Prevalence and Genetic Characterization of Second-Line Drug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in Rural China

This study aimed to investigate the prevalence of resistance to second-line antituberculosis (anti-TB) drugs and its association with resistance-related mutations in Mycobacterium tuberculosis isolated in China. In the present study, we collected 380 isolates from a population-based study in China and tested the drug susceptibility to first- and selected second-line drugs. These results were compared with polymorphisms in the DNA sequences of genes associated with drug resistance and MIC values of the studied second-line drugs. Of 43 multidrug-resistant M. tuberculosis isolates, 13 showed resistance to fluoroquinolones or injectable second-line drugs (preextensively drug-resistant TB [pre-XDR-TB]), and 4 were resistant to both and thus defined as extensively drug-resistant TB (XDR-TB). Age and previous TB therapy, including use of second-line drugs, were two independent factors associated with increased resistance to both first- and second-line drugs. Molecular analysis identified the most frequent mutations in the resistance-associated genes: D94G in gyrA (29.1%) and A1401G in rrs (30.8%). Meanwhile, all 4 XDR-TB isolates had a mutation in gyrA, and 3 of them carried the A1401G mutation in rrs. Mutations in gyrA and rrs were associated with high-level resistance to fluoroquinolones and the second-line injectable drugs. In addition to the identification of resistance-associated mutations and development of a rapid molecular test to diagnose the second-line drug resistance, it should be a priority to strictly regulate the administration of second-line drugs to maintain their efficacy to treat multidrug-resistant TB.     

Mycobacterium tuberculosis rrs A1401G mutation correlates with high-level resistance to kanamycin,amikacin, and capreomycin in clinical isolates from mainland China

Mutations correlating phenotypic resistance level with the injectable second-line anti-tuberculosis drugs (SLDs) including kanamycin (KAN), amikacin (AMK), and capreomycin (CAP) remain elusive. A collection of 114 Mycobacterium tuberculosis clinical isolates from mainland China was analyzed. The minimum inhibitory concentration (MIC) of each strain was determined and the sequences of rrs, tlyA, promoter of eis as well as 5′ untranslated region (UTR) of whiB7 were amplified and sequenced. No mutation in tlyA, promoter of eis and 5′ UTR of whiB7, was found to be associated with resistance among these samples. Sequencing data of 1400 rrs region demonstrated the A1401G mutation in rrs was prevalent, which presented in 84% of the KAN resistant isolates while only in about 50% of the AMK or CAP resistant isolates. Furthermore, most of the resistant isolates with A1401G mutation showed high-level resistance to these injectable SLDs. In conclusion, our results suggest the rrs A1401G mutation was related to high-level resistance to KAN, AMK, and CAP in M. tuberculosis isolates from mainland China.     

Disparities in Capreomycin Resistance Levels Associated with the rrs A1401G Mutation in Clinical Isolates of Mycobacterium tuberculosis

As the prevalence of multidrug-resistant and extensively drug-resistant tuberculosis strains continues to rise, so does the need to develop accurate and rapid molecular tests to complement time-consuming growth-based drug susceptibility testing. Performance of molecular methods relies on the association of specific mutations with phenotypic drug resistance and while considerable progress has been made for resistance detection of first-line antituberculosis drugs, rapid detection of resistance for second-line drugs lags behind. The rrs A1401G allele is considered a strong predictor of cross-resistance between the three second-line injectable drugs, capreomycin (CAP), kanamycin, and amikacin. However, discordance is often observed between the rrs A1401G mutation and CAP resistance, with up to 40% of rrs A1401G mutants being classified as CAP susceptible. We measured the MICs to CAP in 53 clinical isolates harboring the rrs A1401G mutation and found that the CAP MICs ranged from 8 μg/ml to 40 μg/ml. These results were drastically different from engineered A1401G mutants generated in isogenic Mycobacterium tuberculosis, which exclusively exhibited high-level CAP MICs of 40 μg/ml. These data support the results of prior studies, which suggest that the critical concentration of CAP (10 μg/ml) used to determine resistance by indirect agar proportion may be too high to detect all CAP-resistant strains and suggest that a larger percentage of resistant isolates could be identified by lowering the critical concentration. These data also suggest that differences in resistance levels among clinical isolates are possibly due to second site or compensatory mutations located elsewhere in the genome.     

