为HIV感染阳性。 多聚酶链式反应(PCR)主要用于检测血浆中HIV的RNA含量，目前主要用于预测母亲将HIV传染给胎儿的可能性以及新生儿的HIV感染状况。此外，尚可用于判断病人的预后及监测抗病毒治疗的效果。 （一）HIV抗体的初筛检测 初筛试验的要求是敏感性高，理论上要求达到100%，尽可能避免漏掉可能阳性的对象，相对来说，对特异性要求不是太严，允许有少量假阳性，这些假阳性可以通过重复试验和确证试验排除。 HIV抗体初筛检测的方法很多，如酶联免疫吸附实验(enzyme linked immunosorbent assay, ELISA)、明胶颗粒凝集实验(gelatine particle agglutination assay, PA)、乳胶凝集实验(latex agglutination assay, LA)、各种快速检测实验(rapid tests)等。 1．酶联免疫吸附实验检测HIV1/2 型抗体，酶联免疫吸附实验（ELISA）是最常见的HIV抗体检测方法，它具有准确性高、价格低廉、判断结果有客观标准、结果便于记录和保存等优点，适合于大批量标本的检测，是献血员筛选和临床诊断最常用的方法。 3．胶体金法检测HIV1/2抗体实验 胶体金法检测HIV抗体是一种不需要任何仪器设备的血清／血浆检测法，它利用免疫层析分析原理来快速检测血清／血浆中是否含有HIV抗体，从而用于判断人体是否受到HIV1型／HIV2型病毒感染。 注意：由于样本中HIV抗体滴度的不同，测试区（T）内的红色条带会显现出不同深浅的颜色。但是，本试剂盒的测试结果不能做为判定样本中抗体滴度高低的依据。 (二)、初筛试验每一次检测阴性者可报告阴性，若第一次检测阳性，则需进行复测，复测时（最好用不同类型的试剂）可同时测定两孔，判定方法如下：

HIV-1病毒载量检测及质量保证指南

本指南由中国疾病预防控制中心性病艾滋病预防控制中心艾滋病参比实验室组织有关专家撰写，由中国疾病预防控制中心批准执行。 编写主持人：蒋岩 编写人员：潘品良蒋岩李敬云钟平尚红李太生 审核咨询专家及技术人员：邵一鸣康来仪朱效科王佑春汪宁朱红郭志宏季阳 刘海林肖瑶邱茂锋马春涛 编写单位：中国疾病预防控制中心性病艾滋病预防控制中心 参编单位：中国人民解放军军事医学科学院 上海市疾病预防控制中心 中国医科大学 北京协和医院 技术顾问：Mike Ussery, Hao Zhang 美国国立卫生研究院过敏及传染病研究所 James W. Bremer 美国Rush 大学医学中心VQA实验室 Trever Peter 克林顿基金中国艾滋病防治项目实验室专家 Donald J. Brambilla 美国华盛顿大学新英格兰研究所 Francis F. Mandy 加拿大公共卫生局疾病预防控制中心国家免疫学实验室

