Genotypic Study of Pneumocystis jirovecii in Human Immunodeficiency Virus-Positive Patients in Thailand

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1 JOURNAL OF CLINICAL MICROBIOLOGY, May 2005, p Vol. 43, No /05/$ doi: /jcm Copyright 2005, American Society for Microbiology. All Rights Reserved. Genotypic Study of Pneumocystis jirovecii in Human Immunodeficiency Virus-Positive Patients in Thailand Suradej Siripattanapipong, 1 Jeerapun Worapong, 2 Mathirut Mungthin, 3 Saovanee Leelayoova, 3 and Peerapan Tan-ariya 1 * Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Rd., 1 and Department of Parasitology, Phramongkutklao College of Medicine, Ratchawithi Rd., 3 Bangkok 10400, and Center for Biotechnology and Department of Biotechnology, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Nakornpratom 73170, 2 Thailand Received 18 October 2004/Returned for modification 3 November 2004/Accepted 17 January 2005 Pneumocystis jirovecii is one of the common opportunistic infections in human immunodeficiency virus (HIV)-infected patients in Thailand. Information regarding genotypic and epidemiological of this organism in Thai patients is not available. We analyzed the genotypes of 28 P. jirovecii-positive specimens from bronchoalveolar lavage and sputum samples from HIV-infected Thai patients based on nucleotide variations of the internal transcribed spacer regions 1 and 2 of the rrna gene. Thirteen genotypes were the same as previously reported outside Thailand. Ten genotypes, which included Bp, Er, Eq, Ic, Ir, Ip, Rc, Rp, Qb, and Qq, were new. Ir and Rp were unique and dominant types observed in HIV-infected Thai patients. Thirteen specimens (46.4%) were infected with a single type of P. jirovecii, and fifteen (53.6%) were mixed infections. These differences may be used as genotypic markers for studying the epidemiology and transmission of P. jirovecii in the Thai population. Pneumocystis jirovecii is an opportunistic fungus that causes pneumonia in immunocompromised individuals (6). The incidence of Pneumocystis pneumonia (PcP) has increased dramatically with the epidemic of human immunodeficiency virus (HIV) infection (1, 13, 15). P. jirovecii is also associated with pneumonia in other immunocompromised conditions, e.g., patients undergoing organ transplantation or chemotherapy for malignant diseases (10). The introduction of highly active antiretroviral therapy (HAART), which is the most effective treatment for HIV-infected patients, has resulted in many patients having fewer opportunistic infections, which allows patients to discontinue PcP prophylaxis (17). HAART, however, has not been widely used in Thailand due to the expensive cost (17). In Thailand, PcP has been reported as one of the common opportunistic infections in HIV-infected patients, but it has not been identified for the genotypes (16). In 1998, Lee et al. reported that there are three genotypes of P. jirovecii isolated from only three samples of Thai HIV-infected patients based on nucleotide sequence variation in internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) (8). These three genotypes are Ea, Eg, and Gg (8). The genotypic data for this organism in Thai HIV patients have been limited to understand its patterns of transmission and methods of intervention. The molecular epidemiological data are important for understanding the patterns of transmission and developing methods of intervention. More epidemiological data are needed for defining control and prevention strategies; therefore, we cloned and sequenced the ITS1 and ITS2 and defined the * Corresponding author. Mailing address: Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Rd., Phayathai, Bangkok 10400, Thailand. Phone: (662) Fax: (662) scptn@mahidol.ac.th. genotypes of P. jirovecii from Thai HIV patients by using scoring methods previously described by Lee et al. (8). In addition, the diversity of epidemiological and phylogenetic patterns observed in the Thai representative types was determined. MATERIALS AND METHODS Specimens. The P. jirovecii-positive specimens from bronchoalveolar larvage (BAL) or sputum (SP) specimens were obtained from 28 different HIV-positive patients diagnosed as having PcP at Phramongkutklao Hospital, Bangkok, Thailand, from 1997 to 2003 (Table 1). P. jirovecii was stained with Toluidine Blue O and indirect immunofluorescence assay. BAL specimens were centrifuged at 1,500 g for 10 min, and sediments were used for DNA extraction. Sputum was treated with equal volumes of 0.3% dithiothreitol and 0.02 M EDTA, vortexed, and incubated at 37 C for 10 min. Specimens were centrifuged