Updated Confirmatory Diagnosis for Mucopolysaccharidoses in Taiwanese Infants and the Application of Gene Variants.

Mucopolysaccharidosis (MPS) is a lysosomal storage disease caused by genetic defects that result in deficiency of one specific enzyme activity, consequently impairing the stepwise degradation of glycosaminoglycans (GAGs). Except for MPS II, the other types of MPS have autosomal recessive inheritance in which two copies of an abnormal allele must be present in order for the disease to develop. In this study, we present the status of variant alleles and biochemistry results found in infants suspected of having MPS I, II, IVA, and VI. A total of 324 suspected infants, including 12 for MPS I, 223 for MPS II, 72 for MPS IVA, and 17 for MPS VI, who were referred for MPS confirmation from newborn screening centers in Taiwan, were enrolled. In all of these infants, one specific enzyme activity in dried blood spot filter paper was lower than the cut-off value in the first blood sample, as well asin a second follow-up sample. The confirmatory methods used in this study included Sanger sequencing, next-generation sequencing, leukocyte enzyme fluorometric assay, and GAG-derived disaccharides in urine using tandem mass spectrometry assays. The results showed that five, nine, and six infants had MPS I, II, and IVA, respectively, and all of them were asymptomatic. Thus, a laboratory diagnosis is extremely important to confirm the diagnosis of MPS. The other infants with identified nucleotide variations and reductions in leukocyte enzyme activities were categorized as being highly suspected cases requiring long-term and intensive follow-up examinations. In summary, the final confirmation of MPS depends on the most powerful biomarkers found in urine, i.e., the quantification of GAG-derived disaccharides including dermatan sulfate, heparan sulfate, and keratan sulfate, and analysis of genetic variants can help predict outcomes and guide treatment.

Taiwan National Newborn Screening Program by Tandem Mass Spectrometry for Mucopolysaccharidoses Types I, II, and VI.

OBJECTIVE: To evaluate the initial cutoff values, rates of screen positives, and genotypes for the large-scale newborn screening program for multiple mucopolysaccharidoses (MPS) in Taiwan. STUDY DESIGN: More than 100 000 dried blood spots were collected consecutively as part of the national Taiwan newborn screening programs. Enzyme activities were measured by tandem mass spectrometry from dried blood spot punches. Genotypes were obtained when a second newborn screening specimen again had a decreased enzyme activity. Additional clinical evaluation was then initiated based on enzyme activity and/or genotype. RESULTS: Molecular genetic analysis for cases with low enzyme activity revealed 5 newborns with pathogenic alpha-L-iduronidase mutations, 3 newborns with pathogenic iduronate-2-sulfatase mutations, and 1 newborn was a carrier of an arylsulfatase B mutation. Several variants of unknown pathogenic significance were also identified, most likely causing pseudodeficiency. CONCLUSIONS: The highly robust tandem mass spectrometry-based enzyme assays for MPS-I, MPS-II, and MPS-VI allow for high-throughput newborn screening for these lysosomal storage disorders. Optimized cutoff values combined with second tier testing could largely eliminate false-positive results. Accordingly, newborn screening for these lysosomal storage disorders is possible.

Distribution of Staphylococcal Cassette Chromosome (SCC) mec Element Types in Fusidic Acid-Resistant Staphylococcus epidermidis and Identification of a Novel SCC7684 Element.

We analyzed the staphylococcal cassette chromosome mec (SCCmec) types of 143 fusidic acid- and methicillin-resistant Staphylococcus epidermidis isolates. The most frequent SCCmec type was SCCmec III/SCCHg (53%), followed by SCCmec IV (29%). Clonal spreading of SCCmec III/SCCHg strains contributed to the increased prevalence of SCCmec III. A novel non-mec SCC structure, SCC7684, adjacent to SCCmec III, which carries a new ccrC allotype (ccrC3 allele 1) and contains heavy metal resistance genes, was identified in 14 isolates.

Emergence of a small colony variant of vancomycin-intermediate Staphylococcus aureus in a patient with septic arthritis during long-term treatment with daptomycin.

