Genetic diversity of Pneumocystis jirovecii isolates among Turkish population based on mitochondrial large subunit ribosomal RNA and dihydropteroate synthase gene typing.

Pneumocystis jirovecii (P. jirovecii) is an atypical fungus that can cause severe interstitial pneumonia in immunocompromised patients. In this study, mitochondrial large subunit ribosomal RNA (mtLSU-rRNA) and dihydropteroate synthase (DHPS) gene polymorphism in P. jirovecii isolates were investigated in Western Turkey's Izmir province and its surroundings. For this purpose, a total of 157 P. jirovecii isolates obtained from bronchoalveolar lavage samples of hospitalized cases and lung tissue samples of autopsy cases who died outside hospital were examined. Genotypes were identified by direct sequencing of mtLSU-rRNA restriction fragment length polymorphism analysis of the DHPS gene amplicons. The mtLSU-rRNA analysis revealed that genotype 2 was the most common genotype with 58%. The following genotypes were genotype 3 (13%), genotype 1 (11.6%) and genotype 4 (5.1%), while genotype 5 (0.7%) was detected in only one autopsy case. In addition, 16 (11.6%) cases had dual or triple different genotypes (mixed infection). It was observed that the genotype distribution was not affected by characteristics such as age, gender and immune status. However, the predominance of genotype 2 in solid organ tumors and the predominance of mixed infection in patients with chronic pulmonary disease were statistically significant. On the other hand, DHPS gene amplification was positive in 137 (87.3%) of 157 samples. While no mutation was observed in 135 samples, the association of wild-type and 57th codon mutation was detected in two hospitalized cases (1.5%). In this study, important epidemiological data on the distribution of mtLSU-rRNA genotypes were obtained. Also the existence of DHPS gene mutations associated with potential drug resistance in our community was shown for the first time. Further studies are needed to evaluate the possible effects of genotypes on the prognosis of the disease to help with the clinician's treatment decisions. LAY ABSTRACT: Pneumocystis jirovecii (P. jirovecii) is an atypical fungus that can cause life-threatening pneumonia in immunocompromised patients. In this study, we investigated the mtLSU-rRNA and DHPS gene polymorphisms in P. jirovecii isolates from both hospital and autopsy cases.

[Dihydropteroate Synthase (DHPS) Gene Mutations and Mitochondrial Large Subunit (mtLSU) rRNA Genotype Variations in Pneumocystis jirovecii Strains]. / Pneumocystis jirovecii Izolatlarinin Dihidropteroat Sentaz (DHPS) Gen Mutasyonlari ve Mitokondriyal Büyük Alt Birim (mtLSU) rRNA Genotip Varyasyonlarinin Belirlenmesi.

Pneumocystis jirovecii is an atypical fungus that causes Pneumocystis pneumonia (PCP) in HIV/AIDS and immunocompromised patients. Antibiotics containing sulfa and sulfone groups are widely used in PCP prophylaxis and treatment. Especially, long-term use of trimethoprim sulfamethoxazole (TMP-SMX) is known to cause certain point mutations associated with drug resistance in the P.jirovecii dihydropteroate synthase (DHPS) gene. In addition, DHPS and mitochondrial large subunit (mtLSU) rRNA genotype characterization provides important data on the epidemiology of P.jirovecii. In this study, it was aimed to investigate the DHPS and mtLSU rRNA gene polymorphisms of P.jirovecii strains isolated from immunocompromised patients in Mersin University Hospital. In this study, 16 P.jirovecii positive samples, which isolated from 96 patients samples, between August 2016 and February 2018, were included. P.jirovecii mtLSU rRNA genotypes were determined by sequence analysis according to polymorphisms at the 85th and 248th nucleotide positions. Nested PCR and RFLP method was applied for mutation analysis of DHPS locus, 165th and 171st nucleotide positions. In the DHPS mutation analysis, 12/16 (75%) wild type (W165/W171) and 4/16 (25%) mutant type (M165/W171) were detected. Two mutant types belonged to HIV/AIDS positive patients with PCP and had a history of prophylaxis; the other 2 mutant types belonged to patients with colonization. In the study, a history of prophylaxis in 3 (19%) of the 16 patients were recorded, and mutant type was detected in these 2 of 3 patients. According to mtLSU-rRNA analysis, 3 different genotypes were obtained from 16 P.jirovecii isolates. In our region, genotype 2 (43.75%; n= 7) was the most common genotype, genotype 1 (37.5%; n= 6) was the second common and genotype 3 (18.75%; n= 3) was the least one. Genotype 4 was not detected in our region. When DHPS and mtLSU-rRNA were evaluated as multilocus, five different genotypes were observed. As a result, these findings provided important data on P.jirovecii epidemiology in our region and potential drug-resistant strains showed a risk of transmission in immunosuppressive patients. Multicenter studies involving more P.jirovecii isolates are needed to better define the epidemiology of P.jirovecii in our region and in our country.

