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Efficient and large-scale expansion of mesenchymal stem/stromal cells (MSCs) has always been a formidable challenge to researchers in cell-based therapies and regenerative medicine. To reconcile major drawbacks of 2D planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on GMP-grade microcarrier for culture of umbilical cord-mesenchymal stem/stromal cells (UCMSCs), in accordance with the criteria of stem cell bank. ACISCP system is a fully closed system, which employs different models of vivaSPIN bioreactors (CytoNiche Biotech, China) for scale-up cell culture and vivaPREP (CytoNiche Biotech, China) for automated cell harvesting and cell dosage preparation. To realize industrial scale expansion of UCMSCs, a three-stage expansion was conducted with 1 L, 5 and 15 L vivaSPIN bioreactors. Using 3D TableTrixsup®/supand ACISCP system, we inoculated 1.5 × 10sup7/supof UCMSCs into 1 L vivaSPIN bioreactor and finally scaled to two 15 L bioreactor. A final yield of 2.09 × 10sup10/supcells with an overall expansion factor of 1975 within 13 days. The cells were harvested, concentrated, washed and prepared automatically with vivaPREP. The entire process was realized with ACISCP platform and was totally enclosed. Critical quality attributes (CQA) assessments and release tests of MSCs, including sterility, safety, purity, viability, identity, stability and potency were performed accordingly. The quality of cells harvested from 3D culture on the ACISCP and conventional 2D planar culture counterpart has no significant difference. This study provides a bioprocess engineering platform, harnessing GMP-grade 3D TableTrixsup®/supmicrocarriers and ACISCP to achieve industrial-scale manufacturing of clinical-grade hMSCs.

The outbreak of COVID-19 has posed a huge threat to global health and economy. Countermeasures have revolutionized norms for working, socializing, learning, and travel. Importantly, vaccines have been considered as most effective tools to combat with COVID-19. As of the beginning of 2021, >200 COVID-19 vaccine candidates, covering nearly all existing technologies and platforms, are being research and development (R&D) by multiple manufacturers worldwide. This has posed a huge obstacle to the quality control and evaluation of those candidate vaccines, especially in China, where five vaccine platforms are deployed in parallel. To accelerate the R&D progress of COVID-19 vaccines, the guidances on R&D of COVID-19 vaccine have been issued by National Regulatory Authorities or organizations worldwide. The Center for Drug Evaluation and national quality control laboratory in China have played a leading role in launching the research on quality control and evaluation in collaboration with relevant laboratories involved in the vaccine R&D, which greatly supported the progression of vaccines R&D, and accelerated the approval for emergency use and conditional marketing of currently vaccine candidates. In this paper, the progress and experience gained in quality control and evaluation of COVID-19 vaccines developed in China are summarized, which might provide references for the R&D of current and next generation of COVID-19 vaccines worldwide.

'Requirements for Primary Human Hepatocyte' is the first set of guidelines on Primary Human Hepatocyte in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements and transportation requirements for Primary Human Hepatocyte, which is applicable to the quality control for Primary Human Hepatocyte. It was originally released by the China Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols and accelerate the international standardization of Primary Human Hepatocyte for applications.

Lamellar bodies (LBs) are lysosome-related organelles (LROs) of surfactant-producing alveolar type 2 (AT2) cells of the distal lung epithelium. Trafficking pathways to LBs have been understudied but are likely critical to AT2 cell homeostasis given associations between genetic defects of endosome to LRO trafficking and pulmonary fibrosis in Hermansky Pudlak syndrome (HPS). Our prior studies uncovered a role for AP-3, defective in HPS type 2, in trafficking Peroxiredoxin-6 to LBs. We now show that the P4-type ATPase ATP8A1 is sorted by AP-3 from early endosomes to LBs through recognition of a C-terminal dileucine-based signal. Disruption of the AP-3/ATP8A1 interaction causes ATP8A1 accumulation in early sorting and/or recycling endosomes, enhancing phosphatidylserine exposure on the cytosolic leaflet. This in turn promotes activation of Yes-activating protein, a transcriptional coactivator, augmenting cell migration and AT2 cell numbers. Together, these studies illuminate a mechanism whereby loss of AP-3-mediated trafficking contributes to a toxic gain-of-function that results in enhanced and sustained activation of a repair pathway associated with pulmonary fibrosis.

