Development and preliminary assessment of a CRISPR-Cas12a-based multiplex detection of Mycobacterium tuberculosis complex.

Since the onset of the COVID-19 pandemic in 2020, global efforts towards tuberculosis (TB) control have encountered unprecedented challenges. There is an urgent demand for efficient and cost-effective diagnostic technologies for TB. Recent advancements in CRISPR-Cas technologies have improved our capacity to detect pathogens. The present study established a CRISPR-Cas12a-based multiplex detection (designated as MCMD) that simultaneously targets two conserved insertion sequences (IS6110 and IS1081) to detect Mycobacterium tuberculosis complex (MTBC). The MCMD integrated a graphene oxide-assisted multiplex recombinase polymerase amplification (RPA) assay with a Cas12a-based trans-cleavage assay identified with fluorescent or lateral flow biosensor (LFB). The process can be performed at a constant temperature of around 37°C and completed within 1 h. The limit of detection (LoD) was 4 copies µL-1, and no cross-reaction was observed with non-MTBC bacteria strains. This MCMD showed 74.8% sensitivity and 100% specificity in clinical samples from 107 patients with pulmonary TB and 40 non-TB patients compared to Xpert MTB/RIF assay (63.6%, 100%). In this study, we have developed a straightforward, rapid, highly sensitive, specific, and cost-effective assay for the multiplex detection of MTBC. Our assay showed superior diagnostic performance when compared to the widely used Xpert assay. The novel approach employed in this study makes a substantial contribution to the detection of strains with low or no copies of IS6110 and facilitates point-of-care (POC) testing for MTBC in resource-limited countries.

Respiratory microbiota imbalance in children with Mycoplasma pneumoniae pneumonia.

Although previous studies have reported the dysregulation of respiratory tract microbiota in infectious diseases, insufficient data exist regarding respiratory microbiota imbalances in the lower respiratory tracts (LRTs) of children with Mycoplasma pneumoniae pneumonia (MPP). Here, we analysed the microbial community using 16S rRNA gene sequencing. Finally, bronchoalveolar lavage fluid (BALF) samples from 158 children with MPP and 29 with bacterial or viral pneumonia (control group) were collected. The diversity of the microbial community was significantly different between the two groups. A significantly increased abundance of Tenericutes and Mycoplasma was detected in the MPP group, exceeding 67% and 65% of the total bacterial population, respectively. Using Mycoplasma abundance as the diagnostic method, the sensitivity and specificity of the model was 97.5% and 96.6%, respectively. Compared to the mild MPP group, lower alpha diversity and significantly increased Mycoplasma abundance were found in the severe MPP group (P < 0.01). The abundance of Mycoplasma was positively correlated with complications and clinical indices in children with severe MPP compared with children with mild MPP. Our study describes the features of the LRT microbiota of children with MPP and uncovered its association with disease severity. This finding may offer insights into the pathogenesis of MPP in children.

The clinical significance of macrolide resistance in pediatric Mycoplasma pneumoniae infection during COVID-19 pandemic.

Background: Mycoplasma pneumoniae (MP) is a commonly occurring pathogen causing community-acquired pneumonia (CAP) in children. The global prevalence of macrolide-resistant MP (MRMP) infection, especially in Asian regions, is increasing rapidly. However, the prevalence of MRMP and its clinical significance during the COVID-19 pandemic is not clear. Methods: This study enrolled children with molecularly confirmed macrolide-susceptible MP (MSMP) and MRMP CAP from Beijing Children's Hospital Baoding Hospital, Capital Medical University between August 2021 and July 2022. The clinical characteristics, laboratory findings, chest imaging presentations, and strain genotypes were compared between patients with MSMP and MRMP CAP. Results: A total of 520 hospitalized children with MP-CAP were enrolled in the study, with a macrolide resistance rate of 92.7%. Patients with MRMP infection exhibited more severe clinical manifestations (such as dyspnea and pleural effusion) and had a longer hospital stay than the MSMP group. Furthermore, abnormal blood test results (including increased LDH and D-dimer) were more common in the MRMP group (P<0.05). Multilocus variable-number tandem-repeat analysis (MLVA) was performed on 304 samples based on four loci (Mpn13-16), and M3562 and M4572 were the major types, accounting for 74.0% and 16.8% of the strains, respectively. The macrolide resistance rate of M3562 strains was up to 95.1%. Conclusion: The prevalence of MRMP strains in hospitalized CAP patients was extremely high in the Baoding area, and patients infected with MRMP strains exhibited more severe clinical features and increased LDH and D-dimer. M3562 was the predominant resistant clone.