Clinical characteristics and prevalence of dihydropteroate synthase gene mutations in Pneumocystis jirovecii-infected AIDS patients from low endemic areas of China.

Pneumocystis pneumonia (PCP) is an opportunistic and potentially life-threatening infection of AIDS patients caused by the fungus Pneumocystis jirovecii (P. jirovecii). Trimethoprim-sulfamethoxazole (TMP-SMX) is the most commonly used drug combination in the treatment and prophylaxis of PCP. However, with long-term use of this combination, mutations in the dihydropteroate synthase (DHPS) gene of P. jirovecii bring about the development of resistance. Data on the prevalence of P. jirovecii and its DHPS mutants in China, especially in low endemic areas, are still limited. Thus, in the present study, we measured the P. jirovecii infection rate among HIV-positive and AIDS (HIV/AIDS) patients with suspected PCP and investigated the relationship between CD4+ T cell count and PCP occurrence. As well as the polymerase chain reaction (PCR) analysis and sequencing, the restriction fragment length polymorphism (RFLP) method was used to analyze DHPS point mutation in P. jirovecii strains. P. jirovecii was detected in 40.82% of cases. The clinical symptoms and signs of PCP were not typical; with decreasing CD4+ T cell counts, PCP infection in HIV/AIDS patients increased. In only one case (1.67%), the patients' DHPS gene could not be cut by the Acc I restriction enzyme. Furthermore, mutation at codon 171 was detected in 11 cases and no mutation was found at codon 57. Patients treated with sulfamethoxazole combined with Voriconazole or Caspofungin exhibited favorable results. After treatment, the symptoms of dyspnea were alleviated, and chest computed tomography findings showed the improvement of lung shadows. These indicated that the prevalence of DHPS mutations in P. jirovecii isolates in AIDS-PCP patients in the region was low. Thus, the contribution of gene mutations to treatment failure requires further research.

Targeting species specific amino acid residues: Design, synthesis and biological evaluation of 6-substituted pyrrolo[2,3-d]pyrimidines as dihydrofolate reductase inhibitors and potential anti-opportunistic infection agents.

To combine the potency of trimetrexate (TMQ) or piritrexim (PTX) with the species selectivity of trimethoprim (TMP), target based design was carried out with the X-ray crystal structure of human dihydrofolate reductase (hDHFR) and the homology model of Pneumocystis jirovecii DHFR (pjDHFR). Using variation of amino acids such as Met33/Phe31 (in pjDHFR/hDHFR) that affect the binding of inhibitors due to their distinct positive or negative steric effect at the active binding site of the inhibitor, we designed a series of substituted-pyrrolo[2,3-d]pyrimidines. The best analogs displayed better potency (IC50) than PTX and high selectivity for pjDHFR versus hDHFR, with 4 exhibiting a selectivity for pjDHFR of 24-fold.

Pneumocystis Cytochrome b Mutants Associated With Atovaquone Prophylaxis Failure as the Cause of Pneumocystis Infection Outbreak Among Heart Transplant Recipients.