at 1,500 g for 10 min, and the pellets were used for DNA extraction. All specimens were stored at 80 C until used. All portions of this study were done with the approval of the Ethics Committee of the Royal Thai Army Medical Department. DNA extraction. A total of 14 l of BAL or sputum was placed on a 6-mmdiameter FTA disk (Whatman Bioscience), and the filter disk was air dried overnight. The one-fourth piece of FTA disk was washed twice with 200 l of FTA purification reagent (Whatman Bioscience) for 15 min and then washed twice with 200 l of TE buffer (10 mm Tris-HCl, 0.1 mm EDTA [ph 8.0]; Amresco) for 15 min and dried at 80 C. The washed paper was used directly as the DNA template. PCR amplification. A nested PCR was performed on all specimens to amplify a 560-bp region of the ITS1-5.8S-ITS2 gene by using primers 1724F2, ITS2R, and ITS1F (3, 8, 22). The primary PCR was carried out in a 50- l reaction mixture containing 50 mm KCl 10 mm Tris-HCl (ph 9.0) at 25 C, 0.1% Triton X-100, 3 mm MgCl 2, 0.2 mm concentrations of each deoxynucleoside triphosphate, 1 U of Taq DNA polymerase, and 20 pmol of each primer, 1724F2 and ITS2R. Primary thermocyclic conditions were as follows: (i) 3 min at 94 C; (ii) 35 cycles of 1 min at 94 C, 1 min at 47 C, and 2 min at 72 C; and (iii) 5 min at 72 C. The secondary PCR was carried out in a 100- l reaction mixture that contained 5 l of 1:100-diluted primary PCR product with the same concentrations as those of the primary PCR, except for 3.5 mm MgCl 2,2UofTaq DNA polymerase, and 40 pmol of each primer, ITS1F and ITS4. The secondary thermocyclic conditions were as follows: (i) 3 min at 94 C; (ii) 40 cycles of 30 s at 94 C, 1 min at 56 C, and 1 min at 72 C; and (iii) 5 min at 72 C. The amplified products were electropho- 2104

2 VOL. 43, 2005 P. JIROVECII GENOTYPES IN HIV-POSITIVE THAI PATIENTS 2105 TABLE 1. Identification of P. jirovecii ITS types in 28 HIV-infected Thai patients Specimen no. Yr of specimen collection No. of clones P. jirovecii ITS type(s) a Eb, Ec, Rc Ef, Eg, Jf Ir Ne Ir Eb, Eg Ir Ea, Ir Rp Gg, Nb Er, Ir Ea Rp Ip Ai, Bb, Bi, Eb Ir, Rp Eq, Gb, Nb Ne Ir Ip Eb, Qb Bi Eb, Eq Ec, Er, Ic, Ir Bi, Bp, Ip Qq, Ec Eg Eb, Eg, Rp a P. jirovecii ITS type identification by using the score of Lee et al. (8)., type newly described in this study. resed on a 1.25% agarose gel and purified by using a QIAquick gel extraction kit (Qiagen) according to the manufacturer s instruction. Cloning and sequencing. The purified PCR product was ligated into the pdrive cloning vector (Qiagen). Ligated products were introduced into Escherichia coli strain JM109 by transformation. A minimum of two clones from each recombinant plasmid were isolated by using the QIAprep Spin Minipreps kit (Qiagen) and bidirectionally sequenced by T7 and SP6 promoters flanking the cloning region. DNA sequencing was conducted by Macrogen, Inc., Seoul, Korea, and the Bioservice Unit, Bangkok, Thailand. Multiple alignment was performed by using CLUSTAL X, version 1.81 (5, 19), and edited manually. Molecular typing. ITS1 and ITS2 alleles were identified by using the scoring positions and typing method (8). In brief, the scoring positions of ITS1 are composed of 17 nucleotides at positions 6, 12, 15, 21, 23 to 24, 28, 34, 42, 53 to 54, 80 to 81, and 115 to 118. The scoring positions of ITS2 are composed of 28 nucleotides at positions 48 to 49, 52 to 57, 62 to 66, 68 to 72, 76, 122, 160, 166 to 171, and 173. A new sequence variation at any position within scoring positions was accepted as a distinct allele because at least two clones were confirmed from different specimens. The sequences with variation found outside the scoring position were accepted as a new scoring position. These sequences were identified as a new allele when there were at least three totally identical sequences detected from different specimens. P. jirovecii ITS types were defined by a combination of both ITS1 and ITS2 alleles. Phylogenetic analysis. Comparison and alignment of ITS sequences was done by using CLUSTAL X, version 1.81 (5, 19), and then manually aligned. Phylogenetic relationships were examined by using the PHYLIP (distributed by J. Felsenstein, Department of Genetics, University of Washington, Seattle) and the TREEVIEW programs (distributed by R. D. M. Page, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom). Nucleotide sequence accession numbers. The accession numbers of identified ITS1 and ITS2 alleles registered in GenBank are as follows: ITS1 allele Q, AY550106; ITS1 allele R, AY550105; ITS2 allele p, AY550108; ITS2 allele q, AY550109; and ITS2 allele r, AY RESULTS