[Effects of four efflux pump inhibitors on the activities of clarithromycin against Mycobacterium abscessus].

Objective: To detect the effects of four efflux pump inhibitors on the minimum inhibitory concentration of clarithromycin (CLA) against Mycobacterium abscessus (M. abscessus) in vitro, and to explore the role of efflux pump in CLA resistance of M. abscessus. Methods: Four frequently-used efflux pump inhibitors (Carbonyl Cyanide 3-chlorophenylhydrazone, CCCP, N, N'-dicyclohexylcarbodiimide, DCC, Verapamil, VP, Reserpine, RSP) were evaluated in this study. The minimum inhibitory concentration (MIC) values of clarithromycin against M. abscessus reference strain and 60 clinical strains with or without efflux pump inhibitors were detected by Alamar Blue method. Sequence analysis of erm(41) and rrl genes known to be associated with CLA resistance in M. abscessus was performed to analyze the correlation between the effect of efflux pump inhibitors on MIC and mutation of resistance-related genes. Results: CCCP, DCC, VP and RSP could reduce the MIC of M. abscessus to CLA, and the effect of RSP was weaker than the other three efflux pump inhibitors. Among the sixty M. abscessus clinical strains, ten strains were resistant to clarithromycin, seven of which had rrl gene mutation. The CLA resistance rate of smooth phenotype isolates was higher than that of rough phenotype isolates. At 3 day of clarithromycin incubation, the MICs of resistant strains were all reduced by efflux pump inhibitors. Compared with the strains with rrl gene mutation, efflux pump inhibitors had a greater effect on the strains without rrl gene mutation. At 14 day of clarithromycin incubation, 83% of M. abscessus subsp. abscessus, were induced to be resistant, and all of them were T28 sequence type of erm(41). With the occurrence of induced drug resistance, the effect of efflux pump inhibitor on CLA MIC decreased. Efflux pump inhibitors had no statistically significant diffence in the effect of effcux pump inhibitors on CLA MIC levels in different phenotypes of isolates. Conclusions: Efflux pump is involved in the resistance process of M. abscessus to CLA. Efflux pump inhibitors reduce the drug resistance to clarithromycin against M. abscessus in different degrees. The use of efflux pump inhibitors may provide a new way to alleviate the drug resistance of M. abscessus.

[Analysis of lactate dehydrogenase gene polymorphisms and prediction of B cell epitopes in four human Plasmodium species].

OBJECTIVE: To analyze the polymorphism of Plasmodium lactate dehydrogenase (pLDH) gene and predict B-cell epitopes in pLDH peptides in four species of human malaria parasites. METHODS: The blood samples and epidemiological characteristics were collected from malaria cases in Yunnan Province registered in the National Notifiable Disease Report System. The pLDH genes of four human Plasmodium species were amplified using nested PCR assay and sequenced. The polymorphisms of pLDH genes was analyzed using the software MEGA version 7.0.26 and DnaSP version 5.10, and the B-cell epitopes were predicted in pLDH peptides using the Immune Epitope Database (IEDB). RESULTS: The sequences of P. vivax LDH (PvLDH), P. falciparum LDH (PfLDH), P. ovale LDH (PoLDH) and P. malariae LDH (PmLDH) genes were obtained from 153, 29, 17 and 11 blood samples from patients with P. vivax, P. falciparum, P. ovale and P. malariae malaria, respectively, which included 15, 2, 4 and 2 haplotypes and had a nucleotide diversity (π) of 0.104. A high level of intra-species differentiation was seen in the PoLDH gene (π = 0.012), and the π values were all < 0.001 for PvLDH, PfLDH and PmLDH genes. Active regions of B-cell antigen were predicted in the pLDH peptide chain of four human malaria parasites, of 4 to 5 in each chain, and the activity score was approximately 0.430. Among these peptide chains, the "86-PGKSDKEWNRD-96" short-peptide was a B-cell epitope shared by all four species of human malaria parasites, and the "266-GQYGHS (T)-271" short-peptide was present in PvLDH and PoLDH peptide chains, while "212-EEVEGIFDR-220" was only found in the PvLDH peptide chain, and "208-LISDAE-213" was only seen in the PfLDH peptide chain. CONCLUSIONS: The PoLDH gene polymorphism may be derived from the weak negative purification selection, while PvLDH, PfLDH and PmLDH genes may maintain a relatively conservative state. There may be two B-cell epitopes "212-EEVEGIFDR-220" and "208-LISDAE-213" in the proximal region of the C terminal in the pLDH peptide chain, which is feasible to differentiate between P. vivax and P. falciparum infections.