前言 随着HIV-1病毒载量检测在艾滋病临床辅助诊断和抗病毒治疗监测以及艾滋病科研工作中的广泛应用，我国开展该项检测工作的实验室日益增多。自1997年艾滋病实验室首次引进HIV-1病毒载量检测技术，截至2007年底，全国已经配备了近120台HIV-1病毒载量检测仪，覆盖了疾控、医院、检疫、军队等系统。目前，虽然许多实验室已经将病毒载量检测纳入艾滋病相关辅助诊断和治疗监测工作，但大多数实验室开展工作的时间较短，经验不足，因此，急需加强该领域的培训和质量控制工作，以保证检测结果的准确性和可靠性。 为了加强质控和保证检测质量，并得到国际上的认可，艾滋病参比实验室2004年开始参加美国NIH组织的HIV-1病毒载量检测能力验证工作。在通过考核和积累经验的基础上，2005年启动了国内HIV-1病毒载量检测能力验证工作。在积极组织实验室能力验证的同时，艾滋病参比实验室也对实验室技术人员进行培训，积极开展室内质控。这些工作对提高HIV-1病毒载量的检测技术，保证质量起到了重要作用。为了进一步规范艾滋病实验室HIV-1病毒载量检测，建立标准化质量控制程序，准确开展各项检测及质量控制工作，特制定本指南。 本指南是根据各种HIV-1病毒载量检测方法的技术要求，结合现行的国内外相关技术规范而制定。本指南针对HIV-1病毒载量检测的实验室设置、样品管理、检测技术、质量控制、生物安全等方面提出指导。 本指南自2006年初正式开始编写，性病艾滋病预防控制中心多次组织了有国内外专家参加的指南编写专项技术咨询会，与会专家对指南提出了很多建设性意见和建议，美国NIH 病毒载量检测质量保证实验室专家多次对指南进行认真的审阅和具体指导。指南编写过程中，也认真听取了实验室一线检测人员的具体需求和建议，并补充了大量的实验数据，包括HIV-1病毒载量检测值的稳定性、冻融影响实验与不同检测方法之间比较的实验数据等。在此基础上，经过多次修订、补充和审定，于2007年11月完成《HIV-1病毒载量检测及质量保证指南》的最后定稿。 由于国内外还没有可作为参考的HIV-1病毒载量检测及质量控制的相关指南，因此本指南主要提出一些原则上的参考建议，其中定有不妥之处，希望广大同仁提出意见，以便不断完善。 致谢 在本指南的编写过程中，世界卫生组织、克林顿基金中国艾滋病防治项目和美国NIH 等国际组织与机构给予了大力支持，全国HIV-1病毒载量检测实验室一线操作人员提出了许多建设性的意见和具体需求，艾滋病参比实验室工作人员毛玮、陶晓霞进行了大量的补充实验和文字编辑工作，才保证本指南的编写顺利完成，在此一并向他们表示真诚的感谢。 中国疾病预防控制中心性病艾滋病预防控制中心主任吴尊友研究员在本指南编写过程中提出了重要的修改意见，在此特别致谢。

hiv1病毒载量测定及质量保证指南

病毒载量测定及质量保证指南 （版） 中国疾病预防控制中心 年月 前言 随着病毒载量检测在艾滋病临床辅助诊断、抗病毒治疗监测及相关科研工作中的广泛应用，我国开展该项检测工作的实验室日益增多。自年艾滋病实验室首次引进病毒载量检测技术以来，截至年底全国已经配备台病毒载量检测仪，覆盖疾控、医院、检疫、军队等系统，艾滋病流行重点地区及少数县级实验室均已配备病毒载量检测仪，每年检测量已达万人份以上。由于病毒载量检测质量对艾滋病的早期诊断及治疗有重要影响，因此急需加强该领域的培训和质量控制，规范实验室操作，以保证检测结果的准确性和可靠性。 自年开始实施《病毒载量测定及质量保证指南》（试行，以下简称《指南》）以来，参与病毒载量能力验证工作的实验室从年的家增加到年的家，占已开展工作实验室的百分之九十以上。通过综合分析近几年能力验证发现的问题与检测技术发展的实际情况，考虑各种试剂的检测要求，结合专家意见和一线实验室工作人员的经验与需求，对本指南进行了适当修改、调整和补充，主要修改内容包括：（）增加“人员要求”章节；（）修改“常用病毒载量检测方法”；（）修改“病毒载量检测能力验证（）”；（）补充常见问题分析和处理等内容。 修订后的《指南》共分八章，包括：总则，人员要求，实验室环境与设施，样品的采集、处理保存运输，常用检测方法简介，室内质量控制，实验室检测能力验证（），实验室生物安全，以及四个附表。 本指南在修改过程中，接纳了国内专家、同行的意见和建议，经过多次修改编写成《指南》（）版。本指南由中国疾病预防控制中心性病艾滋病预防控制中心艾滋病参比实验室组织有关专家撰写，由中国疾病预防控制中心批准执行，并在病毒载量检测实验室网络内发布实施。 由于国内各实验室条件不同，不能采纳所有实验室的建议，因此本指南主要提出一些原则上的参考建议，其中定有不妥之处，希望广大同仁提出意见，以便不断完善。

领料岗位标准操作规程

分发部门 综合部[ ] 采购储运部[ ] 生产部[ ] 工程项目部[ ] 财务部[ ] 工艺技术部[ ] 研发部[ ] 市场策划部[ ] 销售服务部[ ] 销售事业部[ ]