OBJECTIVES: Small colony variants (SCVs) of Staphylococcus aureus are associated with persistent and drug-resistant infections. We demonstrated for the first time the emergence of SCVs in a patient with vancomycin-intermediate S. aureus (VISA) infection during long-term treatment with daptomycin. METHODS: A 73-year-old man with septic arthritis was infected with VISA. The patient was treated with daptomycin; however, the patient remained infected with VISA, with continuous isolation of VISA from his blood during long-term treatment. Five VISA isolates were characterized by: PFGE; genotyping including staphylococcal cassette chromosome mec (SCCmec), spa and MLST; antimicrobial susceptibility testing; and scanning and transmission electron microscopy. WGS and fatty acid analysis were also performed. RESULTS: The five VISA isolates were from a single clone of ST239/spa3(t037) and, of these, the first three were SCCmecIII positive and daptomycin susceptible, whereas the last two were SCCmecIII negative and daptomycin resistant and exhibited the characteristics of SCVs. The first and last isolates showed 13 remarkable genetic differences in SCCmec and the mprF, cls2, clpX and fabF genes. Of these, mutation of fabF (encoding the fatty acid synthase) seemed to be partially responsible for the slow growth and ultrastructural features, including an abnormal intercellular substance, and for the daptomycin resistance of SCVs. CONCLUSIONS: For the first time, we identified SCVs of VISA in a patient with septic arthritis during long-term treatment with daptomycin. Daptomycin-resistant SCVs of VISA were evolved in a stepwise manner and the mutation of fabF is likely responsible for the physical and ultrastructural characteristics and daptomycin resistance.

A novel fusidic acid resistance determinant, fusF, in Staphylococcus cohnii.

OBJECTIVES: To determine MICs of fusidic acid for and identify genetic determinants of resistance in Staphylococcus cohnii isolates. METHODS: Susceptibility to fusidic acid was determined by the standard agar dilution method in 24 S. cohnii subsp. urealyticus clinical isolates, 7 S. cohnii subsp. cohnii clinical isolates and 2 reference strains. Sequencing of a novel resistance determinant, fusF, and its flanking regions was performed by long and accurate PCR and inverse PCR. To evaluate the function of fusF, the MIC of fusidic acid was determined for recombinant Staphylococcus aureus carrying a plasmid expressing fusF. RESULTS: A total of 25 S. cohnii subsp. urealyticus (24 clinical isolates and 1 reference strain) and 2 S. cohnii subsp. cohnii displayed low-level resistance to fusidic acid (MICs 2-16 mg/L). Sequencing of a 4259 bp fragment from S. cohnii subsp. urealyticus ATCC 49330 revealed a novel resistance gene, designated fusF, which displayed 70.5% nucleotide and 67.3% amino acid identity to fusD. Expression of fusF in S. aureus confers resistance to fusidic acid. CONCLUSIONS: A novel FusB-family gene, fusF, was identified as a major resistance determinant in S. cohnii clinical isolates resistant to fusidic acid.

A novel staphylococcal cassette chromosomal element, SCCfusC, carrying fusC and speG in fusidic acid-resistant methicillin-resistant Staphylococcus aureus.

A high prevalence of fusC (16/46, 59%) was found in fusidic acid-resistant methicillin-resistant Staphylococcus aureus isolates collected from 2008 to 2010. Nucleotide sequencing of fusC and flanking regions revealed a novel staphylococcal cassette chromosome (SCC) structure, SCCfusC, which was integrated into rlmH and located upstream from SCCmec. The SCCfusC element contained speG, which may contribute to the polyamine resistance.

Gemella parahaemolysans sp. nov. and Gemella taiwanensis sp. nov., isolated from human clinical specimens.

Four Gram-staining-positive, catalase-negative, coccoid isolates, designated NTUH_1465(T), NTUH_2196, NTUH_4957 and NTUH_5572(T), were isolated from human specimens. The four isolates displayed more than 99.6% 16S rRNA gene sequence similarity with Gemella haemolysans ATCC 10379(T), and 96.7 to 98.6% similarity with Gemella sanguinis ATCC 700632(T), Gemella morbillorum ATCC 27824(T) or Gemella cuniculi CCUG 42726(T). However, phylogenetic analysis of concatenated sequences of three housekeeping genes, groEL, rpoB and recA, suggested that the four isolates were distinct from G. haemolysans ATCC 10379(T) and other species. Isolates NTUH_2196, NTUH_4957 and NTUH_5572(T) clustered together and formed a stable monophyletic clade. DNA-DNA hybridization values among strains NTUH_1465(T) and NTUH_5572(T) and their phylogenetically related neighbours were all lower than 49%. The four isolates could be distinguished from G. haemolysans and other species by phenotypic characteristics. Based on the phylogenetic and phenotypic results, two novel species Gemella parahaemolysans sp. nov. (type strain NTUH_1465(T) = BCRC 80365(T) = JCM 18067(T)) and Gemella taiwanensis sp. nov. (type strain NTUH_5572(T) = BCRC 80366(T) = JCM 18066(T)) are proposed.

