Rapid Molecular Phenotypic Antimicrobial Susceptibility Test for Neisseria gonorrhoeae Based on Propidium Monoazide Viability PCR.

Neisseria gonorrhoeae (NG) is an urgent threat to antimicrobial resistance (AMR) worldwide. NG has acquired rapid resistance to all previously recommended treatments, leaving ceftriaxone monotherapy as the first and last line of therapy for uncomplicated NG. The ability to rapidly determine susceptibility, which is currently nonexistent for NG, has been proposed as a strategy to preserve ceftriaxone by using alternative treatments. Herein, we used a DNA-intercalating dye in combination with NG-specific primers/probes to generate qPCR cycle threshold (Ct) values at different concentrations of 2 NG-relevant antimicrobials. Our proof-of-concept dual-antimicrobial logistic regression model based on the differential Ct measurements achieved an AUC of 0.93 with a categorical agreement for the susceptibility of 84.6%. When surveying the performance against each antimicrobial separately, the model predicted 90 and 75% susceptible and resistant strains, respectively, to ceftriaxone and 66.7 and 83.3% susceptible and resistant strains, respectively, to ciprofloxacin. We further validated the model against the individual replicates and determined the accuracy of the model in classifying susceptibility agnostic of the inoculum size. We demonstrated a novel PCR-based approach to determine phenotypic ciprofloxacin and ceftriaxone susceptibility information for NG with reasonable accuracy within 30 min, a significant improvement compared to the conventional method which could take multiple days.

Value of rifampicin-resistant real-time fluorescence quantitative nucleic acid amplification detection technology in the fast diagnosis of pulmonary tuberculosis / 中国热带医学

@#Abstract: Objective　To evaluate the value and significance of rifampicin-resistant real-time fluorescence quantitative nucleic acid amplification detection technology (GeneXpert MTB/RIF) in the diagnosis of pulmonary tuberculosis. 　　Methods　The clinical data of 228 patients with suspected pulmonary tuberculosis, who admitted to Hebei Chest Hospital from January 2018 to December 2019, were analyzed retrospectively. The sputum was collected for GeneXpert MTB/RIF, sandwich cup liquid-based bacterial acid-fast staining smear microscopy (referred to as “sandwich cup method”) and Loop-Mediated isothermal amplification (referred to as “LAMP method”) and the results were statistically analyzed by SPSS 17.0 software. Results　Among the 228 patients with suspected cases, 200 cases were clinically diagnosed as pulmonary tuberculosis and 28 were non-tuberculosis. The positive detection rate of GeneXpert MTB/RIF (81.0%, 162/200) was significantly higher than that of sandwich cup method (62.5%, 125/200) and LAMP method (72.5%,145/200) (χ2=16.885, 4.049, P<0.05). Taking clinical diagnosis as gold standard, the sensitivity of GeneXpert MTB/RIF (80.00%,160/200) was significantly higher than that of sandwich cup method (60.00%, 120/200) and LAMP method (70.50%, 141/200) (χ2=19.048, 4.846, P<0.05). The diagnostic consistency of GeneXpert MTB/RIF (K=0.73) was higher than that of sandwich cup method (K=0.39) and LAMP method (K=0.56). Conclusions The GeneXpert MTB/RIF detection method is rapid and simple, and can diagnose pulmonary tuberculosis rapidly and simultaneously detect rifampicin resistance of Mycobacterium tuberculosis with high sensitivity. It has high clinical value for early diagnosis of pulmonary tuberculosis and guidance of treatment in general and specialized hospitals.

Evaluation of CLIA plus NAT for detecting HBsAg single-ELISA-reactive samples of blood donors / 中国输血杂志