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3.2 应采用三层容器对样品进行包装，随样品应附有与样品唯一性编码相对应的送检单。送检单应标明受检者姓名、样品种类等信息，并应放置在第二层和第三层容器之间。 3.2.1 第一层容器：直接装样品，应防渗漏。样品应置于带盖的试管内，试管上应有明显的标记，标明样品的唯一性编码或受检者姓名、种类和采集时间。在试管的周围应垫有缓冲吸水材料，以免碰碎。 3.2.2 第二层容器：容纳并保护第一层容器，可以装若干个第一层容器。要求不易破碎、带盖、防渗漏、容器的材料要易于消毒处理。 3.2.3 第三层容器：容纳并保护第二层容器的运输用外层包装箱。外面要贴上醒目的标签，注明数量、收样和发件人及联系方式，同时要注明“小心轻放、防止日晒、小心水浸、防止重压”等字样，还应易于消毒。 3.2.4 用于抗体检测的血清和血浆样品应在冻存条件下运送。每一件包装的体积以不超过50mL为宜。 3.2.5 运送样品必须有记录。 4 HIV抗体初筛实验中的假阳性分析： 4.1血液恶性病变 4.2自身免疫性疾病 4.3多发性胆汁型肝硬化 4.4酒精性肝炎 4.5疫苗接种（流感、乙肝） 4.6多发性骨髓瘤 4.7肾移植或慢性肾衰竭 4.8疟疾或丝虫病 4.9其它 5 HIV抗体初筛实验中的假阴性分析： 5.1窗口期 5.2免疫移植 5.3换血输血 5.4恶性肿瘤 5.5免疫功能丧失（B细胞功能缺陷）及其它 6如遇被检查者拒绝留本人身份信息，其初筛可能阳性结果的标本2周后消毒处理后按医疗垃圾丢弃。 2016年5月25日

常用的HIV诊断方法

HIV/AIDS艾滋病诊断是一个十分严肃的问题，任何诊断的错误结果无论是假阴性还是假阳性，对家庭和社会都会造成十分严重的后果。HIV检测是诊断HIV感染及艾滋病的重要实验室依据之一，要知道是否感染了HIV可以通过检测病毒的抗体、抗原、核酸或通过病毒培养来确定。常用于诊断HIV/AIDS的方法是HIV抗体检测，它是诊断HIV/AIDS的主要指标，也是世界卫生组织和我国卫生部推荐使用的HIV感染诊断方法。为了最大限度地保证诊断结果准确，HIV诊断采用特殊的检测策略，即先用敏感性高的初筛试剂进行初筛，如果阳性再用特异性强的确认试剂进行确认。检测HIV抗体常用的初筛方法有酶联免疫吸附试验(ELISA)、凝集试验和各种快速诊断方法，确认的方法有蛋白印迹法(WB)、放射免疫沉淀试验(RIP)、间接免疫荧光试验(IFA)等。一般先用ELISA做初筛试验;如果阳性，再用蛋白印迹法来确认。以下介绍一下较常用的HIV抗体测定的方法。 一．初筛试验方法 1．酶联免疫吸附试验（ELISA） 酶联免疫吸附试验简称酶标法，是最常用初筛检测方法，在HIV抗体的测定中有四种模式：间接法、双抗原夹心法、捕获法和竞争法，其中以间接法用的最多。 1）间接法：将HIV抗原纯化后固定于微孔板上，然后加入待测标本，清洗后加酶标记抗人免疫球蛋白（IgG），清洗后加入底物，产生有色反应即为阳性。（见图1） 2）双抗原夹心法：将HIV抗原固定于微孔板上,然后加入待测标本，清洗后加入酶标记的HIV抗原，清洗后加入底物，产生有色反应即为阳性。（见图2）

3）捕获法：将非特异性的兔抗人免疫球蛋白抗体固定于微孔板上，然后加入待测标本,清洗后加入酶标记的HIV抗原，清洗后加入底物，产生有色反应即为阳性。（见图3） 4）竞争法：：将HIV抗原固定于微孔板上,然后同时加入待测标本和一定数量的酶标HIV抗体，如果标本中有HIV抗体,即与加入的酶标HIV抗体竞争结合固定在微孔板上的抗原，标本中HIV抗体量越多，结合在固相上的酶标抗体就愈少，清洗后加入底物。结果判断与间接法相反，产生有色反应即为阴性，无色反应的为阳性（见图4）

