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Prevalence of dihydropteroate synthase mutations in Spanish patients with HIV-associated Pneumocystis pneumonia
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Diagnostic Microbiology and Infectious Disease 64 (2009) 104 – 105 www.elsevier.com/locate/diagmicrobio
Prevalence of dihydropteroate synthase mutations in Spanish patients with HIV-associated Pneumocystis pneumonia To the Editor: Sulfa drugs, trimethoprim–sulfamethoxazole and dapsone, are mainstays for the prophylaxis and treatment of Pneumocystis pneumonia (PcP), a life-threatening disease in immunosuppressed patients. The inability to culture Pneumocystis prevents routine susceptibility testing and detection of drug resistance. Instead, molecular techniques have been used to detect Pneumocystis jirovecii dihydropteroate synthase (DHPS) mutations that cause sulfa resistance in other microorganisms. The most frequent DHPS mutations occur at nucleotide positions 165 and 171, which lead to an amino acid change at positions 55 (Thr to Ala) and 57 (Pro to Ser). These mutations are located in the sulfabinding site and may appear as either a single or double mutation in the same isolate. The lack of a standardized culture system makes that the clinical significance of these DHPS gene mutations must be inferred from correlating the clinical outcome with the presence of DHPS gene mutations in patients with PcP (Huang et al., 2004). Several studies have consistently demonstrated a significant association between the presence of DHPS gene mutations and failure of sulfa prophylaxis (Kazanjian et al., 1998; Nahimana et al., 2003). However, the effect of DHPS mutations on treatment outcome has not been well studied, and the few studies that have been conducted are inconsistent even as to the presence or absence of an association (Stein et al., 2004). In the September 2008 issue of Diagnostic Microbiology and Infectious Diseases, Alvarez-Martínez et al. (2008) reported the lack of association between DHPS gene mutations and a worse outcome in Spanish patients with HIV-associated PcP. However, in this study, only 7 (3.7%) of 188 patients with PcP had DHPS gene mutations. This low prevalence of DHPS mutations could have limited the ability to detect an association between these mutations and adverse outcome as it is discussed by the authors. The prevalence of DHPS mutations observed by AlvarezMartínez et al. (2008) is the lowest reported in a developed country, and it is inconsistent with previous reports from Spanish PcP patients (Calderón et al., 2004b). They 0732-8893/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
commented on the difference between the data from their study and the prevalence report in our study (Montes-Cano et al., 2004). However, they made a grave mistake with our data in Table 1 and in the first paragraph of the Discussion section. They wrote in the article that our reported prevalence of Pneumocystis DHPS mutation was 73.3%, but this is not the frequency of DHPS mutation in our study. This value is the amplification rate of fragments of DHPS gene obtained in patients with PcP, which is similar to that of other published studies. The real prevalence of DHPS mutation in PcP from Spain in our study was 35.5% (Montes-Cano et al., 2004), similar to the data published by our group in other articles (Calderón et al., 2004a; Calderón et al., 2004b; Esteves et al., 2008). Nevertheless, this prevalence is higher than that reported by Alvarez-Martínez et al. (2008), and it is difficult to explain the cause. Alvarez-Martínez et al. (2008) gave as explanation that DHPS mutations were more frequent in our area, but they included in their study 15 samples from our same area and only found patients who harbored DHPS mutations from a region on Northeast Spain. We think that this discrepancy may be due to technical reasons. We identified DHPS mutations in our studies using touchdown polymerase chain reaction and a