Elsevier

The Lancet

Volume 354, Issue 9187, 16 October 1999, Pages 1347-1351
The Lancet

Early Report
Effects of mutations in Pneumocystis carinii dihydropteroate synthase gene on outcome of AIDS-associated P carinii pneumonia

https://doi.org/10.1016/S0140-6736(99)03320-6Get rights and content

Summary

Background

Sulpha drugs are widely used for the treatment and long-term prophylaxis of Pneumocysfis carinii pneumonia (PCP) in HIV-1-infected individuals. Sulpha resistance in many microorganisms is caused by point mutations in dihydropteroate synthase (DHPS), an enzyme that is essential for folate biosynthesis. We assessed whether mutations in the DHPS gene of P carinii were associated with exposure to sulpha drugs and influenced outcome from PCP.

Methods

We studied bronchoalveolar samples collected in 1989–99 from a prospective cohort of HIV-1-infected patients who had PCP. In 144 patients with 152 episodes of PCP, we analysed portions of DHPS using PCR and direct sequencing. The relation between survival, P carinii DHPS mutations, and other predictors of treatment failure was assessed by Kaplan-Meier and multivariate Cox regression analysis.

Findings

P carinii DHPS mutations were found in 31 (20·4%) of 152 PCP episodes. 3-month survival was significantly lower in patients infected with mutant P carinii DHPS strains than in those with wild-type strains (p=0·002). After adjustment for other prognostic variables, presence of DHPS mutations remained the most important predictor of mortality (hazard ratio 3·1 [95% CI 1·2–8·1]). DHPS mutations were significantly more common in patients who had previous exposure to sulpha drugs (18 of 29 [62%]) than in those who had no exposure (13 of 123 [10·5%]; p<0·0001). A significant increase with time in the rate of DHPS mutations (p=0·01 for trend) was closely correlated with the rate of previous or current use of sulpha drugs as chemoprophylaxis.

Interpretation

Mutations in DHPS are associated with impaired prognosis in PCP, and may develop as a result of exposure to sulpha drugs.

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.

Section snippets

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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