Clinical Characteristics of Pulmonary Tuberculosis in Children Tested by Xpert MTB/RIF Ultra.

BACKGROUND: The Xpert MTB/rifampicin Ultra (Xpert Ultra) assay improves the early diagnosis of active tuberculosis (TB) in children. Clinical evaluation is paramount for the interpretation of any positive Xpert Ultra test, especially those with low quantities of DNA. METHODS: In this study, 391 children with suspected TB who were tested with Xpert Ultra were enrolled. The clinical characteristics and Xpert Ultra results were further analyzed. RESULTS: The sensitivity and specificity of Xpert Ultra were 45.0% (149/331) and 96.7% (58/60), respectively. Children with higher semiquantitative scales of Xpert Ultra showed higher percentages of a positive MTB culture, positive acid-fast bacilli staining, severe type of disease, fever, cough and expectoration, a higher white blood cell count and higher C-reactive protein concentrations (all P < 0.01). Among 44 children with an Xpert Ultra trace result, there were no differences in clinical characteristics between confirmed cases and unconfirmed TB cases. CONCLUSIONS: The prevalence of trace is relatively high and can be considered positive in paucibacillary children. Clinical presentations are associated with bacterial load quantified by Xpert Ultra. The interpretation of Xpert Ultra trace results based on clinical information is important for the diagnosis of TB.

Recombinase Polymerase Amplification Combined with Real-Time Fluorescent Probe for Mycoplasma pneumoniae Detection.

Mycoplasma pneumoniae (M. pneumoniae) is one of the major causes of community-acquired pneumonia, accounting for 20-40% of total cases. Rapid and accurate detection of M. pneumoniae is crucial for the diagnosis and rational selection of antibiotics. In this study, we set up a real-time recombinase polymerase amplification (RPA) assay to detect the conserved gene CARDS of M. pneumoniae. The amplification can be finished in 20 min at a wide temperature range from 37-41 °C. The limit of detection of RPA assay was 10 fg per microliter. Cross-reaction with commonly detected respiratory pathogens was not observed using RPA assay. Among clinical sputum samples, the detection rate of RPA assay and real-time PCR assay was 48.4% (92/190) and 46.3% (88/190), respectively (p = 0.68). Therefore, the RPA assay for M. pneumoniae detection is rapid and easy to use and may serve as a promising test for early diagnosis of M. pneumoniae infection.

A Novel Cross-Priming Amplification-Based Assay for Tuberculosis Diagnosis in Children Using Gastric Aspirate.

Low detection rates of Mycobacterium tuberculosis (MTB) by culture and smear microscopy prevent early diagnosis of tuberculosis (TB) in children. Therefore, developing rapid and accurate diagnostic techniques are critical to achieving the global aim of minimizing childhood TB. The present study was performed to evaluate the diagnostic effectiveness of the novel cross-priming amplification-based EasyNAT MTB complex assay (EasyNAT) in childhood TB. Five hundred and six children with suspected TB were enrolled from January 2018 to October 2021. Gastric aspirate (GA) samples were tested by bacterial culture, acid-fast bacillus microscopy, EasyNAT, Xpert MTB/RIF (Xpert), or Xpert MTB/RIF Ultra (Xpert Ultra). Among 239 children simultaneously tested by EasyNAT and Xpert methods, both assays showed similar sensitivities in total active TB cases [22.6% (31/137) vs. 26.3% (36/137), p = 0.441] and in bacteriologically confirmed TB cases [both 60.0% (9/15)]. The two assays presented similar specificities of 98.0% (100/102) and 99.0% (101/102), respectively (p = 1.000). Among 267 children who were simultaneously tested with EasyNAT and Xpert Ultra, Xpert Ultra demonstrated higher sensitivity than EasyNAT in total active TB cases [50.9% (89/175) vs. 30.3% (53/175), p < 0.001]. EasyNAT and Xpert Ultra yielded similar specificities, at 97.8% (90/92) and 100.0% (92/92), respectively (p = 0.155). These findings indicated that Xpert Ultra was superior to EasyNAT despite its higher cost and EasyNAT was not inferior to Xpert in the diagnosis of childhood TB using GA samples. EasyNAT may therefore be a suitable alternative diagnostic method for childhood TB based on its cost-effectiveness, speed, and accuracy.