New structure of phage-related islands carrying fusB and a virulence gene in fusidic acid-resistant Staphylococcus epidermidis.

Nucleotide sequencing of the fusB-flanking regions in two fusidic acid-resistant Staphylococcus epidermidis isolates with the type IV aj1-leader peptide (LP)-fusB structure (lacking aj1) revealed that their fusB gene was located on novel phage-related islands inserted downstream of smpB and are here referred to as SeRIfusB-3692 and SePIfusB-857. The novel SePIfusB-857 structure was followed by SeCI857, forming a composite pathogenicity island which contained a putative virulence gene, vapE. The linkage of fusB and vapE may contribute to bacterial adaption.

Associations between Early Thyroid-Stimulating Hormone Levels and Morbidities in Extremely Preterm Neonates.

INTRODUCTION: High-end cutoffs of thyroid-stimulating hormone (TSH) have been emphasized for hypothyroidism therapy in extremely preterm infants, but the significance of low TSH levels remains unknown. This study hypothesized that the spectrum of TSH levels by newborn screening after birth signifies specific morbidities in extremely preterm neonates. METHODS: The multicenter population cohort analyzed 434 extremely preterm neonates receiving TSH screening at 24-96 h of age in 2008-2019. Neonates were categorized by blood TSH levels into group 1: TSH <0.5 µU/mL, group 2: 0.5 ≤ TSH <2 µU/mL, group 3: 2 ≤ TSH <4 µU/mL, and group 4: TSH ≥4 µU/mL. Neonatal morbidities were categorized using the modified Neonatal Therapeutic Intervention Scoring System. RESULTS: The four groups differed in gestational age, birth weight, and the postnatal age at blood sampling so did the proportions of mechanical ventilation usage (p = 0.01), hypoxic respiratory failure (p = 0.005), high-grade intraventricular hemorrhage (p = 0.007), and periventricular leukomalacia (p = 0.048). Group 1 had higher severity scores for respiratory distress syndrome (RDS; effect size 0.39 [95% confidence interval [CI]: 0.18-0.59]) and brain injury (0.36 [0.15-0.57]) than group 2, which remained significant after adjusting for gestational age, birth weight, dopamine usage, and the postnatal age at TSH screening (RDS: mean + 0.45 points [95% CI: 0.11-0.79]; brain injury: +0.32 [0.11-0.54]). CONCLUSIONS: Low TSH levels in extremely preterm neonates are associated with severe RDS and brain injuries. Studies recruiting more neonates with complete thyroid function data are necessary to understand central-peripheral interactions of the hypothalamic-pituitary-thyroid axis.

Multiplex Lysosomal Enzyme Activity Assay on Dried Blood Spots Using Tandem Mass Spectrometry.

Deficiencies of the enzymes in lysosomes result in the accumulation of undegraded materials and subsequently cellular dysfunction. Early identification of deficiencies can lead to better clinical outcomes before irreversible organ and tissue damages occur. In this chapter, lysosomal enzymes are extracted from dried blood spots and incubated with the commercialized and multiplexed enzyme cocktail containing corresponding substrates and internal standards. After incubation, the enzymatic reactions are quenched, and the mixtures of the reaction products are prepared using liquid/liquid extractions. Multiple enzymes are quantified simultaneously using selected ion monitoring on liquid chromatography-mass spectrometry (LC-MS/MS) system.

Newborn Screening Program for Mucopolysaccharidosis Type II and Long-Term Follow-Up of the Screen-Positive Subjects in Taiwan.

