Early ReportEffects of mutations in Pneumocystis carinii dihydropteroate synthase gene on outcome of AIDS-associated P carinii pneumonia
Introduction
Pneumocystis carinii pneumonla (PCP) is one of the most common opportunistic infections in patients with AIDS. Although the incidence of PCP has declined as a result of specific chemoprophylaxis and highly active antiretroviral therapy, PCP is still an important cause of morbidity and mortality.1 Several prognostic indicators are associated with the severity and outcome of PCP, which include direct measures of the severity of pneumonia (eg, partial pressure of arterial oxygen [PaO2] and serum lactate dehydrogenase) and the degree of inflammation in the lung.2, 3, 4 However, no virulence factors in the P carinii genome have been described.
Co-trimoxazole, the combination of sulphamethoxazole with trimethoprim (ratio five to one), is the drug of choice for treatment and prophylaxis of PCP.5, 6 This combination is more effective than agents such as pentamidine, dapsone, or clindamycin-pyrimethamine. Lifelong prophylaxis is recommended for all HIV-1-positive patients with a CD4-cell count less than 200/μL. In animals, the effect of co-trimoxazole is almost entirely attributable to sulphamethoxazole.7 Because resistance has been observed in many microbial pathogens exposed to sulphonamides, there have been concerns about whether resistance could develop in P carinii. However, because P carinii f sp hominis cannot be cultured in vitro, traditional methods of measuring drug resistance cannot be used. The target of sulphonamides is dihydropteroate synthase (DHPS), which catalyses the condensation of p-aminobenzoic acid and 6-hydroxymethyl-7,8-dihydropterin pyrophosphate to form dihydropteroate. In Escherichia coli, Neisseria meningitidis, Streptococcus pneumoniae, and Plasmodium falciparum, point mutations in conserved regions of the DHPS gene confer sulphonamide resistance.8, 9, 10 In P carinii, DHPS is part of a trifunctional protein with two other enzymes in the folicacid biosynthesis pathway, encoded by fas.11
Point mutations in the DHPS gene of P carinii isolates from patients have now been identified by PCR and sequenced.12, 13, 14 However, few cases of PCP have been examined and the clinical importance of DHPS mutations is unknown. We therefore investigated the possible impact of DHPS mutations on chemoprophylaxis failure and mortality in a large and well-characterised cohort of patients with PCP.
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Patients and therapy
Since 1989, data and samples have been collected from bronchoscopies done at the Department of Infectious Diseases in Hvidovre Hospital (Copenhagen, Denmark).4 From November, 1989, to January, 1999, 237 bronchoscopies were done on 188 HIV-1-infected patients diagnosed with PCP. 225 samples were available for analysis, and we sequenced the DHPS gene in 168 samples from 144 (77%) patients with 152 episodes of PCP (136 men, eight women; median age 37 years [range 22–76]; median CD4-cell count
Analysis
We extracted DNA from frozen unstained bronchoalveolar lavage slides or from frozen bronchoalveolar fluid, as previously described.17 PCR primers Dp15 (5′-TCTGAATTTTATAAAGCGCCTACAC-3′) and Dp800 (5′-ATTTCATAAACATCATGAACCCG-3′) were synthesised and based on consensus end sequences of P carinii f sp hominis DHPS in GenBank (accession numbers U662878-U662883). PCR was done with reagents from the AmpliTaq Gold kit (Perkin Elmer, Allerøod, Denmark). In a volume of 50 μL, 5 μL 10x PCR buffer, 2 μL
Statistical analysis
We analysed data with SPSS (version 8·0). Kruskal-Wallis and Fisher's exact tests were used to compare continuous and discrete measures, respectively. All tests were two-sided and a p value of less than 0·05 was used. Mutants were defined as all DHPS sequences containing either a mutation or a mixture of wild-type and mutant sequences. Kaplan-Meier and log-rank test were used to estimate survival, which was dated from the first bronchoalveolar lavage in the last episode of PCP recorded in the
Genotypes
Five different sequence patterns (300–340 bp) were obtained (table 1). We detected wild-type DHPS in 121 (80%) PCP episodes. In the remaining 31 PCP episodes, four different mutation patterns with non-synonymous nucleotide changes were identified. To assess the stability of DHPS genotypes, we typed samples from 15 patients who had repeated bronchoscopies during single episodes of PCP that lasted 4–28 days. No variation in DHPS type was found among 14 patients, including one patient who had four
Exposure to sulpha drugs
DHPS mutations were significantly more common in patients exposed to sulpha drugs (table 1): 18 of 29 patients exposed had DHPS mutations compared with 13 of 123 patients not exposed (p<0·0001).
In 27 episodes of PCP, the infection occurred despite current prophylaxis. 20 patients had PCP on non-sulpha prophylaxis: 18 patients were on pentamidine and two patients were on clindamycin. In seven episodes, breakthrough occurred on a sulpha-containing drug (four on co-trimoxazole and three on
Recurrent PCP
Eight patients had recurrent episodes of PCP with more than 4 months between each episode. A genotype change was observed in four of these patients with a switch from wild-type in the first PCP episode to DHPS mutation in a later episode; all patients had been treated with cotrimoxazole in their first episode. In four patients, of whom two had the codon 55 mutant and two had wildtype DHPS, the same genotypes were detected in both episodes. (Further information on prophylaxis and treatment in
Clinical characteristics and survival
The clinical characteristics of patients according to P carinii DHPS type and 3-month survival after the last episode of PCP are shown in table 2. Among 144 patients, 115 carried wild-type and 29 carried mutant P carinii DHPS gene. In 128 patients, the PCP episode was the illness that defined AIDS. In the other 17 patients, AIDS had been previously diagnosed. The higher proportion of previous AIDS-defining illnesses in patients with DHPS mutations (24 vs 9%) was mainly accounted for by patients
Discussion
Mutations in the P carinii DHPS gene were independently associated with significantly lower 3-month survival after diagnosis of PCP, and an increased frequency of mutations was seen among patients who received chemoprophylaxis. We found an increase of mutations after 1991, which was closely correlated with previous or current exposure to sulpha drugs as chemoprophylaxis.
Co-trimoxazole remains the drug of choice for the management of PCP.6 Compared with other drugs, it is cheap and associated
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