Detection of pulmonary tuberculosis in children using the Xpert MTB/RIF Ultra assay on sputum: a multicenter study.

Microbiological confirmation is rare in children with active tuberculosis; therefore, a more accurate test is needed to detect pulmonary tuberculosis in children. In this multicenter study, we evaluated the utility of the Xpert MTB/RIF Ultra (Ultra) on sputum, an assay recommended by the World Health Organization to test for childhood tuberculosis in high-burden settings. Children with symptoms suggestive of tuberculosis were enrolled at three hospitals in China and categorized as having active tuberculosis or nontuberculosis. The sensitivity and specificity of Ultra were 42.1% (48/114) and 99.0% (208/210), respectively. Using three MTB culture results as the reference, the sensitivity of Ultra in the subset of 38 children with culture-positive and 76 children with culture-negative was 68.4% (26/38) and 28.9% (22/76), respectively(p < 0.001). A single MTB culture combined with a single Ultra could detect 54 (54/114,47.4%) cases with active TB, while repeated MTB culture combined with a single Ultra detected 60 (60/114, 52.6%) cases with active TB(p = 0.427). Among 155 children (58 with TB and 97 with RTIs) simultaneously tested with the Ultra and Xpert MTB/RIF (Xpert), the sensitivity of the Xpert (24.1%, 14/58) was lower than that of the Ultra (41.4%, 24/58; p = 0.048). Eight children were found to have rifampin-resistant MTB strains. The Xpert MTB/RIF Ultra assay should be implemented to test for pulmonary tuberculosis in children to achieve higher confirmation rates.

Use of Xpert MTB/RIF Ultra assay on stool and gastric aspirate samples to diagnose pulmonary tuberculosis in children in a high-tuberculosis-burden but resource-limited area of China.

OBJECTIVES: Our study analyzed the performance of Xpert MTB/RIF Ultra (Ultra) on stool and gastric aspirate (GA) samples for the diagnosis of pediatric pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis in a high-burden area of China. METHODS: Children with presumptive TB were enrolled in two hospitals in Sichuan Province (July 2019-Oct 2020). Because of the unavailability of sputum for etiological testing, gastric samples were aspirated and tested by bacterial culture, acid-fast bacillus microscopy, and Ultra. Stool samples were tested simultaneously using Ultra and Xpert. RESULTS: In total, 141 children with active TB and 34 with non-TB respiratory tract infections were enrolled. Ultra-stool (60.3%, 85/141) and Ultra-GA (52.5%, 74/141) tests were similarly sensitive (p = 0.187). Among the subset of 48 children with confirmed TB, Ultra testing was equally sensitive on stool and GA samples (85.4%, 41/48). The agreement between Ultra-stool and Ultra-GA was moderate in children with active TB (kappa value = 0.527). After integrating Ultra-GA and Ultra-stool outcomes, 70.9% (100/141) of the children were considered to have confirmed TB. The specificities of Ultra-stool and Ultra-GA were 97.1% (33/34) and 100% (34/34), respectively (p = 0.314). CONCLUSIONS: In children, stools can be used as alternative samples to GAs for Ultra tests. Stool- and GA-based Ultra tests are appropriate for bacteriological TB confirmation.

Tuberculosis infection screening in children with close contact: a hospital-based study.

BACKGROUND: Identifying and prioritizing at-risk populations is critical for pediatric tuberculosis control. We aimed to identify a latent tuberculosis infection (LTBI) screening strategy that is appropriate for the Chinese context among children with different TB exposure levels and to explore its clinical importance. METHODS: During 2013-2015, we enrolled hospitalized children with suspected respiratory infectious disease (RID) for LTBI screening using the tuberculin skin test (TST) and interferon-γ release assay (IGRA) T-SPOT.TB as part of a work up for their RID. Participants with confirmed diagnosis were classified into three subgroups according to level of exposure to TB: no reported contact risk, with household contact risk, and with non-household contact risk. RESULTS: A total 6202 children (median age: 4.76 years; interquartile range: 1.0-8.0 years) were enrolled. Children with no reported contact risk had the lowest proportions of positive results for the IGRA (0.7%) and TST (3.3%). The proportion of positive results for each test was higher for household contacts than non-household contacts. The TST positive proportion was much higher than that for the IGRA in all three groups. Children with IGRA+/TST+ results had larger indurations than those with IGRA- /TST+ results (15 mm vs. 13 mm, P = 0.02). For IGRA, older age (> 5 years) and non-household or household contact risk were associated with a positive result. CONCLUSIONS: Positive IGRA results in children with a contact risk can serve as a critical reference for LTBI management. IGRA can be used, in preference to TST, for Chinese children with a TB exposure risk.

