Longitudinal non-cystic fibrosis trends of pulmonary Mycobacterium abscessus disease from 2010 to 2017: spread of the "globally successful clone" in Asia.

BACKGROUND: Mycobacterium abscessus (MAB) has emerged as the predominant pulmonary non-tuberculous mycobacterial pathogen in parts of Asia, including Taiwan. The reasons for the significant increase in MAB infections in the non-cystic fibrosis (CF) populations are poorly understood. The study aimed to elucidate whether this increase is related to the spread of the globally successful clone of MAB. METHODS: We performed multilocus sequence typing of 371 nonduplicated MAB pulmonary isolates from 371 patients sampled between 2010-2017 at seven hospitals across Taiwan. RESULTS: In total, 183 (49.3%) isolates were M. abscessus subsp. abscessus (MAB-a), 187 (50.4%) were M. abscessus subsp. massiliense (MAB-m), and 1 (0.3%) was M. abscessus subsp. bolletii (MAB-b). MAB-a sequence type (ST)1 (23.7%) and ST127 (3.8%), followed by MAB-m ST48 (16.2%), ST117 (15.1%), ST23 (8.6%) were most common overall. Of MAB-a strains, 50 (27.3%) belonged to novel STs and 38 (10.2%) were singleton strains, while of MAB-m strains, only 10 (5.3%) were novel and 8 (2.2%) were singletons. From 2010 to 2017, the frequency of the historically dominant ST1 declined from 28.6% to 22.5%, whereas the recently emerged globally successful clonal cluster 3, ST23 and ST48, increased from 14.3% to 40.0%. CONCLUSIONS: The dominance of ST1 particularly in the last 2 years of this study appears to be declining, while ST23, reported in outbreaks among CF and post-surgical cohorts across the Americas and Europe, alongside the closely related ST48, is present among non-CF populations in Taiwan. These trends need to be confirmed with further ongoing studies to track the molecular epidemiology of clinical MAB isolates worldwide.

Therapeutic drug monitoring study on the switch from coformulated 600-mg efavirenz, tenofovir disoproxil fumarate, and emtricitabine to coformulated 400-mg efavirenz, tenofovir disoproxil fumarate, and lamivudine among HIV-positive patients with viral suppression.

OBJECTIVES: This study evaluated the efavirenz (EFV) mid-dose plasma concentration (C12), clinical efficacy, and safety after the switch to a single-tablet regimen containing tenofovir disoproxil fumarate (TDF), lamivudine (3TC), and 400-mg EFV in virally suppressed HIV-positive Taiwanese who were receiving co-formulated TDF, emtricitabine (FTC), and 600-mg EFV. METHODS: In this single-arm, open-label study, HIV-positive adults who had undetectable plasma HIV RNA load (<50 copies/ml) for 6 months or longer while receiving co-formulated TDF, FTC, and 600-mg EFV with EFV C12 of ≥1 mg/L were enrolled. The participants were switched to co-formulated TDF, 3TC, and 400-mg EFV and followed for 24 weeks. The primary endpoint was the proportion of participants with EFV C12 ≥ 1 mg/L at Week 4. The secondary endpoints included virologic response and change of CD4 lymphocyte count up to Week 24. Specific adverse effects associated with EFV were recorded before and after the switch. RESULTS: From December 2018 to January 2019, 50 participants were enrolled. EFV C12 remained ≥1 mg/L in 48 (96.0%) participants with a median reduction of 38.9% (interquartile range 29.0-44.4) at Week 4 after switch. All participants had undetectable plasma HIV RNA by Week 12, whereas 96.0% of them remained so at Week 24. Significant increases of CD4 lymphocyte count were observed at Weeks 12 and 24. Thirty-three participants (66.0%) reported improvement of pre-existing adverse effects. CONCLUSION: Switch to coformulated TDF, 3TC, and 400-mg EFV in virally suppressed HIV-positive Taiwanese maintained effective EFV concentration and viral suppression while the adverse effects were reduced.

Comparing the Utilities of Different Multilocus Sequence Typing Schemes for Identifying Outbreak Strains of Mycobacterium abscessus subsp. massiliense.