HIV抗体初筛实验流程

HIV初筛与确诊实验操作步骤与注意事项 1检验科采集HIV抗体标本后，按下列流程图1进行初筛与复检试验。 图1 HIV抗体筛查检测流程 2筛查试验呈阳性或一阴一阳反应，该样品务必转送至市疾控中心作确证试验。初筛“阳性或一阴一阳”标本→临床医生核对取得其姓名和身份证及电话号码→必要时采第二份血样→标本三层包装冷冻→持HIV抗体复检报告单→医院办公室派车→专人集中每周一送往无锡市疾控中心三楼季主任→无锡市疾控中心每周二作确诊试验→结果由疾控中心电话报告被检查者 送无锡市疾控中心作确诊试验，需要核对身份，补充个人信息（如姓名和身份证号码及电话号码），必要时采集第二份血样，持HIV抗体筛查报告，持HIV 抗体复检试验用附表2送检。HIV抗体复检报告需由1名检验人员和1名审核人员签字。

3 样品的运送：每周一是送检日。 3.1 应符合生物安全要求；要获得相应部门批准并由具有资质的人员专程护送。 3.2 应采用三层容器对样品进行包装，随样品应附有与样品唯一性编码相对应的送检单。送检单应标明受检者姓名、样品种类等信息，并应放置在第二层和第三层容器之间。 3.2.1 第一层容器：直接装样品，应防渗漏。样品应置于带盖的试管内，试管上应有明显的标记，标明样品的唯一性编码或受检者姓名、种类和采集时间。在试管的周围应垫有缓冲吸水材料，以免碰碎。 3.2.2 第二层容器：容纳并保护第一层容器，可以装若干个第一层容器。要求不易破碎、带盖、防渗漏、容器的材料要易于消毒处理。 3.2.3 第三层容器：容纳并保护第二层容器的运输用外层包装箱。外面要贴上醒目的标签，注明数量、收样和发件人及联系方式，同时要注明“小心轻放、防止日晒、小心水浸、防止重压”等字样，还应易于消毒。 3.2.4 用于抗体检测的血清和血浆样品应在冻存条件下运送。每一件包装的体积以不超过50mL为宜。 3.2.5 运送样品必须有记录。 4 HIV抗体初筛实验中的假阳性分析： 4.1血液恶性病变 4.2自身免疫性疾病 4.3多发性胆汁型肝硬化 4.4酒精性肝炎 4.5疫苗接种（流感、乙肝） 4.6多发性骨髓瘤 4.7肾移植或慢性肾衰竭 4.8疟疾或丝虫病 4.9其它 5 HIV抗体初筛实验中的假阴性分析： 5.1窗口期 5.2免疫移植 5.3换血输血 5.4恶性肿瘤 5.5免疫功能丧失（B细胞功能缺陷）及其它 6无锡市疾控中心报告日期：每周二作确诊试验。 7如遇被检查者拒绝留本人身份信息，其初筛可能阳性结果的标本2周后消毒处理后按医疗垃圾丢弃。 2012.2.11

HIV抗体初筛实验室管理规章制度模板

HIV 抗体初筛实验室管理规章制度 为营造整洁的工作环境, 建立良好的工作秩序, 保障实验室工作的正常运行和检测过程中人身安全, 特制定本制度。 1、非本实验室工作人员未经许可不得随意出入本初筛实验室 2、工作人员从事本实验室工作前必须经过严格培训, 熟悉本实验 室规章制度, 获得上岗证后才能正式工作。 3、进入实验室操作时, 应穿工作服、带手套、做好隔离、安全防护措 施, 出实验室前要脱掉手套、洗手, 及时把工作服脱下, 放入实验室。 4、实验室工作人员必须认真负责, 严格遵守操作规程, 做好试验的各项 原始记录, 务必准确按时地发出阴性报告。遇筛查阳性时应送市 CDC 进行确认实验。 5、保持实验室清洁, 严禁带手套触摸门把手。 6、实验完毕后必须对实验区域进行必要的清洁处理。废弃物处理应严格 按照实验室废弃物处理制度的规定执行。 7、实验结束离开实验室时应检查水、电等设施, 严格执行安全措 施, 确保实验室安全。 8、要严肃、认真、负责的态度, 对某些检验对象和阳性结果应采取必 要的保密措施以免对本人及家属产生不利影响。 9、实验室内严禁吸烟或饮食, 以免影响检测结果和自身健康; 应注意保 持实验室的整洁卫生, 定期做好清洁、消毒工作。