Increased Macrolide Resistance Rate of M3562 Mycoplasma pneumoniae Correlated With Macrolide Usage and Genotype Shifting.

To characterize Mycoplasma pneumoniae (MP) strains and to clarify the continuous high rates of macrolide resistance, 1,524 oropharyngeal swabs collected from children in Beijing Children's Hospital infected with MP during 2016-2019 were analyzed. Among the 1,524 samples, 1,386 harbored mutations associated with macrolide resistance; 1,049 samples were successfully classified into 11 genotypes using multiple locus variable-number tandem-repeat analysis (MLVA). The proportion of the predominant type, M4572, decreased from 84.49 to 70.77% over the time period examined, while that of M3562 increased from 11.63 to 24.67%. Notably, we also found that the frequency of macrolide resistance in M3562 drastically increased, from 60% in 2016 to 93.48% in 2019. Clinical data suggested that the frequency of resistant M3562 was higher in the macrolide usage group than in the nondrug usage group (90.73 vs 53.57%, P<0.0001), while the resistance rate of M4572 was not substantially affected by previous macrolide exposure. These findings validated that antimicrobial application and clonal expansion of resistant MP strains play important roles in the high rates of macrolide resistance.

Development and Preliminary Application of Multiplex Loop-Mediated Isothermal Amplification Coupled With Lateral Flow Biosensor for Detection of Mycobacterium tuberculosis Complex.

The aim of this study was to develop a simple and reliable method to detect Mycobacterium tuberculosis complex (MTBC) and verify its clinical application preliminarily. A loop-mediated isothermal amplification method coupled with lateral flow biosensor (LAMP-LFB) assay, was developed and evaluated for detection of MTBC. Two sets of primers, which targeted IS6110 and IS1081 sequences of MTBC, were designed for establishment of multiplex LAMP-LFB assay. The amplicons were labelled with biotin and fluorescein isothiocyanate (FITC) by adding FITC labelled primer and biotin-14-dATP and biotin-14-dCTP and could be visualized using LFB. The optimal reaction conditions of multiplex LAMP-LFB assay confirmed were 66°C for 50 min. The analytical sensitivity of multiplex LAMP-LFB is 10 fg of genomic templates using pure culture, and no cross-reactivity with other common bacteria and non-tuberculous mycobacteria strains was obtained. A total of 143 clinical samples collected from 100 TB patients (62 definite TB cases and 38 probable TB cases) and 43 non-TB patients were used for evaluating the feasibility of multiplex LAMP-LFB assay. The multiplex LAMP-LFB (82.0%, 82/100) showed higher sensitivity than culture (47.0%, 47/100, P < 0.001) and Xpert MTB/RIF (54.0%, 54/100, P < 0.001). Importantly, the multiplex LAMP-LFB assay detected additional 28 probable TB cases, which increased the percentage of definite TB cases from 62.0% (62/100) to 90.0% (90/100). The specificity of multiplex LAMP-LFB assay in patients without TB was 97.7% (42/43). Therefore, multiplex LAMP-LFB assay is a simple, reliable, and sensitive method for MTBC detection, especially in probable TB cases and resource limited settings.

Multiple Cross Displacement Amplification Combined With Real-Time Polymerase Chain Reaction Platform: A Rapid, Sensitive Method to Detect Mycobacterium tuberculosis.

