Clinical utility of target amplicon sequencing test for rapid diagnosis of drug-resistant Mycobacterium tuberculosis from respiratory specimens.

An in-house-developed target amplicon sequencing by next-generation sequencing technology (TB-NGS) enables simultaneous detection of resistance-related mutations in Mycobacterium tuberculosis (MTB) against 8 anti-tuberculosis drug classes. In this multi-center study, we investigated the clinical utility of incorporating TB-NGS for rapid drug-resistant MTB detection in high endemic regions in southeast China. From January 2018 to November 2019, 4,047 respiratory specimens were available from patients suffering lower respiratory tract infections in Hong Kong and Guangzhou, among which 501 were TB-positive as detected by in-house IS6110-qPCR assay with diagnostic sensitivity and specificity of 97.9 and 99.2%, respectively. Preliminary resistance screening by GenoType MTBDRplus and MTBDRsl identified 25 drug-resistant specimens including 10 multidrug-resistant TB. TB-NGS was performed using MiSeq on all drug-resistant specimens alongside 67 pan-susceptible specimens, and demonstrated 100% concordance to phenotypic drug susceptibility test. All phenotypically resistant specimens with dominating resistance-related mutations exhibited a mutation frequency of over 60%. Three quasispecies were identified with mutation frequency of less than 35% among phenotypically susceptible specimens. They were well distinguished from phenotypically resistant cases and thus would not complicate TB-NGS results interpretations. This is the first large-scale study that explored the use of laboratory-developed NGS platforms for rapid TB diagnosis. By incorporating TB-NGS with our proposed diagnostic algorithm, the workflow would provide a user-friendly, cost-effective routine diagnostic solution for complicated TB cases with an average turnaround time of 6 working days. This is critical for timely management of drug resistant TB patients and expediting public health control on the emergence of drug-resistant TB.

Acupuncture for de Quervain's tenosynovitis: A randomized controlled trial.

BACKGROUND: Acupuncture has been an alternative approach for de Quervain's tenosynovitis (DQt), but trial evidence is still lacking. PURPOSE: This study aimed to assess the efficacy of acupuncture in patients with DQt. STUDY DESIGN: A randomized controlled trial. METHODS: A total of 68 subjects with DQt were recruited from outpatients of Department of Orthopaedics and Traumatology, and Chinese medicine clinics, The University of Hong Kong, and were randomized into the acupuncture group (n = 34) and the waitlist group (n = 34). Subjects in the acupuncture group received 5 acupuncture sessions over 2 weeks, followed by a 10-week follow-up. The waitlist control group received assessments only in the first 6 weeks of the waiting period and received the same acupuncture treatment and follow-up as the treatment group in the next 12 weeks. The primary outcome was the general pain intensity using the Visual Analogue Scale (VAS) at the end of treatment (week 2). Secondary outcomes were grip and pinch strengths of affected hands, the quick Disabilities of the Arm, Shoulder and Hand Score (Q-DASH), and the World Health Organization Quality of Life-brief Questionnaire (WHOQOL-BREF) at weeks 2 and 6. RESULTS: From baseline to 2 weeks, the mean VAS score decreased by 19.5 points in the acupuncture group and by 3.4 points in the waitlist group. The difference for acupuncture vs waitlist control was -16.2 points (95% CI, -26.7 to -5.6, p = 0.003). Acupuncture effects sustained for 10 weeks (mean difference compared with baseline, -30.6; 95% CI, -39.6 to -21.7). Secondary outcomes showed that acupuncture reduced pain intensity, improved grip and pinch strength of affected hands, and Q-DASH scores, but not the scores of WHOQOL-BREF in patients. No serious adverse event occurred during the study period. CONCLUSIONS: Our findings support that 2-week of acupuncture is safe and effective in the reduction of pain intensity, and improvement of strengths and disabilities of hand in DQt patients. Acupuncture also has long-term effects on DQt. TRIAL REGISTRATION: This study was registered at clinicaltrials.gov (NCT03472443).

High-resolution weather network reveals a high spatial variability in air temperature in the Central valley of California with implications for crop and pest management.