restriction fragment length polymorphism assay (Helweg-Larsen et al., 2000), whereas in the study of Alvarez-Martínez et al., DHPS mutations were determined only by sequencing, and subcloning was not performed. On the other hand, Alvarez-Martínez et al. used only frozen aliquots or alcohol-fixed slides of the PcP clinical specimens previously confirmed microscopically, whereas we used sputum or bronchoalveolar lavage (BAL) samples, in which P. jirovecii infection was confirmed by molecular techniques. Thus, PcP could only be confirmed by molecular techniques in 42% of the cases in one of our studies because conventional staining methods were negative (Calderón et al., 2004b). This kind of samples would be ruled out in the study of Alvarez-Martínez et al., and it could be a cause of selection bias that could explain the low prevalence of DHPS mutations identified in Spanish patients with HIV-associated PcP. Vicente Friaza Marco A. Montes-Cano Nieves Respaldiza Rubén Morilla
Letter to the Editor / Diagnostic Microbiology and Infectious Disease 64 (2009) 104–105
Enrique J. Calderón Carmen de la Horra Instituto de Biomedicina de Sevilla Virgen del Rocío University Hospital Seville, Spain CIBER de Epidemiología y Salud Pública Virgen del Rocío University Hospital Seville, Spain E-mail address: [email protected] doi:10.1016/j.diagmicrobio.2009.01.015 References Alvarez-Martínez MJ, Moreno A, Miró JM, Valls ME, Rivas PV, de Lazzari E, Sued O, Benito N, Domingo P, Ribera E, Santín M, Sirera G, Segura F, Vidal F, Rodríguez F, Riera M, Cordero ME, Arribas JR, Jiménez de Anta MT, Gatell JM, Wilson PE, Meshnick SR, Spanish PCP Working Group (2008) Pneumocystis jirovecii pneumonia in Spanish HIVinfected patients in the combined antiretroviral therapy era: prevalence of dihydropteroate synthase mutations and prognostic factors of mortality. Diagn Microbiol Infect Dis 62:34–43. Calderón EJ, de la Horra C, Medrano FJ, López-Suarez A, Montes-Cano MA, Respaldiza N, Elvira-González J, Martin-Juan J, Bascuñana A, Varela JM (2004a) Pneumocystis jiroveci isolates with dihydropteroate synthase (DHPS) mutations in patients with chronic bronchitis. Eur J Clin Microbiol Infect 23:545–549.
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Calderón EJ, de la Horra C, Montes-Cano MA, Respaldiza N, Martín-Juan J, Varela JM (2004b) Resistencia genotípica a sulfamidas en pacientes con neumonía por Pneumocystis jiroveci. Med Clin (Barc) 122:617–619. Esteves F, Montes-Cano MA, de la Horra C, Costa MC, Calderon EJ, Antunes F, Matos O (2008) Pneumocystis jirovecii multilocus genotyping profiles in patients from Portugal and Spain. Clin Microbiol Infect 14:356–362. Helweg-Larsen J, Eugen-Olsen J, Lundgren B (2000) Rapid detection of dihydropteroate polymorphism in AIDS-related Pneumocystis carinii pneumonia by restriction fragment length polymorphism. Scand J Infect Dis 32:481–483. Huang L, Crothers K, Atzori C, Benfield T, Miller R, Rabodonirina M, Helweg-Larsen J (2004) Dihydropteroate synthase gene mutations in Pneumocystis and sulfa resistance. Emerg Infect Dis 10: 1721–1728. Kazanjian P, Locke AB, Hossler PA, Lane BR, Bartlett MS, Smith JW, Cannon M, Meshnick SR (1998) Pneumocystis carinii mutations associated with sulfa and sulfone prophylaxis failures in AIDS patients. 12:873–878. Montes-Cano MA, de la Horra C, Martín-Juan J, Varela JM, Torronteras R, Respaldiza N, Medrano FJ, Calderón EJ (2004) Pneumocystis jiroveci genotypes in Spanish population. Clin Infect Dis 39:545–549. Nahimana A, Rabodonirina M, Zanetti G, Meneau I, Francioli P, Bille J, Hauser PM (2003) Association between a specific Pneumocystis jiroveci Dihydropteroate synthase mutation and failure of pyrimethamine/ sulfadoxine prophylaxis in human immunodeficiency virus-positive and -negative patients. J Infect Dis 188:1017–1023. Stein CR, Poole C, Kazanjian P, Meshnick SR (2004) Sulfa use, dihydropteroate synthase mutations, and Pneumocystis jirovecii pneumonia. Emerg Infect Dis 10:1760–1765.