[Mycobacterium xenopi pulmonary disease:report of 3 cases and literature review].

Objective: To analyze the clinical manifestations, radiographic characteristics and prognosis of Mycobacterium xenopi pulmonary disease, in order to improve diagnosis and treatment of the disease. Methods: Using "Mycobacterium xenopi, pulmonary disease" as the search term, from February 15, 2007 to February 21, 2021, a total of 1 264 cases were retrieved in the PubMed database. In the Wanfang database, using "Mycobacterium xenopi, pulmonary disease" as the search term, from February 15, 2007 to February 21, 2021, no related document was retrieved. In the CNKI database, "Mycobacterium xenopi, pulmonary disease" was used as the search term, and one relevant case report was retrieved, but did not meet the diagnostic criteria of Mycobacterium xenopi pulmonary disease issued by American Thoracic Society in 2007. The 1 264 cases from the literature and 3 cases of our institution were used for review. Results: Our 3 cases were elderly males complaining of cough and expectoration, and had underlying lung diseases. The imaging examination showed cavitary lesions. All of them had positive sputum smear for acid-fast bacillus and negative Xpert MTB/RIF examination. Mycobacterium xenopi was isolated at least 2 times from sputum samples. Although prescribed with chemotherapy, case 1 and case 2 died 4 years and 2 years later, respectively, after the diagnosis. Case 3 got sputum conversion, symptom improvement and radiographic responses after 30-month chemotherapy. Conclusions: The clinical manifestations of Mycobacterium xenopi pulmonary disease are atypical. For patients with positive sputum smear for acid-fast bacillus and negative Xpert MTB/RIF examination and conventional mycobacterial culture, Mycobacterium xenopi pulmonary disease should be considered. The disease deserves further attention from clinicians due to poor prognosis.

[In vitro evaluation of the antibacterial activity of nemonoxacin against Mycobacterium tuberculosis, Mycobacterium intracellulare and Mycobacterium abscessus].

Objective: To test the in vitro antibacterial activity of nemonoxacin against clinically isolates of Mycobacterium tuberculosis complex(MTBC), Mycobacterium intracellulare(MI) and Mycobacterium abscessus(MA). Methods: Totally 128, 80 and 50 isolates of MTBC, M.intracellulare and M.abscessus were tested, respectively. The minimum inhibitory concentrations (MICs) of nemonoxacin and levofloxacin against the strains of the three most frequently isolated mycobacterium species were measured by double dilution method with micro-well plate. Results: The MICs of 104(81.2%) strains of MTBC isolates against levofloxacin were ≤ 1 µg/ml. Whereas 112 (87.5%) strains of MTBC isolates had MICs against nemonoxacin than>1 µg/ml, furthermore, the MICs of 88(68.8%)strains of MTBC isolates against nemonoxacin were≥4 µg/ml. The median MIC of M. intracellulare isolates against levofloxacin and nenofloxacin were 16 and 32 µg/ml, separately, while were 16 µg/ml and 8 µg/ml for M. abscessus, respectively. The ratios of nemonoxacin MIC/levofloxacin MIC of M. abscessus were between 0.125-1.000. Conclusions: Nemonoxacin presented weaker inhibitory activity than levofloxacin against M. tuberculosis, whereas it had better activity than levofloxacin against M. abscessus.

[Incremental cost-effectiveness of the second Xpert MTB/RIF assay for detection of Mycobacterium tuberculosis].