Summary Background Although several COVID-19 vaccines have been developed so far, they will not be sufficient to meet the global demand. Development of a wider range of vaccines, with different mechanisms of action, could help control the spread of SARS-CoV-2 globally. We developed a protein subunit vaccine against COVID-19 using a dimeric form of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein as the antigen. We aimed to assess the safety and immunogenicity of this vaccine, ZF2001, and determine the appropriate dose and schedule for an efficacy study. Methods We did two randomised, double-blind, placebo-controlled, phase 1 and phase 2 trials. Phase 1 was done at two university hospitals in Chongqing and Beijing, China, and phase 2 was done at the Hunan Provincial Center for Disease Control and Prevention in Xiangtan, China. Healthy adults aged 18–59 years, without a history of SARS-CoV or SARS-CoV-2 infection, an RT-PCR-positive test result for SARS-CoV-2, a history of contact with confirmed or suspected COVID-19 cases, and severe allergies to any component of the vaccine were eligible for enrolment. In phase 1, participants were randomly assigned (2:2:1) to receive three doses of the vaccine (25 μg or 50 μg) or placebo intramuscularly, 30 days apart. In phase 2, participants were randomly assigned (1:1:1:1:1:1) to receive the vaccine (25 μg or 50 μg) or placebo intramuscularly, 30 days apart, in either a two-dose schedule or a three-dose schedule. Investigators, participants, and the laboratory team were masked to group allocation. For phase 1, the primary outcome was safety, measured by the occurrence of adverse events and serious adverse events. For phase 2, the primary outcome was safety and immunogenicity (the seroconversion rate and the magnitude, in geometric mean titres [GMTs], of SARS-CoV-2-neutralising antibodies). Analyses were done on an intention-to-treat and per-protocol basis. These trials are registered with ClinicalTrials.gov ( NCT04445194 and NCT04466085 ) and participant follow-up is ongoing. Findings Between June 22 and July 3, 2020, 50 participants were enrolled into the phase 1 trial and randomly assigned to receive three doses of placebo (n=10), the 25 μg vaccine (n=20), or the 50 μg vaccine (n=20). The mean age of participants was 32·6 (SD 9·4) years. Between July 12 and July 17, 2020, 900 participants were enrolled into the phase 2 trial and randomly assigned to receive two doses of placebo (n=150), 25 μg vaccine (n=150), or 50 μg vaccine (n=150), or three doses of placebo (n=150), 25 μg vaccine (n=150), or 50 μg vaccine (n=150). The mean age of participants was 43·5 (SD 9·2) years. In both phase 1 and phase 2, adverse events reported within 30 days after vaccination were mild or moderate (grade 1 or 2) in most cases (phase 1: six [60%] of ten participants in the placebo group, 14 [70%] of 20 in the 25 μg group, and 18 [90%] of 20 in the 50 μg group; phase 2: 37 [25%] of 150 in the two-dose placebo group, 43 [29%] of 150 in the two-dose 25 μg group, 50 [33%] of 150 in the two-dose 50 μg group, 47 [31%] of 150 in the three-dose placebo group, 72 [48%] of 150 in the three-dose 25 μg group, and 65 [43%] of 150 in the three-dose 50 μg group). In phase 1, two (10%) grade 3 or worse adverse events were reported in the 50 μg group. In phase 2, grade 3 or worse adverse events were reported by 18 participants (four [3%] in the two-dose 25 μg vaccine group, two [1%] in the two-dose 50 μg vaccine group, two [1%] in the three-dose placebo group, four [3%] in the three-dose 25 μg vaccine group, and six [4%] in the three-dose 50 μg vaccine group), and 11 were considered vaccine related (two [1%] in the two-dose 25 μg vaccine group, one [1%] in the two-dose 50 μg vaccine group, one [1%] in the three-dose placebo group, two [1%] in the three-dose 25 μg vaccine group, and five [3%] in the three-dose 50 μg vaccine group); seven participants reported serious adverse events (one [1%] in the two-dose 25 μg vaccine group, one [1%] in the two-dose 50 μg vaccine group, two [1%] in the three-dose placebo group, one [1%] in the three-dose 25 μg vaccine group, and two [1%] in the three-dose 50 μg vaccine group), but none was considered vaccine related. In phase 2, on the two-dose schedule, seroconversion rates of neutralising antibodies 14 days after the second dose were 76% (114 of 150 participants) in the 25 μg group and 72% (108 of 150) in the 50 μg group; on the three-dose schedule, seroconversion rates of neutralising antibodies 14 days after the third dose were 97% (143 of 148 participants) in the 25 μg group and 93% (138 of 148) in the 50 μg group. In the two-dose groups in phase 2, the SARS-CoV-2-neutralising GMTs 14 days after the second dose were 17·7 (95% CI 13·6–23·1) in the 25 μg group and 14·1 (10·8–18·3) in the 50 μg group. In the three-dose groups in phase 2, the SARS-CoV-2-neutralising GMTs 14 days after the third dose were 102·5 (95% CI 81·8–128·5) in the 25 μg group and 69·1 (53·0–90·0) in the 50 μg group. Interpretation The protein subunit vaccine ZF2001 appears to be well tolerated and immunogenic. The safety and immunogenicity data from the phase 1 and 2 trials support the use of the 25 μg dose in a three-dose schedule in an ongoing phase 3 trial for large-scale evaluation of ZF2001's safety and efficacy. Funding National Program on Key Research Project of China, National Science and Technology Major Projects of Drug Discovery, Strategic Priority Research Program of the Chinese Academy of Sciences, and Anhui Zhifei Longcom Biopharmaceutical. Translation For the Chinese translation of the abstract see Supplementary Materials section.

Additional file 1: Fig. S1. Workflow for data search and selection. Fig. S2. Overview of data collected for transcriptome variation analysis. (A) Boxplot showing correlation coefficient of transcriptome expression for each sample to others in the dataset before data selection step 3. 0.9 was selected as a cutoff for filtering out samples with lower median Pearson’s r.(B) Barplot showing number of samples in each study. (C) Histograms showing number of reads (up) and mapping results (down) across samples. Some MSCs derived from different anatomical parts of placenta, AM CM, CP, and CV. AD: Adipose tissue; AM: Amniotic membrane; BM: Bone marrow; CM: Chorionic membrane; CP: Chorionic plate; CV: Chorionic villi; ED: Endometrial; DP: Dental pulp; PL: Placenta; UC: Umbilical cord. Fig. S3. Transcriptome variation across MSC samples. (A) Scatter plot showing DM value and mean expression (CPM) for each expressed gene. (B) Scatter plot showing DM value and gene length for each expressed gene. (C) GESA positive results showing enrichment in GO cellular component (left) and molecular function (right) gene sets database based on ranked genes list in descending order by the DM value. (D) GESA negative results showing enrichment in GO gene sets database based on ranked genes list in descending order by the DM value. Only the top 20 terms with highest NES were presented (p

SummaryBackgroundA safe and effective coronavirus disease 2019 (COVID-19) vaccine is urgently needed to control the ongoing pandemic. Although progress has been made recently with several candidates reporting positive efficacy results, COVID-19 vaccines developed so far cannot meet the global vaccine demand. We developed a protein subunit vaccine against COVID-19, using dimeric form of receptor-binding domain (RBD) as the antigen. We aimed to assess the safety and immunogenicity of this vaccine in humans and determine the appropriate dose and schedule for an efficacy study.MethodsWe did two randomized, double-blind, placebo-controlled, phase 1 and 2 trials for an RBD-based protein subunit vaccine, ZF2001. In phase 1 study, 50 healthy adults aged 18-59 years were enrolled and randomly allocated to three groups to receive three doses of vaccine (25 μg or 50 μg RBD-dimer, with adjuvant) or placebo (adjuvant-only) intramuscularly, 30 days apart. In phase 2 study, 900 healthy adults aged 18-59 years were enrolled and randomly allocated to six groups to receive vaccine (25 μg or 50 μg RBD-dimer, with adjuvant) or placebo (adjuvant-only) intramuscularly, with the former 3 groups given two doses and the latter 3 groups given three doses, 30 days apart. For phase 1 trial, the primary outcome was safety, as measured by the occurrence of adverse events and serious adverse events. The secondary outcome was immunogenicity as measured by the seroconversion rate and magnitude of antigen-binding antibodies, neutralizing antibodies and T-cell cytokine production. For phase 2 trial, the primary outcome included both safety and immunogenicity. These trials are registered with ClinicaTrials.gov, NCT04445194 and NCT04466085.FindingsBetween June 22 and September 15, 2020, 50 participants were enrolled to the phase 1 study (mean age 32.6 years) and 900 participants were enrolled to phase 2 study (mean age 43.5 years), to receive vaccine or placebo with a two-dose or three-dose schedule. For both trials, local and systemic adverse reactions were absent or mild in most participants. There were no serious adverse events related to vaccine in either trial. After three doses, neutralizing antibodies were detected in all participants receiving either 25 μg or 50 μg dose of vaccine in phase 1 study, and in 97% (the 25 μg group) and 93% (the 50 μg group) of participants, respectively, in phase 2 study. The SARS-CoV-2-neutralizing geometric mean titres (GMTs) were 94.5 for the 25 μg group and 117.8 for the 50 μg group in phase 1, and 102.5 for the 25 μg group and 69.1 for the 50 μg group in phase 2, exceeding the level of a panel of COVID-19 convalescent samples (GMT, 51). Vaccine induced balanced TH1 and TH2 responses. The 50 μg group did not show enhanced immunogenicity compared with the 25 μg group.InterpretationThe protein subunit vaccine ZF2001 is well-tolerated and immunogenic. The safety and immunogenicity data from phase 1 and 2 trials for ZF2001 support the use of 25 μg vaccine dose with three-dose schedule to an ongoing phase 3 large-scale evaluation for safety and efficacy.FundingNational Program on Key Research Project of China, National Science and Technology Major Projects of Drug Discovery, Strategic Priority Research Program of the Chinese Academy of Sciences, and Anhui Zhifei Longcom Biopharmaceutical.