High Level of Cross-Resistance between Kanamycin,Amikacin, and Capreomycin among Mycobacterium tuberculosis Isolates from Georgia and a Close Relation with Mutations in the rrs Gene

The aminoglycosides kanamycin and amikacin and the macrocyclic peptide capreomycin are key drugs for the treatment of multidrug-resistant tuberculosis (MDR-TB). The increasing rates of resistance to these drugs and the possible cross-resistance between them are concerns for MDR-TB therapy. Mutations in the 16S rRNA gene (rrs) have been associated with resistance to each of the drugs, and mutations of the tlyA gene, which encodes a putative rRNA methyltransferase, are thought to confer capreomycin resistance in Mycobacterium tuberculosis bacteria. Studies of possible cross-resistance have shown variable results. In this study, the MICs of these drugs for 145 clinical isolates from Georgia and the sequences of the rrs and tlyA genes of the isolates were determined. Of 78 kanamycin-resistant strains, 9 (11.5%) were susceptible to amikacin and 16 (20.5%) were susceptible to capreomycin. Four strains were resistant to capreomycin but were susceptible to the other drugs, whereas all amikacin-resistant isolates were resistant to kanamycin. Sequencing revealed six types of mutations in the rrs gene (A514C, C517T, A1401G, C1402T, C1443G, T1521C) but no mutations in the tlyA gene. The A514C, C517T, C1443G, and T1521C mutations showed no association with resistance to any of the drugs. The A1401G and C1402T mutations were observed in 65 kanamycin-resistant isolates and the 4 capreomycin-resistant isolates, respectively, whereas none of the susceptible isolates showed either of those mutations. The four mutants with the C1402T mutations showed high levels of resistance to capreomycin but no resistance to kanamycin and amikacin. Detection of the A1401G mutation appeared to be 100% specific for the detection of resistance to kanamycin and amikacin, while the sensitivities reached 85.9% and 94.2%, respectively.Although the first-line anti-tuberculosis (anti-TB) drugs rifampin (RMP; rifampicin), isoniazid (INH), ethambutol (EMB), pyrazinamide (PZA), and streptomycin (SM) were discovered several decades ago, they are still used today in standard short-course regimens for the treatment of TB. These regimens are, however, ineffective for the treatment of multidrug-resistant (MDR) TB (defined as resistance to at least the two most powerful anti-TB drugs, RMP and INH), leading to the use of less effective and more toxic second-line drugs (SLDs). Injectable drugs such as kanamycin (KAN), amikacin (AMK), and capreomycin (CAP) are the key SLDs for the treatment of MDR-TB (17). The emergence of extensively drug-resistant TB, defined as MDR-TB with additional resistance to any fluoroquinolone and at least one of the injectable drugs (10), once again underlines the importance of fast and reliable testing for susceptibility to these antibiotics.Mutations in the 3′ part of the 16S rRNA gene (rrs), particularly at positions 1401, 1402, and 1484 (1, 7, 11, 12), have been associated with resistance to each of the drugs. It has also been suggested that mutations in the tlyA gene are responsible for resistance to CAP (8). Additionally, reports of cross-resistance among various aminoglycosides and CAP have been variable (1, 4, 6, 16). Most of the previous investigations were done with laboratory-generated mutants and with only a limited number of clinical isolates. In this work, we investigated the correlation between mutations in the rrs and tlyA genes and the in vitro resistance to the three injectable drugs of clinical Mycobacterium tuberculosis isolates.     

Analysis of drug resistance and mutation profiles in Mycobacterium tuberculosis isolates in a surveillance site in Beijing,China

ObjectiveThis study analyzed drug resistance and mutations profiles in Mycobacterium tuberculosis isolates in a surveillance site in Huairou District, Beijing, China.MethodsThe proportion method was used to assess drug resistance profiles for four first-line and seven second-line anti-tuberculosis (TB) drugs. Molecular line probe assays were used for the rapid detection of resistance to rifampicin (RIF) and isoniazid (INH).ResultsAmong 235 strains of M. tuberculosis, 79 (33.6%) isolates were resistant to one or more drugs. The isolates included 18 monoresistant (7.7%), 19 polyresistant (8.1%), 28 RIF-resistant (11.9%), 24 multidrug-resistant (MDR) (10.2%), 7 pre-extensively drug-resistant (XDR, 3.0%), and 2 XDR strains (0.9%). A higher rate of MDR-TB was detected among previously treated patients than among patients with newly diagnosed TB (34.5% vs. 6.8%). The majority (62.5%) of RIF-resistant isolates exhibited a mutation at S531L in the DNA-dependent RNA polymerase gene. Meanwhile, 62.9% of INH-resistant isolates carried a mutation at S315T1 in the katG gene.ConclusionOur results confirmed the high rate of drug-resistant TB, especially MDR-TB, in Huairou District, Beijing, China. Therefore, detailed drug testing is crucial in the evaluation of MDR-TB treatment.     