实验室安全防护制度 1、检测人员上岗前应接受相关的生物安全与个人防护培训。 2、为检测人员提供一次性乳胶手套、口罩、帽子。 3、检测人员工作前应修剪长的带刺的指甲, 以免刺破手套。 4、避免在检测样本时进食、饮水、吸烟、和化妆。 5、出现手部皮肤有开放性伤口及其它不适于检测工作的情况应暂停 工作。 6、操作过程中, 如发现工作服被污染, 应立即更换; 如手套破损, 应立即丢弃、洗手并换上新手套。 7、避免用戴手套的手触摸暴露的皮肤、口唇、眼睛、耳朵、 头发和清洁工作区域。 8、禁止手套清洗或消毒后再次使用, 因为使用表面活性剂清洗可使 手套对水的通透性增加, 消毒剂能够引起手套的破损。

物资退库管理办法

物资退库管理办法 1、目的 为了规范各项目现场退返物资的管理，保证退返物资质量情况得到鉴别使之合理再利用，节省项目成本，特制定本办法。 2、适用范围 本办法适用于各项目现场退返的物资及公司生产车间退库物资的处理。 3、职责 3.1 工程部负责提出物资退库申请；负责落实返回物资的清理清点工作；工程部经理或副经理负责核实申请单的描述与实物一致性和完整性；负责完成不良品的维修工作。 3.2 产品事业部负责退库申请单的核准并判断退库库别；负责指导工程部维修人员完成不良品的维修工作。 3.3 质量安全部负责对核准的退返物资进行检验并通知入库； 3.4 物资部负责对退返的物资进行账务审核；负责与供应商协调保质期内物资的维修方案和交期；负责退返物资的入库。 3.5 财务部负责退返物资的成本核算 3.6 公司其他相关部门在有需要时应配合责任部门共同完成退返物资入库工作。 4、流程及说明 4.1.流程图

退返物资流程图 工程部产品事业部工程部维修人员质量安全部仓库财务部公司高层会议售后维修更换/项目整体改造评审 生产余料退库 Y 财务成本 预估 项目售后人员或车间生产人员收集整理退返物 资 发回或带回 公司Y 工程师判定 N N 退供应商返修 检验指导 维修检验入库成本核算 Y 需 维修指导维 修入库 退返物资清记录 理、清点 工程师核准 并判定退库别 报废 填写退库申请 单无需维修 N Y维修记录检验记录报废记录 工程部主管审核 4.2流程说明 流程内容执行部门流程说明及注意事项 工程部、产品事业部根据需求下达物资退返指令。工程部门维修更换物资的退返需物资退返指令下达 工程部 /要产品工程师做技术判定，判定无需退返的物资可由现场人员做报废处理并通知物 产品事业部资部门入帐；产品事业部下达的整体改造指令需要提交评估报告至公司高层会议审 批并通报财务部门做成本评估，评审通过后以《技术更改通知单》形式下达执行。 财务成本预估财务部当产品事业部下达整体改造指令时财务部需要对改造成本做评估并上报公司高层退返物资收集整理工程部 工程部现场售后人员 /车间生产人员接到退返指令后按照要求对需要退返的物资进 行收集整理，确定退返数量及原因。 物资退返工程部 需要退返的物资由售后人员寄回或直接带回公司，要做好物资的保管工作。采用寄 送方式时应保证物资的包装完好，如有贵重物资需要退返时可申请专人专车运送退返物资清理清点工程部退返物资返回公司后由工程部进行清理清点，核实物资数量与完好性 填写退库申请单工程部退返物资清点无误后由工程部相关人员填写《物资退库申请单》，申请单应填写详细，请退物资的名称、型号、数量、退库原因及现状态必须写清楚。 工程部主管必须对提交的《退库申请单》进行审核，确保请退物资名称、型号、数 主管审核工程部量及现状态如实无误，并对物资退返原因进行确认。确认无误后在《退库申请单》 上签字并将申请单提交至产品事业部核准。 退库核准及判定产品事业部 产品事业部工程师对提交的《退库申请单》进行核准并对退返物资的处理方式进行 判定。 工程部维修人员在产品事业部工程师指导下对需要进行维修处理的物资进行维修退返物资维修工程部作业，维修时应做好相应的维修记录以备后期追溯。需要时可以申请设计人员协助 完成维修工作。 退返物资检验质量安全部质量安全部按照技术要求对退返物资进行检验，需要时应请求产品事业部指导完