Background Mesenchymal stromal cell (MSC)-based therapies are being actively investigated in various inflammatory disorders. However, functional variability among MSCs cultured in vitro will lead to distinct therapeutic efficacies. Until now, the mechanisms behind immunomodulatory functional variability in MSCs are still unclear. Methods We systemically investigated transcriptomic variations among MSC samples derived from multiple tissues to reveal their effects on immunomodulatory functions of MSCs. We then analyzed transcriptomic changes of MSCs licensed with INFγ to identify potential molecular mechanisms that result in distinct MSC samples with different immunomodulatory potency. Results MSCs were clustered into distinct groups showing different functional enrichment according to transcriptomic patterns. Differential expression analysis indicated that different groups of MSCs deploy common regulation networks in response to inflammatory stimulation, while expression variation of genes in the networks could lead to different immunosuppressive capability. These different responsive genes also showed high expression variability among unlicensed MSC samples. Finally, a gene panel was derived from these different responsive genes and was able to regroup unlicensed MSCs with different immunosuppressive potencies. Conclusion This study revealed genes with expression variation that contribute to immunomodulatory functional variability of MSCs and provided us a strategy to identify candidate markers for functional variability assessment of MSCs.

Objective: The purpose of the study was to investigate the therapeutic effects and safety of genetic modified mesenchymal stem cells (MSCs) in type 2 diabetes mellitues (T2DM).Methods: MSCs derived from adipose tissue were modified with over-expression of FGF21 and GLP1, which was achieved through lentiviral particles transduction. The cells were transplanted to the BKS.Cg-Dock7m+/+Leprdb/Nju mice (T2DM mice model). The injections of physiological saline (0.1 mL), Liraglutide (0.5mg/kg) were employed as negative and positive controls respectively. The protein analyses were performed using ELISA or western blot analysis, while quantitative real-time PCR was adopted for gene expression.Results: The genetic modification had no effects on morphology, differentiation ability, or immunophenotype of MSCs. Moreover, MSC-FGF21+GLP1 cells exhibited significantly increased expression of FGF21 and GLP1. In T2DM model mice, the transplantation of MSC-FGF21+GLP1 cells ameliorated blood glucose and weight, promoted the secretion of insulin, enhanced the recovery of liver structures, and improved lipid profiles. Moreover, FGF21 and GLP1 exhibited synergistic effects in regulation of glucolipid metabolism through controlling the expression of insulin, srebp1 and srebp2.Conclusion: The stem cell treatment based on MSCs modified by FGF21 and GLP1 genes is an effective approach for treatment of T2DM.

Objective The purpose of this study was to investigate the therapeutic effects of genetically modified mesenchymal stem cells (MSCs) in the treatment of type 2 diabetes mellitus (T2DM) in order to identify a new method for treating diabetes that differs from traditional medicine and to provide a new means by which to fundamentally improve or treat diabetes. Methods MSCs derived from adipose tissue were modified to overexpress FGF21 and GLP1, which was achieved through lentiviral particle transduction. The cells were transplanted into BKS.Cg-Dock7m+/+Leprdb/Nju mice (T2DM mouse model). Injections of physiological saline (0.1 mL) and liraglutide (0.5 mg/kg) were used as negative and positive controls, respectively. ELISA or Western blotting was used for protein analysis, and quantitative real-time PCR was used for gene expression analysis. Results Genetic modification had no effects on the morphology, differentiation ability, or immunophenotype of MSCs. Moreover, MSC-FGF21+GLP1 cells exhibited significantly increased secretion of FGF21 and GLP1. In the T2DM mouse model, the transplantation of MSC-FGF21+GLP1 cells ameliorated the changes in blood glucose and weight, promoted the secretion of insulin, enhanced the recovery of liver structures, and improved the profiles of lipids. Moreover, FGF21 and GLP1 exerted synergistic effects in the regulation of glucolipid metabolism by controlling the expression of insulin, srebp1, and srebp2. Conclusion Stem cell treatment based on MSCs modified to overexpress the FGF21 and GLP1 genes is an effective approach for the treatment of T2DM.

Defining the mechanisms of cellular pathogenesis in rare lung diseases such as Hermansky-Pudlak syndrome (HPS) is often complicated by loss of the differentiated phenotype of cultured primary alveolar type 2 (AT2) cells, as well as by a lack of durable cell lines that are faithful to both AT2-cell and rare disease phenotypes. We used CRISPR/Cas9 gene editing to generate a series of HPS-specific mutations in the MLE-15 cell line. The resulting MLE-15/HPS cell lines exhibit preservation of AT2 cellular functions, including formation of lamellar body–like organelles, complete processing of surfactant protein B, and known features of HPS specific to each trafficking complex, including loss of protein targeting to lamellar bodies. MLE-15/HPS1 and MLE-15/HPS2 (with a mutation in Ap3β1) express increased macrophage chemotactic protein-1, a well-described mediator of alveolitis in patients with HPS and in mouse models. We show that MLE-15/HPS9 and pallid AT2 cells (with a mutation in Bloc1s6) also express increased macrophage chemotactic protein-1, suggesting that mice and humans with BLOC-1 mutations may also be susceptible to alveolitis. In addition to providing a flexible platform to examine the role of HPS-specific mutations in trafficking AT2 cells, MLE-15/HPS cell lines provide a durable resource for high-throughput screening and studies of cellular pathophysiology that are likely to accelerate progress toward developing novel therapies for this rare lung disease.