Objective: To study the incremental cost-effectiveness of the second Xpert assay in detection of Mycobacterium tuberculosis (Mtb) and rifampicin (RIF) resistance. Methods: We continuously collected 2 896 specimens from suspected tuberculosis patients who had undergone 2 Xpert tests in a week from March 2015 to March 2018, including 2 402 suspected tuberculosis patients with 1 523 males and 879 females, with an average age of 50 years. Among them, 2 144 specimens of sputum and 258 cases of bronchoalveolar lavage fluid were collected. We also enrolled 494 patients with suspected extrapulmonary tuberculosis, 318 males and 176 females, with an average age of 42 years. Among them, 157 pleural effusion specimens, 106 cerebrospinal fluid specimens, 34 urine specimens and 197 pus specimens were collected. All specimens were subjected to two Xpert tests, smear microscopy, liquid rapid culture (BACTEC MGIT 960), and positively cultured bacteria were tested for drug susceptibility. Results: Among the 2 896 specimens from suspected tuberculosis patients, either one of the two Xpert test results was positive (including both tests were positive, the same below) in 1 639 patients, and 1 502 (91.6%) were positive in the first Xpert tests. The additional 137 (8.4%) test results were positive in the second tests. According to the smear test results, all specimens were divided into the smear negative group and the smear positive group. The second Xpert test was significantly higher than the smear-positive group (14.86%, 3.2%, P<0.001), and the extrapulmonary tuberculosis group was higher than the tuberculosis group (11.2%, 8.0%, P=0.12).Of the susceptibility test results, a total of 371 were rifampicin-resistant specimens. The first Xpert detected 91.4% (339/371), and the second Xpert detected the additional 8.1% (30/371).The cost increase of the second test was very significant. Tests were calculated at 650 yuan per time, the tuberculosis group was 1 184 yuan and 13 696 yuan(P<0.001); the extrapulmonary tuberculosis group was 1 755 yuan and 13 961 yuan(P<0.001). In the test of specimens of tuberculosis and extrapulmonary tuberculosis, the smear-negative specimen cost increase of the second Xpert test was lower than that of the smear-positive specimen. Conclusion: The second xpert test showed significant value-added cost-effectiveness in the diagnosis of tuberculosis.

[Antimicrobial susceptibility and genotyping of Mycobacterium intracellulare].

Objective: To investigate the antimicrobial susceptibility and genotyping of Mycobacterium intracellulare. Methods: A total of 150 M. intracellulare isolates were collected. The susceptibility against 15 antimicrobial agents widely used for treatment of non-tuberculosis mycobacteria (NTM) infections, was tested by broth microdilution assay. Variable number of tandem repeats (VNTR) assay was also performed using the 16-loci genotyping method. Results: The drug susceptibility test revealed that clarithromycin (97.3%, 146/150), moxifloxacin (94.0%, 141/150) and amikacin (90.0%, 135/150) had the best antimicrobial activities in vitro against the M. intracellulare isolates. Secondly, 75.3%(113/150), 64.0%(96/150), 52.7%(79/150) and 8.7%(13/150) of the strains were susceptible to rifampicin, linezolid, capreomycin, and ethambutol, respectively. The MIC(50) and MIC(90) values of the 3 injectable anti-tuberculosis drugs were as follows: amikacin 4 mg/L and 16 mg/L, streptomycin 4 mg/L and 16 mg/L, capreomycin 8 mg/L and 16 mg/L. The MIC(50) and MIC(90) values of the 5 different fluoroquinolones were 0.5 mg/L and 2 mg/L for moxifloxacin , 1 mg/L and 8 mg/L for ciprofloxacin, 1 mg/L and 8ug/ml for levofloxacin, 2 mg/L and 16 mg/L for antoflolxacin, 2 mg/L and 16 mg/L for ofloxacin. The Hunter-Gaston Discriminatory Index (HGDI) value for the 16-loci VNTR typing of M. intracellulare isolates was 0.994. VNTR differentiated the 150 isolates into 21 clusters and acquired a total of 121 unique patterns. Drug resistance profile was not independently associated with cluster strains. Conclusions: Clarithromycin, moxifloxacin and amikacin had the best antimicrobial activities in vitro against M. intracellulare isolates. The 16-loci VNTR typing revealed a highly discriminatory power and drug resistance profile was not independently associated with cluster strains.

[Evaluation of a high-intensity fluorescent fluorophage method for diagnosis of drug-resistance in tuberculosis].