什么是病毒载量

什么是病毒载量？其意义是什么？ 人类免疫缺陷病毒（HIV）感染机体后，主要侵害CD4 T淋巴细胞，并且以CD4 T淋巴细胞为宿主细胞，大量复制繁殖新的HIV，而后, 新的HIV又去感染未被感染的CD4 T 细胞，进入病毒复制的又一循环。这样反复循环，体内就产生了大量的HIV，产生的病毒大部分都是以游离的形式存在于血液中，一部分则藏匿于静止细胞或淋巴结中，而停止复制增生，但它们也随时会再次“出击”，去感染新的细胞。我们在了解HIV/AIDS的相关知识中，经常会看到或听到“病毒载量”这个词，所谓病毒载量即是定量机体内游离病毒的含量。它是一个计数单位, 表示每毫升血浆中含多少拷贝数的HIV-RNA。可以通过此项检测了解机体内血浆中游离病毒的水平。 有三种方法检测病毒载量HIV RNA： 1, 分支DNA检测（bDNA 拜耳公司） 2, 逆转录-聚合酶链反应（RT-PCR, 罗氏公司） 3, 核酸序列扩增检测（NASBA, 阿克苏公司）。

它们之间的基本原理是不同的，对于核酸水平的检测，PCR的技术是基于探针的放大而bDNA的方法是采用信号 放大。由于它们的方法学不同，显示血浆病毒载量的计数单位,也就有所不同，因此在对同一患者进行疾病进展观察及疗效随访监测时，应该采用同样的检测方法，便于对患者检测结果的比较，和分析具有统一的标准。 自90年代初，直接检测血浆病毒RNA 以更敏感，更快速和更方便的途径在血浆中可以定量病毒浓度, 利用病毒载量检测: a. 急性HIV-1感染，病人急性HIV感染期间，处于空窗期时即可检测出高水平的病毒RNA含量。急性期病毒复制高峰期RNA量每毫升可达到9×10 7拷贝。随着抗体的产生，RNA逐步下降与p24抗原的降低相一致。但直到一年或一年之后，仍可发现每毫升血浆中可含有2-4×10 4拷贝的水平，而p24抗原则在1-3个月后即不能测得。提示bDNA方法远较p24抗原检测法敏感。 b. 判定病人疾病的进程和进展，血浆中HIV RNA的定量分析，可表现出病毒复制动力学，也反应血浆中游离病毒的浓度，是病毒增殖和免疫清除机制共同作用的结果，CD4+T细胞越少的病人显示RNA含量越高。在同一病人的系列标本检测中可以看到bDNA值与CD4+T细胞数呈明显的负相关。因而在判定疾病进程和判定临床治疗效果上具有极大的价

HIV抗体初筛实验室SOP文件

HIV抗体初筛实验室SOP文件 安国市医院检验科

目录 第一部分工作制度.................................................................................................................... - 2 - 实验室工作制度.................................................................................................................. - 2 - HIV标本采集与接收登记制度.......................................................................................... - 3 - 艾滋病实验室保密制度...................................................................................................... - 4 - 艾滋病筛查实验室常规消毒清理工作制度 ...................................................................... - 5 - 差错事故处理制度.............................................................................................................. - 6 - 报告单签发审核制度.......................................................................................................... - 7 - 设备管理制度...................................................................................................................... - 8 - 试剂管理制度...................................................................................................................... - 9 - 艾滋病筛查实验室安全防护制度.................................................................................... - 10 - 艾滋病病毒职业暴露预防处理方案................................................................................ - 11 - 第二部分标准操作规程.......................................................................................................... - 14 - 第一章样品采集与处理.................................................................................................. - 14 - 第二章标准操作程序...................................................................................................... - 15 - 第三章结果报告与注意事项.......................................................................................... - 17 - 第三部分设备SOP文件......................................................................................................... - 19 - 一、START FAX—2100酶标分析仪 .............................................................................. - 19 - 二、RT3000洗板机.......................................................................................................... - 21 - 三、电热恒温水箱............................................................................................................ - 26 - 第四部分流程图- 27 - 一、HIV抗体筛查流程图................................................................................................ - 27 - 二、暴露级别的评估........................................................................................................ - 28 -