Objective Necrotizing enterocolitis (NEC) is the most common surgical emergency in preterm infants, and pathogenesis associates with changes in the fecal microbiome. As fecal samples incompletely represent microbial communities in intestinal mucosa, we sought to determine the NEC tissue-specific microbiome and assess its contribution to pathogenesis. Design We amplified and sequenced the V1-V3 hypervariable region of the bacterial 16S rRNA gene extracted from intestinal tissue and corresponding fecal samples from 12 surgical patients with NEC and 14 surgical patients without NEC. Low quality and non-bacterial sequences were removed, and taxonomic assignment was made with the Ribosomal Database Project. Operational taxonomic units were clustered at 97%. We tested for differences between NEC and non-NEC samples in microbiome alpha- and beta-diversity and differential abundance of specific taxa between NEC and non-NEC samples. Additional analyses were performed to assess the contribution of other demographic and environmental confounding factors on the infant tissue and fecal microbiome. Results The fecal and tissue microbial communities were different. NEC was associated with a distinct microbiome, which was characterized by low diversity, higher abundances of Staphylococcus and Clostridium_sensu_stricto, and lower abundances of Actinomyces and Corynebacterium. Infant age and vancomycin exposure correlated with shifts in the tissue microbiome. Conclusion The observed low diversity in NEC tissues suggests that NEC is associated with a bacterial bloom and a distinct mucosal bacterial community. The exact bacterial species that constitute the bloom varied by infant and were strongly influenced by age and exposure to vancomycin.

Bacterial DNA has been reported in the placenta and amniotic fluid by several independent groups of investigators. However, it's taxonomic overlap with fetal and maternal bacterial DNA in different sites has been poorly characterized. Here, we determined the presence of bacterial DNA in the intestines and placentas of fetal mice at gestational day 17 (n = 13). These were compared to newborn intestines (n = 15), maternal sites (mouth, n = 6; vagina, n = 6; colon, n = 7; feces, n = 8), and negative controls to rule out contamination. The V4 region of the bacterial 16S rRNA gene indicated a pattern of bacterial DNA in fetal intestine similar to placenta but with higher phylogenetic diversity than placenta or newborn intestine. Firmicutes were the most frequently assignable phylum. SourceTracker analysis suggested the placenta as the most commonly identifiable origin for fetal bacterial DNA, but also over 75% of fetal gut genera overlapped with maternal oral and vaginal taxa but not with maternal or newborn feces. These data provide evidence for the presence of bacterial DNA in the mouse fetus.

Recombinant adeno-associated virus serotype 1 (AAV1) has attracted tremendous interest as a promising vector for gene therapy and vaccine applications. However, the presence of AAV1 neutralizing antibodies as a consequence of exposure to wild type AAV1 can limit significantly effective gene transfer for biologics based AAV1 vector. Prior studies have reported that a prevalence of AAV1 neutralizing antibodies ranged from 10% to 50% in different countries around the world, and up to 79% in Dutch subjects. However, few studies have reported on the AAV1 neutralizing antibody prevalence in Chinese subjects. In this study, a high-throughput luciferase-based virus neutralization assay was established and standardized for critical parameters, including the appropriate cell line, and the optimal viral infection dose, and the infection time with homologous AAV1 vaccinated mice and guinea pig sera. Then, a total of 500 healthy individual serum samples from two separate regions of China were screened for the AAV1 neutralizing antibodies by conducting a non-randomized, cross-sectional analysis. Interestingly, a high prevalence of AAV1 neutralizing antibody (69.8%) was found in all individuals. There was significant difference observed for prevalence by gender (P = 0.042), age range (P = 0.011) and geographic origin (P 10) in teenagers (year

As pulmonary fungal infections continue to increase due to an increasing number of immunocompromised patients, rapid detection and accurate identification of these fungal pathogens are critical. A broad fungal assay was developed by incorporating broad-range multilocus PCR amplification and electrospray ionization/mass spectrometry (PCR/ESI-MS) to detect and identify fungal organisms directly from clinical specimens. The aims of this study were to evaluate the performance of PCR/ESI-MS for detection, identification, and determination of the distribution of fungal organisms in bronchoalveolar lavage (BAL) fluid specimens. The BAL fluid specimens submitted for fungal culture at Vanderbilt University Medical Center between May 2005 and October 2011 were included. Cultures and identification were done using standard procedures. In addition, DNA was extracted from BAL fluid specimens, and fungal DNA amplification/identification were performed by PCR/ESI-MS. The results were compared with those of the standard cultures. A total of 691 nonduplicated BAL fluid specimens with sufficient leftover volume for molecular testing were evaluated using PCR/ESI-MS. Among them, 134 specimens (19.4%) were positive for fungi by both culture and PCR/ESI-MS testing. Of the dual-positive specimens, 125 (93.3%) were positive for Candida and Aspergillus species, with concordances between culture and PCR/ESI-MS results being 84 (67.2%) at the species level and 109 (87.2%) at the genus level. In addition, 243 (35.2%) and 30 (4.3%) specimens were positive only by PCR/ESI-MS or by culture, respectively (odds ratio [OR] = 11.95, 95% confidence interval [CI] = 7.90 to 18.17, P = 0.0000). Codetection of fungal organisms was noted in 23 (3.3%) specimens by PCR/ESI-MS, which was significantly higher than the 4 (0.6%) in which they were noted by culture (OR = 5.91, 95% CI = 1.93 to 20.27, P = 0.0002). Among 53 specimens in which cultures failed because of bacterial overgrowth, at least one fungus was identified in 26 specimens (47.3%) by PCR/ESI-MS. PCR/ESI-MS provides an advanced tool for rapid and sensitive detection, identification, and determination of the distribution of fungal organisms directly from BAL fluid specimens. Moreover, it detected fungal organisms in specimens in which cultures failed because of bacterial overgrowth. The clinical relevance of the significantly higher detection rate of fungal organisms by PCR/ESI-MS merits further investigation.

The Bruker Biotyper and BD Phoenix systems were evaluated for identification of 1,024 bacterial urinary tract isolates. The Biotyper and Phoenix systems correctly identified 99.9% and 99.5% to the genus level and 99.1% and 98.5% to the species level, respectively. Both systems provide reliable results, and the Biotyper system offers a rapid tool for urine bacterial isolate identification.

Background The current limitations of laboratory testing for the detection of human herpesvirus virus 6 (HHV-6) in clinical specimens with low HHV-6 viral loads make this area a priority for further research and development. Objectives To develop and validate a sensitive qualitative assay for simultaneous HHV-6 detection and variant differentiation. Methods We developed a diagnostic procedure, which combines a magnetic bead-based nucleic acid extraction, PCR amplification, and colorimetric microtiter plate identification (MAG-PCR-EIA), for the sensitive detection of HHV-6 and the simultaneous differentiation of HHV-6A and HHV-6B. Results Analytic sensitivities of the MAG-PCR-EIA assay were 10 copies per reaction for both HHV-6A and HHV-6B variants, which is equivalent to 20 copies/ml when 1 ml of clinical specimen was processed. A proficiency panel containing 11 blinded specimens covering HHV-6A viral loads from 0 to 100,000 copies was tested, and the MAG-PCR-EIA was able to detect the lowest concentration at one copy in 200 μl. A panel of 27 urine specimens, which were collected from patients with and without chronic fatigue syndrome, was tested by the MAG-PCR-EIA. HHV-6 was detected in two (HHV-6A) patients who have chromosomally integrated HHV-6A and in one (HHV-6B) patient who was a healthy control and diagnosed as cervical cancer later on. The HHV-6 results did not correlate with results previously determined by HHV-6 antigenemia in urine. Conclusion With large specimen volumes processed and an additional signal amplification incorporated, the MAG-PCR-EIA provides a sensitive and qualitative system for HHV-6 detection and simultaneous variant differentiation. Clinical relevance of the assay awaits further investigation.