Objective: To evaluate the high-intensity green fluorescent protein fluorophage Φ(2)GFP10 method for drug susceptibility testing of tuberculosis for isoniazid(INH), rifampin(RIF), and streptomycin(SM). Methods: A total of 128 clinical M. tuberculosis strains were isolated from patients with suspected drug-resistant tuberculosis visiting Beijing Chest Hospital (Beijing, China) from April to June 2014.All of the isolates were tested by the phage assay, while conventional drug susceptibility tests were performing on Lwenstein-Jensen culture medium as reference. Results: The sensitivities of Φ(2)GFP10 assay for INH, RIF, and SM resistance detection were 100.0%, 98.1%(53/54), and 92.6%(50/54), respectively, while their specificities were 84.8%(56/66), 91.9%(68/74), and 91.9%(68/74), respectively. The agreement between the phage assay and the conventional assay for detecting INH, RIF, and SM resistance was 0.92, 0.95 and 0.92, respectively. The Φ(2) GFP10-phage assay could be done within 2 days for RIF and SM, and 3 days for INH. Conclusions: The Φ(2)GFP10-phage method for drug susceptibility test is very sensitive and specific. The method has the potential to be a valuable, rapid and economical screening method for detecting drug-resistant tuberculosis.

Trends in drug-resistant tuberculosis in China: data from a clinical tuberculosis centre.

OBJECTIVE: To describe the trends in prevalence of drug-resistant tuberculosis (TB) among in-patients in Beijing Chest Hospital, Beijing, China, using a 10-year retrospective study. DESIGN: From 2005 to 2014, 18 310 in-patients with TB were recruited for the study, most of whom were referrals; no distinction was made between new and previously treated cases. Drug susceptibility testing (DST) was performed in culture-positive cases using the proportion method to determine multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB). Risk factors associated with drug resistance were identified. RESULTS: A total of 5141 (28.0%) samples were culture-positive. DST results showed that 860 (16.7%) cases were MDR-TB and 176 (3.4%) were XDR-TB. MDR-TB and XDR-TB were detected in respectively 21.2% and 12.5% of new cases. The rate of MDR-TB and XDR-TB gradually increased from 2005, with MDR-TB reaching a peak in 2008 and XDR-TB in 2009. These data closely mirror national survey data on this region, patient age and occupation. CONCLUSION: Trends in MDR-TB and XDR-TB prevalence during the past decade and their inflection points were determined, which complemented reports from previous national surveys. This information is useful for fighting TB in China.

[Discriminatory power of variable number on tandem repeats loci for genotyping Mycobacterium tuberculosis strains in China].

Objective: Using the standard genotype method, variable number of tandem repeats (VNTR), we constructed a VNTR database to cover all provinces and proposed a set of optimized VNTR loci combinations for each province, in order to improve the preventive and control programs on tuberculosis, in China. Methods: A total of 15 loci VNTR was used to analyze 4 116 Mycobacterium tuberculosis strains, isolated from national survey of Drug Resistant Tuberculosis, in 2007. Hunter-Gaston Index (HGI) was also used to analyze the discriminatory power of each VNTR site. A set combination of 12-VNTR, 10-VNTR, 8-VNTR and 5-VNTR was respectively constructed for each province, based on 1) epidemic characteristics of M. tuberculosis lineages in China, with high discriminatory power and genetic stability. Results: Through the completed 15 loci VNTR patterns of 3 966 strains under 96.36% (3 966/4 116) coverage, we found seven high HGI loci (including QUB11b and MIRU26) as well as low stable loci (including QUB26, MIRU16, Mtub21 and QUB11b) in several areas. In all the 31 provinces, we found an optimization VNTR combination as 10-VNTR loci in Inner Mongolia, Chongqing and Heilongjiang, but with 8-VNTR combination shared in other provinces. Conclusions: It is necessary to not only use the VNTR database for tracing the source of infection and cluster of M. tuberculosis in the nation but also using the set of optimized VNTR combinations in monitoring those local epidemics and M. tuberculosis (genetics in local) population.

Sensititre® MYCOTB MIC plate for drug susceptibility testing of Mycobacterium tuberculosis complex isolates.

