Analysis of multistage pooling studies of biological specimens for estimating disease incidence and prevalence

The testing of pooled samples of biological specimens for the purpose of estimating disease prevalence may be more cost effective than testing individual samples, particularly if the prevalence of disease is low. Multistage pooling studies involve testing pools and then sequentially subdividing and testing the positive pools. A simple estimator of disease prevalence and its variance are derived for general multistage pooling studies and are shown to be natural generalizations of Thompson's (1962) original estimators for single-stage pooling studies. The reduction in variance associated with each additional stage is calibrated. The results are extended to estimating disease incidence rates. The methods are used to estimate HIV incidence rates from a prevalence study of early HIV infection using a PCR assay for HIV RNA.     

HIV-1 P24抗原国家参考品的研制

收集正常人、HIV感染者和疑似感染者血浆以及HIV-1病毒培养裂解液,对其进行HIV抗体、HIVP24抗原、HCV抗体和HBsAg检测,对HIVP24抗原阳性者进行HIVRNA检测,并对部分样品进行基因分型。以NIBSCP24抗原标准品的系列稀释样品作为线性灵敏度参考品。经过实验筛选出20份阴性参考品,10份阳性参考品,10份线性灵敏度参考品,2份精密性参考品,共同组成HIV-1P24抗原国家参考品,经多家不同的试剂进行标定,制定了相应的标准。稳定性研究结果表明,反复冻融三次对该参考品的稳定性没有影响。由此,初步建立了HIV-1P24抗原国家参考品,该参考品将对HIV-1P24抗原、HIV抗体/P24抗原联合检测试剂的质量控制提供重要依据。     

The Implementation and Appraisal of a Novel Confirmatory HIV-1 Testing Algorithm in the Microbicides Development Programme 301 Trial (MDP301)

We describe the application of a novel HIV confirmatory testing algorithm to determine the primary efficacy endpoint in a large Phase III microbicide trial. 9385 women were enrolled between 2005 and 2009. Of these women, 537 (6%) had at least one positive HIV rapid test after enrolment. This triggered the use of the algorithm which made use of archived serum and Buffy Coat samples. The overall sample set was >95% complete. 419 (78%) of the rapid test positive samples were confirmed as primary endpoints using a combination of assays for the detection of HIV-specific antibodies (EIA''s and Western Blot), and for components of the virus itself (PCR for the detection of nucleic acids and EIA for p24 antigen). 63 (12%) cases were confirmed as being HIV-positive at screening or enrolment and 55 (10%) were confirmed as HIV negative. The testing algorithm confirmed the endpoint at the same visit as that of the first positive rapid test in 90% of cases and at the time of the preceding visit in 10% of cases. Of the 63 cases which were subsequently confirmed to be HIV-1 positive at or before enrolment, 54 specimens contained no detectable HIV antibodies at screening or enrolment. However, 43 were positive using an EIA which detects both HIV antigen and antibody and also had a positive p24 antigen or HIV PCR test, which was highly suggestive of acute infection. There were 6 unusual cases which had undetectable HIV-1 DNA or RNA. In 4 of the 6 cases the presence of HIV-1-specific antibodies was confirmed by Western Blot. One of these cases with an indeterminate Western Blot was a previous vaccine trial participant. The algorithm served the objectives of the study well and can be recommended for use in determining HIV as an endpoint in clinical trials.

Trial Registration

ISRCTN.org ISRCTN 64716212     

Low-Cost HIV-1 Diagnosis and Quantification in Dried Blood Spots by Real Time PCR

Background

Rapid and cost-effective methods for HIV-1 diagnosis and viral load monitoring would greatly enhance the clinical management of HIV-1 infected adults and children in limited-resource settings. Recent recommendations to treat perinatally infected infants within the first year of life are feasible only if early diagnosis is routinely available. Dried blood spots (DBS) on filter paper are an easy and convenient way to collect and transport blood samples. A rapid and cost effective method to diagnose and quantify HIV-1 from DBS is urgently needed to facilitate early diagnosis of HIV-1 infection and monitoring of antiretroviral therapy.

Methods and Findings

We have developed a real-time LightCycler (rtLC) PCR assay to detect and quantify HIV-1 from DBS. HIV-1 RNA extracted from DBS was amplified in a one-step, single-tube system using primers specific for long-terminal repeat sequences that are conserved across all HIV-1 clades. SYBR Green dye was used to quantify PCR amplicons and HIV-1 RNA copy numbers were determined from a standard curve generated using serially diluted known copies of HIV-1 RNA. This assay detected samples across clades, has a dynamic range of 5 log₁₀, and %CV <8% up to 4 log₁₀ dilution. Plasma HIV-1 RNA copy numbers obtained using this method correlated well with the Roche Ultrasensitive (r = 0.91) and branched DNA (r = 0.89) assays. The lower limit of detection (95%) was estimated to be 136 copies. The rtLC DBS assay was 2.5 fold rapid as well as 40-fold cheaper when compared to commercial assays. Adaptation of the assay into other real-time systems demonstrated similar performance.