BACKGROUND: Mucopolysaccharidosis II (MPS II) is an X-linked disorder resulting from a deficiency in lysosomal enzyme iduronate-2-sulfatase (IDS), which causes the accumulation of glycosaminoglycans (GAGs) in the lysosomes of many tissues and organs, leading to progressive cellular dysfunction. An MPS II newborn screening program has been available in Taiwan since 2015. The aim of the current study was to collect and analyze the long-term follow-up data of the screen-positive subjects in this program. METHODS: From August 2015 to April 2022, 548,624 newborns were screened for MPS II by dried blood spots using tandem mass spectrometry, of which 202 suspected infants were referred to our hospital for confirmation. The diagnosis of MPS II was confirmed by IDS enzyme activity assay in leukocytes, quantitative determination of urinary GAGs by mass spectrometry, and identification of the IDS gene variant. RESULTS: Among the 202 referred infants, 10 (5%) with seven IDS gene variants were diagnosed with confirmed MPS II (Group 1), 151 (75%) with nine IDS gene variants were classified as having suspected MPS II or pseudodeficiency (Group 2), and 41 (20%) with five IDS gene variants were classified as not having MPS II (Group 3). Long-term follow-up every 6 months was arranged for the infants in Group 1 and Group 2. Intravenous enzyme replacement therapy (ERT) was started in four patients at 1, 0.5, 0.4, and 0.5 years of age, respectively. Three patients also received hematopoietic stem cell transplantation (HSCT) at 1.5, 0.9, and 0.6 years of age, respectively. After ERT and/or HSCT, IDS enzyme activity and the quantity of urinary GAGs significantly improved in all of these patients compared with the baseline data. CONCLUSIONS: Because of the progressive nature of MPS II, early diagnosis via a newborn screening program and timely initiation of ERT and/or HSCT before the occurrence of irreversible organ damage may lead to better clinical outcomes. The findings of the current study could serve as baseline data for the analysis of the long-term effects of ERT and HSCT in these patients.

Liquid Chromatography-Tandem Mass Spectrometry in Newborn Screening Laboratories.

Tandem mass spectrometry (MS/MS) is the most universal platform currently available for the analysis of enzymatic activities and biomarkers in dried blood spots (DBS) for applications in newborn screening (NBS). Among the MS/MS applications in NBS, the most common is flow-injection analysis (FIA-) MS/MS, where the sample is introduced as a bolus injection into the mass spectrometer without the prior fractionation of analytes. Liquid chromatography combined with MS/MS (LC-MS/MS) has been employed for second-tier tests to reduce the false-positive rate associated with several nonspecific screening markers, beginning two decades ago. More recently, LC-MS/MS has been applied to primary screening for new conditions for which FIA-MS/MS or other methods, including genomic screening, are not yet adequate. In addition to providing a list of the currently used LC-MS/MS-based assays for NBS, the authors share their experience regarding the maintenance requirements of LC-MS/MS vs. FIA-MS/MS systems. The consensus is that the maintenance of LC-MS/MS and FIA-MS/MS instrumentation is similar, and LC-MS/MS has the advantage of allowing for a larger number of diseases to be screened for in a multiplex, cost-effective fashion with a high throughput and an adequate turnaround time.

Identification of fusB-mediated fusidic acid resistance islands in Staphylococcus epidermidis isolates.

To understand the high prevalence of fusB genes in fusidic acid-resistant Staphylococcus epidermidis, analysis of resistance elements in 34 isolates was performed. First, sequence analysis of the aj1-LP-fusB region indicated that at least three types were present. Type I contained full-length aj1, type II contained a partial aj1 truncated from nucleotide position 93 to 421, and type III contained a more truncated aj1 that retained only the last 37 bp. Isolates with type I or type II aj1 displayed slightly higher levels of resistance to fusidic acid (MICs, 8 to 32 µg/ml) than did those with type III aj1 (MICs, 4 to 16 µg/ml). Subsequent sequencing of the flanking regions of fusB from four selected isolates carrying different types of aj1-LP-fusB regions revealed that the fusB genes were all located on phage-related resistance islands (RIs), referred to as SeRI(fusB)(-2793), SeRI(fusB)(-704), SeRI(fusB)(-5907), and SeRI(fusB)(-7778), respectively. Among them, three islands (SeRI(fusB)(-2793), SeRI(fusB)(-704), and SeRI(fusB)(-5907)) were located downstream of groEL (corresponding to the 44-min position based on Staphylococcus aureus whole genomic sequences), and one (SeRI(fusB)(-7778)) was located downstream of rpsR (corresponding to the 8-min position). All of the RIs were inserted into integrase-recognized att sites. Among 34 isolates, the insertion sites of fusB RIs were mostly (28/34, 82%) located downstream of groEL and two were located downstream of rpsR, but four remained unidentified. The pulsotype distribution indicated that fusB-containing S. epidermidis isolates were heterogeneous. In conclusion, the fusB resistance determinant in S. epidermidis was highly associated with phage-related RIs. This is the first report of fusB RI in S. epidermidis.

Nationwide Newborn Screening Program for Mucopolysaccharidoses in Taiwan and an Update of the "Gold Standard" Criteria Required to Make a Confirmatory Diagnosis.

