Rapid SARS-CoV-2 antigen detection assay in comparison with real-time RT-PCR assay for laboratory diagnosis of COVID-19 in Thailand.

BACKGROUND: The Coronavirus disease 2019 (COVID-19) pandemic continues to spread across the world. Hence, there is an urgent need for rapid, simple, and accurate tests to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Performance characteristics of the rapid SARS-CoV-2 antigen detection test should be evaluated and compared with the gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) test for diagnosis of COVID-19 cases. METHODS: The rapid SARS-CoV-2 antigen detection test, Standard™ Q COVID-19 Ag kit (SD Biosensor®, Republic of Korea), was compared with the real-time RT-PCR test, Allplex™ 2019-nCoV Assay (Seegene®, Korea) for detection of SARS-CoV-2 in respiratory specimens. Four hundred fifty-four respiratory samples (mainly nasopharyngeal and throat swabs) were obtained from COVID-19 suspected cases and contact individuals, including pre-operative patients at Siriraj Hospital, Bangkok, Thailand during March-May 2020. RESULTS: Of 454 respiratory samples, 60 (13.2%) were positive, and 394 (86.8%) were negative for SARS-CoV-2 RNA by real-time RT-PCR assay. The duration from onset to laboratory test in COVID-19 suspected cases and contact individuals ranged from 0 to 14 days with a median of 3 days. The rapid SARS-CoV-2 antigen detection test's sensitivity and specificity were 98.33% (95% CI, 91.06-99.96%) and 98.73% (95% CI, 97.06-99.59%), respectively. One false negative test result was from a sample with a high real-time RT-PCR cycle threshold (Ct), while five false positive test results were from specimens of pre-operative patients. CONCLUSIONS: The rapid assay for SARS-CoV-2 antigen detection showed comparable sensitivity and specificity with the real-time RT-PCR assay. Thus, there is a potential use of this rapid and simple SARS-CoV-2 antigen detection test as a screening assay.

Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA.

Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes coronavirus disease 2019 (COVID-19)-in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.

Rapid Sequencing of Multiple RNA Viruses in Their Native Form.

Long-read nanopore sequencing by a MinION device offers the unique possibility to directly sequence native RNA. We combined an enzymatic poly-A tailing reaction with the native RNA sequencing to (i) sequence complex population of single-stranded (ss)RNA viruses in parallel, (ii) detect genome, subgenomic mRNA/mRNA simultaneously, (iii) detect a complex transcriptomic architecture without the need for assembly, (iv) enable real-time detection. Using this protocol, positive-ssRNA, negative-ssRNA, with/without a poly(A)-tail, segmented/non-segmented genomes were mixed and sequenced in parallel. Mapping of the generated sequences on the reference genomes showed 100% length recovery with up to 97% identity. This work provides a proof of principle and the validity of this strategy, opening up a wide range of applications to study RNA viruses.

Lymphocyte subsets and natural killer cell cytotoxicity in intravenous drug users with HIV-1 infection among Thai population.

BACKGOUND: Intravenous drug users (IVDUs) are among the high-risk groups who are most vulnerable to HIV infection. Several illicit drugs alter host immune function with increased incidence of infections including that of HIV. Many studies of the immune response of NK cells in HIV-1 seronegative IVDUs and HIV-1 seropositive IVDUs have been published from the Western countries and yet no data is available from Thailand. OBJECTIVE: To determine natural killer cell cytotoxicity and lymphocyte subsets in Thai HIV-1 infected intravenous drug users. METHODS: The NK cell cytotoxic function was determined using our well-established EGFP-K562 flow cytometric assay in 30 IVDUs with HIV-1 infection (IVH) comparing with those from the same number of non-infected IVDUs (IVX), HIV-1 seropositive individuals (HIV-1+ve) and healthy controls. The percentage and the absolute number of NK cells, helper CD4+ T cells and cytotoxic CD8+ T cells were also investigated. RESULTS: Among the study groups, IVH showed not only the lowest percentage of lytic activity by NK cells, but also a decline in the percentage and absolute count of NK cells. A decline in helper CD4+ T cells and an increase of cytotoxic CD8+ T cells of IVH group when compared to those of other 3 groups were also demonstrated. CONCLUSIONS: The failure of innate immune NK cell function and their number in IVH may support the involvement of additional components of the immune system in the control of HIV-1 disease.