Sequence Analyses of Just Four Genes To Detect Extensively Drug-Resistant Mycobacterium tuberculosis Strains in Multidrug-Resistant Tuberculosis Patients Undergoing Treatment

The rapid detection of Mycobacterium tuberculosis isolates resistant to second-line drugs is crucial for the institution of appropriate treatment regimens as early as possible. Although molecular methods have successfully been used for the rapid detection of resistance to first-line drugs, there are limited data on mutations that confer resistance to second-line drugs. To address this question, we analyzed Mycobacterium tuberculosis strains resistant to ofloxacin (n = 26) and to capreomycin and/or amikacin (n = 48) from Uzbekistan for variations in target genes (gyrA, gyrB, rrs, and tlyA). Strains susceptible to ofloxacin (n = 49) and capreomycin and/or amikacin (n = 39) were included as controls. Mutations in gyrA or gyrB were found in 96% (25/26 strains) of the ofloxacin-resistant strains, while none of the susceptible strains displayed mutations in those two genes. The most common mutation occurred in gyrA at codon 94 (17/26 strains [65.4%]), followed by mutations at codons 90 and 91. Two strains showed a mutation in gyrB, at codons 485 and 543, respectively; both mutations have not been reported previously. The most frequent mutation in strains resistant to both amikacin and capreomycin was A1401G in rrs (34/40 strains [85.0%]). Three strains had mutations in tlyA, of which two (at codons 18 and 118) were associated with resistance to capreomycin alone. Overall, none of the 10 resistant strains (5 amikacin-resistant and capreomycin-susceptible strains) and none of the 39 susceptible control strains had mutations in the genes investigated. Our results clearly demonstrate the potential of sequence analyses of short regions of relatively few target genes for the rapid detection of resistance to second-line drugs among strains isolated from patients undergoing treatment for multidrug-resistant tuberculosis. The mechanisms that confer amikacin resistance in this setting remain unclear.With 9.2 million new cases and 1.7 million deaths in 2006, tuberculosis (TB) remains one of the most serious infectious diseases worldwide (28). In many settings, the emergence and transmission of drug-resistant Mycobacterium tuberculosis strains further threaten TB control efforts. TB fails to respond to treatment with the standard first-line drugs isoniazid (INH) and rifampin (RIF; rifampicin) in patients infected with so-called multidrug-resistant (MDR) Mycobacterium tuberculosis strains. MDR-TB is particularly difficult to treat because the second-line drugs used are less potent and more expensive (12).Disease caused by extensively drug-resistant (XDR) M. tuberculosis isolates, defined as MDR with resistance to any fluoroquinolone and at least one of three injectable second-line drugs (amikacin [AMK], capreomycin [CM], or kanamycin), is associated with very poor treatment outcomes (9).The rapid detection of microbial resistance to second-line drugs prior to and during treatment is therefore of essential importance for the implementation of increased infection control measures that may well limit the spread of TB to other patients.Compared to conventional liquid medium-based drug susceptibility testing, which still takes about 7 to 10 days, beginning from the time that a positive culture is obtained (18), the detection of genetic variants which mediate resistance to certain antimicrobial agents represents a more rapid alternative. Tests for the detection of MDR M. tuberculosis that detect mutations in the rpoB and katG/inhA regions, which confer resistance to RIF and INH, respectively, are now commercially available (3, 8, 13).Resistance to fluoroquinolones, such as ofloxacin (OFX), commonly used to treat MDR-TB is thought to be mediated by mutations (single nucleotide polymorphisms [SNPs]) in the target genes gyrA and, less frequently, gyrB, which encode the respective subunits of the DNA topoisomerase gyrase (24). Most mutations conferring resistance to quinolones are known to accumulate in a short discrete region of the gyrA and gyrB genes termed the quinolone resistance-determining region (QRDR) (2).Resistance to the aminoglycoside AMK and to CM are associated with SNPs in the 16S rRNA gene (rrs), especially in the region between nucleotides 1400 and 1500 (1, 23). Resistance to CM is thought to be additionally mediated by mutations located anywhere in the tlyA gene, which encodes a 2′-O-methyltransferase (10, 15).In the study described here, we aimed to determine if molecular analyses of target genes could be used for the rapid, specific, and sensitive detection of resistance to second-line drugs among new cases of TB and among previously treated patients in a high-incidence setting.Sequence analyses of the genes associated with fluoroquinolone resistance (gyrA and gyrB) and aminoglycoside or CM resistance (rrs and tlyA) were carried out with a panel of MDR- and XDR-TB strains arising from an MDR-TB treatment program in Karakalpakstan, a region in Uzbekistan with a high incidence of anti-TB drug resistance. Genetic data were then correlated with the results of phenotypic resistance testing.     