Outbreaks of infections by Mycobacterium abscessus, particularly subspecies massiliense, are increasingly reported worldwide. Several multilocus sequence typing (MLST) protocols for grouping international outbreak strains have been developed but not yet directly compared. Using the three-gene (hsp65, rpoB, and secA1), seven-gene (argH, cya, glpK, gnd, murC, pta, and purH) and thirteen-gene (all of the preceding genes plus gdhA, pgm, and pknA) MLST schemes, we identified 22, 38, and 40 unique sequence types (STs), respectively, among a total of 139 nonduplicated M. abscessus isolates. Among subspecies massiliense, three-gene MLST not only clustered all outbreak strains together (in 100% agreement with the seven-gene and thirteen-gene schemes), but it also distinguished between two new STs that would have been grouped together by the seven-gene MLST but were distinct by the thirteen-gene MLST owing to differences in hsp65, rpoB, and pknA Here, we show that an abbreviated MLST may be useful for simultaneous identification of M. abscessus the subspecies level and screening M. abscessus subsp. massiliense isolates with outbreak potential.

In Vitro Synergism of Rifabutin with Clarithromycin, Imipenem, and Tigecycline against the Mycobacterium abscessus Complex.

Infections caused by the difficult-to-treat bacterium Mycobacterium abscessus are increasing in frequency. Rifabutin, in contrast to rifampin, appears to be active in vitro against M. abscessus, especially against clarithromycin-resistant strains. However, explorations for potential synergy between rifabutin and available antimicrobials are currently limited. In vitro synergism between rifabutin and 10 antimicrobials was evaluated in 31 mycobacterial strains by the checkerboard method. The fractional inhibitory concentration index (FICI) was calculated for each rifabutin-based combination. The colony morphology was recorded. Molecular methods for determination of the M. abscessus subspecies and analysis of macrolide resistance were performed by sequencing of the secA1, rpoB, hsp65, erm(41), and rrl genes. Rifabutin yielded an MIC50 of 16 mg/liter (range, 2 to 32 mg/liter) against 26 clinical M. abscessus isolates (comprising 13 M. abscessus subsp. abscessus and 13 M. abscessus subsp. massiliense isolates) and 5 reference strains, including M. abscessus subsp. abscessus ATCC 19977, M. abscessus subsp. bolletii BCRC 16915, M. abscessus subsp. massiliense BCRC 16916, M. chelonae ATCC 35752, and M. peregrinum ATCC 700686. Significant synergism, classified by an FICI of ≤0.5, was demonstrated for the combinations of rifabutin and imipenem in 100% of M. abscessus subsp. abscessus and 69% of M. abscessus subsp. massiliense isolates, and significant synergism for rifabutin and tigecycline was demonstrated in 77% of M. abscessus subsp. abscessus and 69% of M. abscessus subsp. massiliense isolates. Among the 6 clarithromycin-resistant (MICs ≥ 8 mg/liter) M. abscessus subsp. abscessus isolates, the combination of rifabutin and clarithromycin was 100% synergistic. Rifabutin showed promising in vitro synergism with first-line anti-M. abscessus agents, especially for macrolide-resistant M. abscessus subsp. abscessus isolates.

Digynic triploidy in a fetus presenting with semilobar holoprosencephaly.

OBJECTIVE: We present digynic triploidy in a fetus with semilobar holoprosencephaly (HPE). CASE REPORT: A 32-year-old, gravid 1, para 0, woman underwent prenatal ultrasound examination at 12 weeks of gestation, and the ultrasound showed relative macrocephaly, a small non-cystic placenta, and a fetus with absent nasal bone and semilobar HPE. The pregnancy was terminated subsequently, and a 50-g fetus was delivered with a relatively enlarged head and premaxillary agenesis. The placenta was small and non-cystic. Postnatal cytogenetic analysis of the umbilical cord revealed a karyotype of 69, XXX. Postnatal DNA marker analysis using quantitative fluorescent polymerase chain reaction assays and the polymorphic short tandem repeat markers for chromosome 18 and 20 on the placental tissues showed a diallelic pattern with a dosage of 1:2 (paternal allele to maternal allele ratio), indicating a maternal origin of the triploidy. CONCLUSION: Fetuses with digynic triploidy may present relative macrocephaly, semilobar HPE and a small placenta on prenatal ultrasound.