Background: Although trimethoprim-sulfamethoxazole is the more efficient drug for prophylactic and curative treatment of pneumocystosis, atovaquone is considered a second-line prophylactic treatment in immunocompromised patients. Variations in atovaquone absorption and mutant fungi selection after atovaquone exposure have been associated with atovaquone prophylactic failure. We report here a Pneumocystis jirovecii cytochrome b (cyt b) mutation (A144V) associated with such prophylactic failure during a pneumocystosis outbreak among heart transplant recipients. Methods: Analyses of clinical data, serum drug dosage, and molecular modeling of the P. jirovecii Rieske-cyt b complex were performed to investigate these prophylactic failures. Results: The cyt b A144V mutation was detected in all infected, heart transplant recipient patients exposed to atovaquone prophylaxis but in none of 11 other immunocompromised, infected control patients not treated with atovaquone. Serum atovaquone concentrations associated with these prophylactic failures were similar than those found in noninfected exposed control patients under a similar prophylactic regimen. Computational modeling of the P. jirovecii Rieske-cyt b complex and in silico mutagenesis indicated that the cyt b A144V mutation might alter the volume of the atovaquone-binding pocket, which could decrease atovaquone binding. Conclusions: These data suggest that the cyt b A144V mutation confers diminished sensitivity to atovaquone, resulting in spread of Pneumocystis pneumonia among heart transplant recipients submitted to atovaquone prophylaxis. Potential selection and interhuman transmission of resistant P. jirovecii strain during atovaquone prophylactic treatment has to be considered and could limit its extended large-scale use in immucompromised patients.

Evaluation of a new commercial real-time PCR assay for diagnosis of Pneumocystis jirovecii pneumonia and identification of dihydropteroate synthase (DHPS) mutations.

The PneumoGenius® real-time PCR assay is a new commercial multiplex real-time PCR method, which detects the Pneumocystis mitochondrial ribosomal large subunit (mtLSU) and two dihydropteroate synthase (DHPS) point mutations. To evaluate the clinical performance of this new real-time PCR assay we tested 120 extracted DNA samples from bronchoalveolar lavage specimens. These set of extracted DNA samples had already tested positive for Pneumocystis and patients had been classified in probable and unlikely PCP in a previous study. To evaluate de accuracy of the DHPS mutant's identification, an "in house" PCR and sequencing was performed. The sensitivity and specificity of PneumoGenius® PCR in discriminating between probable and unlikely Pneumocystis pneumonia (PCP) were 70% and 82% respectively. PneumoGenius® PCR was able to genotype more samples than "in house" DHPS PCR and sequencing. The same DHPS mutations were observed by both methods in four patients: two patients with a single mutation in position 171 (Pro57Ser) and two patients with a double mutation in position 165 (Thr55Ala) and in position 171 (Pro57Ser). A low rate of P. jirovecii (4.5%) harboring DHPS mutations was found, comparable to rates observed in other European countries. The PneumoGenius® real-time PCR is a suitable real-time PCR for PCP diagnosis and detection of DHPS mutants. The added value of DHPS mutation identification can assist in understanding the role of these mutations in prophylaxis failure or treatment outcome.

Molecular detection of DHFR gene polymorphisms in Pneumocystis jirovecii isolates from Indian patients.

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.

Structure-activity correlations for three pyrido[2,3-d]pyrimidine antifolates binding to human and Pneumocystis carinii dihydrofolate reductase.