Conclusions

The accuracy, reliability, genotype inclusivity and affordability, along with the small volumes of blood required for the assay suggest that the rtLC DBS assay will be useful for early diagnosis and monitoring of pediatric HIV-1 infection in resource-limited settings.     

Are seroepidemiological surveys for human immunodeficiency virus infection based on tests on pools of serum specimens accurate and cost-effective?

Serum specimens (n = 17668) from UK antenatal patients in the Thames Regions were tested by Wellcozyme HIV 1/2 EIA singly and in pools of 6, 12 and 24: 35 (0.2%, 1 in 505) were confirmed as anti-HIV positive. The pools of 12 were also tested for anti-HIV 1/2 by IAF Biochem, Behring and Diagnostics Pasteur EIAs. All 35 positive specimens were easily detectable after pooling in groups of 12. The false positive rate for Wellcozyme was nearly halved compared with individual testing (1 in 309 false positive compared with 1 in 174). For the other assays false positive rates on pools of 12 were: IAF Biochem 1 in 193, Behring 1 in 140, Diagnostics Pasteur 1 in 1547. Twenty-two known anti-HIV 2-positive sera were detected by all four EIAs when diluted as in pools of 6 and 12, but by only three EIAs in pools of 24 and 48. Pooling in groups of 6 did not seem to delay detection of HIV 1 seroconversion, but pooling in groups of 12, 24 and 48 might delay it by 1, 2 and 3 weeks respectively. For this study the effect of pooling in groups of 12 would have been a reagent saving of 87-91% and a labour saving of about 50%. Because of the low HIV incidence and rarity of specimens collected around seroconversion in UK, little, if any, loss of sensitivity would result from it. Pooling in groups of 12 has therefore been chosen for the screening of anonymous antenatal specimens in the UK.     

用PCR检测临床血标本中人类细小病毒B19 DNA

用ＰＣＲ检测临床血标本中人类细小病毒Ｂ１９　ＤＮＡ杨洪江,张桦,林书祥,牛艾茹（天津市儿童医院儿研所,天津３０００７４）关键词聚合酶链反应（ＰＣＲ）,人类细小病毒Ｂ１９几种人类细小病毒中,只有Ｂ１９病毒是人类的病原体。由于该病毒不能在体外繁殖,获得该...     

Evaluation of Dried Blood Spots with a Multiplex Assay for Measuring Recent HIV-1 Infection

Laboratory-based HIV tests for recent infection (TRIs), which primarily measure a specific serological biomarker(s) that distinguishes recent from long-term HIV infection, have facilitated the estimation of population-based incidence. Dried blood spots (DBS) on filter paper are an attractive sample source for HIV surveillance, given the simplified and cost-effective methods of specimen collection, storage, and shipment. Here, we evaluated the use of DBS in conjunction with an in-house multiplex TRI, the HIV-1-specific Bio-Plex assay, which measures direct antibody binding and avidity to multiple HIV-1 analytes. The assay performance was comparable between matched plasma and DBS samples from HIV-1 infected individuals obtained from diverse sources. The coefficients of variation, comparing the median antibody reactivity for each analyte between plasma and DBS, ranged from 2.78% to 9.40% and the correlation coefficients between the two sample types ranged from 0.89 to 0.97, depending on the analyte. The correlation in antibody reactivity between laboratory and site-prepared DBS for each analyte ranged from 0.87 to 0.98 and from 0.90 to 0.97 between site-prepared DBS and plasma. The correlation in assay measures between plasma and DBS indicate that the sample types can be used interchangeably with the Bio-Plex format, without negatively impacting the misclassification rate of the assay.     

