A 2-year prospective evaluation of the Prostate Health Index in guiding biopsy decisions in a large cohort.

OBJECTIVES: To prospectively evaluate how the Prostate Health Index (PHI) impacts on clinical decision in a real-life setting for men with a prostate-specific antigen (PSA) level between 4 and 10 ng/mL and normal digital rectal examination. PATIENTS AND METHODS: Since 2016, the PHI has been available at no cost to eligible men in all Hong Kong public hospitals. All eligible patients who received PHI testing in all public Urology units (n = 16) in Hong Kong between May 2016 and August 2017 were prospectively included and followed up. All included men had a PHI test, with its result and implications explained; the subsequent follow-up plan was then decided via shared decision-making with urologists. Patients were followed up for 2 years, with outcomes including prostate biopsy rates and biopsy findings analysed in relation to the initial PHI measurements. RESULTS: A total of 2828 patients were followed up for 2 years. The majority (82%) had PHI results in the lower risk range (score <35). Knowing the PHI findings, 83% of the patients with elevated PSA decided not to undergo biopsy. In all, 11% and 45% opted for biopsy in the PHI score <35 and ≥35 groups, respectively. The initial detection rate of International Society of Urological Pathology (ISUP) Grade Group (GG) ≥2 cancer was higher in the PHI score ≥35 group (23%) than in the PHI score <35 group (7.9%). Amongst patients with no initial positive biopsy findings, the subsequent positive biopsy rate for ISUP GG ≥2 cancer was higher in the PHI score ≥35 group (34%) than the PHI score <35 group (13%) with a median follow-up of 2.4 years. CONCLUSION: In a real-life setting, with the PHI incorporated into the routine clinical pathway, 83% of the patients with elevated PSA level decided not to undergo prostate biopsy. The PHI pathway also improved the high-grade prostate cancer detection rate when compared to PSA-driven strategies. Higher baseline PHI predicted subsequent biopsy outcome at 2 years. The PHI can serve as a tool to individualise biopsy decisions and frequency of follow-up visits.

Development of a Novel, Genome Subtraction-Derived, SARS-CoV-2-Specific COVID-19-nsp2 Real-Time RT-PCR Assay and Its Evaluation Using Clinical Specimens.

The pandemic novel coronavirus infection, Coronavirus Disease 2019 (COVID-19), has affected at least 190 countries or territories, with 465,915 confirmed cases and 21,031 deaths. In a containment-based strategy, rapid, sensitive and specific testing is important in epidemiological control and clinical management. Using 96 SARS-CoV-2 and 104 non-SARS-CoV-2 coronavirus genomes and our in-house program, GolayMetaMiner, four specific regions longer than 50 nucleotides in the SARS-CoV-2 genome were identified. Primers were designed to target the longest and previously untargeted nsp2 region and optimized as a probe-free real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. The new COVID-19-nsp2 assay had a limit of detection (LOD) of 1.8 TCID50/mL and did not amplify other human-pathogenic coronaviruses and respiratory viruses. Assay reproducibility in terms of cycle threshold (Cp) values was satisfactory, with the total imprecision (% CV) values well below 5%. Evaluation of the new assay using 59 clinical specimens from 14 confirmed cases showed 100% concordance with our previously developed COVID-19-RdRp/Hel reference assay. A rapid, sensitive, SARS-CoV-2-specific real-time RT-PCR assay, COVID-19-nsp2, was developed.

GNPTAB c.2404C > T nonsense mutation in a patient with mucolipidosis III alpha/beta: a case report.

BACKGROUND: Mucolipidosis alpha/beta is an inborn error of metabolism characterized by deficiency of GlcNAc-1-phosphotransferase, in which essential alpha/beta subunits are encoded by the GNPTAB gene. The autosomal recessive condition is due to disruptions of hydrolase mannose 6-phosphate marker generation, defective lysosomal targeting and subsequent intracellular accumulation of non-degraded material. Clinical severity depends on residual GlcNAc-1-phosphotransferase activity, which distinguishes between the milder type III disease and the severe, neonatal onset type II disease. CASE PRESENTATION: We report the clinical, biochemical and genetic diagnosis of mucolipidosis III alpha/beta in a two-year-old Chinese boy who initially presented with poor weight gain, microcephaly and increased tone. He was confirmed to harbor the common splice site mutation c.2715 + 1G > A and the nonsense variant c.2404C > T (p.Q802*). Clinically, the patient had multiple phenotypic features typical of mucopolysaccharidosis including joint contractures, coarse facial features, kypho-lordosis, pectus carinatum and umbilical hernia. However, the relatively mild developmental delay compared to severe type I and type II mucopolysaccharidosis and the absence of macrocephaly raised the possibility of the less commonly diagnosed mucolipidosis alpha/beta. Critical roles of lysosomal enzyme activity assay, which showed elevated α-iduronidase, iduronate sulfatase, galactose-6-sulphate sulphatase, arylsulfatase B and α-hexosaminidase activities; and genetic study, which confirmed the parental origin of both mutations, were highlighted. CONCLUSIONS: The recently reported nonsense variant c.2404C > T in the GNPTAB gene is further recognized and this contributes to the genotype-phenotype spectrum of mucolipidosis alpha/beta.