Prenatal diagnosis of a familial 15q11.2 (BP1-BP2) microdeletion encompassing TUBGCP5, CYFIP1, NIPA2 and NIPA1 in a fetus with ventriculomegaly, microcephaly and intrauterine growth restriction on prenatal ultrasound.

OBJECTIVE: We present prenatal diagnosis of a 15q11.2 (BP1-BP2) microdeletion encompassing TUBGCP5, CYFIP1, NIPA2 and NIPA1 in a fetus with ventriculomegaly, microcephaly and intrauterine growth restriction (IUGR) on prenatal ultrasound. CASE REPORT: A 30-year-old, gravida 3, para 2, woman was referred to the hospital for amniocentesis because of fetal ventriculomegaly on prenatal ultrasound. Her husband was 31 years old. The couple had two healthy daughters, and there was no family history of mental disorders and congenital malformations. Amniocentesis revealed a karyotype of 46,XX. Array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniocytes revealed a 451.89-kb 15q11.2 microdeletion or arr 15q11.2 (22,765,628-23,217,514) × 1.0 [GRCh37 (hg19)] encompassing TUBGCP5, CYFIP1, NIPA2 and NIPA1. The parental karyotypes were normal. aCGH analysis on the DNAs extracted from parental bloods revealed a 402-kb 15q11.2 microdeletion or arr 15q11.2 (22,815,577-23,217,514) × 1.0 (hg19) encompassing TUBGCP5, CYFIP1, NIPA2 and NIPA1 in the phenotypically normal father. The mother did not have any genomic imbalance. Level II ultrasound at 21 weeks of gestation revealed microcephaly and IUGR. The parents elected to terminate the pregnancy at 22 weeks of gestation, and a female fetus was delivered with a body weight of 448 g (10th centile) and a body length of 26 cm (3rd-10th centile) but no gross abnormalities. CONCLUSION: Fetuses with a 15q11.2 (BP1-BP2) microdeletion may present ventriculomegaly, microcephaly and IUGR on prenatal ultrasound, and aCGH is helpful for prenatal diagnosis under such a circumstance.

Prenatal diagnosis of a familial 5p14.3-p14.1 deletion encompassing CDH18, CDH12, PMCHL1, PRDM9 and CDH10 in a fetus with congenital heart disease on prenatal ultrasound.

OBJECTIVE: We present prenatal diagnosis of a familial 5p14.3-p14.1 deletion in a fetus with congenital heart disease on prenatal ultrasound. CASE REPORT: A 33-year-old woman underwent amniocentesis at 18 weeks of gestation because of fetal ventricular septal defect (VSD) and echogenic bowel on prenatal ultrasound. Amniocentesis revealed a karyotype of 46,XX,del (5) (p14p14). Simultaneous array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniocytes revealed a 5.589-Mb 5p14.3-p14.1 deletion or arr 5p14.3p14.1 (19, 497, 649-25,086,268) × 1.0 [GRCh37 (hg19)] encompassing CDH18, CDH12, PMCHL1, PRDM9 and CDH10. Cytogenetic and aCGH analyses of the parents showed that the phenotypically normal mother carried the 5p14.3-p14.1 deletion. The father did not have such a deletion. The parents elected to continue the pregnancy, and a 3426-g female baby was delivered at 38 weeks of gestation with no gross abnormalities. The infant postnatally manifested VSD, atrial septal defect and patent ductus areriosus, and underwent cardiac surgery to treat the congenital heart disease. When follow-up at age 1 year and 4 months, she had a body weight of 8.8 Kg (50th-75th centile), a body height of 75.6 cm (85th-95th centile) and normal psychomotor development. CONCLUSION: Fetuses with a 5p14.3-p14.1 deletion may present congenital heart disease on prenatal ultrasound, and aCGH is helpful for prenatal diagnosis under such a circumstance.