In this study, we evaluated the diagnostic accuracy of multiple cross displacement amplification (MCDA) combined with real-time PCR platform in pulmonary tuberculosis (PTB) patients. Total 228 PTB patients and 141 non-TB cases were enrolled. Based on the analysis of the first available sample of all participants, MCDA assay showed a higher overall sensitivity (64.0%), with a difference of more than 10% compared with Xpert MTB/RIF (Xpert) assay (51.8%, P < 0.05) and combined liquid and solid culture (47.8%, P < 0.001) for PTB diagnosis. In particular, MCDA assay detected 31 probable TB patients, which notably increased the percentage of confirmed TB from 57.9% (132/228) to 71.5% (163/228). The specificities of microscopy, culture, Xpert and MCDA assay were 100% (141/141), 100% (141/141), 100% (141/141), and 98.6% (139/141), respectively. Among the patients with multiple samples, per patient sensitivity of MCDA assay was 60.5% (52/86) when only the first available sputum sample was taken into account, and the sensitivity increased to 75.6% (65/86) when all samples tested by MCDA assay were included into the analysis. Therefore, MCDA assay established in this study is rapid, accurate and affordable, which has the potential in assisting the accurate and rapid diagnosis of PTB and speed up initiation of TB treatment in settings equipped with real-time PCR platform.

Graphene oxide and self-avoiding molecular recognition systems-assisted recombinase polymerase amplification coupled with lateral flow bioassay for nucleic acid detection.

A new nucleic acid detection technique, termed Nano-SAMRS-RPA, is reported which employed carbon nanomaterial (graphene oxide, GO) and self-avoiding molecular recognition systems (SAMRS) to improve the specificity of recombinase polymerase amplification (RPA). In the presence of GO and SAMRS primers, the assay artifacts, including primer-dimers, nonspecific products, off-target hybrids, and non-canonical folds, are completely suppressed and eliminated, which makes the creation of RPA-based methods faster by simplifying the primer design and eliminating the need for primer optimization and complex probe. Moreover, a lateral flow bioassay (LFB) was also devised for simply and rapidly indicating the Nano-SAMRS-RPA results. Particularly, the new detection system only requires a single-labeled primer, eliminating the false-positive result from hybridization (the labeled probe and reverse primer) and the use of real-time instrument, more complex enzymatic solutions, and probes. As a result, GO, SAMRS primers, and LFB convert RPA from a technique suited only for the research laboratory into one that has a practical value in clinical settings, field environments, and at points-of-care testing. Human papillomaviruses (HPV) genotypes 16 and 18 were applied as model analytes to test the assay's availability. The initial data indicated that Nano-SAMRS-RPA could detect down to 10 copies per reaction, and the sensitivity (14/14 samples collected from HPV16 and HPV 18 patients) and specificity (75/75 samples collected from non-HPV patients) for clinical sample detection were 100%. The proof-of-concept technique can be reconfigured to detect various nucleic acid sequences by redesigning the specific RPA primers.Graphical abstract.

Evaluation of Xpert MTB/RIF Ultra Assay for Diagnosis of Childhood Tuberculosis: a Multicenter Accuracy Study.

A multicenter study was performed to evaluate the value of testing gastric aspirate (GA) with Xpert MTB/RIF Ultra assay (Ultra) for childhood tuberculosis (TB) detection in China. In total, 129 children with active TB and 173 children without TB were enrolled. The sensitivity of Ultra in bacteriologically confirmed TB and probable TB cases was 87.5% (42/48) and 44.4% (36/81), respectively. The specificity of Ultra was high (99.4%, 172/173). When Ultra, culture, and acid-fast bacilli outcomes were integrated as a composite reference standard, the percentage of children with definite TB increased from 37.2% (48/129) to 67.4% (87/129). The sensitivity of Ultra is 80.0% (40/50) in children aged <4 years, which is significantly higher than that in older children (48.1%, 38/79) (P < 0.001). Ultra conducted using GA samples can provide faster results, allowing an early and accurate TB diagnosis, especially in younger children with difficulty producing sputum.

Development of loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor assay for Mycoplasma pneumoniae detection.

Mycoplasma pneumoniae (MP) is one of the most common pathogens causing respiratory tract infection, especially for community-acquired pneumonia (CAP) in school-age children. There was considerable amount of studies on loop-mediated isothermal amplification (LAMP) assay for MP detection. However, the result interpretation of these developed LAMP assays was sophisticated and subjective. Therefore, we developed and evaluated a LAMP coupled with nanoparticle-based lateral flow biosensor (LFB) assay (LAMP-LFB) for simple, reliable, and objective identification of MP (MP-LAMP-LFB). Six primers specific to P1 gene of MP were designed, and the preferred temperature for this assay was confirmed to be 65 °C. The amplification products could be visually interpreted by LFB within 2 min. The MP-LAMP-LFB assay specifically identified DNA templates of MP, and no cross-reactivity with other pathogens was obtained. The limit of the detection for this assay was 600 fg of DNA templates in pure cultures, which was in complete accordance with colorimetric indicator detection and agarose gel electrophoresis analysis. This assay was applied to 209 oropharyngeal swab specimens collected from children with acute respiratory tract infection for clinical evaluation, and compared to real-time PCR detection. Using the LAMP-LFB and real-time PCR assay, the positive rates of MP were 47.8% and 31.6%, respectively. Results suggested that the LAMP-LFB assay displayed high sensitivity compared to real-time PCR method. In summary, LAMP-LFB assay established here was a simple, objective, and sensitive assay for MP detection, which can be widely applied in clinical settings, especially in rural areas.