Diagnostic Microbiology and Infectious Disease 64 (2009) 104 – 105 www.elsevier.com/locate/diagmicrobio
Prevalence of dihydropteroate synthase mutations in Spanish patients with HIV-associated Pneumocystis pneumonia To the Editor: Sulfa drugs, trimethoprim–sulfamethoxazole and dapsone, are mainstays for the prophylaxis and treatment of Pneumocystis pneumonia (PcP), a life-threatening disease in immunosuppressed patients. The inability to culture Pneumocystis prevents routine susceptibility testing and detection of drug resistance. Instead, molecular techniques have been used to detect Pneumocystis jirovecii dihydropteroate synthase (DHPS) mutations that cause sulfa resistance in other microorganisms. The most frequent DHPS mutations occur at nucleotide positions 165 and 171, which lead to an amino acid change at positions 55 (Thr to Ala) and 57 (Pro to Ser). These mutations are located in the sulfabinding site and may appear as either a single or double mutation in the same isolate. The lack of a standardized culture system makes that the clinical significance of these DHPS gene mutations must be inferred from correlating the clinical outcome with the presence of DHPS gene mutations in patients with PcP (Huang et al., 2004). Several studies have consistently demonstrated a significant association between the presence of DHPS gene mutations and failure of sulfa prophylaxis (Kazanjian et al., 1998; Nahimana et al., 2003). However, the effect of DHPS mutations on treatment outcome has not been well studied, and the few studies that have been conducted are inconsistent even as to the presence or absence of an association (Stein et al., 2004). In the September 2008 issue of Diagnostic Microbiology and Infectious Diseases, Alvarez-Martínez et al. (2008) reported the lack of association between DHPS gene mutations and a worse outcome in Spanish patients with HIV-associated PcP. However, in this study, only 7 (3.7%) of 188 patients with PcP had DHPS gene mutations. This low prevalence of DHPS mutations could have limited the ability to detect an association between these mutations and adverse outcome as it is discussed by the authors. The prevalence of DHPS mutations observed by AlvarezMartínez et al. (2008) is the lowest reported in a developed country, and it is inconsistent with previous reports from Spanish PcP patients (Calderón et al., 2004b). They 0732-8893/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
commented on the difference between the data from their study and the prevalence report in our study (Montes-Cano et al., 2004). However, they made a grave mistake with our data in Table 1 and in the first paragraph of the Discussion section. They wrote in the article that our reported prevalence of Pneumocystis DHPS mutation was 73.3%, but this is not the frequency of DHPS mutation in our study. This value is the amplification rate of fragments of DHPS gene obtained in patients with PcP, which is similar to that of other published studies. The real prevalence of DHPS mutation in PcP from Spain in our study was 35.5% (Montes-Cano et al., 2004), similar to the data published by our group in other articles (Calderón et al., 2004a; Calderón et al., 2004b; Esteves et al., 2008). Nevertheless, this prevalence is higher than that reported by Alvarez-Martínez et al. (2008), and it is difficult to explain the cause. Alvarez-Martínez et al. (2008) gave as explanation that DHPS mutations were more frequent in our area, but they included in their study 15 samples from our same area and only found patients who harbored DHPS mutations from a region on Northeast Spain. We think that this discrepancy may be due to technical reasons. We identified DHPS mutations in our studies using touchdown polymerase chain reaction and a restriction fragment length polymorphism assay (Helweg-Larsen et al., 2000), whereas