【Objective】 To study the detection performance of HBsAg single-ELISA-reactive samples of blood donors. 【Methods】 Two kinds of ELISA reagents from different manufacturers (named as reagents A and B) were used for HBsAg screening. A total of 276 samples, from January 2017 to May 2021, with HBsAg single-ELISA-reactive results were collected for further nucleic acid detection technology (NAT) and chemiluminescence immunoassay (CLIA) testing, to undergo HBV-DNA and five hepatitis B tests, respectively. The relationship between HBsAg single-ELISA-reactivity, NAT and CLIA was statistically analyzed. 【Results】 Among the 276 HBsAg single-ELISA-reactive samples, 14 were NAT reactive, with the positive rate of 5.07% (14/276). Fisher′s exact test was used to compare the compliance of reagents A and B with NAT reactivity, and the difference was not statistically significant (P<0.05). Among 14 HBsAg+ /NAT+ samples retested by CLIA, 2 were HBsAg reactive(14.29%, 2/14), 13 were anti-HBc reactive (92.86%, 13/14), 9 had the quantitative value of anti-HBs <10 mIU/mL, 5 had the quantitative value of anti-HBs between 10 to 100mIU/ mL. A total of 5 serological patterns were detected, and anti-HBe+ /anti-HBc+ pattern was the dominant. There were 262 cases of HBsAg+ /NAT- samples, but only 1 (0.38%, , 1/262) case was HBsAg reactive by CLIA, 100 were anti-HBc reactive (38.17%, 100/262), 144 (54.96%, 144/262) were anti-HBs reactive, and 1 was HBeAg reactive. A total of 8 serological patterns were detected. 【Conclusion】 Most of HBsAg single-ELISA-reactive results are false, and NAT could effectively reduce the residual risk of transfusion transmitted diseases.

Nucleic acid amplification techniques for the detection of Schistosoma mansoni infection in humans and the intermediate snail host: a structured review and meta-analysis of diagnostic accuracy.

BACKGROUND: Schistosomiasis is a parasitic disease caused by hematodes of genus Schistosoma. This review evaluated the available nucleic acid amplification techniques for diagnosing S. mansoni infections in humans, intermediate host snails, and presumed rodent reservoirs. METHODS: Sensitivity, specificity, diagnostic odds ratio (DOR), and 95% CI were calculated based on available literature. The potential of PCR, nPCR, PCR-ELISA, qPCR, and LAMP was compared for diagnosing S. mansoni infections. RESULTS: A total of 546 published records were identified. Quality assessment by QUADAS-2 revealed an uncertain risk in most studies, and 21 references were included in the final. For human samples, the four nucleic acid amplification techniques showed an overall sensitivity of 89.79% (95% CI: 83.92%-93.67%), specificity of 87.70% (95% CI: 72.60%-95.05%), and DOR of 37.73 (95% CI: 21.79-65.33). LAMP showed the highest sensitivity, followed by PCR-ELISA, PCR, and qPCR, while this order was almost reversed for specificity; qPCR had the highest AUC. For rodent samples, qPCR showed modest sensitivity (68.75%, 95% CI: 43.32%-86.36%) and high specificity (92.45%, 95% CI: 19.94%-99.83%). For snail samples, PCR and nPCR assays showed high sensitivity of 90.06% (95% CI: 84.39%-93.82%) and specificity of 85.51% (95% CI: 54.39%-96.69%). CONCLUSION: Nucleic acid amplification techniques had high diagnostic potential for identifying S. mansoni infections in humans.

Strategy and validation of a consistent and reproducible nucleic acid technique for mycoplasma detection in advanced therapy medicinal products.

Advanced therapy medicinal products (ATMP) are required to maintain their quality and safety throughout the production cycle, and they must be free of microbial contaminations. Among them, mycoplasma contaminations are difficult to detect and undesirable in ATMP, especially for immunosuppressed patients. Mycoplasma detection tests suggested by European Pharmacopoeia are the "culture method" and "indicator cell culture method" which, despite their effectiveness, are time consuming and laborious. Alternative methods are accepted, provided they are adequate and their results are comparable with those of the standard methods. To validate a novel in-house method, we performed and optimized, a real time PCR protocol, using a commercial kit and an automatic extraction system, in which we tested different volumes of matrix, maximizing the detection sensitivity. The results were compared with those obtained with the gold standard methods. From a volume of 10 ml, we were able to recognize all the mycoplasmas specified by the European Pharmacopoeia, defined as genomic copies per colony forming unit ratio (GC/CFU). Our strategy allows to achieve faster and reproducible results when compared with conventional methods and meets the sensitivity and robustness criteria required for an alternative approach to mycoplasmas detection for in-process and product-release testing of ATMP.

Laboratory Diagnostic Tools for Syphilis: Current Status and Future Prospects.