Weather is the most important driver of crop development. However, spatial variability in weather makes it hard to obtain reliable high resolution datasets across large areas. Most growers rely on data from a single station that can be up to 50km away to make decisions about irrigation, pest management and penology-associated cultural practices at the block level. In this regard, we hypothesize that kriging a large network of weather stations can improve thermal time data quality compared to using the closest station. This study aims to explore the spatial variability in California's Central Valley and what is the relationship between the density of weather stations used and the error in the measurement of temperature related metrics and derived models. For this purpose, we used temperature records from January 1st 2020 to March 1st 2021 collected by the California Irrigation Management Information System (CIMIS) and a system of 731 weather stations placed above the canopy of trees in commercial orchards (in-orchard). We observed large discrepancies (>300 GDDTb0) in thermal time accumulation between using an interpolation of all stations available and just using the closest CIMIS station. Our data suggests these differences are not systematic bias but true differences in mesoclimate. Similar results were observed for chill accumulation in areas especially prone to not meeting pistachio chill requirements where the discrepancies between using the site-specific in-orchard weather station network and not using them were up to 10 CP. The use of this high resolution network of weather stations revealed spatial patterns in grape, almond, pistachio and pests phenology not reported before. Whereas previous studies have been focused on predictions at the county or state or regional level, our data suggests that a finer resolution can result in major improvements in the quality of data crucial for crop decision making.

Targeted-Sequencing Workflows for Comprehensive Drug Resistance Profiling of Mycobacterium tuberculosis Cultures Using Two Commercial Sequencing Platforms: Comparison of Analytical and Diagnostic Performance, Turnaround Time, and Cost.

BACKGROUND: The emergence of Mycobacterium tuberculosis with complex drug resistance profiles necessitates a rapid and comprehensive drug susceptibility test for guidance of patient treatment. We developed two targeted-sequencing workflows based on Illumina MiSeq and Nanopore MinION for the prediction of drug resistance in M. tuberculosis toward 12 antibiotics. METHODS: A total of 163 M. tuberculosis isolates collected from Hong Kong and Ethiopia were subjected to a multiplex PCR for simultaneous amplification of 19 drug resistance-associated genetic regions. The amplicons were then barcoded and sequenced in parallel on MiSeq and MinION in respective batch sizes of 24 and 12 samples. A web-based bioinformatics pipeline, BacterioChek-TB, was developed to translate the raw datasets into clinician-friendly reports. RESULTS: Both platforms successfully sequenced all samples with mean read depths of 1,127× and 1,649×, respectively. The variant calling by MiSeq and MinION could achieve 100% agreement if variants with an allele frequency of <40% reported by MinION were excluded. Both workflows achieved a mean clinical sensitivity of 94.8% and clinical specificity of 98.0% when compared with phenotypic drug susceptibility test (pDST). Turnaround times for the MiSeq and MinION workflows were 38 and 15 h, facilitating the delivery of treatment guidance at least 17-18 days earlier than pDST, respectively. The higher cost per sample on the MinION platform ($71.56) versus the MiSeq platform ($67.83) was attributed to differences in batching capabilities. CONCLUSION: Our study demonstrates the interchangeability of MiSeq and MinION platforms for generation of accurate and actionable results for the treatment of tuberculosis.

Diagnostic evaluation of an in-house developed single-tube, duplex, nested IS6110 real-time PCR assay for rapid pulmonary tuberculosis diagnosis.