Characterization of mutations in multi- and extensive drug resistance among strains of Mycobacterium tuberculosis clinical isolates in Republic of Korea

In order to characterize molecular mechanisms of first- and second-line drug resistance in Mycobacterium tuberculosis and to evaluate the use of molecular markers of resistance, we analyzed 62 multidrug-resistant, 100 extensively drug-resistant, and 30 pan-susceptible isolates from Korean tuberculosis patients. Twelve genome regions associated with drug resistance, including katG, ahpC, and inhA promoter for isoniazid (INH); embB for ethambutol (EMB), rpoB for rifampin (RIF), pncA for pyrazinamide (PZA), gyrA for fluoroquinolones; rpsL, gidB, and rrs for streptomycin; rrs and eis for kanamycin (KM); rrs and tylA for capreomycin (CAP); and rrs for amikacin (AMK) were amplified simultaneously by polymerase chain reaction, and the DNA sequences were determined. We found mutations in 140 of 160 INH-resistant isolates (87.5%), 159 of 162 RIF-resistant isolates (98.15%), 127 of 143 EMB-resistant isolates (88.8%), 108 of 123 ofloxacin-resistant isolates (87.8%), and 107 of 122 PZA-resistant isolates (87.7%); 43 of 51 STM-resistant isolates (84.3%), 15 of 17 KM-resistant isolates (88.2%), and 14 of 15 (AMK and CAP)-resistant isolates (93.3%) had mutations related to specific drug resistance. In addition, the sequence analyses of the study revealed many novel mutations involving these loci. This result suggests that mutations in the rpoB531, katGSer315Thr, and C-15T in the inhA promoter region, and gyrA94, embB306, pncA159, rpsL43, and A1401G in the rrs gene could serve as useful markers for rapid detection of resistance profile in the clinical isolates of M. tuberculosis in Korea, with potentials for the new therapeutic benefits in actual clinical practice.     

耐多药结核分枝杆菌临床分离株30株对氟喹诺酮类及二线注射类抗结核药敏感性的分析

目的 了解耐多药结核分枝杆菌对氟喹诺酮类和（或）二线注射类抗结核药物的敏感性情况.方法 收集2011年6～9月采用Bactec-MGIT 960检测的30株耐多药结核分枝杆菌临床分离株,检测其对氟喹诺酮类及二线注射类抗结核药的药敏结果并进行分析.结果 30株耐多药结核分枝杆菌菌株对氟喹诺酮类和二线注射类抗结核药物耐药共21株（70％）.单药耐药依次为：氧氟沙星耐药19株（63.33％）,莫西沙星耐药13株（耐药率43.33％）,左氧氟沙星耐药10株（耐药率33.33％）,阿米卡星耐药9株（耐药率30％）,卷曲霉素最少（26.67％）.氧氟沙星耐药率高于左氧氟沙星耐药和三种氟喹诺酮类药同时耐药,差异有统计学意义（P=0.038）.氟喹诺酮类任意耐药及两种注射类药物任意耐药共8株[即广泛耐药结核病（XDR-TB）].氟喹诺酮类任意耐药及两种注射类药物敏感为11株,氟喹诺酮类均敏感及两种注射类药物任意耐药为2株,相比差异有统计学意义（P =0.001）.结论 耐多药结核病（MDR-TB）临床分离株对氟喹诺酮类药物耐药严重,氟喹诺酮类药物的耐药也是早期XDR-TB菌株的耐药主要形式.因此,氧氟沙星不建议作为MDR-TB的治疗用药.而MDR-TB临床分离株对阿米卡星和卷曲霉素敏感性较好,推荐可用于MDR-TB的首选药物.     

Identification of two novel FUT1 mutations in people with Bombay phenotype from Iran

Background and purposeBombay and Para-Bombay phenotypes are characterized by FUT1 gene mutation and lack of H antigen expression in red blood cells. ABH antigens are not present in the body secretions of Bombay individuals, while they are expressed in the secretions of para-Bombay. The aim of this study was to investigate the molecular basis of FUT1 and FUT2 genes in Iranians with the Bombay or Para-Bombay phenotype.Materials and MethodsABO phenotype analysis and routine serological tests were performed on 11 people with Bombay and Para-Bombay phenotypes. The coding regions of FUT1 and FUT2 genes were amplified by PCR followed by sequencing. The ABO genotypes were also determined by sequencing exons 6 and 7 of the ABO gene. Results: Serological investigations confirmed the Bombay phenotype in 8 samples and the Para-Bombay phenotype in 3 samples. Family members with the Bombay phenotype had the classic c 0.725 T > G mutation in the FUT1 gene, accompanied by deletion of the FUT2 gene. Other samples had c.653 A>G, c 0.661 C>T, c 0.652 C>G, and c.722 A>C mutations in the FUT1 while FUT2 was silenced by c 0.461 G>A. Conclusion: In this research, we identified two novel mutations in the FUT1 gene in individuals with the Bombay phenotype. This and previous works confirm the variety of FUT1 mutations.     