Establishment and Application of a Multiple Cross Displacement Amplification Coupled With Nanoparticle-Based Lateral Flow Biosensor Assay for Detection of Mycoplasma pneumoniae.

Mycoplasma pneumoniae (M. pneumoniae) is responsible for pneumonia, and is a causative agent of other respiratory tract infections (e.g., bronchiolitis and tracheobronchitis). Herein, we established and applied a multiple cross displacement amplification (MCDA) coupled with a nanoparticle-based lateral flow biosensor (LFB) assay (MCDA-LFB) for rapid, simple, and reliable detection of target pathogen. A set of 10 primers was designed based on M. pneumoniae-specific P1 gene, and optimal reaction conditions were found to be 30 min at 65°C. The detection results were visually reported using a biosensor within 2 min. The M. pneumoniae-MCDA-LFB method specifically detected only M. pneumoniae templates, and no cross-reactivity was generated from non-M. pneumoniae isolates. The analytical sensitivity for this assay was 50 fg of genomic templates in the pure cultures, as obtained from colorimetric indicator and real-time turbidimeter analysis. The assay was applied to 197 oropharyngeal swab samples collected from children highly suspected of M. pneumoniae infection, and compared to culture-based method and real-time PCR assay. The detection rates of M. pneumoniae using a culture-based method, real-time PCR assay, and MCDA-LFB assay were 8.1%, 33.0%, and 52.3%, respectively, which indicated that the MCDA-LFB assay was superior to the culture-based method and real-time PCR method for detection of target agent. Using this protocol, 25 min for rapid template extraction followed by MCDA reaction (30 min) combined with LFB detection (2 min) resulted in a total assay time of ~60 min. In conclusion, the MCDA-LFB assay established in this report was a simple, rapid, sensitive, and reliable assay to detect M. pneumoniae strains, and can be used as a potential diagnostic tool for M. pneumoniae in basic and clinical laboratories.

Development and Clinical Validation of Multiple Cross Displacement Amplification Combined With Nanoparticles-Based Biosensor for Detection of Mycobacterium tuberculosis: Preliminary Results.

Tuberculosis is still a major threat to global public health. Here, a novel diagnosis assay, termed as multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor (MCDA-LFB), was developed to simultaneously detect IS6110 and IS1081 of Mycobacterium tuberculosis (MTB) in DNA extracted from reference strain H37Rv and clinical samples. The amplification can be finished within 30 min at a fixed temperature (67°C), thus the whole procedure, including rapid template preparation (15 min), isothermal reaction (30 min) and result reporting (2 min), can be completed within 50 min. The limit of detection of multiplex MCDA assay was 10 fg per reaction. By using the multiplex MCDA protocol, cross-reaction with non-mycobacteria and non-tuberculous mycobacteria (NTM) strains was not observed. Among clinically diagnosed TB patients, the sensitivity of liquid culture, Xpert MTB/RIF and multiplex MCDA assay was 42.0% (50/119), 49.6% (59/119), and 88.2% (105/119), respectively. Among culture positive samples, the sensitivity of Xpert MTB/RIF and multiplex MCDA assay was 86.0% (43/50) and 98.0% (49/50), respectively. Among culture negative samples, the sensitivity of Xpert MTB/RIF and multiplex MCDA assay was 23.2% (16/69) and 81.2% (56/69), respectively. The specificity was 100% (60/60) for Xpert MTB/RIF and 98.3% (59/60) for multiplex MCDA. Therefore, the multiplex MCDA assay for MTB detection is rapid, sensitive and easy to use and may be a promising test for early diagnosis of TB.

Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction.