Prenatal diagnosis of a familial 1q21.1-q21.2 microdeletion in a fetus with polydactyly of left foot on prenatal ultrasound.

OBJECTIVE: We present prenatal diagnosis of a familial 1q21.1-q21.2 microdeletion in a fetus with polydactyly of left foot on prenatal ultrasound. CASE REPORT: A 30-year-old, gravida 2, para 1, woman underwent amniocentesis at 22 weeks of gestation because of fetal polydactyly of left foot and echogenic heart foci on prenatal ultrasound. She and her husband and the 2-year-old son were healthy, and there was no family history of mental disorders, skeletal abnormalities and congenital malformations. Amniocentesis revealed a karyotype of 46,XX. Simultaneous array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniocytes revealed a 1.317-Mb 1q21.1-q21.2 microdeletion encompassing PRKAB2, FMO5, CHD1L, BCL9, ACP6, GJA5, GJA8 and GPR89B. aCGH analysis of the family members revealed that the phenotypically normal father and elder son carried the same 1q21.1-q21.2 microdeletion. The mother did not have such a deletion. The parents elected to continue the pregnancy, and a 3416-g female baby was delivered at 40 weeks of gestation with neither facial dysmorphism nor gross abnormalities except postaxial polydactyly of the left foot. CONCLUSION: Fetuses with a 1q21.1-q21.2 microdeletion may present polydactyly on prenatal ultrasound, and aCGH is helpful for prenatal diagnosis under such a circumstance.

Prenatal diagnosis and molecular cytogenetic characterization of chromosome 22q11.2 deletion syndrome associated with congenital heart defects.

OBJECTIVE: To report prenatal diagnosis of 22q11.2 deletion syndrome in a pregnancy with congenital heart defects in the fetus. CASE REPORT: A 26-year-old, primigravid woman was referred for counseling at 24 weeks of gestation because of abnormal ultrasound findings of fetal congenital heart defects. The Level II ultrasound revealed a singleton fetus with heart defects including overriding aorta, small pulmonary artery, and ventricular septal defect. Cordocentesis was performed. The DNA extracted from the cord blood was analyzed by multiplex ligation-dependent amplification (MLPA). The MLPA showed deletion in the DiGeorge syndrome (DGS) critical region of chromosome 22 low copy number repeat (LCR) 22-A∼C. Conventional cytogenetic analysis revealed a normal male karyotype. Repeated amniocentesis and cordocentesis were performed. Whole-genome array comparative genomic hybridization (aCGH) on cord blood was performed. aCGH detected a 3.07-Mb deletion at 22q11.21. Conventional cytogenetic analysis of cultured amniocytes revealed a karyotype 46,XY. Metaphase fluorescence in situ hybridization (FISH) analysis on cultured amniocytes confirmed an interstitial 22q11.2 deletion. CONCLUSION: Prenatal ultrasound findings of congenital heart defects indicate that the fetuses are at increased risk for chromosome abnormalities. Studies for 22q11.2 deletion syndrome should be considered adjunct to conventional karyotyping. Although FISH has become a standard procedure for diagnosis of 22q11.2 deletion syndrome, MLPA can potentially diagnose a broader spectrum of abnormalities, and aCGH analysis has the advantage of refining the 22q11.2 deletion breakpoints and detecting uncharacterized chromosome rearrangements or genomic imbalances.

Lack of resistance-associated mutations in UL54 and UL97 genes of circulating cytomegalovirus strains isolated in a medical center in Taiwan.

Human cytomegalovirus (HCMV) is a large DNA virus and a member of the betaherpesvirus family. HCMV infection is extremely common in human populations and can cause severe diseases in immunocompromised hosts. Ganciclovir is the most widely used antiviral drug for cytomegalovirus infection and works by blocking the amplification of HCMV. HCMV strains resistant to ganciclovir have been detected in recent decades and mainly result from mutations in UL97 (protein kinase) and UL54 (DNA polymerase) genes. In order to understand the prevalence of resistance of HCMV in Taiwan, we studied 40 clinical isolates to detect the mutations of UL97 and UL54 that might be related to resistance. The results showed that no mutation known to cause ganciclovir resistance was detected in any strain, but some polymorphisms (N685S, A688V, A885T, N898D in UL54; D605E in UL97) were frequently observed. Our results suggest that resistant HCMV strains are not prevalent in Taiwan.