SETTING: National Tuberculosis Clinical Laboratory, China. OBJECTIVE: To evaluate the accuracy and feasibility of the MYCOTB MIC plate in anti-tuberculosis drug susceptibility testing. DESIGN: MYCOTB testing of Mycobacterium tuberculosis isolates, the Löwenstein-Jensen (LJ) proportion method and the resurazine microtitre assay (REMA), which is extensively used for in-house assay for minimal inhibition concentration (MIC) testing, were performed simultaneously for comparison. A total of 126 clinical isolates were tested using both MYCOTB and the LJ proportion method against 12 anti-tuberculosis drugs; 80 were also tested using REMA. RESULTS: Categorical agreement between MYCOTB and the LJ proportion method was 99.2% for rifampicin, ofloxacin, amikacin, kanamycin and cycloserine, and 98.4% for isoniazid and para-aminosalicylic acid; ethambutol (EMB) had the lowest agreement (86.5%). The overall categorical agreement between MYCOTB and REMA ranged between 98.8% and 100%. MYCOTB outcomes, interpreted on day 10 and 21, were stable for all drugs except EMB. CONCLUSION: MYCOTB is a rapid, convenient, quantitative and accurate method for testing both first- and second-line anti-tuberculosis drugs.

Modified cytospin slide microscopy method for rapid diagnosis of smear-negative pulmonary tuberculosis.

OBJECTIVE: To evaluate a modified method of the Ziehl-Neelsen stain and determine whether it improves the detection rate of acid-fast bacilli (AFB) in bronchoalveolar lavage fluid (BALF) specimens. DESIGN: Bronchoscopy of patients with suspected smear-negative pulmonary tuberculosis (PTB) patients was conducted to collect BALF to assess the efficacy and accuracy of the modified method for PTB diagnosis. RESULTS: A total of 106 BALF specimens was collected from 74 PTB patients on the basis of BALF samples that were culture-positive for Mycobacterium tuberculosis. When analysed by patient, the sensitivity and specificity of our modified method were respectively 87.8% and 99.6%, while the positive predictive (PPV) and negative predictive values (NPV) were respectively 98.5% and 96.8%. Conversely, the sensitivity of direct smears and concentrated smears was respectively 16.2% and 37.8%, with 100% specificity. On analysing 106 samples, the culture positivity rate of the direct smear and the concentrated smear methods was respectively 76.4%, 13.2% and 34%, while it was 91.5% for the modified method. CONCLUSION: The sensitivity of our modified method was significantly higher than that of direct or concentrated smears. Overall, the modified method improved the detection rate of AFB in BALF specimens, and provided an efficient and accurate diagnosis of PTB in patients with suspected smear-negative PTB.

[Frequency and clinical relevance of rapidly growing mycobacterium isolated from respiratory samples].

OBJECTIVE: To assess the frequency and clinical relevance of rapidly growing mycobacterium (RGM) isolates in a tuberculosis referral center in Beijing, China. METHODS: All isolates were identified by using targeted gene sequencing. RESULTS of species identification for 228 nontuberculous Mycobacterium (NTM) isolates from respiratory samples were analyzed, and available medical files of patients from whom NTM were isolated were reviewed retrospectively. Diagnostic criteria for RGM pulmonary disease issued by the American Thoracic Society (ATS) were used to determine clinical relevance. RESULTS: Isolates of Mycobacterium abscessus (M.abscessus) and Mycobacterium fortuitum (M.fortuitum) accounted for 28.9% (66 isolates) and 8.8% (20 isolates)of NTM isolates, respectively. Sixty-six M. abscessus isolates from 32 patients had evaluable medical files, including 28 cases diagnosed as definite M. abscessus lung disease, and 4 as probable M. abscessus lung disease. Eight M. fortuitum isolates from 8 cases had evaluable medical files, and all of them were diagnosed as unlikely lung disease. Mycobacteria Growth Indicator Tube (MGIT) was more effective to diagnose M. abscessus lung disease, as compared with Lowestein-Jensen medium (23/24 vs 18/28). CONCLUSIONS: RGM is a common NTM in our institute. M. abscessus is mostly associated with RGM lung disease, but M. fortuitum is not.

Clinical and molecular characteristics of bloodstream infections caused by Candida albicans in children from 2003 to 2011.