With the increasing number of patients infected with syphilis in the past 20 years, early diagnosis and early treatment are essential to decline syphilis prevalence. Owing to its diverse manifestations, which may occur in other infections, the disease often makes clinicians confused. Therefore, a sensitive method for detecting T. pallidum is fundamental for the prompt diagnosis of syphilis. Morphological observation, immunohistochemical assay, rabbit infectivity test, serologic tests, and nucleic acid amplification assays have been applied to the diagnosis of syphilis. Morphological observation, including dark-field microscopy, silver-staining, and direct fluorescent antibody staining for T. pallidum, can be used as a direct detection method for chancre specimens in primary syphilis. Immunohistochemistry is a highly sensitive and specific assay, especially in the lesion biopsies from secondary syphilis. Rabbit infectivity test is considered as a sensitive and reliable method for detecting T. pallidum in clinical samples and used as a historical standard for the diagnosis of syphilis. Serologic tests for syphilis are widely adopted using non-treponemal or treponemal tests by either the traditional or reverse algorithm and remain the gold standard in the diagnosis of syphilis patients. In addition, nucleic acid amplification assay is capable of detecting T. pallidum DNA in the samples from patients with syphilis. Notably, PCR is probably a promising method but remains to be further improved. All of the methods mentioned above play important roles in various stages of syphilis. This review aims to provide a summary of the performance characteristics of detection methods for syphilis.

PCR primers designed for new world Leishmania: A systematic review.

Studies of the primers that were designed to detect New World Leishmania were systematically reviewed to report the characteristics of each target, detection limit, specificity of the primers designed and diagnostic sensibility. The papers identified in the databases PubMed and Web of Science involved 50 studies. Minicircle is the most applied target in molecular research for diagnosis, due to its high sensitivity in detecting Leishmania in different clinical samples, a characteristic that can be partially attributed to the higher number of copies of the minicircle per cell. The other molecular targets shown in this review were less sensitive to diagnostic use because of the lower number of copies of the target gene per cell, but more specific for identification of the subgenus and/or species. The choice of the best target is an important step towards the result of the research. The target allows the design of primers that are specific to the genus, subgenus or a particular species and also imparts sensitivity to the method for diagnosis. The findings of this systematic review provide the advantages and disadvantages of the main molecular targets and primers designed for New World Leishmania, offering information so that the researcher can choose the PCR system best suited to their research need. This is a timely and extremely thorough review of the primers designed for New World Leishmania.

Effectiveness of blood donor screening by HIV, HCV, HBV-NAT assays, as well as HBsAg and anti-HBc immunoassays in Germany (2008-2015).

BACKGROUND AND OBJECTIVES: In Germany, in addition to standard blood donor screening, further mandatory tests were introduced for HCV-RNA, HIV-1-RNA and for anti-HBc. Screening for HBV-DNA is optional. This study investigates the benefits of these additional tests for the detection of HIV, HCV, and HBV infections among German blood donors. MATERIALS AND METHODS: From 2008 to 2015 we collected data on blood donations exclusively testing NAT positive (NAT yield) or reactive in only one of the screening assays. Assuming a Poisson distribution, we calculated NAT yield/reactive only rates on a per donation basis (number of yield/reactive only cases divided by the number of donations tested in the period under review) with 95% confidence intervals. RESULTS: Responding establishments covered 95% of the donations. We identified 20 HIV-1-NAT, 61 HCV-NAT and 29 HBV-NAT yield cases among approximately 46 million blood donations tested corresponding to 0·43 HIV-1 NAT, 1·32 HCV-NAT, and 0·64 HBV-NAT yield cases per million blood donations tested. For one HBsAg reactive only case and 23 anti-HBc reactive only cases in repeat donors, infection was confirmed by ID-NAT which translates into 0·02 and 0·55 cases per million donations tested. During the 8-year-observation period, one HIV-1, no HCV and four HBV transmissions associated with donations in the viremic pre-seroconversion window period were reported. CONCLUSION: Annually, NAT screening alone detected 2·5 HIV-1, 7·6 HCV, and 3·6 HBV infectious donations; anti-HBc screening alone identified 2·9 infectious donations of repeat donors with occult HBV infection. Overall, the survey results support that the currently practiced donor HIV/HCV/HBV screening strategy in Germany does ensure a high standard of blood safety.