Emergence and Molecular Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Clinical Isolates from the Delhi Region in India

We screened 194 Mycobacterium tuberculosis strains isolated from tuberculosis (TB) patients in Delhi and neighboring regions in India to identify the prevalence of extensive drug resistance (XDR) in clinical isolates. Among these, 104 isolates were found to be multidrug resistant (MDR), and 6 were identified as XDR isolates, which was later confirmed by antimicrobial susceptibility testing against the respective drug screening panel. Genotyping was carried out by amplifying and sequencing the following genes: rpoB (rifampin), katG (isoniazid), gyrA (fluoroquinolones), and rrs (amikacin, kanamycin, and capreomycin). Our analyses indicated that mutations at the hot spots of these genes were positively correlated with drug resistance in clinical isolates. The key mutation observed for rpoB was in the codon for amino acid position 531 (S531L), and other mutations were seen in the hot spot, including those encoding Q510P, L511H, D516V, and H526Y mutations. We identified S315T and R463L substitutions encoded in the katG locus. An S95T substitution encoded in the gyrA locus was the most common mutation observed in fluoroquinolone-resistant isolates. In addition, we saw D94G and D94N mutations encoded in the QRDR region. The 16S rRNA (rrs) gene encoded mainly the A1401G mutation and an additional mutation, G1484T, resulting in ribosomal modifications. Taken together, the data in this report clearly establish the presence of phenotypically distinct XDR strains in India by molecular profiling and further identify specific mutational hot spots within key genes of XDR-TB strains.In recent years, the control of tuberculosis (TB) has become a global challenge due to the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). With 9.2 million new cases and 1.7 million deaths in 2006, TB remains one of the major life-threatening diseases worldwide (22). XDR-TB isolates are resistant to isoniazid and rifampin, to any fluoroquinolone (FQ), and to at least one of the three injectable second-line drugs (amikacin, kanamycin, and capreomycin) (6). As of June 2008, XDR-TB strains have been found in 49 countries, including the United States (6, 22). Furthermore, a recent report points to an alarming increase in the number of tuberculosis patients in the South Asian subcontinent, with India being singled out as having the greatest burden of XDR-TB, with a poor prognosis and high mortality among HIV-infected individuals (4). The risk of XDR-TB spread across country borders has heightened global concern over a potentially untreatable epidemic that may jeopardize recent advances made in global TB control.The prevalence of XDR-TB in India was reported in 2007, but no further efforts have been made to identify its genotypes or geographical spread (9). The present study was undertaken to characterize mutations prevalent in clinical isolates from India with respect to various drug target loci. We examined the drug target genes for rifampin (rpoB), isoniazid (katG), fluoroquinolones (gyrA), and aminoglycosides (rrs), which are commonly prescribed for the treatment of tuberculosis in India. The loci studied were rpoB (RNA polymerase B subunit), katG (catalase-peroxidase), rrs (16S rRNA), and gyrA (DNA gyrase A). Here we report, for the first time, the molecular characterization of XDR-TB isolates from India. This study confirms the presence of XDR-TB in India and simultaneously raises an alarm about its prevalence among TB patients, as many of them may initially have MDR-TB that slowly progresses and mutates to XDR-TB. Furthermore, the fact that some of these patients have HIV infection or the possibility of later coinfection with HIV has the potential to make this global HIV-TB epidemic untreatable with current therapies.     

Mechanism of Resistance to Amikacin and Kanamycin in Mycobacterium tuberculosis

An A1400G mutation of the rrs gene was identified in Mycobacterium tuberculosis (MTB) strain ATCC 35827 and in 13 MTB clinical isolates resistant to amikacin-kanamycin (MICs, >128 μg/ml). High-level cross-resistance may result from such a mutation since MTB has a single copy of the rrs gene. Another mechanism(s) may account for high-level amikacin-kanamycin resistance in two mutants and lower levels of resistance in four clinical isolates, all lacking the A1400G mutation.     