Rapid Identification of Pathogenic Variants in Two Cases of Charcot-Marie-Tooth Disease by Gene-Panel Sequencing.

Charcot-Marie-Tooth disease (CMT) is a common inherited peripheral neuropathy affecting up to 1 in 1214 of the general population with more than 60 nuclear genes implicated in its pathogenesis. Traditional molecular diagnostic pathways based on relative prevalence and clinical phenotyping are limited by long turnaround time, population-specific prevalence of causative variants and inability to assess multiple co-existing variants. In this study, a CMT gene panel comprising 27 genes was used to uncover the pathogenic mutations in two index patients. The first patient is a 15-year-old boy, born of consanguineous parents, who has had frequent trips and falls since infancy, and was later found to have inverted champagne bottle appearance of bilateral legs and foot drop. His elder sister is similarly affected. The second patient is a 37-year-old woman referred for pre-pregnancy genetic diagnosis. During early adulthood, she developed progressive lower limb weakness, difficulties in tip-toe walking and thinning of calf muscles. Both patients are clinically compatible with CMT, have undergone multiple genetic testings and have not previously received a definitive genetic diagnosis. Patients 1 and 2 were found to have pathogenic homozygous HSPB1:NM_001540:c.250G>A (p.G84R) variant and heterozygous GDAP1:NM_018972:c.358C>T (p.R120W) variant, respectively. Advantages and limitations of the current approach are discussed.

Impacts of semi-transparent window on reflectance from metallic gratings at oblique incidence.

This work both numerically and experimentally investigates the reflectance from the smooth side of a semi-transparent 508-µm-thick window with metallic gratings attached on the other side. The incident light is oblique and linearly polarized, and its wavelength ranges from 2.5 to 25 µm. Three gratings with various levels of profile complexity and periods are investigated. Influences from the intrinsic material absorption, light refraction, total internal reflection, interference, and diffraction on both the specular and hemispherical reflectance are studied. Moreover, the applicability of effective medium theory to our samples is verified.

Automated pangenomic analysis in target selection for PCR detection and identification of bacteria by use of ssGeneFinder Webserver and its application to Salmonella enterica serovar Typhi.

With the advent of high-throughput DNA sequencing, more than 4,000 bacterial genomes have been sequenced and are publicly available. We report a user-friendly web platform, ssGeneFinder Webserver (http://147.8.74.24/ssGeneFinder/), which is updated weekly for the automated pangenomic selection of specific targets for direct PCR detection and the identification of clinically important bacteria without the need of gene sequencing. To apply the ssGeneFinder Webserver for identifying specific targets for Salmonella enterica serovar Typhi, we analyzed 11 S. Typhi genomes, generated two specific targets, and validated them using 40 S. Typhi, 110 non-Typhi Salmonella serovars (serovar Paratyphi A, n = 4; Paratyphi B, n = 1; Typhimurium, n = 5; Enteritidis, n = 12; non-Paratyphi group A, n = 6; non-Paratyphi group B, n = 29; non-Paratyphi group C, n = 12; non-Typhi group D, n = 35; group E and others, n = 6), 115 Enterobacteriaceae isolates (Escherichia, n = 78; Shigella, n = 2; Klebsiella, n = 13; Enterobacter, n = 9; others, n = 13), and 66 human stool samples that were culture negative for S. Typhi. Both targets successfully detected all typical and atypical S. Typhi isolates, including an H1-j flagellin gene mutant, an aflagellated mutant which reacted with 2O Salmonella antiserum, and the Vi-negative attenuated vaccine strain Ty21a. No false positive was detected from any of the bacterial isolates and stool samples. DNA sequencing confirmed the identity of all positive amplicons. The PCR assays have detection limits as low as 100 CFU per reaction and were tested using spiked stool samples. Using a pangenomic approach, ssGeneFinder Webserver generated targets specific to S. Typhi. These and other validated targets should be applicable to the identification and direct PCR detection of bacterial pathogens from uncultured, mixed, and environmental samples.