Application of ELISA combined with nucleic acid testing for blood screening and residual risk analysis in blood donors / 中华实验和临床病毒学杂志

Objective@#To analyze the residual risk of transfusion transmitted hepatitis B virus (HBV) infection by enzyme-linked immunosorbent assay (ELISA) method in hepatitis B surface antigen (HBsAg) negative blood donors, and to assess the infection status.@*Methods@#A total of 45551 samples were collected from blood donors.All samples were tested by 2 different ELISA kids of HBsAg and nucleic acid testing (NAT) individually of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV). Those ELISA HBsAg negative and NAT single reactive (HBsAg-/HBV DNA+ ) specimens were analyzed by quantitative detection of HBV DNA and by serologic testing of HBV antigen and antibody.@*Results@#A total of 44 HBsAg-/HBV DNA+ samples were detected, including 42 occult HBV infections (OBI) and 2 window period infections (WP). The detection rate of OBI rate was 0.90‰, and 32 samples of OBI sample HBV DNA was less than 20 IU/ml, and the OBI detection rate was significantly different between different genders, ages and blood donation times (P<0.05). In the OBI sample, there were 6 serological models, 92.9%(39/42) OBI samples hepatitis B core antibody (HBcAb) positive, and 76.9%(30/39) HBV DNA in HBcAb positive samples were less than 20 IU/ml; 29.5% (13/42) of OBI blood donors hepatitis B e antigen (HBeAb) and HBcAb were also positive, of whom 84.6% (11/13) were HBV DNA quantitatively <20 IU/ml.@*Conclusions@#HBV residual risk of transfusion-transmitted infection may occur through HBsAg- and single NAT reactive blood donors, mainly include OBI, and HBV DNA low level. Blocking of single NAT reactive blood donors could reduce transfusion-transmitted HBV infection.

Strategy for an abbreviated in-house qualification of a commercially available Rapid Microbiology Method (RMM) for canadian regulatory approval.

BACKGROUND AIMS: Cell therapy products (CTP) typically require full sterility, endotoxin and Mycoplasma testing before product release. Often this is not feasible with fresh cells, and sponsors may rely on rapid microbiological methods (RMM). RMM must be qualified in-house using the sponsor's facilities, equipment, consumables, cells and matrices to meet regulatory approval. Herein, we present a cost-effective strategy to conduct an in-house abbreviated qualification of a commercially available RMM kit to meet Health Canada regulatory requirements. METHODS: We performed an abbreviated qualification using a polymerase chain reaction (PCR)-based Mycoplasma testing method involving assay sensitivity and ruggedness, based on an experimental plan that was pre-approved by Health Canada. Briefly, investigational CTPs were tested in-house using a PCR-based Mycoplasma detection kit. Assay sensitivity was determined using a 10-fold dilution series of genomic DNA of only two Mycoplasma species, Mycoplasma arginini and Mycoplasma hominis in the absence of CTP-matrix as the kit had been previously validated against nine species. Matrix interference was measured by testing independent CTP samples. Testing by different operators on different days measured ruggedness. RESULTS: The RMM Mycoplasma qualification exceeded sensitivity (4 genome copies per reaction for M. arginini and 0.12 genome copies per reaction for M. hominis) and met ruggedness requirements without matrix interference, as required by the Pharmacopoeial guidelines (Ph. Eur. 2.6.7 and USP <1223>). DISCUSSION: Our approach represents a minimal qualification that can be performed by an academic institution while ensuring regulatory compliance for implementing RMM testing for in-process and product-release testing of CTPs.

Fast and simple detection methods for the 4-base pair deletion of canine MDR1/ ABCB1 gene by PCR and isothermal amplification.