Genome Sequences of Zika Virus Strains Recovered from Amniotic Fluid, Placenta, and Fetal Brain of a Microcephaly Patient in Thailand, 2017.

We present here the complete genome sequences of Zika virus strains isolated from aborted fetal tissue (brain and placenta) and amniotic fluid of a microcephaly patient in Thailand in 2017. The virus genomes that were sequenced have an average length of 10,807 nucleotides.

Case of Microcephaly after Congenital Infection with Asian Lineage Zika Virus, Thailand.

We sequenced the virus genomes from 3 pregnant women in Thailand with Zika virus diagnoses. All had infections with the Asian lineage. The woman infected at gestational week 9, and not those infected at weeks 20 and 24, had a fetus with microcephaly. Asian lineage Zika viruses can cause microcephaly.

Prevalence of HIV-1 Subtypes and Antiretroviral Drug Resistance Mutations in Nepal.

BACKGROUND: There have been very few reports of HIV-1 subtypes and drug resistance mutations (DRMs) from Nepal which is geographically located between two high-prevalence HIV-1 infection countries, China and India. OBJECTIVE: The aim of this study was to determine prevalence of acquired and transmitted DRMs and HIV-1 subtypes in Nepal. METHODS: Thirty-five HIV-1 seropositive samples from central region of Nepal were collected in 2011. The subjects were divided into two groups, antiretroviral (ARV) drug naïve group (n=15) and antiretroviral treatment (ART) group (n=20), 90% (18/20) of them received zidovudine, lamivudine and nevirapine (AZT/3TC/NVP) regimen. HIV pol (protease and reverse transcriptase regions) nucleotide sequences were analyzed by Viroseq HIV-1 Genotyping System. Nearly full-length genomic (NFLG) sequences of 10 samples were performed. RESULTS: NFLG genotyping revealed that 80% of samples were infected with subtype C and 20% with recombinants (C/D/H and C/A). Phylogenetic analysis of 35 pol sequences from Nepal were subtype C. The prevalence of acquired DRMs to NNRTIs and NRTIs was 15% (3/20). DRMs to NVP, K103N and V179D, and to NRTIs were observed at 11.1% (2/18) and 5% (1/20), respectively. The prevalence of DRMs to rilpivirine for E138A/G was 5.7%. The minor protease inhibitors (PI) associated mutations (A71T/V and T74S) were observed in 5/35 (14.3%) subjects. CONCLUSION: This is the first report of NFLG HIV-1 genomic sequences and DRMs from Nepal. National surveillance of HIV DRMs to ARVs and molecular epidemiology study should be done annually for better prevention and treatment of HIV infection in Nepal.

Epidemiological, Clinical and Virological Characteristics of Influenza B Virus from Patients at the Hospital Tertiary Care Units in Bangkok during 2011-2014.

Influenza B virus, which causes acute respiratory infections, has increased in prevalence in recent years. Based on the nucleotide sequence of the hemagglutinin (HA) gene, influenza B virus can be divided into two lineages, Victoria and Yamagata, that co-circulate during the influenza season. However, analysis of the potential association between the clinical and virological characteristic and the lineage of influenza B viruses isolated in Thailand was lacking. To investigate influenza B virus genetically and determine its neuraminidase (NA) inhibitor susceptibility phenotype, a total of 6920 nasopharyngeal-wash samples were collected from patients with influenza-like illness between the years 2011 and 2014 and were screened for influenza B virus by real-time PCR. Of these samples, 3.1% (216/6920) were confirmed to contain influenza B viruses, and 110 of these influenza viruses were randomly selected for nucleotide sequence analysis of the HA and NA genes. Phylogenetic analysis of the HA sequences showed clustering into various clades: Yamagata clade 3 (11/110, 10%), Yamagata clade 2 (71/110, 64.5%), and Victoria clade 1 (28/110, 25.5%). The analysis of clinical characteristic demonstrated that the Victoria lineage was significantly associated with the duration of hospitalization, number of deceased cases, pneumonia, secondary bacterial infection and underlying disease. When combined with phylogenetic analysis of the NA sequences, four samples showed viruses with reassortant sequences between the Victoria and Yamagata lineages. Statistical analysis of the clinical outcomes and demographic data for the reassortant strains did not differ from those of the other strains in circulation. Oseltamivir-resistant influenza B viruses were not detected. Our findings indicated the co-circulation of the Victoria and Yamagata lineages over the past four cold seasons in Bangkok. We also demonstrated differences in the clinical symptoms between these lineages.

