Molecular diagnosis and detection of Pneumocystis jirovecii DHPS and DHFR genotypes in respiratory specimens from Colombian patients

Muñoz, Camilo; Zuluaga, Alejandra; Restrepo, Ángela; Tobón, Ángela M.; Cano, Luz Elena; González, Ángel

doi:10.1016/j.diagmicrobio.2011.11.015

Cited by 14 publications

(11 citation statements)

References 52 publications

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“…Our results document a high frequency of DHPS mutants (48%) in anti-P. jirovecii sulfa-prophylaxis-naive patients with a first episode of PCP. This frequency is excessively high compared to the low prevalence of DHPS mutations in countries in the same continent, e.g., Brazil (0%) and Colombia (6.6%), that report similar patterns of sulfa drug usage in humans (22,31,32). Therefore, although our findings add further evidence to support the hypothesis of interhuman transmission as a mechanism of acquisition of mutant P. jirovecii types, additional mechanisms of acquisition may be possible.…”

Section: Discussioncontrasting

confidence: 47%

High Prevalence of Pneumocystis jirovecii Dihydropteroate Synthase Gene Mutations in Patients with a First Episode of Pneumocystis Pneumonia in Santiago, Chile, and Clinical Response to Trimethoprim-Sulfamethoxazole Therapy

Ponce

Chabé

George

et al. 2017

Antimicrob Agents Chemother

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Mutations in the dihydropteroate synthase (DHPS) gene of Pneumocystis jirovecii are associated with the failure of sulfa prophylaxis. They can develop by selection in patients receiving sulfa drugs or be acquired via person-to-person transmission. DHPS mutations raise concern about the decreasing efficacy of sulfa drugs, the main available therapeutic tool for Pneumocystis pneumonia (PCP). The prevalence of Pneumocystis DHPS mutations was examined in Pneumocystis isolates from 56 sulfa-prophylaxis-naive adults with a first episode of PCP from 2002 to 2010 in Santiago, Chile. Their clinical history was reviewed to analyze the effect of these mutations on response to trimethoprim-sulfamethoxazole (TMP-SMX) therapy and outcome. Mutant genotypes occurred in 22 (48%) of 46 HIV-infected patients and in 5 (50%) of 10 HIV-uninfected patients. Compared to patients with a wild-type genotype, those with mutant genotypes were more likely to experience sulfa treatmentlimiting adverse reactions and to have a twice-longer duration of mechanical ventilation if mechanically ventilated. Specific genotypes did not associate with death, which occurred in none of the HIV-infected patients and in 50% of the non-HIVinfected patients. Chile has a high prevalence of DHPS mutations, which were presumably acquired through interhuman transmission because patients were not on sulfa prophylaxis. These results contrast with the low prevalence observed in other Latin American countries with similar usage of sulfa drugs, suggesting that additional sources of resistant genotypes may be possible. The twice-longer duration of mechanical ventilation in patients with mutant DHPS genotypes suggests a decreased efficacy of TMP-SMX and warrants collaborative studies to assess the relevance of DHPS mutations and further research to increase therapeutic options for PCP.

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Section: Discussioncontrasting

confidence: 47%

High Prevalence of Pneumocystis jirovecii Dihydropteroate Synthase Gene Mutations in Patients with a First Episode of Pneumocystis Pneumonia in Santiago, Chile, and Clinical Response to Trimethoprim-Sulfamethoxazole Therapy

Ponce

Chabé

George

et al. 2017

Antimicrob Agents Chemother

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“…Studies from other sites have failed to find nonsynonomous mutations in DHFR from P. jirovecii, suggesting failure of therapy is not uniquely linked to mutations in DHFR (7)(8)(9)(10). Although DHPS mutations apparently contribute to clinical failure by conferring resistance to sulfamethoxazole (1,2), the appearance of mutations in both DHPS and DHFR in clinical isolates and the apparently more frequent development of DHFR mutations among patients who did receive prophylaxis that included an inhibitor of DHFR suggest a selection pressure is exerted on DHFR by exposure to trimethoprim or pyrimethamine (2).…”

mentioning

confidence: 99%

“…Mutations leading to amino acid substitutions in DHPS have been linked to resistance to sulfamethoxazole (1). Mutations leading to amino acid substitutions in dihydrofolate reductase of Pneumocystis jirovecii have also been observed worldwide for over a decade, but attempts to link clinical outcomes to these mutations have been inconclusive (2)(3)(4)(5)(6)(7)(8)(9)(10).…”

mentioning

confidence: 99%

Trimethoprim Resistance of Dihydrofolate Reductase Variants from Clinical Isolates of Pneumocystis jirovecii