A total of 82 samples from patients with cervical cancer (Group 1) and 50 samples from patients with other genital diseases (Group 2) were collected in Gansu, China. All 132 samples were tested for HPV DNA with a typing kit that can detect 21 types of HPV, and also tested for neutralizing antibodies against HPV-16, -18, -58, -45, -6, and -11 using pseudovirus-based neutralization assays. The results revealed that 28% (23/82) of sera in Group 1 were positive for type-specific neutralizing antibodies with a titer range of 160-640, of which 23.2% (19/82), 2.4% (2/82), 2.4% (2/82), 1.2% (1/82), and 1.2% (1/82) were against HPV-16, -58, -6, -18, and -45, respectively. Only one serum (2%) in Group 2 was positive for neutralizing antibodies, which were against HPV-6 with a titer of 2,560. Overall, 85.4% (70/82) of samples in Group 1 were HPV DNA-positive, compared with 28% (14/50) of samples in Group 2. The seven most common types detected in Group 1 were HPV-16 (80%), HPV-52 (7.1%), HPV-66 and HPV-11 (5.7% each), and HPV-58, HPV-18, and HPV-33 (4.3% each), while the four most common types in Group 2 were HPV-16 (12%), HPV-52 and HPV-11 (6% each), and HPV-68 (4%). The concordance between HPV DNA and corresponding neutralizing antibodies was 32.9% (27/82) with a significant difference (P < 0.005). More specifically, the concordance was 42.7% (35/82) for HPV-16 in Group 1. The full-length sequences of six HPV types (HPV-16, -58, -33, -59, -11, and -68) were determined and showed 99% identities with their reported genomes.

Aim: SARS-associated coronavirus (SARS-CoV) has been confirmed as the pathogen for severe acute respiratory syndrome (SARS). The aim of our study was to construct a sensitive and specific real-time quantitative fluorescent (QF) reverse transcriptase (RT)-PCR method for the detection of SARS-CoV RNA. Methods: Stored blood specimens from 44 patients with confirmed SARS were used along with blood samples from two sets of controls, 30 healthy volunteers who had no contact with SARS patients, and 30 healthy doctors and nurses who had contact with SARS patients but were without symptoms of SARS. Two pairs of primers were synthesized by the Shanghai Sangon Company according to SARS-CoV BJ01 strain sequence (AY278488), and then a pair of primers were designed and compared with a pair of primers published by WHO. Results: Using serial dilutions of SARS-CoV, the 44 blood samples from SARS patients specimens were tested. Using a 0.01% dilution of SARS-CoV, all 44 clinical samples tested positive in our assay. In comparison, using a 0.1% dilution of SARS-CoV, 26 of the 44 samples tested positive using the WHO primers. In the QF-RT-PCR assay, there was a linear amplification from 100 copies to 108 copies of the control RNA per RT-PCR and at least 10 copies, and sometimes even 1 copy, of target RNA tested positive in our assay. Conclusion: The primer we developed is sufficiently sensitive and specific to diagnose symptomatic SARS-CoV infections and for monitoring virus load.

We used multilocus PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) to determine the genotype and drug resistance profiles for 96 Mycobacterium tuberculosis isolates circulating in regions of high and low tuberculosis (TB) endemicity in China. The dominant principal genetic group (PGG) circulating in China was PGG1, and drug-resistant gene mutations were more diversified in the region of low rather than high TB endemicity.

Fecal sampling is widely utilized to define small intestinal tissue-level microbial communities in healthy and diseased newborns. However, this approach may lead to inaccurate assessments of disease or therapeutics in newborns because of the assumption that the taxa in the fecal microbiota are representative of the taxa present throughout the gastrointestinal tract. To assess the stratification of microbes in the newborn gut and to evaluate the probable shortcoming of fecal sampling in place of tissue sampling, we simultaneously compared intestinal mucosa and fecal microbial communities in 15 neonates undergoing intestinal resections. We report three key results. First, when the site of fecal and mucosal samples are further apart, their microbial communities are more distinct, as indicated by low mean Sorensen similarity indices for each patient's fecal and tissue microbiota. Second, two distinct niches (intestinal mucosa and fecal microbiota) are evident by principal component analyses, demonstrating the critical role of sample source in defining microbial composition. Finally, in contrast to adult studies, intestinal bacterial diversity was higher in tissue than in fecal samples. This study represents an unprecedented map of the infant microbiota from intestinal mucosa and establishes discernable biogeography throughout the neonatal gastrointestinal tract. Our results question the reliance on fecal microbiota as a proxy for the developing intestinal microbiota. Additionally, the robust intestinal tissue-level bacterial diversity we detected at these early ages may contribute to the maturation of mucosal immunity.

Recombinant adeno-associated virus serotype 1 (AAV1) has attracted tremendous interest as a promising vector for gene therapy and vaccine applications. However, the presence of AAV1 neutralizing antibodies as a consequence of exposure to wild type AAV1 can limit significantly effective gene transfer for biologics based AAV1 vector. Prior studies have reported that a prevalence of AAV1 neutralizing antibodies ranged from 10% to 50% in different countries around the world, and up to 79% in Dutch subjects. However, few studies have reported on the AAV1 neutralizing antibody prevalence in Chinese subjects. In this study, a high-throughput luciferase-based virus neutralization assay was established and standardized for critical parameters, including the appropriate cell line, and the optimal viral infection dose, and the infection time with homologous AAV1 vaccinated mice and guinea pig sera. Then, a total of 500 healthy individual serum samples from two separate regions of China were screened for the AAV1 neutralizing antibodies by conducting a non-randomized, cross-sectional analysis. Interestingly, a high prevalence of AAV1 neutralizing antibody (69.8%) was found in all individuals. There was significant difference observed for prevalence by gender (P = 0.042), age range (P = 0.011) and geographic origin (P 0.001). The percentage of positive AAV1 neutralizing antibodies (NT50 10) in teenagers (year18, as of 2012) was significant lower than that of adults (19-56, as of 2012) (P = 0.011), indicating the optimal vaccination period of childhood. The current study provides a useful insight for the future development of AAV1-based vaccination and gene therapy strategies in Beijing and Anhui provinces of China.