Our results identified 23 P. jirovecii ITS types. Among these 23 types, 9 ITS1 alleles and 10 ITS2 alleles were observed. Two new ITS1 alleles and three new ITS2 alleles were also detected. The new ITS1 alleles were designated as alleles Q and R. ITS1 allele Q differed from the allele E by a G-to-A transition at scoring position 81 (Fig. 1). ITS1 allele R differed from the allele I by a G-to-A outside the scoring position at position 49 and a deletion of a T nucleotide outside scoring position 131 (Fig. 1). Three new ITS2 alleles were designated alleles p, q, and r. ITS2 allele p differed from the allele r by having no insertion with T and A nucleotides at scoring positions 60 and 61 (Fig. 2). Along with 13 types previously reported outside of Thailand, 10 unique P. jirovecii types were found in the present study (Table 1). These novel P. jirovecii types were Bp, Er, Eq, Ic, Ir, Ip, Rc, Rp, Qb, and Qq. A three-nucleotide (GAA) insertion event was observed between positions 186 and 190 from five different specimens in clones , , , , , , and , respectively (Fig. 2). Figure 3 summarizes the frequency and distribution of P. jirovecii in 28 HIV-positive patients. The most prevalent type was type Ir, which was found in eight patients (28.6%), i.e., patients 6, 9, 17, 19, 24, 33, 46, and 59. Type Eb was identified in six patients (21.4%), i.e., patients 1, 12, 28, 49, 58, and 70. Type Eg was detected in four patients (14.3%), i.e., patients 4, 12, 69, and 70. Type Rp was found in four patients (14.3%), i.e., patients 22, 26, 33, and 70. Types Bi, Ec, and Ip were each identified in three patients (10.7%), i.e., patients 28, 54, and 61, patients 1, 59, and 67, and patients 27, 48, and 61, respectively. Types Ea, Er, Eq, Nb, and Ne were each found in two patients (7.1%), i.e., patients 19 and 25, 24 and 59, 39 and 58, 23 and 39, FIG. 1. Sequence alignment of new P. jirovecii ITS1 alleles. The first sequence is the consensus sequence of P. jirovecii ITS1 (8). Underlined bases indicate the scoring positions established by Lee et al. (8). Bases indicated by asterisks are implicated in the scoring positions established in the present study. Bases that are identical to those of the consensus sequence are represented by periods, missing bases are represented by hyphens, and bases that are different from those of the consensus sequence are given. The sequence at positions 61 to 70, 101 to 110, and 121 to 130 is not shown because no sequence variations were found in this area.

3 2106 SIRIPATTANAPIPONG ET AL. J. CLIN. MICROBIOL. FIG. 2. Sequence alignment of new P. jirovecii ITS2 alleles. The first sequence is the consensus sequence of P. jirovecii ITS2 (8). Underlined bases are indicated the scoring positions established by Lee et al. (8). Bases that indicated by asterisks are implicated in the scoring positions established in the present study. Bases that are identical to those of the consensus sequence are indicated by periods, missing bases are indicated by hyphens, and bases that are different from those of the consensus sequence are given. The sequence at positions 101 to 120 and 141 to 150 is not shown because no sequence variations were found in this area. and 7 and 45, respectively. Types Ai, Bb, Bp, Ef, Gb, Gg, Ic, Jf, Rc, Qb, and Qq were each identified in only one patient (3.6%), i.e., patients 28, 28, 61, 4, 39, 23, 59, 4, 1, 49, and 67, respectively. From the above results, coinfection of more than one type was observed in 15 of the 28 specimens (53.6%). The coinfection of two types, i.e., Eb and Eg, Ea and Ir, Gg and Nb, Er and Ir, Ir and Rp, Eb and Qb, Eb and Eq, and Ec and Qq, were found in patients 12, 19, 23, 24, 33, 49, 58, and 67, respectively (28.6%). In addition, the mixed infection of three types i.e., Eb, Ec, and Rc; Ef, Eg, and Jf; Eq, Gb, and Nb; Bi, Bp, and Ip; and Eb, Eg, and Rp were found in patients 1, 4, 39, 61, and 70, respectively (17.9%). The mixed infection of four types i.e., Ai, Bb, Bi, and Eb and Ic, Ir, Ec, and Er were observed in patients 28 and 59, respectively (7.1%). A single infection was detected in patients 6, 7, 9, 17, 22, 25, 26, 27, 45, 46, 48, 54, and 69 (46.4%) containing types Ir, Ne, Ir, Ir, Rp, Ea, Rp, Ip, Ne, Ir, Ip, Bi, and Eg, respectively. The 5.8S rrna sequences of 23 types of P. jirovecii found in Thailand and P. jirovecii AF were compared by using CLUSTAL W (Fig. 4). A dinucleotide (CG) point mutation at positions 115 and 116 was observed in 23 Thai types that differed from GC in the P. jirovecii AF S rrna sequence. FIG. 3. Prevalence of P. jirovecii types found in 28 HIV-infected Thai patients.