Dogs with a 4-bp deletion in the MDR1 (or ABCB1) gene show intolerance to certain drugs routinely used in veterinary medicine, such as ivermectin, vincristine, and doxorubicin. The mutation leads to a dysfunctional P-glycoprotein drug transporter, which results in drug accumulation in the brain and severe neurotoxicity. A rapid and accurate in-house test to determine the genotype of patients in cases of acute neurotoxic signs or in tumor patients is desirable. We describe a cost-effective detection method with simple technical equipment for veterinary practice. Two allele-specific methods are presented, which allow discrimination of all genotypes, require little hands-on time, and show the results within ~1 h after DNA sampling. DNA from buccal swabs of 115 dogs with known genotype (no mutation, n = 54; heterozygous for the mutation, n = 37; homozygous for the mutation, n = 24) was extracted either by using a column-based extraction kit or by heating swabs in a simple NaOH-Tris buffer. Amplification was performed either by allele-specific fast polymerase chain reaction or by allele-specific loop-mediated isothermal amplification (LAMP). Analysis was done either on agarose gels, by simple endpoint visualization using ultraviolet light, or by measuring the increase of fluorescence and time to threshold crossing. Commercial master mixes reduced the preparation time and minimized sources of error in both methods. Both methods allowed the discrimination of all 3 genotypes, and the results of the new methods matched the results of the previous genotyping. The presented methods could be used for fast individual MDR1/ ABCB1 genotyping with less equipment than existing methods.

Serological and virological epidemiology characterization of occult hepatitis B virus infection in Jiaxing volunteer blood donors / 中国输血杂志

Objective To research and analyze serological and virological epidemiology charactererization of occult hepatitis B virus infection in Jiaxing volunteer blood donors.Methods 52 698 samples were screened by ELISA(HBsAg、antiHCV 、anti-HIV、anti-TP) and Nucleic acid amplification technique(NAT),then NAT positive samples were further identified to detect virus type.HBsAg-/HBV-DNA+ samples were collected in three different kinds of qualitative HBsAg detection of ELISA kit.The quantitative determination of HBsAg and anti-HBs were used by chemiluminescencemethod.At the same time,real-time fluorescence quantitative PCR (QPCR) was used to measure the viral load of HBV.Further analysis and study on the serological and virological distribution of OBI combined with five markers of hepatitis B virus (HBV),with tracing general epidemiological data (sex,age and age).Results The prevalence rate of OBI was 0.89‰ (1 ∶ 1 121) in all donors with OBI infection,and 2 cases of window period (WP) were found in 52698 donors (1 ∶ 26 349).The results of HBsAg and HBeAg were negative in 49 HBsAg-/HBV-DNA+ samples,and 6OBI serological profiles were found.Anti-HBs quantitative concentration(＞100 mIU/mL)accounted for 27.66％ (13/47),while anti-HBc+ positive rate was 91.49％ (43/47).HBV-DNA nucleic acid quantitative ranged from 4.10 to 1.82× 103(IU/mL) (median of 15.83),whereas HBsAg+/HBV-DNA+positive viral load was in the range of 61.47 to 1.28× 104(IU/mL) (median of 538.15).The difference was significant in viral load between experiment group and control group(P＜0.05).Male donors of more than 40 years were higher in prevalence rate of OBI infection (P＜0.05),meanwhile there was a significant difference in OBI infection rate between repeated blood donors and fnrst blood donors(0.01＜P＜0.05).Conclusion The viral load was low in OBI infected donors,and anti-HBc+ was the main manifestation.NAT had the ability to detect OBI,shorten the window period,and contributed to ensure the safety of clinical blood.

Risk Minimization Measures for Blood Screening HIV-1 Nucleic Acid Amplification Technique Assays in Germany.

BACKGROUND: Several publications describe HIV-1 RNA false-negative results or viral load underquantitation associated with Communauté Européenne(CE)-marked qualitative or quantitative nucleic acid amplification technique (NAT) assays. 6 cases occurred during blood screening in Germany, with 2 of them causing HIV-1 transmissions to recipients of blood components. The implicated NAT assays were mono-target assays amplifying in different viral genome regions (gag or long terminal repeat). METHODS: Specimens characterized by HIV-1 NAT underquantitation or false-negative NAT results were comparatively investigated in CE-marked HIV-1 NAT systems of different design to identify potential reasons. The target regions of the viral nucleic acids were sequenced and these sequences compared to primers and probes of the assays. Potential risk minimization measures were considered for quantitative and blood-screening HIV-1 NAT systems. RESULTS: Nucleotide sequencing of the viral target region in cases of HIV-1 RNA underquantitation or false-negative test results revealed new HIV-1 variants that were mismatched with primers and probes used in some mono-target assays. So far, dualtarget NAT assays have not been associated with mismatch-based false-negative test results. From 2015, the Paul Ehrlich Institute will request HIV-1 NAT assays of dual-target design or an analogous solution for further reducing the risk in blood screening. CONCLUSION: HIV differs from other blood-borne viruses with regard to its fast evolution of new viral variants. The evolution of new sequences is hardly predictable; therefore, NAT assays with only 1 target region appear to be more vulnerable to sequence variations than dual-target assays. The associated risk may be higher for HIV-1 NAT assays used for blood screening compared to quantitative assays used for monitoring HIV-1-infected patients. In HIV-1 screening NAT assays of dual-target design may adequately address the risk imposed by new HIV-1 variants.