OBJECTIVE: To perform a prospective evaluation on the diagnostic performance of an in-house developed, duplex nested IS6110 real-time Polymerase-Chain-Reaction (PCR) assay (IS6110-qPCR assay) for rapid pulmonary TB diagnosis. METHODS: A total of 503 sputum specimens were prospectively collected from July 2016 to November 2016. Diagnostic accuracy and optimal cut-off Cycle-threshold (Ct) value for IS6110-qPCR assay was determined by Receiver Operating Characteristic (ROC) curve. Using the optimal cut-off Ct, diagnostic performance of IS6110-qPCR assay was assessed with reference to both bacteriological and clinical information. Meanwhile, limit of detection (LOD) was calculated using Mycobacterium tuberculosis H37Rv as reference strain. RESULT: ROC curve analysis of IS6110-qPCR assay showed a high Area Under the Curve (AUC) value (0.948) with optimal Ct value at 24.140. Prospective analysis of IS6110-qPCR assay with cut-off Ct = 24.140 showed a high overall sensitivity and specificity of 97.2% and 99.7%, respectively. No cross reactivity was observed among all non-tuberculous mycobacteria specimens in this study. LOD analysis on MTB-spiked sputum showed an average detection limit of 5.0 CFU/mL at Ct = 23.18 (±SD, 0.57). CONCLUSION: IS6110-qPCR assay is a highly accurate and cost-effective assay developed for primary screening of suspected TB cases, which is particularly suitable for regions with limited resources but high TB burden.

Comparative Genomic Analysis of Two Clonally Related Multidrug Resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing.

Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB under in vivo challenge of anti-TB drugs. Result: Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations at rpoB S531L, inhA C-15T, and embB M306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 of rv0888 in pre-treatment strain, and a missense mutation at rv3303c (lpdA) V44I and a 6-bp inframe deletion at codon 67-68 in rv2071c (cobM) in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function. Conclusion: This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations at rv0888, lpdA, and cobM that might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.

Novel mutations in myopathic form of carnitine palmitoyltransferase II deficiency in a Chinese patient.

BACKGROUND: Carnitine palmitoyltransferase II (CPT II) deficiency is one of the most common disorders of oxidative fatty acid metabolism. In this disorder, long-chain acylcarnitines cannot be converted to acyl CoA and there is impairment of ß-oxidation of fatty acids. RESULTS: In the 3 distinct clinical subtypes of CPT II deficiency, adult onset myopathic form shows mild clinical manifestations, characterized by recurrent rhabdomyolysis after intense physical stress. In this study, we report a case with adult myopathic form of CPT II deficiency presenting recurrent exercise-induced myoglobinuria. CONCLUSION: The acylcarnitine profile showed characteristic CPTII deficiency profile and sequencing of the CPT2 gene showed 2 novel missense mutations p. H369Q and p G497S.

Mutational analysis of 65 Wilson disease patients in Hong Kong Chinese: identification of 17 novel mutations and its genetic heterogeneity.

Wilson disease (WD), an autosomal recessive disorder of copper transport, is the most common inherited liver disorder in Hong Kong Chinese. This was the first local study to elucidate the molecular basis and establish an effective DNA-based diagnostic protocol. The ATP7B genes of 65 patients were amplified by polymerase chain reaction (PCR) and sequenced. Haplotype analysis was performed using D13S301, D13S314, and D13S316. The p.L770L/p.R778L status in 660 subjects was determined to estimate WD prevalence. Allele age of p.R778L was determined by the smallest homozygosity region between D13S301 and D13S270. We identified 42 different mutations with 17 being novel. p.R778L (17.3%) was the most prevalent. Exons 2, 8, 12, 13, and 16 harbored 70% mutations. Thirty-two haplotypes were associated with WD chromosomes. The estimated prevalence rate was 1 in 5,400. Three out of 660 normal subjects had p.L770L/p.R778L. In the remaining 657 individuals, neither p.L770L nor p.R778L was found. We characterized a Hong Kong Chinese-specific ATP7B mutation spectrum with great genetic diversity. Exons 2, 8, 12, 13, and 16 should be screened first. The perfect linkage disequilibrium suggested that p.R778L and its private polymorphism p.L770L originated from a single ancestor. This East-Asian-specific mutation p.R778L/p.L770L is aged at least 5,500 years.

Erratum to: Mutational analysis of 65 Wilson disease patients in Hong Kong Chinese: Identification of 17 novel mutations and its genetic heterogeneity.

The correct name of the eighth author should be given as Sik-To Lai, not Jak-Yiu Lai.