Formalin-fixed, paraffin-embedded skin biopsy specimens, including 72 suppurative granulomatous inflammation (SGI) and 47 non-SGI controls, were tested for mycobacteria by using a broad-range PCR and a suspension array identification system. Mycobacterium smegmatis was detected in 13 (18.1%) of the SGI skin biopsy specimens, which was significantly more than 2 (4.3%) in the controls (odds ratio, 5.73; 95% confidence interval, 1.21 to 27.06; P = 0.028).

Background The vaccinia virus Guang9 strain (VG9), derived from the vaccinia virus Tian Tan strain (VTT) has been found to be less virulent than VTT. Methodology/Principal Findings To investigate whether VG9 could be a potential replicating virus vector, the TK genes in VG9 and VTT were replaced with the HIV-1 envelope gene via homologous recombination, resulting in the recombinant viruses, VG9-E and VTT-E. The biology, virulence, humoral and cellular immunological responses of VG9-E and VTT-E were evaluated. Our results indicated no obvious difference in range of host cells and diffusion between two recombinant viruses. Neurovirulence for VG9-E in weanling and suckling mice, and skin virulence in rabbits, were lower than that of VTT-E. The humoral immune responses, including binding antibody and neutralizing antibody responses, induced by VG9-E were not significantly different from those for VTT-E whilst IFN-γ response which represented cellular immune response induced by VG9-E was significantly higher than that did by VTT-E. Conclusions/Significance Our results indicated that VG9-E was less virulent, yet induced higher cellular immune response than VTT-E. Therefore, it could be an ideal replicating vaccinia vector for HIV vaccine research and development.

Background Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China. Methodology/Principal Findings A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30–55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin. Conclusion A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in Beijing and Anhui provinces of China.

Background Several commercially available HIV-1 viral load assays based on real-time detection technology and automated platforms are available. It is not clear how the diversity of HIV-1 genotypes impacts the ability to consistently detect HIV-1 viral loads. Objectives To examine whether the diversity of HIV-1 genotypes impacts the ability of the Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 (CAP/CTM v2.0), its version 1.0 (CAP/CTM v1.0) and the NucliSens EasyQ HIV-1 version 2.0 (EasyQ v2.0) assays to consistently determine the viral loads. Study design The three assays were used to measure the viral load in 178 plasma samples with diverse genotypes from treatment-naive patients. Results CAP/CTM v2.0 showed significant correlation and high agreement with CAP/CTM v1.0 and EasyQ v2.0. CAP/CTM v2.0 showed excellent detection of clade B samples compared with CAP/CTM v1.0 and EasyQ v2.0. However, significant differences were observed when using CAP/CTM v2.0 to test clade BC and AE samples. The HIV-1 load measured by CAP/CTM v2.0 differed by >0.5logIU/ml in 59.52% and 72.62% of clade BC samples, and in 57.14% and 85.71% of clade AE samples, compared with CAP/CTM v1.0 and EasyQ v2.0, respectively. CAP/CTM v2.0 was more precise (13.18%) than EasyQ v2.0 (29.21%), and both assays showed good linearity ( R ≥0.9926). Conclusions The three assays may not deliver consistent results for samples belonging to clades BC and AE. It is strongly suggested that the version of the HIV-1 viral load assay used initially is also used at follow-up.

Rapid detection and identification of Ehrlichia species improves clinical outcome for patients suspected of ehrlichiosis. We describe an assay that employs multilocus PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) to detect and identify Ehrlichia species directly from blood specimens. The results were compared to those of a colorimetric microtiter PCR enzyme immunoassay (PCR-EIA) used as a diagnostic assay. Among 213 whole-blood samples collected from patients who were clinically suspected of ehrlichiosis from 1 May to 1 August 2008 at Vanderbilt University Hospital, 40 were positive for an Ehrlichia species by PCR/ESI-MS, giving a positive rate of 18.8%. In comparison to the PCR-EIA, PCR/ESI-MS possessed a sensitivity, a specificity, and positive and negative predictive values of 95.0%, 98.8%, 95.0%, and 98.8%, respectively. The 38 specimens that were positive for Ehrlichia by both PCR/ESI-MS and the PCR-EIA were further characterized to the species level, with 100% agreement between the two assays. In addition, Rickettsia rickettsii was detected by PCR/ESI-MS from four specimens that were confirmed retrospectively by serology and PCR-EIA. In three specimens, the PCR/ESI-MS assay identified Pseudomonas aeruginosa , Neisseria meningitidis , and Staphylococcus aureus ; these were confirmed by culture and/or clinical diagnosis as being clinically relevant. From specimen processing to result reporting, the PCR/ESI-MS assay can be completed within 6 h, providing another laboratory tool for the diagnosis of ehrlichiosis. Moreover, this system may provide rapid detection and identification of additional pathogens directly from blood specimens.

A standardized molecular test for the detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid (CSF) would assist the further assessment of the association of C. pneumoniae with multiple sclerosis (MS). We developed and validated a qualitative colorimetric microtiter plate-based PCR assay (PCR-EIA) and a real-time quantitative PCR assay (TaqMan) for detection of C. pneumoniae DNA in CSF specimens from MS patients and controls. Compared to a touchdown nested-PCR assay, the sensitivity, specificity, and concordance of the PCR-EIA assay were 88.5%, 93.2%, and 90.5%, respectively, on a total of 137 CSF specimens. PCR-EIA presented a significantly higher sensitivity in MS patients (p = 0.008) and a higher specificity in other neurological diseases (p = 0.018). Test reproducibility of the PCR-EIA assay was statistically related to the volumes of extract DNA included in the test (p = 0.033); a high volume, which was equivalent to 100 microl of CSF per reaction, yielded a concordance of 96.8% between two medical technologists running the test at different times. The TaqMan quantitative PCR assay detected 26 of 63 (41.3%) of positive CSF specimens that tested positive by both PCR-EIA and nested-PCR qualitative assays. None of the CSF specimens that were negative by the two qualitative PCR methods were detected by the TaqMan quantitative PCR. The PCR-EIA assay detected a minimum of 25 copies/ml C. pneumoniae DNA in plasmid-spiked CSF, which was at least 10 times more sensitive than TaqMan. These data indicated that the PCR-EIA assay possessed a sensitivity that was equal to the nested-PCR procedures for the detection of C. pneumoniae DNA in CSF. The TaqMan system may not be sensitive enough for diagnostic purposes due to the low C. pneumoniae copies existing in the majority of CSF specimens from MS patients.