4 VOL. 43, 2005 P. JIROVECII GENOTYPES IN HIV-POSITIVE THAI PATIENTS 2107 FIG. 4. Alignment of 5.8S rrna sequences of P. jirovecii ITS types in 28 HIV-infected Thai patients and of P. carinii f. sp. hominis (AF013954).

5 2108 SIRIPATTANAPIPONG ET AL. J. CLIN. MICROBIOL. FIG. 5. Phylogenetic relationships of P. jirovecii ITS types in 28 HIV-infected Thai patients constructed from the aligned ITS 496 bp by the neighbor-joining analyses of the PHYLIP program (distributed by J. Felsenstein, Department of Genetics, University of Washington, Seattle). Numbers on the branches are bootstrap values with 5,000 replicates of neighbor-joining analysis. The scale bar length represents 0.01 substitution. To examine the genetic relationship of all 23 types of P. jirovecii found in Thailand, inferred trees under molecular phylogenetic methods were constructed from 498 aligned nucleotide positions of ITS1-5.8S-ITS2 sequences. Under the distance method, a neighbor-joining inferred tree with bootstrap values on each node showed new genotypes of P. jirovecii clustered with old types of P. jirovecii (Fig. 5). Within the first clade, types Ne, Nb, Eb, Ea, Gb, and Ec clustered together with low bootstrap values, but types Nb and Ne were sister types with a bootstrap value of 66. The second branch was formed by types Bb, Qb, Eq, and Qq with a bootstrap value of 28, and the third cluster was represented by types Er, Ef, Bp, Eg, Gg, Bi, and Ai with a bootstrap value of 29. Within the second and the third clades, several subclades with quite high bootstrap values were observed (53, 40, 50, and 95%), although the basal branches between these subclades did not receive high bootstrap support. Types Ic, Ir, Jf, Ip, Rc, and Rp grouped the last clade with a bootstrap value of 54. Within the last clade, type Ic formed with types Rp and Rc with a bootstrap value of 52%, whereas type Ir formed with an internode of types Ip and Jf with 45% of the bootstrap value. DISCUSSION The spectrum of polymorphism of the P. jirovecii ITS types found in Thai patients was similar to previous reports (4, 7, 8, 12, 20, 21), indicating a high diversity of P. jirovecii ITS genotypes. Among these types, the combination of ITS1 allele I and ITS2 allele c defining type Ic was first demonstrated. The novel ITS2 allele q sequence is not similar to any sequence patterns previously reported (8, 12, 14, 20). The ITS2 allele r should be mentioned as a new ITS2 allele because it was found to be identical to the sequence of the clone E reported in 1998, which was not defined in their original report (8). When the sequences of 28 P. jirovecii-positive specimens were compared to the consensus sequences, sporadic changes were observed at positions other than scoring positions. They were not considered to have any typing utility in the present study since these variations did not show any specific patterns. The three-nucleotide (GAA) insertion event, which was observed at positions 187, 188, and 190 from five different specimens, was not included in the scoring system because none of the clone sequences shows 100% identical according to the requirement of the typing criteria. If the presence of these three nucleotides is confirmed in future studies, they will probably have to be taken into account to define a new scoring system. Among the 23 types of P. jirovecii, type Ir was the most common (28.6%) in Thai HIV patients. The second and third common types were type Eb (21.4%) and types Eg and Rp (14.3%), respectively. This observation was in contrast to that reported by Lee et al. (8). In that study, type Eg was the most common type (20.3%), followed by types Ne (14.8%) and Eb (8.6%). The highest prevalence (up to 73.7%) of type Eg was also reported in patients from South Africa, followed by types