We investigated the clinical and molecular characteristics of Candida albicans bloodstream infection (BSI) in children from a tertiary-level medical centre in Taiwan over a 9-year period from January 2003 to December 2011. We performed multilocus sequence typing (MLST) to investigate the genetic relatedness of these C. albicans BSI isolates. A total of 79 episodes of C. albicans BSI in 76 paediatric patients were identified, including 41 (51.9%) from the paediatric intensive care unit, 24 (30.4%) from the neonatal intensive care unit and 14 (17.7%) from general wards. More than half (59.5%) of these patients had underlying chronic co-morbidities, and the majority (94.9%) had a catheter or some other artificial device. All the isolates were susceptible to the antifungal agents tested. Only 32.9% (26/79) received effective antifungal agents within 24 h of onset of candidaemia. Twenty-five (31.6%) patients had persistent candidaemia (>3 days after the start of antifungal treatment) and candidaemia-attributable mortality rate was 22.8% (18/79). The 72 isolates available for MLST yielded 53 unique diploid sequence types (DSTs). Forty-five DSTs were singletons and eight DSTs were shared by 27 (37.5%) isolates. Seventy-one (98.6%) isolates were clustered within previously known clades. Based on the definition of two or more strains with shared DST occurring within a period of 90 days, 10.1% of the infections were categorized as nosocomial clusters, most commonly identified in the intensive care units. Although cluster-associated candidaemia was not associated with a higher mortality rate, none of the clusters were identified by the hospital infection control team.

Predictors of clinical and microbiological treatment failure in neonatal bloodstream infections.

This study aimed to identify independent predictors of clinical and microbiological treatment failure and develop a predictive model for neonates with bloodstream infection (BSI). This study included 1087 episodes of BSIs in 793 neonates in a tertiary-level neonatal intensive care unit of northern Taiwan between 2004 and 2012. Patient demographics, underlying chronic comorbidities, clinical features, antimicrobial treatment and microbiological characteristics were evaluated. The presence of underlying congenital anomalies (odds ratio [OR] 2.12, 95% confidence interval [CI] 1.09 to 4.10) and pulmonary hypertension (OR 3.63, 95% CI 1.70 to 7.74), infections caused by multidrug-resistant gram-negative bacteria (OR 2.89, 95% CI 1.23 to 6.79), group B Streptococcus (OR 3.15, 95% CI 1.33 to 7.46), and fungi (OR 4.13, 95% CI 2.02 to 8.46), a Neonatal Therapeutic Intervention Scoring System score of ≥ 23 (OR 6.96, 95% CI 2.55 to 28.58), inappropriate antibiotics (OR 2.13, 95% CI 1.41 to 3.23), and concomitant meningitis (OR 4.25, 95% CI 2.08 to 8.69) and ventilator-associated pneumonia (OR 2.73, 95% CI 1.22 to 6.13) were identified as independent risk factors for 28-day treatment failure in neonatal BSI. A risk score model was created by adding the points for each independent risk factor, and had a c-statistic of 0.83. Patients with risk scores of 0, 4, 8, 12 and 15 had estimated 28-day treatment failure rates of approximately 3.5%, 17.0%, 53.5%, 86.6% and 95.9%, respectively. This predictive model, calculated after documentation of a BSI, reflects a spectrum of BSI severity and was associated with subsequent treatment failure through illness severity score and case mix variables. This simple score could prove useful in clinical and research settings, and practical in estimating the prognosis.

Recurrent late-onset sepsis in the neonatal intensive care unit: incidence, clinical characteristics and risk factors.