Hepatitis A virus (HAV) is the major pathogen responsible for acute infectious hepatitis A, a disease that is prevalent worldwide. Although HAV immunization effectively prevents infection, primary immunizations must be administered at least 2 weeks prior to HAV exposure. In contrast, passive immunization with pooled human immunoglobulin (Ig) can provide immediate and rapid protection from HAV infection. Because the use of human sera-derived Igs carries the risk of contamination, we sought to develop recombinant HAV-neutralizing human antibodies. We prepared a combinatorial phage display library of recombinant human anti-HAV antibodies from RNA extracted from the blood lymphocytes of a convalescent hepatitis A patient. Two recombinant human IgG antibodies, HAIgG16 and HAIgG78, were screened from the antibody library by their ability to bind with high affinity to purified, inactivated HAV virions. These antibodies recognized different epitopes of the HAV virion capsid, and competed with both patient sera and well-characterized neutralizing mouse monoclonal antibodies. A cocktailed mixture of HAIgG16 and HAIgG78 at a 3:1 ratio was prepared to compare its combined biological activity with that conferred by each antibody individually. The cocktailed antibodies displayed a stronger neutralizing activity in vitro than that observed with either HAIgG16 and HAIgG78 alone. To determine the in vivo neutralizing abilities of these antibodies, rhesus monkeys were inoculated with cocktailed antibodies and challenged with HAV. Whereas control animals developed hepatitis A and seroconverted to the HAV antibody, animals receiving cocktailed antibodies were protected either from viral infection or from developing clinical hepatitis. These results demonstrate that recombinant human antibody preparations could be used to prevent or treat early-stage HAV infection.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are two important viral pathogens that cause respiratory tract infections in the pediatric population. The rapid detection of these agents allows the prompt isolation and treatment of infected patients. In the present prospective study, we evaluated the performances of four rapid antigen detection assays, including a rapid chromatographic immunoassay (CIA) for RSV (Directigen EZ RSV; Becton Dickinson, Sparks, MD), a direct fluorescent-antibody assay (DFA) for RSV (Bartels; Trinity Biotech, Carlsbad, CA), and two DFAs for hMPV manufactured by Diagnostic Hybrids Inc. (DHI; Athens, OH) and Imagen (Oxoid Ltd., Basingstoke, Hampshire, United Kingdom). The clinical specimens tested comprised 515 nasopharyngeal aspirates submitted to the Clinical Microbiology Laboratory at Hartford Hospital from 1 November 2006 to 21 April 2007. Compared to the results of real-time reverse transcription-PCR (RT-PCR), the CIA had a sensitivity of 79.8% and a specificity of 89.5%. The RSV DFA with Bartels reagents showed a sensitivity of 94.1% and a specificity of 96.8%. For hMPV, the sensitivity and specificity were 62.5% and 99.8%, respectively, for the DHI DFA and 63.2% and 100%, respectively, for the Imagen DFA. The hands-on and test turnaround times for CIA were 10 and 30 to 60 min, respectively, and the hands-on and test turnaround times for the RSV and hMPV DFAs were 30 and 105 min, respectively. We conclude that while the RSV CIA is user-friendly, it lacks sensitivity and specificity, especially during off-peak months. In contrast, the RSV DFA is more sensitive and specific, but interpretation of its results is subjective and it demands technical time and expertise. Similarly, both hMPV DFAs are highly specific in comparison to the results of RT-PCR, but their sensitivities await further improvements.

Rapid detection of the Mycobacterium tuberculosis complex is important in patient management in terms of initiating appropriate antimycobacterial therapy as well as controlling the spread of this pathogen. Although mycobacterial culture continues to be a valuable diagnostic tool, the results are not rapid. The Centers for Disease Control and Prevention as well as the American Thoracic Society recommends obtaining mycobacterial cultures of at least three sputum specimens from patients in whom tuberculosis is suspected. In contrast to the increased time required for mycobacterial culture, acid-fast bacillus (AFB) staining generally provides rapid evidence for the presence of mycobacteria in a clinical specimen. AFB staining is used by most health care facilities to determine when patients with suspected tuberculosis should be isolated. However, AFB smears will be positive in only 60% of tuberculosis cases (7). Moreover, a recent study found that only 8.6% of patients placed in isolation proved to have tuberculosis yet 19% of patients with pulmonary tuberculosis were not isolated on the first day after hospital admission (9). Although AFB staining and mycobacterial culture are still the primary diagnostic tests in most laboratories, molecular tests based on nucleic acid amplification techniques have been available for the detection of the M. tuberculosis complex in a variety of clinical specimens (2, 4, 6). We used a colorimetric microtiter plate PCR-enzyme immunoassay (PCR-EIA) (8) targeting two specific genes for the rapid detection of the M. tuberculosis complex. One primer set (IS-1, 5′-CCT GCG AGC GTA GGC GTC GG-3′, and IS-2, 5′-CTC GTC CAG CGC CGC TTC GG-3′) was designed to target IS6110, an insertion sequence within the chromosome of M. tuberculosis (2). Another primer set (MPB-1, 5′-GAG GAG TTG AAA GGC ACC GAT-3′, and MPB-2, 5′-CGC GTC TGG GCG ATG TA-3′) was designed to target an M. tuberculosis gene which encodes the MPB64 protein (6). Two M. tuberculosis complex-specific 5′-biotinylated capture probes (IS-p, 5′-GGC GAA CCC TGC CCA GGT CGA CAC-3′, and MPB-p, 5′-CGG CCA GGC GTG CCA GAT TC-3′) were incorporated into a colorimetric signal amplification procedure to detect and confirm the amplification products as previously described (8). A similar format was used separately to amplify the human β-actin gene to assure the quality of extracted DNA samples. During a study period from December 2001 until January 2002, a total of 782 respiratory specimens, mainly sputa, were sent to the Diagnostic Laboratory Services, Inc., in Honolulu, Hawaii, for AFB staining and mycobacterial culture. Standard sample decontamination with N-acetyl-l-cysteine-NaOH (2% final concentration) and concentration by centrifugation were performed as described previously (5, 10). The volume of the final sediment was approximately 2 ml. Part of the sediment was used for the AFB smear, which was then stained using auramine-rhodamine fluorochrome staining (10). Part of the sediment was inoculated onto Middlebrook 7H10 solid medium and into BACTEC 12B broth bottles that were monitored for growth using the BACTEC 460TB instrument (Becton Dickinson, Sparks, Md.). Of these specimens, all 15 M. tuberculosis culture-positive specimens and 60 M. tuberculosis culture-negative specimens (which represented 7.8% of all collected M. tuberculosis-negative specimens) were included in the study. Of 75 specimens, 26 (34.7%) grew one or more types of mycobacteria. Of the positive mycobacterial cultures, 15 had the M. tuberculosis complex, 5 had the M. avium complex, 2 had M. simiae, 2 had M. chelonae, 1 had M. fortuitum, and 1 had the M. avium complex and M. chelonae. Nine of 15 specimens which grew the M. tuberculosis complex upon culture were positive by AFB staining, giving a sensitivity of 60%. A specimen that grew the M. avium complex upon culture was also positive by AFB staining, giving a specificity of 98%. The remaining specimens, having grown non-M. tuberculosis complex mycobacterial species, were negative by AFB staining. Nucleic acid was then extracted directly from 0.2 ml of N-acetyl-l-cysteine-NaOH-decontaminated and concentrated sediments by using IsoQuick (Orca Research, Inc., Bothell, Wash.) according to the manufacturer's instructions, as previously described (8). Extracted DNA was resuspended in 25 μl of water, and 10 μl of extracts was included in a PCR master mixture for PCR amplification (8). A housekeeping gene, human β-actin, was detectable in all DNA samples, indicating that these DNA samples were free of amplification inhibitors. All extracted DNA samples were tested for the M. tuberculosis complex by PCR-EIA targeting both IS6110 and MPB64 genes. M. tuberculosis complex-specific DNA was detected in 13 of 15 specimens that were culture positive for the M. tuberculosis complex, giving a sensitivity of 87%. Only two culture-positive specimens were PCR-EIA negative; both were also negative by AFB staining. All 60 respiratory specimens that were negative by culture were also negative by PCR-EIA, giving a specificity of 100%. These data indicated that PCR-EIA provides more rapid detection of the M. tuberculosis complex in clinical specimens than does AFB staining, with sensitivity improving from 60 to 87% and specificity improving from 98 to 100%. While our study population in Hawaii still has a relatively high incidence of tuberculosis, other hospitals now face a change in tuberculosis epidemiology. Tuberculosis has become a relatively uncommon disease in the continental United States in recent years. As a result, physicians have less experience with the disease and may have difficulty recognizing it, a circumstance potentially leading to misdiagnosis and spread of the infection (3). As the incidence of tuberculosis declines, more resources will be used inappropriately for patients whose culture specimens grow mycobacteria other than M. tuberculosis. A recent study prospectively assessed the management of patients with suspected tuberculosis in an area with a high prevalence of mycobacteria other than M. tuberculosis and a low incidence of tuberculosis. The high prevalence of mycobacteria other than M. tuberculosis in specimens has a substantial impact on the management of suspected tuberculosis. Clinicians' assessments were sensitive for tuberculosis but had poor predictive value. Owing to a high rate of isolation of mycobacteria other than M. tuberculosis, AFB staining was a weak predictor of tuberculosis, with a positive predictive value of only 33% (1). Therefore, development of additional techniques to enhance the early diagnostic predictive values given by AFB staining will be of benefit in addressing this problem. Although several nucleic acid amplification-based M. tuberculosis complex detection kits are commercially available, they are relatively expensive and the procedure is time-consuming (4). PCR-EIA as described in this report is simple, rapid, and user friendly. The whole procedure, including specimen processing, DNA extraction, PCR amplification, and amplicon identification, can be completed within an 8-h shift. An additional signal amplification step included in the microtiter plate system enhances test sensitivity, making this test an alternative to the AFB smear test in the clinical setting for tuberculosis patient management.