Mucopolysaccharidoses (MPSs) are a group of lysosomal storage diseases (LSDs) caused by an inherited gene defect. MPS patients can remain undetected unless the initial signs or symptoms have been identified. Newborn screening (NBS) programs for MPSs have been implemented in Taiwan since 2015, and more than 48.5% of confirmed cases of MPS have since been referred from these NBS programs. The purpose of this study was to report the current status of NBS for MPSs in Taiwan and update the gold standard criteria required to make a confirmative diagnosis of MPS, which requires the presence of the following three laboratory findings: (1) elevation of individual urinary glycosaminoglycan (GAG)-derived disaccharides detected by MS/MS-based assay; (2) deficient activity of a particular leukocyte enzyme by fluorometric assay; and (3) verification of heterogeneous or homogeneous variants by Sanger sequencing or next generation sequencing. Up to 30 April 2021, 599,962 newborn babies have been screened through the NBS programs for MPS type I, II, VI, and IVA, and a total of 255 infants have been referred to MacKay Memorial Hospital for a confirmatory diagnosis. Of these infants, four cases were confirmed to have MPS I, nine cases MPS II, and three cases MPS IVA, with prevalence rates of 0.67, 2.92, and 4.13 per 100,000 live births, respectively. Intensive long-term regular physical and laboratory examinations for asymptomatic infants with confirmed MPS or with highly suspected MPS can enhance the ability to administer ERT in a timely fashion.

Fusidic acid resistance determinants in Staphylococcus aureus clinical isolates.

A total of 71 fusidic acid-resistant Staphylococcus aureus (45 methicillin-resistant and 26 methicillin-susceptible) isolates were examined for the presence of resistance determinants. Among 45 fusidic acid-resistant methicillin-resistant S. aureus (MRSA), isolates, 38 (84%) had fusA mutations conferring high-level resistance to fusidic acid (the MIC was ≥128 µg/ml for 22/38), none had fusB, and 7 (16%) had fusC. For 26 fusidic acid-resistant methicillin-susceptible S. aureus (MSSA), only 3 possessed fusA mutations, but 15 (58%) had fusB and 8 (31%) had fusC. Low-level resistance to fusidic acid (MICs ≤ 32 µg/ml) was found in most fusB- or fusC-positive isolates. For 41 isolates (38 MRSA and 3 MSSA), with fusA mutations, a total of 21 amino acid substitutions in EF-G (fusA gene) were detected, of which R76C, E444K, E444V, C473S, P478S, and M651I were identified for the first time. The nucleotide sequencing of fusB and flanking regions in an MSSA isolate revealed the structure of partial IS257-aj1-LP-fusB-aj2-aj3-IS257-partial blaZ, which is identical to the corresponding region in pUB101, and the rest of fusB-carrying MSSA isolates also show similar structures. On the basis of spa and staphylococcal cassette chromosome mec element (SCCmec) typing, two major genotypes, spa type t037-SCCmec type III (t037-III; 28/45; 62%) and t002-II (13/45; 29%), were predominant among 45 MRSA isolates. By pulsed-field gel electrophoresis analysis, 45 MRSA isolates were divided into 12 clusters, while 26 MSSA isolates were divided into 15 clusters. Taken together, the distribution of fusidic acid resistance determinants (fusA mutations, fusB, and fusC) was quite different between MRSA and MSSA groups.

Distribution of emm types and genetic characterization of the mgc locus in group G Streptococcus dysgalactiae subsp. equisimilis from a hospital in northern Taiwan.

A total of 274 Streptococcus dysgalactiae subsp. equisimilis isolates was analyzed by emm typing and by determining the organization of their mgrC loci. Three of the most frequent emm types were stG485.0 (45/274, 16.4%), stG6.1 (43/274, 15.7%), and stC839.0 (32/274, 11.7%), in decreasing order. The cpdB-positive mgrC locus appears to be predominant in some emm types.

Use of groESL as a target for identification of Abiotrophia, Granulicatella, and Gemella species.