Evaluation of Pyrosequencing for Detecting Extensively Drug-Resistant Mycobacterium tuberculosis among Clinical Isolates from Four High-Burden Countries

Reliable molecular diagnostics, which detect specific mutations associated with drug resistance, are promising technologies for the rapid identification and monitoring of drug resistance in Mycobacterium tuberculosis isolates. Pyrosequencing (PSQ) has the ability to detect mutations associated with first- and second-line anti-tuberculosis (TB) drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations. The aim of this project was to evaluate the performance of PSQ in predicting phenotypic drug resistance in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) clinical isolates from India, South Africa, Moldova, and the Philippines. A total of 187 archived isolates were run through a PSQ assay in order to identify M. tuberculosis (via the IS6110 marker), and to detect mutations associated with M/XDR-TB within small stretches of nucleotides in selected loci. The molecular targets included katG, the inhA promoter and the ahpC-oxyR intergenic region for isoniazid (INH) resistance; the rpoB core region for rifampin (RIF) resistance; gyrA for fluoroquinolone (FQ) resistance; and rrs for amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance. PSQ data were compared to phenotypic mycobacterial growth indicator tube (MGIT) 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP. PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first- and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.     

Mutation scanning of exon 20 of the BRCA1 gene by high-resolution melting curve analysis

Objectives5382insC frameshift mutation along with 5331G>A (G1738R) missense mutation, both found in exon 20 of the BRCA1 gene, are relatively frequent among the Greek breast and ovarian cancer population (46%). Our goal was to develop a novel, reliable and rapid genotyping/scanning method for mutation detection of the exon 20 of the BRCA1 gene, using high-resolution melting curve analysis.Design and methodsThe developed methodology was based on real-time PCR and high-resolution melting curve analysis in the presence of LCGreen I dye. Two amplicons on the exon 20 of BRCA1 gene were designed (157 bp and 100 bp), one flanking the exon's boundaries, and one embracing the 5382insC mutation. Our methodology was first optimized and validated by using genomic DNA samples with the 5382insC and 5331G>A (G1738R) mutations and wild-type. In total, the developed methodology was applied on 90 peripheral blood and 127 formalin-fixed paraffin-embedded breast tissue samples.ResultsSensitivity studies with gDNA isolated from peripheral blood showed that mutated DNA could be reliably detected in the presence of wild-type DNA at 5% and 0.5% ratio with the larger and the smaller amplicon, respectively. By using the developed methodology we successfully identified 5382insC, 5331G>A and 5370C>T (R1751X) mutations, in genomic DNA isolated from peripheral blood samples and 5382insC mutation in two breast tumors, as verified by DNA sequencing.ConclusionsThe combination of real-time PCR and high-resolution melting curve analysis provides a cost-efficient, simple and rapid approach to successfully scan exon 20 of BRCA1 gene for these clinically important and frequent mutations.     

Prevalence and Molecular Characteristics of Drug-Resistant Mycobacterium tuberculosis in Hunan,China

To determine the prevalence and molecular characteristics of drug-resistant tuberculosis in Hunan province, drug susceptibility testing and spoligotyping methods were performed among 171 M. tuberculosis isolates. In addition, the mutated characteristics of 12 loci, including katG, inhA, rpoB, rpsL, nucleotides 388 to 1084 of the rrs gene [rrs(388–1084)], embB, pncA, tlyA, eis, nucleotides 1158 to 1674 of the rrs gene [rrs(1158–1674)], gyrA, and gyrB, among drug-resistant isolates were also analyzed by DNA sequencing. Our results indicated that the prevalences of isoniazid (INH), rifampin (RIF), streptomycin (SM), ethambutol (EMB), pyrazinamide (PZA), capreomycin (CAP), kanamycin (KAN), amikacin (AKM), and ofloxacin (OFX) resistance in Hunan province were 35.7%, 26.9%, 20.5%, 9.9% 15.2%, 2.3%, 1.8%, 1.2%, and 10.5%, respectively. The previously treated patients presented significantly increased risks for developing drug resistance. The majority of M. tuberculosis isolates belonged to the Beijing family. Almost all the drug resistance results demonstrated no association with genotype. The most frequent mutations of drug-resistant isolates were katG codon 315 (katG₃₁₅), inhA15, rpoB₅₃₁, rpoB₅₂₆, rpoB₅₁₆, rpsL₄₃, rrs₅₁₄, embB₃₀₆, pncA₉₆, rrs₁₄₀₁, gyrA₉₄, and gyrA₉₀. These results contribute to the knowledge of the prevalence of drug resistance in Hunan province and also expand the molecular characteristics of drug resistance in China.     