6 VOL. 43, 2005 P. JIROVECII GENOTYPES IN HIV-POSITIVE THAI PATIENTS 2109 Gg (21.0%), Eu (15.8%), and Gh (10.6%) (14). Type Eb was also found in specimens from Denmark, United States, France, and Portugal. Types Di, Eg, and Gh, which were previously reported in three specimens from Thailand in 1998 (8), were not detected in the present study. Other types, i.e., Ai, Bb, Bi, Ea, Eb, Ec, Ef, Gb, Jf, Nb, and Ne were also reported in specimens from Denmark, United states, Ivory Coast, Italy, France, The Netherlands, Portugal, and Sweden (8). These types were also found in Thai patients together with 10 new types. It is premature to conclude that these types are unique since the sample size was relatively small in the present study. A considerably greater number of specimens are required for further study before any conclusion could be drawn. From the present study, 15 specimens (53.6%) were of mixed infection. The mixed infection was not from the multiple copies of rrna genes since very strong evidences for a single rrna nuclear gene have been reported (2, 11, 18). This observation is consistent with the report of Tsolaki et al. (21) that mixed infection of PcP could be found from 50% of examined specimens. These investigators also concluded that P. jirovecii in immunocompromised patients is not necessarily clonal, and other studies also confirm the occurrence of mixed infections at 25 to 82% (8, 9, 14, 21). The variation of 5.8S rrna nucleotide sequences had no correlation with ITS types of P. jirovecii found in Thailand. Of 23 Thai representative types of P. jirovecii, 14 types possessed different nucleotides at different positions with no linkage or associated with any types of P. jirovecii. For example, types Eq, Gg, Ic, Eb, Ai, and Bp harbored C at positions 5 and 69, G at position 10, A at position 15, G at position 17, T at position 21, and A at position 56, whereas types Jf and Ip had an A at position 67. The variation of this region may imply that there would be recombination events among these coexisting strains, as explained by Robberts et al. (14). More samples of P. jirovecii are needed for further investigations and application of this region for defining strain types within identical ITS populations. The dissection of phylogenetic patterns observed in the present study showed that there were geographical variations of P. jirovecii ITS type and a unique information with regard to the molecular epidemiology among the HIV-infected patients through the geographic origins. The mutation of The ITS region of P. jirovecii would continue to circulate silently because 10 new types found in Thailand and the prevalent types appeared in European and America were clustered together in two clades of the inferred tree. The first clade was of types Bp, Er, Ir, Ip, Ic, Rc, and Rp and the prevalent types Ai, Bi, Eg, Gg, Ef, and Jf found in European and America (8). Another new clade was of types Qb, Eq, Qq, and the preexisting type Bb found in Denmark, France, and America (8). It is interesting that all new types were collected from 2000 to The new genotype Rp, however, has been reported since This correlation may imply that this genotype has the potential to reemerge and cause major epidemics of Pneumocystis pneumonia in Thai HIV-infected patients, with the supportive evidence of epidemiological frequencies, which were 14.28% or the second most prevalent types. With relative frequencies of epidemiology, the most and the third prevalent types, Ir and Rp, fell in the same major clade, whereas the second most frequent type Eb formed a sister clade of ITS types of Nb and Ne. The types Ir, Rp, and Ip may be the crucial genotype to predict the important aspects of their emergence and long-term evolutionary relationships of P. jirovecii in Thai HIV-infected patients. Within the major clade of types Er, Ef, Bp, Eg, Gg, Bi, and Ai, the third most frequent type Eg was a sister type Gg that was reported as the second rank of patients from South Africa (14). In this clade, type Bp found in the present study formed the basal node of types Ai, Bi, Eg, and Gg. This result suggested that the new type Bp had already coexisted in Thailand, even though it was collected in In summary, the present study identifies the considerable divergence of P. jirovecii genotypes in Thai HIV-infected patients by comparing current data with previous reports from immunocompromised patients with PcP. In agreement with other studies, geographical variations of P. jirovecii ITS type can be found. Thus, the distribution of P. jirovecii genotype in each geographical area should be studied as a tool to track the molecular epidemiology of P. jirovecii infection. ACKNOWLEDGMENTS This study was supported by Thailand-Tropical Diseases Research Program (T-2) grant 02-2-HEL and a thesis grant from the Faculty of Graduate Studies, Mahidol University, Mahidol, Thailand. We acknowledge Tawee Naaglor of the Department of Parasitology, Phramongkutklao College of Medicine, for technical assistance. We thank Padungsri Dubbs and Deven R. 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