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

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.3.776-780.2001 ◽

2001 ◽

Vol 45 (3) ◽

pp. 776-780 ◽

Cited By ~ 13

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.

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Typing of Pneumocystis carinii f. sp. hominis by single-strand conformation polymorphism of four genomic regions.

Journal of Clinical Microbiology ◽

10.1128/jcm.35.12.3086-3091.1997 ◽

1997 ◽

Vol 35 (12) ◽

pp. 3086-3091 ◽

Cited By ~ 43

Author(s):

P M Hauser ◽

P Francioli ◽

J Bille ◽

A Telenti ◽

D S Blanc

Keyword(s):

Pneumocystis Carinii ◽

Single Strand Conformation Polymorphism ◽

Single Strand ◽

Genomic Regions ◽

Conformation Polymorphism

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

The Journal of Infection in Developing Countries ◽

10.3855/jidc.914 ◽

2010 ◽

Vol 4 (11) ◽

pp. 761-766 ◽

Cited By ~ 4

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.

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A Unique Method for Mutation Analysis of Tumor Suppressor Genes in Colorectal Carcinomas Using a Crypt Isolation Technique

Archives of Pathology & Laboratory Medicine ◽

10.5858/2000-124-0382-aumfma ◽

2000 ◽

Vol 124 (3) ◽

pp. 382-386 ◽

Cited By ~ 1

Author(s):

Tamotsu Sugai ◽

Wataru Habano ◽

Shin-ichi Nakamura ◽

Noriyuki Uesugi ◽

Shunichi Sasou ◽

...

Keyword(s):

Tumor Suppressor ◽

Genetic Analysis ◽

Tumor Suppressor Genes ◽

Single Strand Conformation Polymorphism ◽

Single Strand ◽

Colorectal Carcinomas ◽

Suppressor Genes ◽

Isolation Technique ◽

Crypt Isolation ◽

Conformation Polymorphism

Abstract Background.—Contamination of nontumor tissue makes genetic analysis difficult. For this reason, it is important to obtain pure tumor tissue to ensure accurate genetic analysis. Objective.—To accurately assess the incidence of mutation of tumor suppressor genes (p53: exon 5–8; APC: mutated cluster region; NF-2 gene: all exons) in 45 colorectal carcinomas. Methods.—We developed an application of the polymerase chain reaction–single-strand conformation polymorphism and DNA sequence by coupling them with crypt isolation. Results.—Mutations of p53 and APC genes were found in 24 and 22 of 45 colorectal carcinomas, respectively. No mutation of the NF-2 gene was observed in this cancer. Single-strand conformation polymorphism using a crypt isolation technique showed a clear migrating band and no false-positive data. Conclusions.—The crypt isolation technique is a useful method for accurately analyzing genetic alterations. Furthermore, our proposed method confirmed the morphological findings obtained before the genetic analysis.

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