In vitro and in vivo antibacterial activity of nitrofurantoin against clinical isolates of E. coli in Japan and evaluation of biological cost of nitrofurantoin resistant strains using a mouse urinary tract infection model

IntroductionNitrofurantoin is a well-established antibiotic, and is an important first-line oral treatment for uncomplicated urinary tract infections. However, little information is available with respect to its antibacterial activity in Japan, in vivo efficacy, or the in vivo biological cost of resistant strains.MethodsWe compared the susceptibility of six representative antibacterial agents—nitrofurantoin, sulfamethoxazole/trimethoprim, fosfomycin, mecillinam, ciprofloxacin, and cefdinir—against E. coli clinically isolated in Japan during 2017. We evaluated the in vivo efficacy of nitrofurantoin using a model of mouse urinary tract infection caused by ciprofloxacin resistant E. coli. We obtained nitrofurantoin resistant isolates through tests generating spontaneous mutations, and assessed the in vivo fitness of nitrofurantoin resistant isolates.ResultsThe MIC₉₀ of nitrofurantoin was 16 μg/mL, and was the lowest among the drugs tested. It was found that, in the mouse urinary tract infection model, 30 mg/kg and 100 mg/kg of nitrofurantoin reduced the count of viable bacterial cells in the kidney, while 100 mg/kg of ciprofloxacin did not. All spontaneous bacterial mutants resistant to nitrofurantoin had deletions in the nfsA gene, and we found that the resistant strain had lower growth in the mouse urinary tract infection model than in the parent strain.ConclusionsWe demonstrated promising in vitro and in vivo activity of nitrofurantoin against E. coli clinical isolates in Japan, and lower in vivo fitness of the resistant strain of nitrofurantoin.     

How Much Oxycodone Is Needed for Adequate Analgesia After Breast Cancer Surgery: Effect of the OPRM1 118A>G Polymorphism

Most clinically used opioids are mu-opioid receptor agonists. Therefore, genetic variation of the OPRM1 gene that encodes the mu-opioid receptor is of great interest for understanding pain management. A polymorphism 118A>G (rs1799971) within the OPRM1 gene results in a missense mutation and affects the function of the receptor. We studied the association between the 118A>G polymorphism and oxycodone analgesia and pain sensitivity in 1,000 women undergoing breast cancer surgery. Preoperatively, experimental cold and heat pain sensitivities were tested. Postoperative pain was assessed at rest and during motion. Intravenous oxycodone analgesia was titrated first by a research nurse and on the ward using a patient-controlled analgesia device. The primary endpoint was the amount of oxycodone needed for the first state of adequate analgesia. For each patient, the 118A>G polymorphism was genotyped using the Sequenom MassARRAY (Sequenom, San Diego, CA). The association between this variant and the pain phenotypes was tested using linear regression. The 118A>G variant was associated significantly with the amount of oxycodone requested for adequate analgesia (P = .003, β = .016). Collectively, oxycodone consumption was highest in individuals having the GG genotype (.16 mg/kg), lowest for those with the AA genotype (.12 mg/kg), and moderate for those having the AG genotype (.13 mg/kg). Furthermore, the G allele was associated with higher postoperative baseline pain ratings (P = .001, β = .44). No evidence of association with other pain phenotypes examined was observed.PerspectiveThis study demonstrates that the OPRM1 118A>G polymorphism was associated with the amount of oxycodone required in the immediate postoperative period. Although a significant factor for determining oxycodone requirement, the 118A>G polymorphism alone explained less than 1% of the variance. No association was found between 118A>G and experimental pain.     

GenoType MTBDRsl for detection of second-line drugs and ethambutol resistance in multidrug-resistant Mycobacterium tuberculosis isolates at a high-throughput laboratory

We assessed the performance of MTBDRsl for detection of resistance to fluoroquinolones, aminoglycosides/cyclic peptides, and ethambutol compared to BACTEC MGIT 960 by subjecting simultaneously to both tests 385 phenotypically multidrug-resistant-Mycobacterium tuberculosis isolates from Sao Paulo, Brazil. Discordances were resolved by Sanger sequencing. MTBDRsl correctly detected 99.7% of the multidrug-resistant isolates, 87.8% of the pre-XDR, and 73.9% of the XDR. The assay showed sensitivity of 86.4%, 100%, 85.2% and 76.4% for fluoroquinolones, amikacin/kanamycin, capreomycin and ethambutol, respectively. Specificity was 100% for fluoroquinolones and aminoglycosides/cyclic peptides, and 93.6% for ethambutol. Most fluoroquinolone-discordances were due to mutations in genome regions not targeted by the MTBDRsl v. 1.0: gyrA_H70R and gyrB_R446C, D461N, D449V, and N488D. Capreomycin-resistant isolates with wild-type rrs results on MTBDRsl presented tlyA mutations. MTBDRsl presented good performance for detecting resistance to second-line drugs and ethambutol in clinical isolates. In our setting, multidrug-resistant. isolates presented mutations not targeted by the molecular assay.     