核酸定量检测试验应用于HIV-1感染实验室诊断的探讨

目的:探讨核酸定量检测在HIV-1感染实验室诊断中的应用。方法:选取145例第四代抗原/抗体联合诊断筛查试验为阳性反应的血浆样本,分别用Western印迹和HIV-1核酸定量方法进行检测,综合对比分析2种方法检测结果。结果:Western印迹检出阳性样本120例,不确定样本17例,阴性样本8例;HIV-1核酸定量试验检出结果大于检测限样本131例,其中包括12例Western印迹不确定样本、2例Western印迹阴性样本;有3例Western印迹阳性样本用HIV-1核酸定量检测试验未能检出。结论:核酸定量检测试验对于HIV-1感染阳性样本是一种有效的实验室诊断方法;对HIV-1核酸定量检测结果为"TND"的样本,建议加做Western印迹或结合其他补充试验结果进行综合诊断。     

Evaluation of DNA pooling for the estimation of microsatellite allele frequencies: a case study using striped bass (Morone saxatilis)

Using striped bass (Morone saxatilis) and six multiplexed microsatellite markers, we evaluated procedures for estimating allele frequencies by pooling DNA from multiple individuals, a method suggested as cost-effective relative to individual genotyping. Using moment-based estimators, we estimated allele frequencies in experimental DNA pools and found that the three primary laboratory steps, DNA quantitation and pooling, PCR amplification, and electrophoresis, accounted for 23, 48, and 29%, respectively, of the technical variance of estimates in pools containing DNA from 2-24 individuals. Exact allele-frequency estimates could be made for pools of sizes 2-8, depending on the locus, by using an integer-valued estimator. Larger pools of size 12 and 24 tended to yield biased estimates; however, replicates of these estimates detected allele frequency differences among pools with different allelic compositions. We also derive an unbiased estimator of Hardy-Weinberg disequilibrium coefficients that uses multiple DNA pools and analyze the cost-efficiency of DNA pooling. DNA pooling yields the most potential cost savings when a large number of loci are employed using a large number of individuals, a situation becoming increasingly common as microsatellite loci are developed in increasing numbers of taxa.     

Development of a sample pooling strategy for the serodiagnosis of small ruminant lentiviral infections using the ELITEST-MVV ELISA

To reduce the costs of using the ELITEST-MVV, we explored the possibilities of sample pooling. Straight forward pooling applying the manufacturer's test conditions resulted in a significant loss of sensitivity. This was solved by using lower pre-dilutions of the samples than prescribed. Although an increase of background signal was encountered, discrimination between positive and negative samples was even better at pre-dilutions up to 12.5× as compared to the standard pre-dilution of 100×. This implied that pooling of up to eight samples was feasible. Receiver operating characteristic (ROC) analysis was used to determine the optimal cut-off value for the testing of pooled serum samples.

A model for cost-benefit analysis of pooling was applied which combines the economics of the technical performance of the modified assay and other additional cost factors connected with pooling such as hands-on time for composing the pools, expected seroprevalence in the test population, sample tracing and testing the individual samples of positive pools.

We concluded that pooling of samples was only feasible for monitoring SRLV-free accredited flocks because of their very low prevalence of infection. A pool consisting of five samples turned out to be the economical optimum although technically pool sizes of 10 samples were permitted.     

Evaluation of a new assay for HBV DNA quantitation in patients with chronic hepatitis B

Background: The Amplicor™ HBV Monitor Test for quantitative determination of serum hepatitis B virus (HBV) DNA has recently been introduced. This assay is based on PCR and a non-radioactive hybridization and detection system on microwell plates.Objective: The performance of the Amplicor™ HBV Monitor Test was evaluated in a routine diagnostic laboratory. The Amplicor™ HBV Monoitor assay was compared to the Digene Hybrid Capture™ System HBV DNA assay for the quantitation of HBV in patient sera.Study design: Sensitivity and reproducibility were determined with 10-fold dilution series of two Eurohep HBV reference plasma specimens. Furthermore, 196 sera from 14 children with chronic HBV infection and interferon therapy were tested with both assays.Results: The detection limit was found to be 10³ copies/ml with the Amplicor™ PCR assay compared to 10⁶ to 10⁷ copies/ml with the Digene™ hybridization assay. Both assays were quasi-linear over the measurable ranges. The new PCR assay proved to be very reliable. With the Amplicor™ PCR assay, 26.2% of the HBV DNA-positive clinical samples were found between 10³ and 10⁷ copies/ml and all of them tested below the detection limit with the hybridization assay.Conclusion: The Amplicor™ HBV Monitor Test shows excellent sensitivity and provides a valuable tool for the detection of HBV DNA in serum. It can be used for recognizing those patients who might benefit from antiviral therapy, for evaluation of the efficacy of anti-HBV therapy, and for validation of blood products.     

Dried Blood Spot Sampling for Hepatitis B Virus Serology and Molecular Testing

Background & Aims

Dried blood spots (DBS) on filter paper have been successfully used to diagnose and monitor several infectious diseases. The aim was to investigate the performance of DBS in hepatitis B virus (HBV) diagnosis using commercial tests in comparison to standard methods.

