scholarly journals Rapid Detection of Mutations in the Human-DerivedPneumocystis carinii Dihydropteroate Synthase Gene Associated with Sulfa Resistance

2001 ◽  
Vol 45 (3) ◽  
pp. 776-780 ◽  
Author(s):  
Liang Ma ◽  
Joseph A. Kovacs
Keyword(s):  
Rapid Detection ◽  
Direct Sequencing ◽  
Pneumocystis Carinii ◽  
Point Mutations ◽  
Single Strand Conformation Polymorphism ◽  
Rapid Identification ◽  
Clinical Isolates ◽  
Minority Population ◽  
Dihydropteroate Synthase ◽  
Dhps Gene

ABSTRACT Recent studies have shown that point mutations in the dihydropteroate synthase (DHPS) gene of human-derivedPneumocystis carinii are related to exposure to sulfa drugs and possibly represent the emergence of sulfa resistance. We developed a simple single-strand conformation polymorphism (SSCP) method to permit rapid detection of these mutations. With plasmid constructs, SSCP was able to detect as little as 10% of a minority population. The SSCP assay was compared to direct sequencing for typing the DHPS gene by examining 37 clinical isolates with known DHPS sequences and 41 clinical isolates with unknown DHPS sequences. The typing results were consistent between these two methods for all isolates except 11 in which mutations were detected by SSCP but not by direct sequencing. Sequencing of individual clones after subcloning confirmed the presence of mutations in a minority population as determined by SSCP. SSCP is a very simple and sensitive method for rapid identification of P. camii DHPS mutations.

scholarly journals Genetic Analysis of Multiple Loci Suggests that Mutations in the Pneumocystis carinii f. sp.hominis Dihydropteroate Synthase Gene Arose Independently in Multiple Strains

2001 ◽  
Vol 45 (11) ◽  
pp. 3213-3215 ◽  
Author(s):  
Liang Ma ◽  
Joseph A. Kovacs
Keyword(s):  
Genetic Analysis ◽  
Pneumocystis Carinii ◽  
Single Strand Conformation Polymorphism ◽  
Single Strain ◽  
Dihydropteroate Synthase ◽  
Multiple Loci ◽  
Dhps Gene ◽  
Multiple Strains ◽  
Genomic Regions ◽  
Conformation Polymorphism

ABSTRACT To determine if mutations in the dihydropteroate synthase (DHPS) gene of Pneumocystis carinii f. sp.hominis arose in a single strain that was subsequently widely disseminated, we examined four genomic regions of 22 P. carinii clinical isolates selected based on the absence or presence of mutations in the DHPS gene. By single-strand conformation polymorphism and DNA sequencing, we found varying genotypes for each of the four regions in isolates with DHPS mutations, suggesting that these mutations occurred independently in multiple strains ofP. carinii. This suggests that exposure to sulfa will select for these mutations in diverse strains.

scholarly journals Dihydropteroate synthase (DHPS) gene mutation study in HIV-Infected Indian patients with Pneumocystis jirovecii pneumonia

2010 ◽  
Vol 4 (11) ◽  
pp. 761-766 ◽  
Author(s):  
Anuj Kumar Tyagi ◽  
Bijay Ranjan Mirdha ◽  
Kalpana Luthra ◽  
Randeep Guleria ◽  
Anant Mohan ◽  
...  
Keyword(s):  
Silver Staining ◽  
Tertiary Care ◽  
Pneumocystis Carinii ◽  
Pneumocystis Jirovecii ◽  
Hiv Positive ◽  
Pneumocystis Jirovecii Pneumonia ◽  
Sulfa Drug ◽  
Dihydropteroate Synthase ◽  
Methenamine Silver ◽  
Dhps Gene

Introduction: Pneumocystis jirovecii dihydropteroate synthase (DHPS) gene mutations' (55th and 57th codon) association with prior sulfa prophylaxis failure has been reported from both developed and developing countries. We conducted a prospective study to determine the prevalence of P. jirovecii DHPS mutations from 2006 to 2009 on P. jirovecii isolates obtained from HIV-infected patients with a clinical diagnosis of Pneumocystis carinii pneumonia (PCP) admitted to our tertiary care reference health center in New Delhi, India. Methodology: Detection of P. jirovecii cysts was performed by direct fluorescent antibody (DFA) staining and by Grocott's-Gomori methenamine silver staining (GMS). DNA detection was performed by polymerase chain reaction (PCR) using primers for the major surface glycoprotein (MSG) gene. P. jirovecii DHPS gene was amplified by nested PCR protocol and sequenced for detecting mutations at the 55th and 57th codons. Results: Out of 147 HIV-positive patients with suspected Pneumocystis pneumonia (PCP), 16 (10.8%) PCP positive cases were detected. Of 16 cases, nine (56.2%) were positive by DFA staining, four (25%) were positive by Grocott's-Gomori methenamine silver staining, and all 16 were positive by MSG PCR. DHPS mutations at the 55th and 57th codons were observed in 6.2% of HIV patients studied, which was relatively low compared to reports from developed nations. Conclusions:  Prevalence of Pneumocystis jirovecii DHPS mutations associated with cotrimoxazole treatment failure may be low in the Indian subpopulation of HIV-positive patients and warrants larger studies to elucidate the true picture of Pneumocystis jirovecii sulfa drug resistance in India.