We determined the groESL sequences of three species of nutritionally variant streptococci (Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans) and three Gemella species (Gemella morbillorum, Gemella haemolysans, and Gemella sanguinis). The nucleotide sequence similarities between the groES and groEL genes of the above genera were 41.7 to 85.9% and 63.7 to 84.3%, respectively. The intraspecies similarities of groESL sequences for the isolates of Abiotrophia and Granulicatella species were 94.4 to 97.8% for groES and 94.0 to 98.2% for groEL. For Ge. morbillorum and Ge. sanguinis, all strains showed the same groESL spacer length (8 bp), and sequence identities within species were >97.8% for groES and >96.1% for groEL. However, higher intraspecies heterogeneity was observed in Ge. haemolysans. Phylogenetic analysis of groEL sequences separated the 6 isolates of Ge. haemolysans into two subgroups. Among these isolates, three isolates with the same groESL spacer region length (45 bp) clustered together but were distant from the ATCC reference strain (with a spacer length of 8 bp). The remaining three isolates, with a spacer length of 50 or 8 bp, clustered together. Although 16S rRNA gene sequence analysis did not provide enough discrimination for the 6 Ge. haemolysans isolates, rpoB gene sequence analysis supported the subgrouping. Based on the obtained groESL sequences, we developed a multiplex PCR that enables simple, rapid, and accurate identification of Abiotrophia, Granulicatella, and Gemella at the genus level. This assay would be helpful for identifying these fastidious and slow-growing organisms in clinical laboratories.

Prediction of functional consequences of the five newly discovered G6PD variations in Taiwan.

Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency; OMIM #300908) is the most common inborn error disorders worldwide. While the G6PD is the key enzyme of removing oxidative stress in erythrocytes, the early diagnosis is utmost vital to prevent chronic and drug-, food- or infection-induced hemolytic anemia. The characterization of the mutations is also important for the subsequent genetic counseling, especially for female carrier with ambiguous enzyme activities and males with mild mutations. While multiplex SNaPshot assay and Sanger sequencing were performed on 500 G6PD deficient males, five newly discovered variations, namely c.187G > A (p.E63K), c.585G > C (p.Q195H), c.586A > T (p.I196F), c.743G > A (p.G248D), and c.1330G > A (p.V444I) were detected in the other six patients. These variants were previously named as the Pingtung, Tainan, Changhua, Chiayi, and Tainan-2 variants, respectively. The in silico analysis, as well as the prediction of the structure of the resultant mutant G6PD protein indicated that these five newly discovered variants might be disease causing mutations.

Applying a multiplexed primer extension method on dried blood spots increased the detection of carriers at risk of glucose-6-phosphate dehydrogenase deficiency in newborn screening program.

BACKGROUND: Patients with glucose-6-phosphate dehydrogenase deficiency might develop acute hemolytic anemia, chronic hemolytic anemia, and neonatal hyperbilirubinemia when exposed to high levels of oxidative stress. Severe hemolysis may occur in not only patients but also female carriers under certain conditions. However, 80%-85% of female carriers were undetected in an existing newborn screening program because of their wide-ranging levels of enzyme activity. METHODS: We developed a cost- and time-efficient multiplex SNaPshot assay using dried blood spots. RESULTS: By detecting 21 common mutations in Taiwan and Southeast Asia, the assay could determine 98.2% of the mutant alleles in our cohort of Taiwanese newborns. The 9 undetermined mutant alleles were consequently detected by Sanger sequencing, of which 5 unpublished variations-c.187G > A (Pingtung), c.585G > C (Tainan), c.586A > T (Changhua), c.743G > A (Chiayi), and c.1330G > A (Tainan-2)-were detected. Furthermore, 13% of mild mutations were missed in male infants whose enzyme levels at 6.1-7.0 U/gHb in the newborn screening program when set the cutoff value at 6.0 U/gHb. We therefore suggest increasing the cutoff value and applying the multiplex SNaPshot assay as the second tier for neonatal screening. CONCLUSIONS: Our approach could significantly increase the detection rate of male patients and female carriers with a reasonable cost and a reasonable number of clinic referrals.

PCR-RFLP assay for species and subspecies differentiation of the Streptococcus bovis group based on groESL sequences.

The sequence diversity of groESL genes among Streptococcus bovis group isolates was analysed, including five reference strains and 36 clinical isolates. Phylogenetic analysis of the groES and groEL sequences showed that the isolates that belonged to the same species or subspecies usually clustered together. The intergenic spacer region between groES and groEL was variable in size (67-342 bp) and sequence and appeared to be a unique marker for species or subspecies determination. Sequence similarities of the groESL genes among species and subspecies ranged from 84.2 to 99.0 % in groES, and from 88.0 to 99.0 % in groEL. Based on the sequences determined, a Streptococcus bovis group-specific PCR assay was developed, which may provide an alternative means of distinguishing the bovis group from other viridans streptococci. Restriction digestion of the amplicon with AclI further differentiated the species and subspecies.