Plasma lipid, lipoprotein and apolipoprotein levels in a random population sample of 2875 Hong Kong Chinese adults and their implications (NCEP ATP-III, 2001 guidelines) on cardiovascular risk assessment.

The age- and sex-related levels of plasma lipids, lipoproteins and apolipoproteins in a random population sample of 2875 Hong Kong Chinese Adults (1397 men and 1478 women aged 25-74) and their implications on cardiovascular risk assessment are reported. Total cholesterol, low-density lipoprotein (LDL)-cholesterol and triglycerides increased with age in both sexes. Postmenopausal women had the worst profiles. They also showed higher triglyceride and non-high density lipoprotein (non-HDL)-cholesterol and had higher percentage of values greater than the desirable limits, compared with men of the same age groups. Overall 39% of men and 29% of women had non-HDL cholesterol of 4.2 mmol/L or greater. Apolipoproteins A-I and B followed the same trends as HDL-cholesterol and LDL-cholesterol, respectively. Apolipoprotein E (apo E) allele frequencies were: epsilon2 8.7, epsilon3 80.4 and epsilon4 10.9% with the genotype having a significant effect on plasma apo E concentration (p < 0.001). Carriers of the epsilon2 allele had higher apo E values than those homozygous for E3. Lipoprotein(a) levels were higher in women than men (geometric mean 152 versus 102 mg/L, p < 0.05) and in women with FSH above versus below 40 IU/L (185 versus 136 mg/L, p < 0.05). With respect to the NCEP ATP-III 2001 guidelines, the prevalence of hyperlipidemia in the Hong Kong population approached those in high CHD prevalence Caucasian communities. Local management guidelines and community-wide programs to reduce fat intake, increase regular moderate exercise and reduce the prevalence of overweight and obesity are urgently required, and hormone replacement therapy for postmenopausal women might be warranted.

Novel insertion 496_497insG creating a stop codon D194X in a Chinese family with X-Linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (XALD, MIM 300100), the commonest inherited peroxisomal disorder, is characterized by central nervous system demyelination, primary adrenal failure and the systemic accumulation of saturated very long chain fatty acids (VLCFAs). The defective gene ABCD1 encodes an ATP-binding cassette (ABC) transport protein named ALDP, which functions as a crucial transporter of VLCFAs into the peroxisomes for beta-oxidation. Here, we report a Chinese man with adrenomyeloneuropathy characterized by Addison's disease and spastic paraparesis. His plasma VLCFA levels, ratios of C24:0/C22:0 and C26:0/C22:0 were all significantly elevated. We performed mutation analysis of the ABCD1 gene in the proband and the family members using direct DNA sequencing and restriction analysis. A novel insertion 496_497insG in exon 1 causing a frame shift and a premature stop codon at amino acid position 194 (D194X) was identified (GenBank accession No. NM_000033). The insertional mutation abolishes an HhaI restriction site. The same mutation was found in his mother and the eldest sister even though their clinical and biochemical abnormalities were milder. Diagnosis of XALD often relies upon the detection of elevated VLCFA levels and ratios of C26:0/C22:0 and C24:0/C22:0 in fasting blood, however, 5-15% of the obligate heterozygotes would give normal values. DNA-based testing thus remains the most reliable tool for heterozygote detection when the disease-causing mutations are known. Using restriction fragment length polymorphism with HhaI, we have devised a rapid method for the identification of the carriers among the proband's family members and possibly for the screening of the mutations in other XALD patients.

Cerebrotendinous xanthomatosis in a Hong Kong Chinese kinship with a novel splicing site mutation IVS6-1G>T in the sterol 27-hydroxylase gene.

We reported a Hong Kong Chinese proband with Cerebrotendinous Xanthomatosis in which a novel acceptor splicing site mutation (IVS6-1G>T) was identified. Family screening revealed the same mutation in his elder brother and the youngest sister. All the three affected siblings were compound heterozygous for IVS6-1G>T and a known missense mutation R372Q (GenBank Accession No. M62401). Significant phenotypic variation was noted among them that the youngest sister was still symptom-free at the time of writing.