To further define the interactions that enhance the selectivity of binding and to directly compare the binding of the most potent analogue {N(6)-methyl-N(6)-(3,4,5-trifluorophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine; compound 26} in the series of bicyclic pyrido[2,3-d]pyrimidine analogues of piritrexim (PTX) with native human (h), Pneumocystis carinii (pc) and Pneumocystis jirovecii (pj) dihydrofolate reductase (DHFR) enzymes, the crystal structures of hDHFR complexed with N(6)-methyl-N(6)-(4-isopropylphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 22), of hDHFR complexed with compound 26 and of pcDHFR complexed with N(6)-methyl-N(6)-1-naphthylpyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 24) are reported as ternary complexes with NADPH. This series of bicyclic pyrido[2,3-d]pyrimidines were designed in which there was a transposition of the 5-methyl group of PTX to the N9 position of the pyrido[2,3-d]pyrimidine. It was hypothesized that the N9-methyl group would preferentially interact with Ile123 of pcDHFR (and Ile123 of pjDHFR), but not with the shorter Val115 in hDHFR. Structure-activity data for this series of antifolates revealed that a trifluoro derivative (26) was the most selective against pjDHFR compared with mammalian DHFR (h/pj = 35.7). Structural data for the hDHFR-26 complex revealed that 26 binds in a different conformation from that observed in the pcDHFR-26 complex. In the hDHFR-26 complex the trifluorophenyl ring of 26 occupies a position near the cofactor-binding site, with close intermolecular contacts with Asp21, Ser59 and Ile60, whereas this ring in the pcDHFR-26 complex is positioned away from the cofactor site and near Ile65, with weaker contacts with Ile65, Phe69 and Ile123. Comparison of the intermolecular contacts between the N9-methyl group with Val115/Ile123 validates the hypothesis that the N9-methyl substituent preferentially interacts with Ile123 compared with Val115 of hDHFR, as the weaker contact with Val115 in the hDHFR structure is consistent with its weaker binding affinity compared with pcDHFR. The results for the structures of hDHFR-22 and pcDHFR-24 show that their inhibitor-binding orientation is similar to that observed in pcDHFR-26 and the pcDHFR variant (F69N) reported previously. The naphthyl moiety of 24 makes several intermolecular contacts with the active-site residues in pcDHFR that help to stabilize the binding, resulting in a more potent inhibitor.

Evidence for Proinflammatory ß-1,6 Glucans in the Pneumocystis carinii Cell Wall.

Inflammation is a major cause of respiratory impairment during Pneumocystis pneumonia. Studies support a significant role for cell wall ß-glucans in stimulating inflammatory responses. Fungal ß-glucans are comprised of d-glucose homopolymers containing ß-1,3-linked glucose backbones with ß-1,6-linked glucose side chains. Prior studies in Pneumocystis carinii have characterized ß-1,3 glucan components of the organism. However, recent investigations in other organisms support important roles for ß-1,6 glucans, predominantly in mediating host cellular activation. Accordingly, we sought to characterize ß-1,6 glucans in the cell wall of Pneumocystis and to establish their activity in lung cell inflammation. Immune staining revealed specific ß-1,6 localization in P. carinii cyst walls. Homology-based cloning facilitated characterization of a functional P. carinii kre6 (Pckre6) ß-1,6 glucan synthase in Pneumocystis that, when expressed in kre6-deficient Saccharomyces cerevisiae, restored cell wall stability. Recently synthesized ß-1,6 glucan synthase inhibitors decreased the ability of isolated P. carinii preparations to generate ß-1,6 carbohydrate. In addition, isolated ß-1,6 glucan fractions from Pneumocystis elicited vigorous tumor necrosis factor alpha (TNF-α) responses from macrophages. These inflammatory responses were significantly dampened by inhibition of host cell plasma membrane microdomain function. Together, these studies indicate that ß-1,6 glucans are present in the P. carinii cell wall and contribute to lung cell inflammatory activation during infection.

Dihydropteroate synthase gene mutation rates in Pneumocystis jirovecii strains obtained from Iranian HIV-positive and non-HIV-positive patients.

The dihydropteroate sulfate (DHPS) gene is associated with resistance to sulfa/sulfone drugs in Pneumocystis jirovecii. We investigated the DHPS mutation rate in three groups of Iranian HIV-positive and HIV-negative patients by polymerase chain reaction-restricted fragment length polymorphism analysis. Furthermore, an association between P. jirovecii DHPS mutations and strain typing was investigated based on direct sequencing of internal transcribed spacer region 1 (ITS1) and ITS2. The overall P. jirovecii DHPS mutation rate was (5/34; 14.7%), the lowest rate identified was in HIV-positive patients (1/16; 6.25%) and the highest rate was in malignancies patients (3/11; 27.3%). A moderate rate of mutation was detected in chronic obstructive pulmonary disease (COPD) patients (1/7; 14.3%). Most of the isolates were wild type (29/34; 85.3%). Double mutations in DHPS were detected in patients with malignancies, whereas single mutations at codons 55 and 57 were identified in the HIV-positive and COPD patients, respectively. In this study, two new and rare haplotypes were identified with DHPS mutations. Additionally, a positive relationship between P. jirovecii strain genotypes and DHPS mutations was identified. In contrast, no DHPS mutations were detected in the predominant (Eg) haplotype. This should be regarded as a warning of an increasing incidence of drug-resistant P. jirovecii strains.