Novel pan-genomic analysis approach in target selection for multiplex PCR identification and detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia complex species: a proof-of-concept study.

Burkholderia pseudomallei, Burkholderia thailandensis, and the Burkholderia cepacia complex differ greatly in pathogenicity and epidemiology. Yet, they are occasionally misidentified by biochemical profiling, and even 16S rRNA gene sequencing may not offer adequate discrimination between certain species groups. Using the 23 B. pseudomallei, four B. thailandensis, and 16 B. cepacia complex genome sequences available, we identified gene targets specific to each of them (a Tat domain protein, a 70-kDa protein, and a 12-kDa protein for B. pseudomallei, B. thailandensis, and the B. cepacia complex, respectively), with an in-house developed algorithm. Using these targets, we designed a robust multiplex PCR assay useful for their identification and detection from soil and simulated sputum samples. For all 43 B. pseudomallei, seven B. thailandensis, and 20 B. cepacia complex (B. multivorans, n = 6; B. cenocepacia, n = 3; B. cepacia, n = 4; B. arboris, n = 2; B. contaminans, B. anthina, and B. pyrrocinia, n = 1 each; other unnamed members, n = 2) isolates, the assay produced specific products of predicted size without false positives or negatives. Of the 60 soil samples screened, 19 (31.6%) and 29 (48.3%) were positive for B. pseudomallei and the B. cepacia complex, respectively, and in four (6.7%) soil samples, the organisms were codetected. DNA sequencing confirmed that all PCR products originated from their targeted loci. This novel pan-genomic analysis approach in target selection is simple, computationally efficient, and potentially applicable to any species that harbors species-specific genes. A multiplex PCR assay for rapid and accurate identification and detection of B. pseudomallei, B. thailandensis, and the B. cepacia complex was developed and verified.

Clinical Mass Spectrometry in the Bioinformatics Era: A Hitchhiker's Guide.

Mass spectrometry (MS) is a sensitive, specific and versatile analytical technique in the clinical laboratory that has recently undergone rapid development. From initial use in metabolic profiling, it has matured into applications including clinical toxicology assays, target hormone and metabolite quantitation, and more recently, rapid microbial identification and antimicrobial resistance detection by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In this mini-review, we first succinctly outline the basics of clinical mass spectrometry. Examples of hard ionization (electron ionization) and soft ionization (electrospray ionization, MALDI) are presented to demonstrate their clinical applications. Next, a conceptual discourse on mass selection and determination is presented: quadrupole mass filter, time-of-flight mass spectrometer and the Orbitrap; and MS/MS (tandem-in-space, tandem-in-time and data acquisition), illustrated with clinical examples. Current applications in (1) bacterial and fungal identification, antimicrobial susceptibility testing and phylogenetic classification, (2) general unknown urine toxicology screening and expanded new-born metabolic screening and (3) clinical metabolic profiling by gas chromatography are outlined. Finally, major limitations of MS-based techniques, including the technical challenges of matrix effect and isobaric interference; and novel challenges in the post-genomic era, such as protein molecular variants, are critically discussed from the perspective of service laboratories. Computer technology and structural biology have played important roles in the maturation of this field. MS-based techniques have the potential to replace current analytical techniques, and existing expertise and instrument will undergo rapid evolution. Significant automation and adaptation to regulatory requirements are underway. Mass spectrometry is unleashing its potentials in clinical laboratories.

Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C).