Queener

Cody

Pace

et al. 2013

Antimicrob Agents Chemother

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dPneumocystis jirovecii is an opportunistic pathogen that causes serious pneumonia in immunosuppressed patients. Standard therapy and prophylaxis include trimethoprim (TMP)-sulfamethoxazole; trimethoprim in this combination targets dihydrofolate reductase (DHFR). Fourteen clinically observed variants of P. jirovecii DHFR were produced recombinantly to allow exploration of the causes of clinically observed failure of therapy and prophylaxis that includes trimethoprim. Six DHFR variants (S31F, F36C, L65P, A67V, V79I, and I158V) showed resistance to inhibition by trimethoprim, with K i values for trimethoprim 4-fold to 100-fold higher than those for the wild-type P. jirovecii DHFR. An experimental antifolate with more conformational flexibility than trimethoprim showed strong activity against one trimethoprim-resistant variant. The two variants that were most resistant to trimethoprim (F36C and L65P) also had increased K m values for dihydrofolic acid (DHFA). The catalytic rate constant (k cat ) was unchanged for most variant forms of P. jirovecii DHFR but was significantly lowered in F36C protein; one naturally occurring variant with two amino acid substitutions (S106P and E127G) showed a doubling of k cat , as well as a K m for NADPH half that of the wild type. The strongest resistance to trimethoprim occurred with amino acid changes in the binding pocket for DHFA or trimethoprim, and the strongest effect on binding of NADPH was linked to a mutation involved in binding the phosphate group of the cofactor. This study marks the first confirmation that naturally occurring mutations in the gene for DHFR from P. jirovecii produce variant forms of DHFR that are resistant to trimethoprim and may contribute to clinically observed failures of standard therapy or prophylaxis.

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“…In addition, it has been documented that PCR is more sensitive than the staining methods [43]. The nested PCR validated in our laboratory has shown a sensitivity of 86% and a specificity of 98% when using deep respiratory samples [15]. This method shows excellent diagnostic value and higher negative predictive value than microscopy, sufficient to confirm or rule out the diagnosis of this disease in high-risk patients [43].…”

Section: Discussionmentioning

confidence: 65%

“…Calculation of number of samples: The total number of samples was calculated by adding the total records of each of the 14 laboratory assays analyzed in this work including: direct examination (wet and nigrosin); special stains (Wright and Grocott-Gomori's methenamine silver [GMS]); culture and hemoculture for deep mycoses, and API 20C AUX for yeast identification; Histoplasma antigen in urine; Cryptococcus antigen in serum and cerebrospinal fluid (CSF); Aspergillus antigen in serum and bronchoalveolar lavage (BAL); Aspergillus, Paracoccidioides and Histoplasma antibodies in serum and CSF, using agar gel immunodiffusion assay (AGID), complement fixation (CF), or both assays simultaneously. Likewise, Histoplasma and Pneumocystis specific Polymerase Chain Reaction (PCR), and a universal PCR for fungi (Panfungal PCR) were analyzed [14][15][16].…”

Section: Methodsmentioning

confidence: 99%

Frequency of Invasive Fungal Disease in Adults: Experience of a Specialized Laboratory in Medellín, Colombia (2009–2015)

Valencia

Cáceres

Bedout

et al. 2020

JoF

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Invasive fungal diseases (IFD) contribute significantly to worldwide morbidity and mortality, but their frequency is not well-described in some countries. The present work describes the frequency of IFD in a specialized laboratory in Colombia. A retrospective, descriptive study was implemented between March 2009 and December 2015. Results: 13,071 patients with clinical suspicion of IFD were referred during the study period, from which 33,516 biological samples were processed and analyzed using 14 laboratory methods. Diagnosis was confirmed in 1425 patients (11%), distributed according to the mycoses of interest analyzed here: histoplasmosis in 641/11,756 patients (6%), aspergillosis in 331/10,985 patients (3%), cryptococcosis in 239/8172 patients (3%), pneumocystosis in 111/1651 patients (7%), paracoccidioidomycosis in 60/10,178 patients (0.6%), and invasive candidiasis in 48/7525 patients (0.6%). From the first year of the study period to the last year, there was a 53% increase in the number of cases of IFD diagnosed. Our laboratory experienced a high frequency of IFD diagnosis, possibly attributable to the availability of a greater range of diagnostic tools. Frequency of IFD in this study was atypical compared with other studies, probably as a result of the single laboratory-site analysis. This demonstrates that implementing educational strategies helps to create a high index of clinical suspicion, while the availability and utilization of appropriate diagnostic assays assure greater reliability in identification of these cases.

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Molecular diagnosis and detection of Pneumocystis jirovecii DHPS and DHFR genotypes in respiratory specimens from Colombian patients

Cited by 14 publications

References 52 publications

High Prevalence of Pneumocystis jirovecii Dihydropteroate Synthase Gene Mutations in Patients with a First Episode of Pneumocystis Pneumonia in Santiago, Chile, and Clinical Response to Trimethoprim-Sulfamethoxazole Therapy

High Prevalence of Pneumocystis jirovecii Dihydropteroate Synthase Gene Mutations in Patients with a First Episode of Pneumocystis Pneumonia in Santiago, Chile, and Clinical Response to Trimethoprim-Sulfamethoxazole Therapy

Trimethoprim Resistance of Dihydrofolate Reductase Variants from Clinical Isolates of Pneumocystis jirovecii

Frequency of Invasive Fungal Disease in Adults: Experience of a Specialized Laboratory in Medellín, Colombia (2009–2015)

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