Pneumocystis jirovecii infection and the associated dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) mutations in HIV-positive individuals from Pune, India.

The present study was undertaken to detect Pneumocystis jirovecii infection among HIV-positive patients presenting with symptoms of lower respiratory tract infection and analyze the associated dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) mutations. P. jirovecii infection was detected in 12.6% cases. We did not find DHPS gene mutations at the commonest positions of codon 55 and 57; however, mutation at codon 171 was detected in two cases. No mutations in DHFR gene were detected. The results indicate low prevalence of DHPS and DHFR mutations in Indian P. jirovecii isolates, suggesting that the selective pressure of sulfa drugs on the local strains has probably not reached the levels found in developed nations.

Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S-Adenosylmethionine:Sterol C-24 Methyltransferase.

The AIDS-associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S-adenosylmethionine:sterol C24-methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C-24 position of the sterol side chain producing both C28 and C29 24-alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild-type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy ((1)H-NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C28 sterol ergosterol as the major sterol, and also resulted in low levels of C29 sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ(24(28)) -sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C29 sterols as that found in P. carinii.

Complementation of a manganese-dependent superoxide dismutase-deficient yeast strain with Pneumocystis carinii sod2 gene.

Manganese-dependent superoxide dismutase (MnSOD) is one of the key enzymes involved in the cellular defense against oxidative stress. Previously, the Pneumocystis carinii sod2 gene (Pcsod2) was isolated and characterized. Based on protein sequence comparison, Pcsod2 was suggested to encode a putative MnSOD protein likely to be targeted into the mitochondrion. In this work, the Pcsod2 was cloned and expressed as a recombinant protein in EG110 Saccharomyces cerevisiae strain lacking the MnSOD-coding gene (Scsod2) in order to investigate the function and subcellular localization of P. carinii MnSOD (PcMnSOD). The Pcsod2 gene was amplified by PCR and cloned into the pYES2.1/V5-His-TOPO(®) expression vector. The recombinant construct was then transformed into EG110 strain. Once its expression had been induced, PcMnSOD was able to complement the growth defect of EG110 yeast cells that had been exposed to the redox-cycling compound menadione. N-term sequencing of the PcMnSOD protein allowed identifying the cleavage site of a mitochondrial targeting peptide. Immune-colocalization of PcMnSOD and yeast CoxIV further confirmed the mitochondrial localization of the PcMnSOD. Heterologous expression of PcMnSOD in yeast indicates that Pcsod2 encodes an active MnSOD, targeted to the yeast mitochondrion that allows the yeast cells to grow in the presence of reactive oxygen species (ROS).

Mutational analysis of Pneumocystis jirovecii dihydropteroate synthase and dihydrofolate reductase genes in HIV-infected patients in China.

We investigated Pneumocystis jirovecii dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) genes for mutations in 25 Chinese HIV-infected patients with P. jirovecii pneumonia. We identified DHPS mutations in 3 (12%) patients and DHFR mutations in 1 (4%) patient. The prevalence of DHPS and DHFR mutations in China remains low, as it does in other developing countries.

The Pneumocystis Ace2 transcription factor regulates cell wall-remodeling genes and organism virulence.