Glycans flanking the hypervariable connecting peptide between the A and B strands of the V1/V2 domain of HIV-1 gp120 confer resistance to antibodies that neutralize CRF01_AE viruses.

Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain ß-sheet structure. Although N136 has previously been implicated in the binding of broadly neutralizing monoclonal antibodies, this glycosylation site appears to inhibit the binding of neutralizing antibodies in plasma from HIV-1 infected subjects. Previous studies have reported that the length of the V1/V2 domain in transmitted founder viruses is shorter and possesses fewer glycosylation sites compared to viruses isolated from chronic infections. Our results suggest that vaccine immunogens based on recombinant envelope proteins from clade CRF01_AE viruses might be improved by inclusion of envelope proteins that lack these glycosylation sites. This strategy might improve the efficacy of the vaccines used in the partially successful RV144 HIV vaccine trial, where the two CRF01_AE immunogens (derived from the A244 and TH023 isolates) both possessed glycosylation sites at N136 and N149.

Comparison of Droplet Digital PCR and Real Time PCR Method for HBV DNA Quantification.

BACKGROUND: HBV infection causes a potential serious public health problem. The ability to detect HBV DNA concentration is an important issue that had been continuously improved. When using quantitative polymerase chain reaction (qPCR), several factors are of concern, for example, sources of material, standard curve calibration, and PCR efficiency. Digital PCR (dPCR) is an alternative PCR-based technique for absolute quantification using Poisson's statistics without requiring a standard curve. OBJECTIVE: Compare the data set of HBV DNA generated between dPCR and qPCR methods. MATERIAL AND METHODS: Fifty-four samples were quantified by Abbot's real time PCR and with 2-6 log10 HBV DNA were selected for comparison with dPCR. RESULTS: Of these 54 samples, there were two outlier samples defined as negative by dPCR, whereas 52 samples were positive by both of these assays. The difference between two assays was less than 0.25 log IU/mL in 24/52 samples (46%) of paired samples; less than 0.5 log IU/mL in 46/52 samples (88%) and less than 1 log in 50/52 samples (96%). The correlation coefficient (r) was 0.788 (p-value < 0.0001). Comparison with qPCR method, data generated by dPCR tend to be an overestimation in the sample with the low level ofHBVDNA concentration and underestimated in the sample with high viral load. The variation of DNA by dPCR measurement might be due to the pre-amplification procedure and PCR template. CONCLUSION: Measurement of HBV DNA by using dPCR, the results ofthe HBV DNA copy number tended to be deviated by over- or under-estimated when comparison to real time PCR method. In addition, a large quantity of DNA was used when compared to qPCR. However, the optimum processes of this assay have to be further investigated.

Prevalence and molecular characterization of human metapneumovirus in influenza a negative sample in Thailand.

BACKGROUND: Human metapneumovirus (hMPV) causes respiratory tract infection in influenza-like illness. The role of hMPV infections in all age groups in Thailand has not yet been investigated. Thus, the objective of this study was to determine prevalence of hMPV infection in all age groups in Thailand during 2011. METHODS: A total of 1,184 nasopharyngeal washes were collected from hospitalized patients and sent to the Department of Microbiology, Siriraj Hospital, for influenza A virus detection. Real-time polymerase chain reaction (PCR) was used to detect hMPV infection. Partially, F gene from hMPV positive samples were sequenced and used for genotyping by phylogenetic tree analysis. RESULTS: The prevalence of hMPV for all age groups was 6.3%. The highest prevalence of hMPV infection was in children aged <2 years. Of 71 hMPV-positive patients, three (4.2%) were coinfected with respiratory syncytial virus (RSV), two with rhinovirus (2.8%), one with coronavirus (1.4%), and one with RSV and adenovirus (1.4%). Phylogenetic analysis of F gene revealed that 96.8% of hMPV detected was subgenotype B1, 1.6% was sublineage A2a, and 1.6% was A2b. Genetic variation of F gene was much conserved. CONCLUSION: We demonstrated the prevalence of hMPV subgenotype B1 circulating in Thailand during 2011.