HBV transmission from an occult carrier with five mutations in the major hydrophilic region of HBsAg to an immunosuppressed plasma recipient.

We describe the case of transmission of an HBsAg-negative hepatitis B infection to an immunosuppressed patient by plasma donation from an HBsAg-negative subject, but with very low serum HBV DNA (about 50 IU/ml) and five mutations in the major hydrophilic region of HBsAg.

World Health Organization International Standard to harmonize assays for detection of hepatitis E virus RNA.

Nucleic acid amplification technique-based assays are a primary method for the detection of acute hepatitis E virus (HEV) infection, but assay sensitivity can vary widely. To improve interlaboratory results for the detection and quantification of HEV RNA, a candidate World Health Organization (WHO) International Standard (IS) strain was evaluated in a collaborative study involving 23 laboratories from 10 countries. The IS, code number 6329/10, was formulated by using a genotype 3a HEV strain from a blood donation, diluted in pooled human plasma and lyophilized. A Japanese national standard, representing a genotype 3b HEV strain, was prepared and evaluated in parallel. The potencies of the standards were determined by qualitative and quantitative assays. Assay variability was substantially reduced when HEV RNA concentrations were expressed relative to the IS. Thus, WHO has established 6329/10 as the IS for HEV RNA, with a unitage of 250,000 International Units per milliliter.

The capacity of MTD method for distinguishing Mycobacterium tuberculosis and nontuberculosis mycobacteria / 中华检验医学杂志

Objective To evaluate the capacity of MTD method to distinguish between Myeobacterium tuberculosis and nontuberculosis mycobacteria.Methods Ten standard strains(including 1 H_(37)Rv strain and 9 nontuberculosis mycobacteria strains),94 clinical strains(including 48 Mycobacterium tuberculosis and 46 nontuberculosis mycobaeteria strains)and 40 sputum specimens were tested by MTD method(AMPLIFIED-MTI))and traditional methods.The results of these methods were compared.Results For all Myeobacterium tuberculosis and nontuberculosis mycobacteria strains,the agreement of MTD method and traditional method was 100%.And the positive detectable rate for sputum samples was 65% that was obviously higher than that for the direct smear(5/40),concentration smear(10/ 40)or culture(5/40).Conclusion MTD is a rapid test for identification of Mycobacterium tuberculosis and nontuberculosis mycobaeteria with high sensitivity and specificity.

Establishment of relative quantification in real time reverse transcription polymerase chain reaction to measure cytokine expression / 中华检验医学杂志

Objective To establish relative quantification in real time reverse transcription polymerase chain reaction to measure cytokine expression.Methods TNF? and GAPDH were used as target gene and internal reference respectively. Two gene fragment were cloned, vectors were purified and quantificated. Standard curves were established using a series dilution of quantificated plasmids to measure the amplification efficiency of TNF? and GAPDH. By changing reaction conditions, the amplification efficiency of two gene were nearly 100%. Ct value of TNF? and GAPDH were measured in 14 samples stimuteneously.Results The method can detected as low as 10 3 copies with the linear range was 10 3～10 9 copies, the intra assay and interassay variation was 1% and 8% respectively. TNF? increased 8 6～9 8 fold with the average 9 2 relative to untreated control group analyzed by the 2 -??Ct methods. Conclusions The method we established has fine sensitivity and reproducibility and the data analysis was simple and reliable and can be apply to any genes.