Argonaute-2 enhances suppression of human cytomegalovirus replication by polycistronic short hairpin RNAs targeting UL46, UL70 and UL122.

BACKGROUND: Human cytomegalovirus (HCMV) is a common human pathogen that causes significant morbidity and mortality. The efficacy of anti-HCMV drugs such as ganciclovir, foscarnet and cidofovir is limited because of drug toxicities and frequent development of resistance. Here, we report an alternative anti-HCMV method using RNA silencing. METHODS: Combinatorial use of second-generation short hairpin RNAs (shRNA-miRs) targeting various transcripts of HCMV and an RNA-silencing endonuclease Argonaute-2 (Ago2) expression vector were applied to inhibit replication of HCMV AD169. Normal human fetal lung MRC-5 fibroblasts were transfected with pSM30-shRNA-miRs harbouring single or multiple shRNA-miR cassettes with or without Ago2 and then infected with HCMV AD169. Production of small interfering RNA (siRNA) was quantified by reverse transcription PCR. Virus secretion was evaluated by plaque reduction assays. RESULTS: The use of shRNA-miRs targeting a single HCMV gene suppressed HCMV AD169 viral titres by 50-70%. Polycistronic shRNA-miRs targeting UL46+UL122 and UL70+UL46+UL122 reached nearly 80% of inhibition. Coexpression of Ago2 with shRNA-miRs targeting UL46+UL122 and UL70+UL46+UL122 achieved a 95% reduction in viral maturation. CONCLUSIONS: Coexpression of Ago2 with shRNA-miRs enhanced the production of mature siRNAs and increased the efficiency of RNA silencing in the suppression of HCMV replication. This strategy may be universally applied to RNA interference-based therapies.

Polymorphisms in metabolic GSTP1 and DNA-repair XRCC1 genes with an increased risk of DNA damage in pesticide-exposed fruit growers.

Pesticide exposure is associated with various neoplastic diseases and congenital malformations. Previous studies have indicated that pesticides may be metabolized by cytochrome P450 3A5 or glutathione S-transferases. DNA-repair genes, including X-ray repair cross-complementing group 1 (XRCC1) and xeroderma pigmentosum group D (XPD), may also be implicated in the process of pesticide-related carcinogenesis. Thus, we investigated whether various metabolic and DNA-repair genotypes increase the risk of DNA damage in pesticide-exposed fruit growers. Using the comet assay, the extent of DNA damage was evaluated in the peripheral blood of 135 pesticide-exposed fruit growers and 106 unexposed controls. The metabolic genotypes CYP3A5 (A(-44)G) and GSTP1 (Ile105Val) and DNA-repair genotypes XRCC1 (Arg399Gln, Arg194Trp, T(-77)C) and XPD (Asp312Asn, Lys751Gln) were identified by polymerase chain reaction. Our multiple regression model for DNA tail moment showed that age, high pesticide exposure, low pesticide exposure, GSTP1 Ile-Ile, and XRCC1 399 Arg-Arg genotype were associated with increased DNA tail moment (DNA damage). Further analysis of interaction between GSTP1 and XRCC1 genes that increase susceptibility revealed a significant difference in DNA tail moment for high pesticide-exposed subjects carrying both GSTP1 Ile-Ile with XRCC1 399 Arg-Arg genotypes (2.49+/-0.09 microm/cell; P=0.004), compared to those carrying GSTP1 Ile-Val/Val-Val with XRCC1 399 Arg-Gln/Gln-Gln genotypes (1.98+/-0.15 microm/cell). These results suggest that individuals with susceptible metabolic GSTP1 and DNA-repair XRCC1 genotypes may be at increased risk of DNA damage due to pesticide exposure.