Dosage Optimization of Efavirenz Based on a Population Pharmacokinetic–Pharmacogenetic Model of HIV-infected Patients in Thailand

PurposeEfavirenz exhibits high interindividual variability in plasma concentrations, leading to unpredictable efficacy and toxicity. Polymorphism of CYP2B6 516G > T has been found to predominantly contribute to efavirenz variability. However, dosage recommendations incorporating CYP2B6 516G > T polymorphism have not been investigated in the Thai population. This study aimed to develop a population model of the pharmacokinetic properties of efavirenz, and to investigate the impact of patients' characteristics and CYP2B6 516G > T polymorphism on the pharmacokinetic properties of efavirenz. Model-based simulations were performed to provide genotype-based dosage optimization in a Thai population.MethodsPlasma efavirenz concentrations measured at 12 h post-dose in 360 Thai HIV-infected patients with and without tuberculosis were analyzed by the nonlinear mixed-effects modeling approach. A 1-compartment model with first-order absorption and elimination was used for describing the pharmacokinetic properties of efavirenz.FindingsThe allele frequency of CYP2B6 516G > T was 34.17%. The efavirenz oral clearance were 11.9, 8.0, and 2.8 L/h in patients weighing 57 kg and having the CYP2B6 516 GG, 516 GT, and 516 TT genotypes, respectively. The use of rifampicin increased efavirenz oral clearance by 28%. The results from the simulations suggest that efavirenz dosages of 400, 300, and 100 mg once daily in Thai HIV mono-infected patients, and 800, 600, and 200 mg once daily in HIV/tuberculosis co-infected patients carrying CYP2B6 516 GG, 516 GT, and 516 TT, respectively.ImplicationThe results from this study provide a rationale for efavirenz dose adjustment based on CYP2B6 516G > T polymorphism in Thai HIV-infected patients, which could help to improve treatment outcomes in this population. ClinicalTrials.gov identifier: NCT01138267.     

Two cases of 5-fluorouracil toxicity linked with gene variants in the DPYD gene

ObjectivesDihydropyrimidine dehydrogenase (DPD) is the initial rate-limiting enzyme in endogenous pyrimidine catabolism and is responsible for the reduction of the pyrimidine analog 5-fluorouracil (5-FU). DPD deficiency is known to cause potentially lethal toxicity in patients receiving 5-FU. We here report a frequency analysis of one of the major splice-site mutations in the DPDY gene, and further two new DPYD gene variants.Design and methodsRestriction fragment length polymorphism (RFLP) and DNA sequence analysis were performed on genomic DNA and mRNA.ResultsIn 400 patients that were diagnosed with cancer and were eligible for 5-FU treatment, 14 patients were found to be heterozygous for the splice-site mutation DPYD IVS14+1G>A, which corresponds to a population frequency of 3.5%. Two novel variants in the DPYD gene were identified. The first case was heterozygous for DPYD c.1796T>C (p.M599T). In the second case, we observed heterozygosity for the splice-site mutation DPYD IVS14+17A>G.ConclusionsWe report two new DPYD gene variants, of which DPYD c.1796T>C is potentially pathogenic, whereas DPYD IVS14+17A>G is suggested as a variant without clinical significance.     

In-vitro activity of ofloxacin against Mycobacterium tuberculosis and its clinical efficacy in multiply resistant pulmonary tuberculosis

The in-vitro susceptibilities to ofloxacin of 159 clinical sputum isolates of Mycobacterium tuberculosis, comprising 95 isolates sensitive to all drugs, 31 isolates resistant to streptomycin or isoniazid or both, 27 isolates resistant to streptomycin, isoniazid and rifampicin and six isolates resistant to rifampicin (and in three cases to other drugs) were determined. Favourable MICs of ofloxacin (0.63-1.25 mg/l) were demonstrated for 147 isolates (92%). Twenty-two patients with resistant strains (including one patient with rifampicin intolerance) were studied: ten were given 300 mg ofloxacin and ten were given 800 mg ofloxacin, once daily in both cases, together with second-line accompanying drugs, for nine months to one year. Two received 800 mg of ofloxacin once daily alone for similar periods. In the 300 mg-ofloxacin group and the 800 mg-ofloxacin group, five and eight patients, respectively, achieved culture conversion; the rest failed. In the former group, the peak serum ofloxacin concentrations were 3.71-8.08 mg/l and the mean sputum/serum ratio was 0.85. In the latter group, the corresponding values were 10-18.7 mg/l, and 0.76, respectively. All patients tolerated the drugs well. Analysing only patients with accompanying drugs, those on ofloxacin 800 mg once daily had more rapid sputum culture conversion than those on ofloxacin 300 mg once daily (Mann-Whitney Wilcoxon Rank Sum Test: P less than 0.05), indicating more rapid bacteriolysis and implying the definite efficacy of ofloxacin when used together with second-line accompanying drugs in the management of resistant tuberculosis.