Cytokine profiles in HIV-1 subtype CRF01_AE infected individuals with different rates of diseases progression: a multiplex immunoassay.

OBJECTIVE: Cytokines play an important role in controlling the homeostasis of the immune system and contribute to the pathogenesis of HIV infection. The measurement soluble cytokines in plasma of HIV-1 infected individuals with different rates of disease progression may provide additional information to complement prognostic markers and understand disease process. The aim of the present study was to determine the cytokine profiles in plasma of Thai HIV-1 CRFO1_AE infected individuals with different rates of disease progression by using a multiplex system for simultaneous detection of 7 cytokines. MATERIAL AND METHOD: The authors used a multiplex immunoassay method to measure 7 cytokines (IL-2, IL-4, IL-6, IL-7, IL-10, IL-15 and IFN-gamma) in plasma of 23 progressors (PRs; symptomatic or AIDS within 5 years and CD4+ < 200/mm3), 23 slower progressors (SPs; asymptomatic more than 5 years and CD4+ > 350/mm3) and 23 normal healthy individuals. RESULTS: Both PRs and SPs demonstrated significantly higher levels of IL-7, IL-10 and IFN-gamma than healthy controls (p < 0.05). No significant difference in IL-6 between SPs and healthy controls but significant difference between RPs and controls were found. Furthermore, PRs showed significantly higher levels of plasma IL-6 (p = 0.001), IL-7 (p = 0.016), IL-10 (p < 0.001) and IFN-gamma (p = 0.026) than SPs. No significant difference in IL-2, IL-4 and IL-15 was found among 3 groups (PRs, SPs and healthy control). CONCLUSION: These results suggested that a Th1 to Th2 cytokine switch did not occur. However, the measurements of plasma levels of cytokines could be used for predicting disease progression.

Comprehensive investigation of common antibody-dependent cell-mediated cytotoxicity antibody epitopes of HIV-1 CRF01_AE gp120.

The antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism involves both innate and adaptive immune systems. While a number of epitope mapping studies of neutralizing (Nt) antibodies and cytotoxic T lymphocyte (CTL) against a variety of HIV-1 clades have been reported, there has been a paucity of similar studies aimed at identifying epitopes of ADCC-inducing antibodies. Herein we screened 35 sera from HIV-1 CRF01_AE-infected blood donors for ADCC antibody activity against gp120 utilizing an EGFP-CEM-NK(r) flow cytometric assay. Eighteen sera with high ADCC antibody activity were then comprehensively examined for ADCC antibody epitopes using the HIV-1 subtype CRF01_AE TH023 gp120 peptide set consisting of 126 peptides of 15 amino acids in length, overlapping by 11 amino acids. This peptide set was divided into five pools (E1-E5). Each positive peptide pool was further investigated for fine epitope mapping of ADCC antibody activity using a 5 by 5 peptide matrix format. Interestingly, six and three peptides from peptide pools E1 and E2, respectively, responded to at least 33.33% of the tested sera. These nine common ADCC epitopes were localized to the C1 and V2 region of gp120. Furthermore, 5/9 epitopes were also shown to serve as full or partial Nt antibody targets for HIV-1 subtypes B and CRF01_AE. We submit these data on epitope mapping of ADCC or dual ADCC-Nt antibodies against HIV-1 gp120 that should be considered in the formulation of vaccines against HIV-1.

Boosted p24 antigen assay for early diagnosis of perinatal HIV infection.