Pneumocystis carinii (Pc) ß-glucans are major components of the organism cell wall; yet, the regulation of Pc cell wall genesis and remodeling is not well understood. Ace2 transcription factors, which are present in many fungi, regulate glucanases and other enzymes needed for cell wall remodeling. The cloning and heterologous expression of PcAce2 in ace2Δ Saccharomyces cerevisiae demonstrated that PcAce2 can restore the defective glucanase and endochitinase gene expression of the mutant as well as regulate cell wall ß-glucan biosynthetic genes. Furthermore, when a reconstructed yeast system was used, PcAce2 activated the transcription of the Pneumocystis gsc1 ß-glucan synthetase, confirming the activity of a Pc transcription factor on a native Pneumocystis promoter and gene for the first time. We further observed that Pneumocystis binding to host extracellular matrix proteins and lung epithelial cells induced the phosphorylation (activation) of the PcAce2 transcription factor. Finally, we present a novel method that confirms the role of PcAce2 in modulating organism virulence using ace2Δ Candida glabrata infection in neutropenic mice. Together, these results indicate that the adherence of Pc to lung matrix proteins and epithelial cells leads to the activation of the Ace2 transcription factor, which regulates cell wall degradation and biosynthesis genes that are required for cell wall remodeling.

Design, synthesis, and molecular modeling of novel pyrido[2,3-d]pyrimidine analogues as antifolates; application of Buchwald-Hartwig aminations of heterocycles.

Opportunistic infections caused by Pneumocystis jirovecii (P. jirovecii, pj), Toxoplasma gondii (T. gondii, tg), and Mycobacterium avium (M. avium, ma) are the principal causes of morbidity and mortality in patients with acquired immunodeficiency syndrome (AIDS). The absence of any animal models for human Pneumocystis jirovecii pneumonia and the lack of crystal structures of pjDHFR and tgDHFR make the design of inhibitors challenging. A novel series of pyrido[2,3-d]pyrimidines as selective and potent DHFR inhibitors against these opportunistic infections are presented. Buchwald-Hartwig coupling reaction of substituted anilines with pivaloyl protected 2,4-diamino-6-bromo-pyrido[2,3-d]pyrimidine was successfully explored to synthesize these analogues. Compound 26 was the most selective inhibitor with excellent potency against pjDHFR. Molecular modeling studies with a pjDHFR homology model explained the potency and selectivity of 26. Structural data are also reported for 26 with pcDHFR and 16 and 22 with variants of pcDHFR.

Kinetic and structural analysis for potent antifolate inhibition of Pneumocystis jirovecii, Pneumocystis carinii, and human dihydrofolate reductases and their active-site variants.

A major concern of immunocompromised patients, in particular those with AIDS, is susceptibility to infection caused by opportunistic pathogens such as Pneumocystis jirovecii, which is a leading cause of pneumonia in immunocompromised patients. We report the first kinetic and structural data for 2,4-diamino-6-[(2',5'-dichloro anilino)methyl]pyrido[2,3-d]pyrimidine (OAAG324), a potent inhibitor of dihydrofolate reductase (DHFR) from P. jirovecii (pjDHFR), and also for trimethoprim (TMP) and methotrexate (MTX) with pjDHFR, Pneumocystis carinii DHFR (pcDHFR), and human DHFR (hDHFR). OAAG324 shows a 9.0-fold selectivity for pjDHFR (Ki, 2.7 nM) compared to its selectivity for hDHFR (Ki, 24.4 nM), whereas there is only a 2.3-fold selectivity for pcDHFR (Ki, 6.3 nM). In order to understand the determinants of inhibitory potency, active-site mutations of pj-, pc-, and hDHFR were explored to make these enzymes more like each other. The most unexpected observations were that the variant pcDHFR forms with K37Q and K37Q/F69N mutations, which made the enzyme more like the human form, also made these enzymes more sensitive to the inhibitory activity of OAAG324, with Ki values of 0.26 and 0.71 nM, respectively. A similar gain in sensitivity was also observed for the hDHFR N64F variant, which showed a lower Ki value (0.58 nM) than native hDHFR, pcDHFR, or pjDHFR. Structural data are reported for complexes of OAAG324 with hDHFR and its Q35K and Q35S/N64F variants and for the complex of the K37S/F69N variant of pcDHFR with TMP. These results provide useful insight into the role of these residues in the optimization of highly selective inhibitors of DHFR against the opportunistic pathogen P. jirovecii.

Dihydropteroate synthase mutations in Pneumocystis pneumonia: impact of applying different definitions of prophylaxis, mortality endpoints and mutant in a single cohort.

