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Published on: March 30, 2014
Molecular detection of DHFR gene polymorphisms in Pneumocystis jirovecii isolates from Indian patients
Yogita Singh1, Bijay Ranjan Mirdha, Randeep Guleria
1All India institute of Medical Sciences, New Delhi, India. yogitasingh1985@gmail.com.
Introduction:
Pneumocystis pneumonia (PCP) is an opportunistic life-threatening infection, especially for immunocompromised individuals. A trimethoprim-sulfamethoxazole (TMP-SMX) combination is commonly used for the treatment of PCP, targeting both dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes. Several studies have already shown that polymorphisms in the DHPS gene are associated with drug resistance. The present study analyzed DHFR gene polymorphisms in Pneumocystis jirovecii recovered from clinical samples from patients admitted to a tertiary care health center in New Delhi, India.
Methodology:
Detection of P. jirovecii was performed using Gomori methenamine silver staining (GMS) and nested polymerase chain reaction (PCR) assay targeting the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) gene. The DHFR gene was amplified using nested PCR protocol and was sequenced for detection of polymorphisms.
Results:
Of 180 clinical samples, only 4% (7/180) were positive by GMS staining, and 10% (18/180) were positive by mt LSU rRNA PCR assay. Of these 18 positive samples, only 77% (14/18) were amplified by the DHFR gene PCR assay. A total of 16 nucleotide substitutions were observed in 42% (6/14) samples targeted for the DHFR gene, of which 8 nucleotide substitutions were synonymous and the rest were non-synonymous.
Conclusions:
The DHFR gene mutations found in this study may possibly indicate an association of process likely to contribute to therapeutic failure or an evolutionary process, and warrant continuous monitoring.
Insights
Dihydrofolate reductase (DHFR) gene mutations were identified in Pneumocystis jirovecii from Indian patients. These mutations may contribute to treatment failure and require ongoing surveillance for Pneumocystis pneumonia (PCP) management.
Area of Science:
- Medical Mycology
- Infectious Diseases
- Molecular Biology
Background:
- Pneumocystis pneumonia (PCP) is a severe opportunistic infection in immunocompromised individuals.
- Trimethoprim-sulfamethoxazole (TMP-SMX) is a primary treatment, targeting dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS).
- DHPS gene polymorphisms are linked to TMP-SMX resistance; DHFR mutations are less understood.
Purpose of the Study:
- To investigate dihydrofolate reductase (DHFR) gene polymorphisms in Pneumocystis jirovecii.
- To assess potential associations with drug resistance or evolutionary changes in clinical isolates from India.
Main Methods:
- Detection of P. jirovecii using Gomori methenamine silver (GMS) staining and PCR targeting the mt LSU rRNA gene.
- Amplification and sequencing of the DHFR gene from positive clinical samples.
- Analysis of nucleotide substitutions, including synonymous and non-synonymous mutations.
Main Results:
- P. jirovecii was detected in 10% of samples by PCR, with 77% of these positive for the DHFR gene.
- Six of 14 (42%) samples analyzed for DHFR showed 16 nucleotide substitutions.
- Eight of these substitutions were non-synonymous, indicating potential functional impact.
Conclusions:
- DHFR gene mutations in P. jirovecii were observed in an Indian cohort.
- These mutations may be associated with therapeutic failure or represent an evolutionary adaptation.
- Continuous monitoring of DHFR polymorphisms is recommended for effective PCP treatment and management.
