| Abstract: | Pneumocystis jirovecii pneumonia (PCP) is an important cause of morbidity and mortality in immunocompromised patients. Sulfa-containing drugs are used for the treatment and prophylaxis of PCP. Mutations in the P. jirovecii fas gene, which encodes dihydropteroate synthase (DHPS), are associated with prior exposure to sulfa drugs, and their appearance suggests the emergence of variants with reduced sulfa susceptibility. The present study examined the prevalence of DHPS mutations in P. jirovecii strains isolated from South African patients with PCP. P. jirovecii infection was investigated by immunofluorescence microscopy and quantitative real-time PCR with respiratory specimens from 712 patients (93% of whom were >15 years of age) with suspected PCP consecutively received for the detection of P. jirovecii over 1 year. PCR amplification and sequencing of the DHPS fas gene was attempted with DNA from the P. jirovecii-positive samples. P. jirovecii infection was confirmed by immunofluorescence microscopy in 168/712 (24%) of the patients. Carriage of the fungus was revealed by real-time PCR in 17% of the patients with negative microscopy results. The P. jirovecii fas gene was successfully amplified from specimens from 151 patients and sequenced. Mutations resulting in the Thr55Ala and/or Pro57Ser amino acid substitution were detected in P. jirovecii strains from 85/151 (56%) patients. The high frequency of PCP episodes with P. jirovecii harboring DHPS mutations in South Africa indicates that populations of this fungus are evolving under the considerable selective pressure exerted by sulfa-containing antibiotics. These results, similar to previous observations of sulfa drug resistance in bacterial populations, underscore the importance of the rational use of sulfa medications either prophylactically against PCP or for the treatment of other infections.Pneumocystis pneumonia (PCP), a major opportunistic infection in immunocompromised patients, is caused by the fungus Pneumocystis jirovecii. The incidence of PCP, which increased dramatically with the advent of the HIV/AIDS pandemic, has decreased in the industrialized world owing to the widespread use of sulfa drug prophylaxis and the introduction of highly active antiretroviral therapy (HAART). However, PCP remains an important cause of morbidity and mortality in HIV/AIDS patients, as well as in immunocompromised non-HIV-infected patients, in whom its incidence is increasing (17, 35). In South Africa, which has a population of 48.5 million, an estimated 5.7 million people were living with HIV in 2007, and 350,000 deaths were attributed to AIDS during the same year (20). The South African government initiated the provision of HAART to the public sector in April 2004, but prior to that, the HIV epidemic was largely untreated. By the end of 2006, the rate of HAART coverage was estimated to be 21% among those needing antiretroviral therapy (20). Studies from African countries report variable incidences of PCP in adult patients with HIV/AIDS and generally higher rates in children (1, 3, 27, 28, 43, 47, 49). In South Africa, where a limited number of laboratories offer testing for P. jirovecii, the vast majority of PCP cases are diagnosed clinically and radiologically.Sulfonamides, usually combined with trimethoprim, as in trimethoprim-sulfamethoxazole (TMP-SMX), and dapsone are used for the treatment and prophylaxis of PCP. There are few alternative drugs for the treatment of this infection. Sulfonamides inhibit the enzyme dihydropteroate synthase (DHPS), an essential component of the folate synthesis pathway (36). In P. jirovecii, two nonsynonymous point mutations in the fas gene, which encodes the DHPS enzyme, are associated with prior exposure to sulfa drugs (5, 15, 16, 22, 26, 32), and concerns have been raised about the possible emergence of resistance to sulfa drugs (38). These mutations, at nucleotide positions 165 and 171, cause the amino acid substitutions Thr55Ala and Pro57Ser in the DHPS protein, respectively. Point mutations in the DHPS-encoding genes of microorganisms such as Plasmodium falciparum, Staphylococcus aureus, Mycobacterium leprae, and Escherichia coli have been shown to confer resistance to sulfonamides (6, 14, 21, 45). As P. jirovecii cannot yet be cultured, conventional in vitro susceptibility tests cannot be utilized; therefore, studies of drug resistance in this organism rely on the use of genetic markers and suitable models. Functional complementation of either DHPS-disrupted E. coli with a mutant P. jirovecii fas gene or FOL1-disrupted Saccharomyces cerevisiae with the fol1 gene mutated at positions analogous to positions 165 and 171 in fas results in the loss of susceptibility to sulfamethoxazole and other sulfa-containing drugs (19, 29).The prevalence of P. jirovecii DHPS mutations reported from countries in the developed world ranges widely, from 4% to 81% (2, 5, 15, 18, 32, 39, 41, 42). In South Africa, a few studies that screened specimens from limited numbers of adults and children have reported mostly low mutation rates (8, 12, 34, 48). Here we present the results of a large laboratory-based study aimed at ascertaining the prevalence in South Africa of P. jirovecii strains harboring mutations at positions 165 and 171 in the fas gene. |