in the study of Alvarez-Martínez et al., DHPS mutations were determined only by sequencing, and subcloning was not performed. On the other hand, Alvarez-Martínez et al. used only frozen aliquots or alcohol-fixed slides of the PcP clinical specimens previously confirmed microscopically, whereas we used sputum or bronchoalveolar lavage (BAL) samples, in which P. jirovecii infection was confirmed by molecular techniques. Thus, PcP could only be confirmed by molecular techniques in 42% of the cases in one of our studies because conventional staining methods were negative (Calderón et al., 2004b). This kind of samples would be ruled out in the study of Alvarez-Martínez et al., and it could be a cause of selection bias that could explain the low prevalence of DHPS mutations identified in Spanish patients with HIV-associated PcP. Vicente Friaza Marco A. Montes-Cano Nieves Respaldiza Rubén Morilla
Letter to the Editor / Diagnostic Microbiology and Infectious Disease 64 (2009) 104–105
Enrique J. Calderón Carmen de la Horra Instituto de Biomedicina de Sevilla Virgen del Rocío University Hospital Seville, Spain CIBER de Epidemiología y Salud Pública Virgen del Rocío University Hospital Seville, Spain E-mail address: [email protected] doi:10.1016/j.diagmicrobio.2009.01.015 References Alvarez-Martínez MJ, Moreno A, Miró JM, Valls ME, Rivas PV, de Lazzari E, Sued O, Benito N, Domingo P, Ribera E, Santín M, Sirera G, Segura F, Vidal F, Rodríguez F, Riera M, Cordero ME, Arribas JR, Jiménez de Anta MT, Gatell JM, Wilson PE, Meshnick SR, Spanish PCP Working Group (2008) Pneumocystis jirovecii pneumonia in Spanish HIVinfected patients in the combined antiretroviral therapy era: prevalence of dihydropteroate synthase mutations and prognostic factors of mortality. Diagn Microbiol Infect Dis 62:34–43. Calderón EJ, de la Horra C, Medrano FJ, López-Suarez A, Montes-Cano MA, Respaldiza N, Elvira-González J, Martin-Juan J, Bascuñana A, Varela JM (2004a) Pneumocystis jiroveci isolates with dihydropteroate synthase (DHPS) mutations in patients with chronic bronchitis. Eur J Clin Microbiol Infect 23:545–549.
105
Calderón EJ, de la Horra C, Montes-Cano MA, Respaldiza N, Martín-Juan J, Varela JM (2004b) Resistencia genotípica a sulfamidas en pacientes con neumonía por Pneumocystis jiroveci. Med Clin (Barc) 122:617–619. Esteves F, Montes-Cano MA, de la Horra C, Costa MC, Calderon EJ, Antunes F, Matos O (2008) Pneumocystis jirovecii multilocus genotyping profiles in patients from Portugal and Spain. Clin Microbiol Infect 14:356–362. Helweg-Larsen J, Eugen-Olsen J, Lundgren B (2000) Rapid detection of dihydropteroate polymorphism in AIDS-related Pneumocystis carinii pneumonia by restriction fragment length polymorphism. Scand J Infect Dis 32:481–483. Huang L, Crothers K, Atzori C, Benfield T, Miller R, Rabodonirina M, Helweg-Larsen J (2004) Dihydropteroate synthase gene mutations in Pneumocystis and sulfa resistance. Emerg Infect Dis 10: 1721–1728. Kazanjian P, Locke AB, Hossler PA, Lane BR, Bartlett MS, Smith JW, Cannon M, Meshnick SR (1998) Pneumocystis carinii mutations associated with sulfa and sulfone prophylaxis failures in AIDS patients. 12:873–878. Montes-Cano MA, de la Horra C, Martín-Juan J, Varela JM, Torronteras R, Respaldiza N, Medrano FJ, Calderón EJ (2004) Pneumocystis jiroveci genotypes in Spanish population. Clin Infect Dis 39:545–549. Nahimana A, Rabodonirina M, Zanetti G, Meneau I, Francioli P, Bille J, Hauser PM (2003) Association between a specific Pneumocystis jiroveci Dihydropteroate synthase mutation and failure of pyrimethamine/ sulfadoxine prophylaxis in human immunodeficiency virus-positive and -negative patients. J Infect Dis 188:1017–1023. Stein CR, Poole C, Kazanjian P, Meshnick SR (2004) Sulfa use, dihydropteroate synthase mutations, and Pneumocystis jirovecii pneumonia. Emerg Infect Dis 10:1760–1765.