Pneumocystis jirovecii dihydropteroate synthase (DHPS) gene mutations are well-reported. Although sulfa prophylaxis generally is associated with DHPS mutant infection, whether mutant infection is associated with poorer clinical outcomes is less clear. The differing definitions of sulfa prophylaxis and the different mortality endpoints used in these studies may be one explanation for the conflicting study results. Applying different definitions of prophylaxis, mortality endpoints and DHPS mutant to 301 HIV-infected patients with Pneumocystis pneumonia, we demonstrate that prophylaxis, irrespective of definition, increased the risk of infection with pure mutant (any prophylaxis: AOR 4.00, 95% CI: 1.83-8.76, P < 0.001) but not mixed genotypes (any prophylaxis: AOR 0.78, 95% CI: 0.26-2.36, P = 0.65). However, infection with mutant DHPS, irrespective of definition, was not associated with increased mortality (all-cause or PCP death) at the three time-intervals examined (all P > 0.05). Future studies should standardize key variables associated with DHPS mutant infection as well as examine DHPS mutant subtypes (pure mutant vs. mixed infections) - perhaps even individual DHPS mutant genotypes - so that data can be pooled to better address this issue.

Low prevalence of Pneumocystis pneumonia (PCP) but high prevalence of pneumocystis dihydropteroate synthase (dhps) gene mutations in HIV-infected persons in Uganda.

Pneumocystis jirovecii pneumonia (PCP) is an important opportunistic infection in patients infected with HIV, but its burden is incompletely characterized in those areas of sub-Saharan Africa where HIV is prevalent. We explored the prevalence of both PCP in HIV-infected adults admitted with pneumonia to a tertiary-care hospital in Uganda and of putative P. jirovecii drug resistance by mutations in fungal dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr). In 129 consecutive patients with sputum smears negative for mycobacteria, 5 (3.9%) were diagnosed with PCP by microscopic examination of Giemsa-stained bronchoalveolar lavage fluid. Concordance was 100% between Giemsa stain and PCR (dhps and dhfr). PCP was more prevalent in patients newly-diagnosed with HIV (11.4%) than in patients with known HIV (1.1%; p = 0.007). Mortality at 2 months after discharge was 29% overall: 28% among PCP-negative patients, and 60% (3 of 5) among PCP-positive patients. In these 5 fungal isolates and an additional 8 from consecutive cases of PCP, all strains harbored mutant dhps haplotypes; all 13 isolates harbored the P57S mutation in dhps, and 3 (23%) also harbored the T55A mutation. No non-synonymous dhfr mutations were detected. PCP is an important cause of pneumonia in patients newly-diagnosed with HIV in Uganda, is associated with high mortality, and putative molecular evidence of drug resistance is prevalent. Given the reliability of field diagnosis in our cohort, future studies in sub-Saharan Africa can investigate the clinical impact of these genotypes.

Circulation of Pneumocystis dihydropteroate synthase mutants in France.

Data on the prevalence of Pneumocystis jirovecii (P. jirovecii) dihydropteroate synthase (DHPS) mutants in France are still limited. In this study, mutant prevalence in the Brest region (western France) was determined. Archival pulmonary specimens from 85 patients infected with P. jirovecii and admitted to our institution (University Hospital, Brest) from October 2007 to February 2010 were retrospectively typed at the DHPS locus using a polymerase chain reaction-restriction fragment length polymorphism assay. Type identification was successful in 66 of 85 patients. Sixty-four patients were infected with a wild type, whereas mutants were found in 2 patients (2/66, 3%). Medical chart analysis revealed that these 2 patients usually lived in Paris. Another patient usually lived on the French Riviera, whereas 63 patients were from the city of Brest. Thus, the corrected prevalence of mutants in patients who effectively lived in our geographic area was 0% (0/63). Taking into account that i) Paris is characterized by a high prevalence of mutants from 18.5% to 40%, ii) infection diagnoses were performed in the 2 Parisians during their vacation <30 days, iii) infection incubation is assumed to last about 2 months, the results provide evidence of mutant circulation from Paris to Brest through infected vacationers. The study shows that the usual city of patient residence, rather than the city of infection diagnosis, is a predictor of mutants and that P. jirovecii infections involving mutants do not represent a public health issue in western France.