OBJECTIVE: The authors evaluated the accuracy of in-house boosted-p24 antigen assay for diagnosis of perinatal HIV infection. MATERIAL AND METHOD: The author has retrospectively reviewed the medical records of infants born to HIV-positive mothers. The infants were tested for boosted-p24 antigen assay at the age of 1-2 months and 4-6 months. HIV infection was defined as positive anti-HIV at the age 18 months or older, or had positive HIV-PCR with clinical signs and symptoms compatible with HIV/AIDS. RESULTS: There were 168 infants included in this review and six were HIV-infected. The boosted-p24 antigen assay had the sensitivity, specificity, positive predictive value, and negative predictive value of 33.33%, 98.27%, 50%, and 95.8%, respectively at 1-2 month-old, and 100%, 98.27%, 71.43%, and 100%, respectively at 4-6 month-old. CONCLUSION: Boosted-p24 antigen assay could be a cheaper alternative test to help diagnosis of perinatal HIV infection in infants. The test was very accurate when performed at 4-6 months.

HIV-1 subtype B Tat gene activities and disease progression in HIV-1 CRF01_AE infection.

HIV-1 tat gene function and immunogenicity of HIV-1 Tat protein from 3 low (PS01, PS40, PS58) and 3 high (PS19, PS65, LP22) viral load infected, untreated and asymptomatic individuals from Thailand were compared. Levels of Tat-dependent chloramphenicol acetyltransferase (CAT) induced in HL3T1 cells with tat1 gene from HIV-1 isolates of high viral load group was significantly higher than those from low viral load group. HIV-1 subtype determination using env (C2-V4) gene demonstrated that 2/3 (PS01 and PS40) and 1/3 (PS58) from low viral load group were CRF01_AE and subtype B, while all 3 HIV-1 isolates from high viral load group were CRF01_AE. However, all 3 HIV-1 tat nucleotide sequences from low viral load group, which contained env CRF01_AE sequence, belonged to subtype B whereas all those from high viral load group contained CRF01_AE sequence. HIV Tat recombinant proteins from these groups were tested for immunogenicity in mice. All recombinant Tat proteins (except from PS58) were immunogenic in a dose-dependent manner, but with significantly differences of the immunogenicity levels between high and low viral load groups. These results indicated that HIV-1 subtype B tat gene activities might be associated with reduced disease progression of HIV-1 CRF01_AE infected individuals.

Structure and function of HIV-1 CRF01_AE envelope proteins from blood and genital fluid isolates.

The recombinant envelope protein (gp120) of the human immunodeficiency virus type 1 (HIV-1) CRF01_AE env gene isolated from the corresponding blood (rgp120-F36PC) and genital fluid (rgp120-F36VC) specimens obtained from HIV infected individuals was successfully produced in both prokaryote and eukaryote cells. The yields of HIV-1 recombinant envelope proteins rgp120-F36PC and rgp120-F36VC produced in E. coli and in mammalian cells were 1.0 and 1.2, and 0.3 and 0.5 mg/ml, respectively. Antibody responses in mice immunized with rgp120-F36VC protein were not significantly higher than those with rgp120-F36PC protein. The level of antibody response in mice immunized with V3 deleted recombinant gp120 proteins from rgp120-F36VC and rgp120-F36PC was not significantly different from wild type rgp120 proteins. beta-strands at the tip of the V3 loop of the HIV-1 envelope protein were predicted for the wild type genital fluid isolate but not for the wild type blood isolate. The replication capacity of both F36PC and F36VC was quite efficient. The infectivity assay of the epithelial cell line for pNL4-3/gp120F36VC was better than for pNL4-3/gp120F36PC. The extra beta-strands in the V3 loop may be involved in cell tropism.

HIV-1 neutralization by monoclonal antibody against conserved region 2 and patterns of epitope exposure on the surface of native viruses.