We aimed to characterize the incidence, clinical features, risk factors and outcomes of recurrent late-onset sepsis (LOS) in the neonatal intensive care unit (NICU). All neonates with LOS from the NICU of a tertiary-level teaching hospital in northern Taiwan between 2004 and 2011 were enrolled for analyses. A case-control study was performed to determine risk factors for recurrence. Of 713 neonates with LOS, 150 (21.0%) experienced recurrence and 48 (6.7%) had >1 recurrences; c. two-thirds of recurrent LOS occurred in infants with birth weight (BW)≦1500 g or gestational age (GA)≦30 weeks. The recurrent LOS episodes were significantly more severe and had a higher sepsis-attributable mortality rate than the first episodes. The overall in-hospital mortality rate was 30.7% for neonates with recurrent LOS and 7.8% for those with single LOS (odds ratio (OR), 5.22; 95% CI, 3.28-8.30). When both BW and GA were controlled, neonates with recurrent LOS had a significantly prolonged hospitalization compared with the controls (median 109 vs. 84 days, p<0.001). After multivariate logistic regression, longer duration of total parenteral nutrition (TPN; OR, 1.30; 95% CI, 1.10-1.52 for every 10-day increment), presence of congenital anomalies (OR, 2.64; 95% CI, 1.10-6.35) and neurological co-morbidities (OR, 4.14; 95% CI, 1.14-15.10) were identified as the independent risk factors for LOS recurrence. We concluded that c. one-fifth of neonates with LOS had recurrence, which significantly resulted in prolonged hospitalization and increased mortality. Longer TPN administration, presence of congenital anomalies and neurological co-morbidities are independently associated with recurrent LOS.

Developmental expression of key steroidogenic enzymes in the brain of protandrous black porgy fish, Acanthopagrus schlegeli.

In the present study, we tested the hypothesis that the brain of the black porgy fish, Acanthopagrus schlegeli, has the capacity for de novo steroidogenesis and that these neurosteroids may impact sex differentiation. Gonadal histology and Dmrt1 gene expression revealed that the fish were not sex differentiated until 155 dah (days after hatching). We further demonstrated the developmental expressions of the mRNAs encoding for four key neurosteroidogenic enzymes, namely, the cytochrome P450 side chain cleavage (CYP11A1), 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3betaHSD), cytochrome P450c17 (CYP17) and aromatase (CYP19b) in the brain at different post-hatching developmental ages. The results indicated that steroidogenic genes are expressed in brain from the earliest sampling time, 60 dah. Quantitative real-time polymerase chain reaction analysis demonstrated significantly higher expression levels of these enzymes at 120 dah compared to 60 dah in all the brain regions. However, the increase for 3betaHSD was significant only in hypothalamus and midbrain, whereas it was significant only in forebrain and hypothalamus for CYP19b. A decline in mRNA levels were observed for all the genes at 155 dah except in midbrain for CYP11A1 and in hindbrain for CYP19b. Analysis of aromatase enzyme activity showed a significant increase in aromatase activity in the forebrain at 120 dah. Thus, the present study demonstrated for the first time an age- and/or region dependent expression of the mRNAs encoding the steroidogenic enzyme genes in the brain of black porgy. The presence of key steroidogenic enzymes as early as 60 dah, before gonadal sex differentiation, demonstrates that steroid biosynthetic capacity in brain precedes histological gonad differentiation. The mRNA transcripts of these genes showed a synchronous peak at 120 dah, suggesting that oestradiol may be locally formed in most parts of the brain. The study suggests an important role for brain aromatase in male black porgy brain sex differentiation, and considers the possibility of a role for this enzyme in neurogenesis.

A new chloro-monoterpene from the mangrove endophytic fungus Tryblidiopycnis sp. (4275).

A new chloro-monoterpene (compound 1) and three known compounds, peroxyergosterol, uracil and methylisocoumarin, were isolated from the ethyl acetate extract of the fermentation broth of the mangrove endophytic fungus Tryblidiopycnis sp. (4275) obtained from Kandelia woody tissue from Mai Po, Hong Kong. Its structure was determined spectroscopically and by X-ray crystallographic analysis.

Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites.

Group II introns, the presumed ancestors of nuclear pre-mRNA introns, are site-specific retroelements. In addition to "homing" to unoccupied sites in intronless alleles, group II introns transpose at low frequency to ectopic sites that resemble the normal homing site. Two general mechanisms have been proposed for group II intron transposition, one involving reverse splicing of the intron RNA directly into an ectopic DNA site, and the other involving reverse splicing into a site in RNA followed by reverse transcription and integration of the resulting cDNA by homologous recombination. Here, by using an "inverted-site" strategy, we show that the yeast mtDNA group II intron aI1 retrotransposes by reverse splicing directly into an ectopic DNA site. This same mechanism could account for other previously described ectopic transposition events in fungi and bacteria and may have contributed to the dispersal of group II introns into different genes.