Infection, Genetics and Evolution 3 (2003) 281–285 Pneumocystis jiroveci in Portuguese immunocompromised patients: association of specific ITS genotypes with treatment failure, bad clinical outcome and childhood Olga Matos a,∗ , Chao-Hung Lee b , Shaoling Jin b , Baozheng Li b , Marina C. Costa a , Luzia Gonçalves c , Francisco Antunes a,d a Instituto de Higiene e Medicina Tropical, Unidade de Protozoários Oportunistas/VIH e outras Protozooses, Unidade de Parasitologia e Microbiologia Médicas (UPMM), Rua da Junqueira 96, 1349-008 Lisboa, Portugal b Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA c Instituto de Higiene e Medicina Tropical, Unidade de Epidemiologia e Bioestat´ ıstica, Lisboa, Portugal d Cl´ ınica das Doenças Infecciosas, Hospital de Santa Maria, Lisboa, Portugal Received 14 January 2003; received in revised form 7 April 2003; accepted 30 June 2003 Abstract We analyzed the genetic variation among isolates of Pneumocystis jiroveci from Portuguese immunocompromised patients with PCP at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon and at the dihydropteroate synthase (DHPS) gene. Pulmonary secretions from 42 patients with PCP corresponding to 43 episodes were studied. Demographic, immunological, and clinical data were obtained from all patients. By combining the two regions ITS1 and ITS2, we found 17 different ITS types of P. jiroveci, two of them were new types (Pb and Pe). The four most prevalent ITS types were Eg (23.3%), Eb and Ne (11.6% each), and Bi (9.3%). A single type was detected in 95.3% of the samples and 4.7% had mixed infections with three different ITS types. DHPS mutants were present in 17 (46%), and the wildtype was present in 20 (54%) of 37 isolates. No association was found between ITS and DHPS types and between DHPS types and therapy or response to anti-PCP treatment. Type Ne presented an association with negative response to anti-PCP treatment (P< 0.001) and with death before 120 days after PCP diagnosis (P = 0.025). Type Eb was significantly more common in children than in adults (P = 0.001). Our data suggest an association of specific ITS genotypes with treatment failure, bad clinical outcome and childhood. © 2003 Elsevier B.V. All rights reserved. Keywords: Genotyping; Portuguese immunocompromised patients; ITS regions; DHPS gene; Pneumocystis jiroveci pneumonia 1. Introduction Pneumocystis jiroveci, formerly named P. carinii f. sp. hominis (Stringer et al., 2002) is an opportunistic agent that causes significant morbidity and mortality in immuno- compromised patients usually in the form of a severe pneumonia. Until now no reliable culture system for the propagation of human-derived Pneumocystis exists and no morphological means for determination of species differ- ence is available. Studies on biodiversity are being con- ducted by molecular typing based on nucleotide sequence variations of P. jiroveci genome. Analysis of DNA sequence divergence among different isolates of P. jiroveci has been reported at several genetic loci. The highest level of se- ∗ Corresponding author. Tel.: +351-213-652600; fax: +351-213-632105. E-mail address: omatos@ihmt.unl.pt (O. Matos). quence variation has been observed at internal transcribed spacer (ITS) regions of the nuclear rRNA operon (Lu et al., 1994; Tsolaki et al., 1996; Lee et al., 1998). Fifteen dif- ferent ITS1 types and 14 different ITS2 types have been found with a total of 210 potential types. Approximately 60 ITS combination types have been reported (Lee et al., 1998). Dihydropteroate synthase (DHPS) gene is another genetic loci that is being studied with much interest due to the potential association of point mutations at codons 55 and 57 with trimethoprim-sulfamethoxazole (TMP-SMZ) resistance (Kazanjian et al., 1998; Mei et al., 1998). In this study, analysis of genetic variation among isolates of P. jiroveci from immunocompromised Portuguese patients was performed at the ITS regions of the nuclear rRNA operon and at the DHPS gene. Investigation of the possible associ- ation between specific P. jiroveci ITS and DHPS types, and between ITS or DHPS types with demographic and clinical parameters was also undertaken. 1567-1348/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1567-1348(03)00092-3