Methods

Paired DBS and plasma samples were collected from 200 patients: 100 patients with HBsAg negative status and 100 patients with HBsAg positive status. In the latter patient, HBeAg reactivity was tested. Ten samples of anti-HBs were collected from people vaccinated against HBV. We also studied 50 patients with positive HBV DNA viral load in plasma and 10 HBV DNA negative patients. HBV genotypes and gene polymerase mutations were determined in 10 randomly selected HBV-infected patients. The DBS sample consisted of 50 µL of whole blood, i.e. a 12-mm paper card.

Results

The sensitivity thresholds of HBsAg and anti-HBs antibody were 0.30±0.08 IU/mL and 18.11±6.05 IU/mL, respectively, for DBS with 98% sensitivity and 100% specificity. Sensitivity was 98% and specificity 100% for the detection of HBV DNA on a blotter, considering an HBV DNA threshold of 914.1±157.8 IU/ml. Ten patients had an HBeAg positive status in plasma, all were detected positive using DBS. HBV genotyping and mutation detection were successfully performed on DBS, with full concordance between the 10 paired DBS and plasma samples.

Conclusion

This study shows DBS is a reliable alternative to plasma specimens for quantifying and detecting HBsAg, anti-HBs, HBeAg and genotyping. DBS may increase the opportunities for HBV testing and treatment follow-up in hard-to-reach individuals.     

HIV-1包膜蛋白gp41优势抗原的构建及可溶性表达

目的:筛选1型人免疫缺陷病毒(HIV-1)中国流行株中包膜蛋白gp41的优势抗原片段,构建具有区域流行代表性的HIV-1 gp41重组抗原,为改进现有HIV-1初筛试剂盒中使用的同类抗原奠定基础。方法:利用免疫斑点杂交和生物信息学方法,从收集自重庆、广州、上海的区域代表性150份HIV-1感染者血清标本中筛选gp41抗原性强的候选样本,利用RT-PCR及巢式PCR方法扩增包含重要抗原表位决定蔟的gp41基因片段,与原核表达载体pQE30连接,转化大肠杆菌M15构建gp41重组抗原表达菌株,表达后经亲和层析纯化、SDS-PAGE和Western印迹鉴定。结果:兔源HRP标记的gp41多抗能识别标本中gp41抗原性差异,得到候选样本,扩增包含gp41主要抗原表位片段;构建了包含gp41抗原表达簇的重组原核表达质粒,表达、纯化后经His标签抗体Western印迹鉴定为阳性。结论:高纯度的重组优势gp41抗原的构建和鉴定,为进一步改进现有HIV初筛诊断奠定了基础。     

Effective High-Throughput Blood Pooling Strategy before DNA Extraction for Detection of Malaria in Low-Transmission Settings

In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost- and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, 20 µl in 1 sample, was optimal, and the parasite density as low as 2 p/µl for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings (<9% positive rate). The results indicated that pooling of the blood samples before DNA extraction followed by usual nested PCR is useful and effective for detection of malaria in screening of hidden cases in low-transmission settings.     

CCR5 Expression Correlates with Susceptibility of Maturing Monocytes to Human Immunodeficiency Virus Type 1 Infection

The chemokine receptor CCR5 and to a lesser extent CCR3 and CCR2b have been shown to serve as coreceptors for human immunodeficiency virus type 1 (HIV-1) entry into blood- or tissue-derived macrophages. Therefore, we examined the expression of the chemokine receptors CCR1, CCR2b, CCR3, CCR5, and CXCR4 as RNAs or as membrane-expressed antigens in monocytes maturing into macrophages and correlated these results with the susceptibility of macrophages to HIV-1 infection, as measured by their concentrations of extracellular p24 antigen and levels of intracellular HIV DNA by quantitative PCR. There was little change in levels of CCR1, CCR2b, and CCR5 RNAs. CCR3 RNA and surface antigen were undetectable throughout maturation of adherent monocytes over 10 days. CXCR4 RNA and membrane antigen were strongly expressed in newly adherent monocytes, but their levels declined at day 7. The amounts of CCR5 RNA remained stable, but the amounts of CCR5 antigen increased from undetectable to peak levels at day 7 and then declined slightly at day 10. Levels of susceptibility to laboratory (HIV-1_BaL) and clinical strains of HIV-1 showed parallel kinetics, peaking at day 7 and then decreasing at days 10 to 14. The concordance of levels of HIV DNA and p24 antigen suggested that the changes in susceptibility with monocyte maturation were at or immediately after entry and correlated well with CCR5 expression and inversely with CXCR4 expression.