Low-Dose Treatment with Sulfadoxine-Pyrimethamine Combinations Selects for Drug-Resistant Plasmodium falciparum Strains

1999 ◽  
Vol 43 (9) ◽  
pp. 2205-2208 ◽  
Author(s):  
Jürgen F. J. Kun ◽  
Leopold G. Lehman ◽  
Bertrand Lell ◽  
Ruprecht Schmidt-Ott ◽  
Peter G. Kremsner
Keyword(s):  
Low Dose ◽  
Point Mutations ◽  
Drug Resistant ◽  
Dhfr Gene ◽  
Dihydropteroate Synthase ◽  
Before And After ◽  
Genes Encoding ◽  
Resistant Strains ◽  
Dhps Gene ◽  
Drug Resistant Strains

ABSTRACT A total of 252 children were enrolled in a drug trial to assess the effect of minimal doses of sulfadoxine (Sdx) and pyrimethamine (Pyr). Parasite samples isolated from these patients were analyzed before and after treatment to investigate the level of drug-resistant strains. The parasite genes encoding dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) were assayed for point mutations that are associated with resistance against drugs. Before treatment, Pyrr genotypes of the DHFR gene were found in 42% of all samples, 8% of the patients harbored a mixed parasite population and 50% had a sensitive DHFR genotype. In terms of the DHPS gene, we found mutations in 45% of the parasites. Twenty-four percent had a Ser436 mutation, and 26% had a Gly437mutation. Recrudescent parasites were highly enriched for both Pyrr and Sdxr strains after treatment (P < 0.001 and P = 0.029, respectively).

scholarly journals A Case of Manifesting Carrier with DMD Phenotype

2009 ◽  
Vol 52 (4) ◽  
pp. 167-170 ◽  
Author(s):  
Akshay Anand ◽  
Monika Vinish ◽  
Sudesh Prabhakar
Keyword(s):  
Polymerase Chain Reaction ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Single Strand Conformation Polymorphism ◽  
Clinical Findings ◽  
Lower Limbs ◽  
Chain Reaction ◽  
History Of ◽  
Polymerase Chain ◽  
Conformation Polymorphism

A case of a 35-year old female with a history of proximal weakness in lower limbs and bulkiness of both calves manifesting before ten years of age was reported. Clinical findings were suggestive of muscular dystrophy. Genetic analysis using polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) and direct sequencing revealed several point mutations, which account for dystrophin dysfunction and DMD type pathogenesis.

scholarly journals Detection of gyrA Mutations among 335Pseudomonas aeruginosa Strains Isolated in Japan and Their Susceptibilities to Fluoroquinolones

1999 ◽  
Vol 43 (2) ◽  
pp. 406-409 ◽  
Author(s):  
Takashi Takenouchi ◽  
Eiko Sakagawa ◽  
Mie Sugawara
Keyword(s):  
Double Point ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Single Strand Conformation Polymorphism ◽  
Fluoroquinolone Resistance ◽  
Polymorphism Analysis ◽  
Novel Mutations ◽  
Conformation Polymorphism Analysis ◽  
High Level ◽  
Conformation Polymorphism

ABSTRACT gyrA point mutations in 335 clinical Pseudomonas aeruginosa isolates were examined mainly by nonisotopic single-strand conformation polymorphism analysis and direct sequencing. Seven types of missense gyrA mutations were observed in 70 of 335 strains (20.9%), and ciprofloxacin MICs were ≥3.13 μg/ml for 63 of 70 strains (90.0%). These included two double point mutations and three novel mutations (Ala-67→Ser plus Asp-87→Gly, Ala-84→Pro, and Gln-106→Leu). Thr-83→Ile mutants were predominantly observed (63 of 70 mutants) and showed high-level fluoroquinolone resistance (ciprofloxacin MIC at which 50% of isolates are inhibited, 25 μg/ml).

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis.