Thiopurine intolerance and treatment-related toxicity, such as fatal myelosuppression, is related to non-function genetic variants encoding thiopurine S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15). Genetic testing of the common variants NUDT15:NM_018283.2:c.415C>T (Arg139Cys, dbSNP rs116855232 T allele) and TPMT: NM_000367.4:c.719A>G (TPMT*3C, dbSNP rs1142345 G allele) in East Asians including Chinese can potentially prevent treatment-related complications. Two complementary genotyping approaches, real-time PCR-high resolution melt (PCR-HRM) and PCR-restriction fragment length morphism (PCR-RFLP) analysis were evaluated using conventional PCR and Sanger sequencing genotyping as the gold standard. Sixty patient samples were tested, revealing seven patients (11.7%) heterozygous for NUDT15 c.415C>T, one patient homozygous for the variant and one patient heterozygous for the TPMT*3C non-function allele. No patient was found to harbor both variants. In total, nine out of 60 (15%) patients tested had genotypic evidence of thiopurine intolerance, which may require dosage adjustment or alternative medication should they be started on azathioprine, mercaptopurine or thioguanine. The two newly developed assays were more efficient and showed complete concordance (60/60, 100%) compared to the Sanger sequencing results. Accurate and cost-effective genotyping assays by real-time PCR-HRM and PCR-RFLP for NUDT15 c.415C>T and TPMT*3C were successfully developed. Further studies may establish their roles in genotype-informed clinical decision-making in the prevention of morbidity and mortality due to thiopurine intolerance.

Novel Tetra-Primer ARMS-PCR Assays for Thiopurine Intolerance Susceptibility Mutations NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C) in East Asians.

Thiopurines are clinically useful in the management of diverse immunological and malignant conditions. Nevertheless, these purine analogues can cause lethal myelosuppression, which may be prevented by prospective testing for variants in the thiopurine S-methyltransferase (TPMT) and, in East Asians, Nudix hydrolase 15 (NUDT15) genes. Two single-tube, tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR) assays were developed to genotype the common loss-of-function variants NUDT15 c.415C>T (rs116855232) and TPMT*3C c.719A>G (rs1142345). In a group of 60 unselected patients, one and seven were found to be homozygous and heterozygous, respectively, for NUDT15 c.415C>T; one was found to be heterozygous for TPMT*3C c.719A>G. There was no non-specific amplification, and the genotypes were 100% concordant with Sanger sequencing. Limit-of-detection for both assays was below 1 ng of heterozygous template per reaction. Time- and cost-effective ARMS-PCR assays, suitable for genotyping East-Asian patients for thiopurine intolerance, were successfully developed and validated.

Laboratory diagnosis of melioidosis: past, present and future.

Melioidosis is an emerging, potentially fatal disease caused by Burkholderia pseudomallei, which requires prolonged antibiotic treatment to prevent disease relapse. However, difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes. Isolation of B. pseudomallei from clinical specimens has been improved with the use of selective media. However, even with positive cultures, identification of B. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. Commercial identification systems may fail to distinguish between B. pseudomallei and closely related species such as Burkholderia thailandensis. Genotypic identification of suspected isolates can be achieved by sequencing of gene targets such as groEL which offer higher discriminative power than 16S rRNA. Specific PCR-based identification of B. pseudomallei has also been developed using B. pseudomallei-specific gene targets such as Type III secretion system and Tat-domain protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolutionary technique for pathogen identification, has been shown to be potentially useful for rapid identification of B. pseudomallei, although existing databases require optimization by adding reference spectra for B. pseudomallei. Despite these advances in bacterial identification, diagnostic problems encountered in culture-negative cases remain largely unresolved. Although various serological tests have been developed, they are generally unstandardized "in house" assays and have low sensitivities and specificities. Although specific PCR assays have been applied to direct clinical and environmental specimens, the sensitivities for diagnosis remain to be evaluated. Metabolomics is an uprising tool for studying infectious diseases and may offer a novel approach for exploring potential diagnostic biomarkers. The metabolomics profiles of B. pseudomallei culture supernatants can be potentially distinguished from those of related bacterial species including B. thailandensis . Further studies using bacterial cultures and direct patient samples are required to evaluate the potential of metabolomics for improving diagnosis of melioidosis.

Burkholderia pseudomallei in soil samples from an oceanarium in Hong Kong detected using a sensitive PCR assay.