Human cytomegalovirus (HCMV) continues to be a significant cause of morbidity and mortality in immunocompromised patients, such as solid organ or bone marrow transplant recipients (21, 36). Effective preemptive antiviral therapy that includes ganciclovir and foscarnet depends on detecting HCMV at an early stage of infection (3, 14, 21, 37), but these drugs must be used carefully because of their side effects. The early and accurate diagnosis of CMV infection status is an essential part of the management of these patients (14, 36). However, because the primary HCMV infection results in a latent life-long infection and because many transplant patients secrete HCMV without any clinical disease, the mere detection of HCMV does not always indicate the need for treatment (3, 21, 36). Quantitation of the systemic HCMV load may provide a highly sensitive and specific method to predict which patients will develop HCMV diseases. Several techniques have been used to monitor HCMV loads. The traditional methods, based on virus isolation as indicated by cytopathic effect, are time-consuming procedures, have poor reproducibility, and have only moderate sensitivity. The antigenemia assay, which detects the viral lower matrix protein pp65 (UL83) in blood leukocytes (4, 11, 13, 34), has been widely used as a tool to monitor HCMV infections. The number of pp65-positive cells has been shown to correlate with HCMV disease, although the threshold for HCMV disease may vary between patient groups (3, 23). This method, however, needs to be processed within 8 h of collection and suffers from its lack of standardization (3, 11, 23). Only virus associated with cells is detected, and free viruses in biological fluids are not detectable by this assay. The reading of the assay depends on technical skill and training and requires a moderate amount of technologist time. Furthermore, this assay cannot be carried out when a patient's leukocyte count is very low (4). The wide application of in vitro amplification techniques for the detection of viral nucleic acids has become the most important innovation in laboratory diagnosis and clinical management of posttransplant HCMV infection. Multiple studies have shown that the real-time quantitative PCR for HCMV DNA in plasma and other biological samples is useful in the rapid diagnosis of HCMV infection and in effective monitoring of clinical course as well as response to therapy (7, 10, 11, 14, 15, 26, 27, 29, 32, 37). The real-time systems measure the accumulation of PCR products with a fluorogenic probe and instantaneous laser scanning (7, 15). Unlike regular PCR procedures performed by using conventional thermocyclers, the real-time PCR system is closed and therefore avoids the important concerns of carryover contamination with the previous product. In addition, the nucleic acid products can be monitored in real time after each cycle of amplification. As a result of shorter thermocycling and simultaneous product detection, the turnaround time of the assay is significantly shorter than that of prior quantitative assays. In this study we developed such a real-time PCR assay that uses the 7700 ABI PRISM Sequence Detector (Applied Biosystems, Foster City, Calif.) (12) to quantitate HCMV loads in whole-blood specimens collected from transplant recipients. We compared this assay with an antigenemia assay that has been in use for years on which we had reliable threshold points for treatment in different patient populations. (This study was presented in part at the 102nd General Meeting of the American Society for Microbiology, Salt Lake City, Utah, 19 to 23 May 2002.)

BACKGROUND: The presence of various levels of Adenovirus serotype 5 neutralizing antibodies (Ad5NAb) is thought to contribute to the inconsistent clinical results obtained from vaccination and gene therapy studies. Currently, two platforms based on high-throughput technology are available for Ad5NAb quantification, chemiluminescence- and fluorescence-based assays. The aim of this study was to compare the results of two assays in the seroepidemiology of Ad5NAb in a local population of donors. METHODOLOGY/PRINCIPAL FINDINGS: The fluorescence-based neutralizing antibody detection test (FRNT) using recombinant Ad5-EGFP virus and the chemiluminescence-based neutralizing antibody test (CLNT) using Ad5-Fluc were developed and standardized for detecting the presence of Ad5NAb in serum samples from the population of donors in Beijing and Anhui provinces, China. First, the overall percentage of people positive for Ad5NAb performed by CLNT was higher than that obtained by FRNT (85.4 vs 69.9%, p