1997 ◽  
Vol 41 (3) ◽  
pp. 540-543 ◽  
Author(s):  
A Scorpio ◽  
P Lindholm-Levy ◽  
L Heifets ◽  
R Gilman ◽  
S Siddiqi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Direct Sequencing ◽  
Single Strand Conformation Polymorphism ◽  
Therapy Resistance ◽  
Sscp Analysis ◽  
Clinical Isolates ◽  
Nucleotide Substitutions ◽  
Resistant Strains ◽  
Made In

Pyrazinamide (PZA) is a first-line drug for short-course tuberculosis therapy. Resistance to PZA is usually accompanied by loss of pyrazinamidase (PZase) activity in Mycobacterium tuberculosis. PZase converts PZA to bactericidal pyrazinoic acid, and the loss of PZase activity is associated with PZA resistance. The gene (pncA) encoding the M. tuberculosis PZase has recently been sequenced, and mutations in pncA were previously found in a small number of PZA-resistant M. tuberculosis strains. To further understand the genetic basis of PZA resistance and determine the frequency of PZA-resistant strains having pncA mutations, we analyzed a panel of PZA-resistant clinical isolates and mutants made in vitro. Thirty-three of 38 PZA-resistant clinical isolates had pncA mutations. Among the five strains that did not contain pncA mutations, four were found to be falsely resistant and one was found to be borderline resistant to PZA. The 33 PZA-resistant clinical isolates and 8 mutants made in vitro contained various mutations, including nucleotide substitutions, insertions, or deletions in the pncA gene. The identified mutations were dispersed along the pncA gene, but some degree of clustering of mutations was found at the following regions: Gly132-Thr142, Pro69-Leu85, and Ile5-Asp12. PCR-single-strand conformation polymorphism (SSCP) analysis was shown to be useful for the rapid detection of pncA mutations in the PZA-resistant strains. We conclude that a mutation in the pncA gene is a major mechanism of PZA resistance and that direct sequencing by PCR or SSCP analysis should help to rapidly identify PZA-resistant M. tuberculosis strains.

scholarly journals p53 gene mutations in multiple myeloma are associated with advanced forms of malignancy

Blood ◽  
1993 ◽  
Vol 81 (1) ◽  
pp. 128-135 ◽  
Author(s):  
A Neri ◽  
L Baldini ◽  
D Trecca ◽  
L Cro ◽  
E Polli ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Gene Mutations ◽  
P53 Gene ◽  
Single Strand Conformation Polymorphism ◽  
P53 Mutation ◽  
Late Event ◽  
Multistep Process ◽  
P53 Gene Mutations

Abstract The frequency and type of p53 gene mutations was investigated in a series of 52 cases of multiple myeloma (MM) representative of the different clinical phases and forms of the disease (indolent, 12 cases; chronic, 24 cases; acute/leukemic, 16 cases). DNAs were analyzed for p53 gene mutations in exons 5 to 9 by polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and direct sequencing of PCR-amplified fragments. Point mutations were detected in 7 of 52 patients (13%) (5 at exon 8; 1 at exon 6; 1 at exon 7), and were specifically associated with the more advanced and clinically aggressive acute/leukemic forms of MM (7 of 16 [43%].) Three of the mutated cases had been evaluated at clinical presentation in earlier phases of the disease (indolent or chronic) and were found to be negative for p53 mutation. Moreover, three patients with p53 mutation had not received chemotherapy at the time of investigation. These results support the notion that the development of MM is a multistep process and suggest that alterations in the p53 gene may represent an important late event in MM tumor progression.

scholarly journals p53 gene mutations in multiple myeloma are associated with advanced forms of malignancy

Blood ◽  
1993 ◽  
Vol 81 (1) ◽  
pp. 128-135 ◽  
Author(s):  
A Neri ◽  
L Baldini ◽  
D Trecca ◽  
L Cro ◽  
E Polli ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Gene Mutations ◽  
P53 Gene ◽  
Single Strand Conformation Polymorphism ◽  
P53 Mutation ◽  
Late Event ◽  
Multistep Process ◽  
P53 Gene Mutations

The frequency and type of p53 gene mutations was investigated in a series of 52 cases of multiple myeloma (MM) representative of the different clinical phases and forms of the disease (indolent, 12 cases; chronic, 24 cases; acute/leukemic, 16 cases). DNAs were analyzed for p53 gene mutations in exons 5 to 9 by polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and direct sequencing of PCR-amplified fragments. Point mutations were detected in 7 of 52 patients (13%) (5 at exon 8; 1 at exon 6; 1 at exon 7), and were specifically associated with the more advanced and clinically aggressive acute/leukemic forms of MM (7 of 16 [43%].) Three of the mutated cases had been evaluated at clinical presentation in earlier phases of the disease (indolent or chronic) and were found to be negative for p53 mutation. Moreover, three patients with p53 mutation had not received chemotherapy at the time of investigation. These results support the notion that the development of MM is a multistep process and suggest that alterations in the p53 gene may represent an important late event in MM tumor progression.

scholarly journals Nucleotide substitutions in the Candida albicans ERG11 gene of azole-susceptible and azole-resistant clinical isolates.