BACKGROUND: Conserved neutralizing epitopes are considered to be a key role for eliciting broadly neutralizing antibody (NAb). Previously, two conserved neutralizing epitopes of HIV-1 CRF01_AE envelope were identified at amino acid 93-112 of the C1 (C1E) and at 218-239 of the C2 (C2E) regions. To access the potency of antibody directed against conserved epitopes, a monoclonal antibody (MAb) specific to the C2E region was developed and characterized. METHODS: The immunogenicity of two epitopes was examined by immunizing BALB/c mice with the matching synthetic peptides. One MAb, C2EB5, directed against peptide C2E, was generated by conventional methods, while C1E1 and C1E2 peptides induced slight antibody response in mice. The neutralizing activity of MAb C2EB5 was examined using a peripheral blood mononuclear cell (PBMC) based method and various HIV-1 subtypes including A, B, C, D, and CRF01_AE; C2EB5 was compared with other known neutralizing MAbs (4E10, 447-52D) and with sCD4. The exposure of the C2 epitope on native virus was investigated using virus capture by these MAbs. RESULTS: The MAb C2EB5 demonstrated cross-neutralization against various HIV-1 subtypes. The overall potency of MAb C2EB5 against 5 subtypes was ranked in the following order: subtype C> CRF01_AE> subtype D> subtype A> subtype B. The epitope exposure for MAb C2EB5 was also correlated with the neutralization properties of each subtype. CONCLUSION: This study demonstrates the cross-clade neutralizing activity of a MAb directed against an epitope located in the C2 region of the HIV-1 env and highlights differences in the exposure of antigenic epitopes on the surface of various HIV-1 subtypes. The epitope for this newly identified neutralizing MAb made against a subtype CRF01_AE peptide is particularly exposed in subtype C viral isolates.

Blood and seminal plasma HIV-1 RNA levels among HIV-1-infected injecting drug users participating in the AIDSVAX B/E efficacy trial in Bangkok, Thailand.

BACKGROUND: We investigated effects of vaccination with AIDSVAX B/E HIV-1 candidate vaccine on blood and seminal plasma HIV-1 RNA viral loads (BVL and SVL, respectively) in vaccine recipients (VRs) and placebo recipients (PRs) who acquired infection. METHODS: Linear mixed models were fitted for repeated measurements of BVL. Generalized estimating equations were used to assess the difference in SVL detectability between VRs and PRs. RESULTS: A total of 196 participants became HIV-1 infected during the trial. Thirty-two (16%) became infected with HIV-1 subtype B and 164 (84%) with HIV-1 subtype CRF01_AE. Per protocol-specified analysis, there were no differences in BVL levels between VRs and PRs. When stratified by HIV-1-infecting subtype, vaccination with AIDSVAX B/E was initially associated with higher BVL among HIV-1 CRF01_AE-infected VRs compared with HIV-1 CRF01_AE-infected PRs; however, this difference did not persist over time. HIV-1 subtype B-infected VRs had slightly higher BVL levels and were more likely to have detectable SVL during the follow-up period than HIV-1 subtype B-infected PRs. CONCLUSIONS: Subtle differences in BVL and SVL were detected between VRs and PRs. These results may help to further understand the dynamics between HIV-1 vaccination, HIV-1-infecting subtypes, and subsequent viral expression in different body compartments.

International network for comparison of HIV neutralization assays: the NeutNet report.

BACKGROUND: Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays. METHODS: Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression. FINDINGS: PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent. CONCLUSIONS: The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing activities. Since it is not known which in vitro assay correlates with in vivo protection, a range of neutralization assays is recommended for vaccine evaluation.

Multi-probe real-time PCR identification of common Mycobacterium species in blood culture broth.

Mycobacterium tuberculosis complex, M. avium, and M. intracellulare are the most common causes of systemic bacterial infection in AIDS patients. To identify these mycobacterial isolates in primary blood culture broths, we developed a multiple hybridization probe-based real-time PCR assay using the LightCycler system. The primers were designed to amplify a 320-bp fragment of Mycobacterium 16S rRNA genes. Reaction specificity was evaluated using PCR amplification curves along with specific melting temperatures of probes on DNA extracted from 13 Mycobacterium species. In this study, results showed 100% accuracy for the selected bacterial panel. Detection limits were 350, 600, and 650 colony-forming unit (CFU)/ml blood culture broths for M. tuberculosis complex, M. avium, and M. intracellulare, respectively (1 to 2 CFU/reaction). To evaluate clinical applicability, 341 acid-fast bacilli in blood culture broths were analyzed. In total, 327 (96%) were positively identified: 54.5% M. tuberculosis complex, 37.5% M. avium, and 3.8% M. intracellulare. Results can be available within 3 hours of receiving a broth sample, which makes this rapid and simple assay an attractive diagnostic tool for clinical use.