The current report devised a novel isothermal diagnostic assay, termed as nanoparticle-based biosensor (NB)- and antarctic thermal sensitive uracil-DNA-glycosylase (ATSU)-supplemented polymerase spiral reaction (PSR; NB-ATSU-PSR). The technique merges enzymatic digestion of carryover contaminants and isothermal nucleic acid amplification technique (PSR) for simultaneous detection of nucleic acid sequences and elimination of carryover contamination. In particular, nucleic acid amplification and elimination of carryover contamination are conducted in a single pot and, thus, the use of a closed-tube reaction can remove undesired results due to carryover contamination. For demonstration purpose, Klebsiella pneumoniae is employed as the model to demonstrate the usability of NB-ATSU-PSR assay. The assay's sensitivity, specificity, and practical feasibility were successfully evaluated using the pure cultures and sputum samples. The amplification products were detectable from as little as 100 fg of genomic DNAs and from ~550 colony-forming unit (CFU) in 1 ml of spiked sputum samples. All K. pneumoniae strains examined were positive for NB-ATSU-PSR detection, and all non-K. pneumoniae strains tested were negative for the NB-ATSU-PSR technique. The whole process, including rapid template preparation (20 min), PSR amplification (60 min), ATSU treatment (5 min), and result reporting (within 2 min), can be finished within 90 min. As a proof-of-concept methodology, NB-ATSU-PSR technique can be reconfigured to detect various target nucleic acid sequences by redesigning the PSR primer set.

Population pharmacokinetics and dosing optimization of latamoxef in neonates and young infants.

OBJECTIVES: There has been recent renewed interest in historical antibiotics because of the increased antibiotic-resistant bacterial strains. Latamoxef, a semi-synthetic oxacephem antibiotic developed in 1980s, has recently been brought back into use for treatment of infections in newborns; however, it is still used off-label in neonatal clinical practice due to the lack of an evidence-based dosing regimen. This study was performed to evaluate the pharmacokinetics of latamoxef in neonates and young infants, and to provide an evidence-based dosing regimen for newborns based on developmental pharmacokinetics-pharmacodynamics (PK-PD). METHODS: Opportunistic blood samples from newborns treated with latamoxef were collected to determine the latamoxef concentration by high-performance liquid chromatography with UV detection. Population PK-PD analysis was conducted using NONMEM and R software. A total of 165 plasma samples from 128 newborns (postmenstrual age range 28.4-46.1 weeks) were available for analysis. RESULTS: A two-compartment model with first-order elimination showed the best fit with the data. Current body weight, birth weight, and postnatal age were identified as significant covariates influencing latamoxef clearance. Simulation indicated that the current dosing regimen (30 mg/kg q12h) is adequate with an MIC of 1 mg/L. For an MIC of 4 mg/L, 30 mg/kg q8h was required to achieve a target rate of 70% of patients having a free antimicrobial drug concentration exceeding the MIC during 70% of the dosing interval. CONCLUSIONS: Based on the developmental PK-PD analysis of latamoxef, a rational dosing regimen of 30 mg/kg q12h or q8h was required in newborns, depending on the pathogen.

Quantitative Analysis of the Global Proteome in Peripheral Blood Mononuclear Cells from Patients with New-Onset Psoriasis.

Psoriasis is a common chronic autoimmune skin disease involving the activation of T cells. To explore the proteomic signature of peripheral blood mononuclear cells, a quantitative analysis of their global proteome was conducted in samples from Chinese patients with new-onset psoriasis (n = 31) and healthy controls (n = 32) using an integrated quantitative approach with tandem mass tag labeling and LC-MS/MS. Protein annotation, unsupervised hierarchical clustering, functional classification, functional enrichment and cluster, and protein-protein interaction analyses were performed. A total of 5178 proteins were identified, of which 4404 proteins were quantified. The fold-change cutoff was set at 1.2 (patients vs controls); 335 proteins were upregulated, and 107 proteins were downregulated. The bioinformatics analysis indicated that the differentially expressed proteins were involved in processes related to the activation of immune cells including the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, cellular energy metabolism, and proliferation. Three upregulated proteins and two phosphorylated proteins in the NF-κB pathway were verified or identified by Western blotting. These results confirm that the NF-κB pathway is critical to psoriasis. In addition, many differentially expressed proteins identified in this study have never before been associated with psoriasis, and further studies on these proteins are necessary.