2013 ◽  
Vol 60 (4) ◽  
Author(s):  
Joanna Katarzyna Strzelczyk ◽  
Anna Slemp-Migiel ◽  
Magdalena Rother ◽  
Karolina Gołąbek ◽  
Andrzej Wiczkowski
Keyword(s):  
Candida Albicans ◽  
Sequence Analysis ◽  
Point Mutations ◽  
Single Strand Conformation Polymorphism ◽  
Clinical Isolates ◽  
Primary Target ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Conformation Polymorphism

One of the mechanisms of Candida albicans resistance to azole drugs used in antifungal therapy relies on increased expression and presence of point mutations in the ERG11 gene that encodes sterol 14α demethylase (14DM), an enzyme which is the primary target for the azole class of antifungals. The aim of the study was to analyze nucleotide substitutions in the Candida albicans ERG11 gene of azole-susceptible and azole-resistant clinical isolates. The Candida albicans isolates represented a collection of 122 strains selected from 658 strains isolated from different biological materials. Samples were obtained from hospitalized patients. Fluconazole susceptibility was tested in vitro using a microdilution assay. Candida albicans strains used in this study consisted of two groups: 61 of the isolates were susceptible to azoles and the 61 were resistant to azoles. Four overlapping regions of the ERG11 gene of the isolates of Candida albicans strains were amplified and sequenced. The MSSCP (multitemperature single strand conformation polymorphism) method was performed to select Candida albicans samples presenting genetic differences in the ERG11 gene fragments for subsequent sequence analysis. Based on the sequencing results we managed to detect 19 substitutions of nucleotides in the ERG11 gene fragments. Sequencing revealed 4 different alterations: T495A, A530C, G622A and A945C leading to changes in the corresponding amino acid sequence: D116E, K128T, V159I and E266D. The single nucleotide changes in the ERG11 gene did not affect the sensitivity of Candida albicans strains, whereas multiple nucleotide substitutions in the ERG11 gene fragments indicated a possible relation with the increase in resistance to azole drugs.

Detection of grlA and gyrA Mutations in 344 Staphylococcus aureus Strains

1998 ◽  
Vol 42 (2) ◽  
pp. 236-240 ◽  
Author(s):  
Tong Wang ◽  
Mayumi Tanaka ◽  
Kenichi Sato
Keyword(s):  
Staphylococcus Aureus ◽  
Direct Sequencing ◽  
Single Point ◽  
Point Mutations ◽  
Single Strand Conformation Polymorphism ◽  
Fluoroquinolone Resistance ◽  
Polymorphism Analysis ◽  
Ciprofloxacin Resistance ◽  
Single Point Mutations ◽  
Length Analysis

ABSTRACT Mutations in the grlA and gyrA genes of 344 clinical strains of Staphylococcus aureus isolated in 1994 in Japan were identified by combinations of single-strand conformation polymorphism analysis, restriction fragment length analysis, and direct sequencing to identify possible relationships to fluoroquinolone resistance. Five types of single-point mutations and four types of double mutations were observed in the grlA genes of 204 strains (59.3%). Four types of single-point mutations and four types of double mutations were found in the gyrA genes of 188 strains (54.7%). Among them, the grlA mutation of TCC→TTC or TAC (Ser-80→Phe or Tyr) and the gyrAmutation of TCA→TTA (Ser-84→Leu) were principal, being detected in 137 (39.8%) and 121 (35.9%) isolates, respectively. ThegrlA point mutations of CAT→CAC (His-77 [silent]), TCA→CCA (Ser-81→Pro), and ATA→ATT (Ile-100 [silent]) were novel, as was the GAC→GGC (Asp-73→Gly) change in gyrA. A total of 15 types of mutation combinations within both genes were related to ciprofloxacin resistance (MIC ≥ 3.13 μg/ml) and were present in 193 mutants (56.1%). Strains containing mutations in both genes were highly resistant to ciprofloxacin (MIC at which 50% of the isolates are inhibited [MIC50] = 50 μg/ml). Those with the Ser-80→Phe or Tyr alteration in grlA but wild-typegyrA showed a lower level of ciprofloxacin resistance (MIC50 ≤ 12.5 μg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlAin the presence or absence of a concomitant mutation at codon 73, 84, or 88 in gyrA (MIC ≤ 6.25 μg/ml). The new fluoroquinolone DU-6859a showed good activity with 186 of 193 isolates (96.4%) for which the MIC was ≤6.25 μg/ml.

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