Melioidosis, caused by Burkholderia pseudomallei, is an emerging infectious disease with an expanding geographical distribution. Although assessment of the environmental load of B. pseudomallei is important for risk assessment in humans or animals in endemic areas, traditional methods of bacterial culture for isolation have low sensitivities and are labor-intensive. Using a specific polymerase chain reaction (PCR) assay targeting a Tat domain protein in comparison with a bacterial culture method, we examined the prevalence of B. pseudomallei in soil samples from an oceanarium in Hong Kong where captive marine mammals and birds have contracted melioidosis. Among 1420 soil samples collected from various sites in the oceanarium over a 15-month period, B. pseudomallei was detected in nine (0.6%) soil samples using bacterial culture, whereas it was detected in 96 (6.8%) soil samples using the specific PCR assay confirmed by sequencing. The PCR-positive samples were detected during various months, with higher detection rates observed during summer months. Positive PCR detection was significantly correlated with ambient temperature (P<0.0001) and relative humidity (P=0.011) but not with daily rainfall (P=0.241) or a recent typhoon (P=0.787). PCR-positive samples were obtained from all sampling locations, with the highest detection rate in the valley. Our results suggest that B. pseudomallei is prevalent and endemic in the oceanarium. The present PCR assay is more sensitive than the bacterial culture method, and it may be used to help better assess the transmission of melioidosis and to design infection control measures for captive animals in this unique and understudied environment.

Romance of the three domains: how cladistics transformed the classification of cellular organisms.

Cladistics is a biological philosophy that uses genealogical relationship among species and an inferred sequence of divergence as the basis of classification. This review critically surveys the chronological development of biological classification from Aristotle through our postgenomic era with a central focus on cladistics. In 1957, Julian Huxley coined cladogenesis to denote splitting from subspeciation. In 1960, the English translation of Willi Hennig's 1950 work, Systematic Phylogenetics, was published, which received strong opposition from pheneticists, such as numerical taxonomists Peter Sneath and Robert Sokal, and evolutionary taxonomist, Ernst Mayr, and sparked acrimonious debates in 1960-1980. In 1977-1990, Carl Woese pioneered in using small subunit rRNA gene sequences to delimitate the three domains of cellular life and established major prokaryotic phyla. Cladistics has since dominated taxonomy. Despite being compatible with modern microbiological observations, i.e. organisms with unusual phenotypes, restricted expression of characteristics and occasionally being uncultivable, increasing recognition of pervasiveness and abundance of horizontal gene transfer has challenged relevance and validity of cladistics. The mosaic nature of eukaryotic and prokaryotic genomes was also gradually discovered. In the mid-2000s, high-throughput and whole-genome sequencing became routine and complex geneologies of organisms have led to the proposal of a reticulated web of life. While genomics only indirectly leads to understanding of functional adaptations to ecological niches, computational modeling of entire organisms is underway and the gap between genomics and phenetics may soon be bridged. Controversies are not expected to settle as taxonomic classifications shall remain subjective to serve the human scientist, not the classified.

Identification of a novel bat papillomavirus by metagenomics.

The discovery of novel viruses in animals expands our knowledge of viral diversity and potentially emerging zoonoses. High-throughput sequencing (HTS) technology gives millions or even billions of sequence reads per run, allowing a comprehensive survey of the genetic content within a sample without prior nucleic acid amplification. In this study, we screened 156 rectal swab samples from apparently healthy bats (n = 96), pigs (n = 9), cattles (n = 9), stray dogs (n = 11), stray cats (n = 11) and monkeys (n = 20) using a HTS metagenomics approach. The complete genome of a novel papillomavirus (PV), Miniopterus schreibersii papillomavirus type 1 (MscPV1), with L1 of 60% nucleotide identity to Canine papillomavirus (CPV6), was identified in a specimen from a Common Bent-wing Bat (M. schreibersii). It is about 7.5kb in length, with a G+C content of 45.8% and a genomic organization similar to that of other PVs. Despite the higher nucleotide identity between the genomes of MscPV1 and CPV6, maximum-likelihood phylogenetic analysis of the L1 gene sequence showed that MscPV1 and Erethizon dorsatum papillomavirus (EdPV1) are most closely related. Estimated divergence time of MscPV1 from the EdPV1/MscPV1 common ancestor was approximately 60.2-91.9 millions of years ago, inferred under strict clocks using the L1 and E1 genes. The estimates were limited by the lack of reliable calibration points from co-divergence because of possible host shifts. As the nucleotide sequence of this virus only showed limited similarity with that of related animal PVs, the conventional approach of PCR using consensus primers would be unlikely to have detected the novel virus in the sample. Unlike the first bat papillomavirus RaPV1, MscPV1 was found in an asymptomatic bat with no apparent mucosal or skin lesions whereas RaPV1 was detected in the basosquamous carcinoma of a fruit bat Rousettus aegyptiacus. We propose MscPV1 as the first member of the novel Dyolambda-papillomavirus genus.