Pneumocystis jirovecii

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Pneumocystis jirovecii dihydropteroate synthase gene mutations. AIDS 19, 801–805. (2005). 13. Valerio A, Tronconi E, Mazza F, Cargnel A,. Fantoni G, Atzori C: ...

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Olga Matos†1 & Francisco Esteves1 Unidade de Protozoários Oportunistas/VIH e Outras Protozooses, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal † Author for correspondence: Tel.: +351 213 652 638 n Fax: +351 213 632 105 n [email protected] 1

Pneumocystis jirovecii pneumonia (PcP) remains a major cause of respiratory illness among immunocompromised patients, especially patients infected with HIV, but it has also been isolated from immunocompetent persons. This article discusses the application of multilocus genotyping ana­lysis to the study of the genetic diversity of P. jirovecii and its epidemiological and clinical parameters , and the important concepts achieved to date with these approaches. The multilocus typing studies performed until now have shown that there is an important genetic diversity of stable and ubiquitous P. jirovecii genotypes; infection with P. jirovecii is not necessarily clonal, recombination between some P. jirovecii multilocus genotypes has been suggested. P. jirovecii- specific multilocus genotypes can be associated with severity of PcP. Patients infected with P. jirovecii, regardless of the form of infection they present with, are part of a common human reservoir for future infections. The CYB, DHFR, DHPS, mtLSU rRNA, SOD and the ITS loci are suitable genetic targets to be used in further epidemiological studies focused on the identification and characterization of P. jirovecii haplotypes correlated with drug resistance and PcP outcome.

Pneumocystis jirovecii (formerly Pneumocystis carinii f. sp. hominis) causes severe interstitial pneumonia (PcP) in immunocompromised patients across the world, especially those with HIV [1–3] . P. jirovecii has also been identified in pulmonary specimens from patients with rheumatologic pathologies or inflammatory bowel disease, patients with chronic pulmonary diseases as well as in immunocompetent persons [4–11] . As P. jirovecii cannot be cultured reliably, molecular typing has been used to describe differences between organisms. Despite the application of molecular methods to the study of this organism, until recently, attempts to associate specific genotypes with the clinical course or outcome of PcP gave inconsistent results. PcP outcome is a complex issue, with several clinical variables affecting the patient’s prognosis  [12–18] . Advances in genetic characterization of P. jirovecii have shown that specific polymorphisms are correlated with the epidemiological profile of the organism, including the geographic distribution, drug resistance, virulence of some genotypes, modes of transmission and population genetics  [7,9,12,19–27] . Multilocus genotyping approaches are currently being used for the characterization of pathogenic microorganisms. The chief advantage of multilocus typing using PCR is that there is a greater sensitivity for detecting differences when several loci are examined concurrently than when 10.2217/FMB.10.75 © 2010 Future Medicine Ltd


Future Microbiology

Pneumocystis jirovecii multilocus gene sequencing: findings and implications

a single locus is examined  [28,29] . In addition, by looking at polymorphisms at multiple loci, further information is available for strain characterization and strain typing is achieved, rather than simply gene typing [30] . These data suggest that factors such as virulence or drug resistance may be dependent on multiple P. jirovecii genotypes and therefore on the association of multiple polymorphisms that occur in various regions of the genome of the organism. The different associations of polymorphisms, or haplotypes, could give the organism distinct characteristics, influencing the presentation and clinical course of PcP. However, these genetic polymorphisms may or may not be associated with the existence of distinct, stable genetic strains, which also may or may not be associated with different clinical outcomes. Genetic diversity of Pneumocystis jirovecii & epidemiological & clinical parameters

In the last 20 years, the combination of epidemiologic studies with genotyping techniques has contributed to a better understanding of the characteristics of Pneumocystis. Genetic diversity in P.  jirovecii, based on identification of genotypes at single or multiple loci, has been described by using PCR followed by DNA sequencing, restriction fragment length Future Microbiol. (2010) 5(8), 1257–1267

Keywords clinical course n diversity genotype n multilocus approach n Pneumocystis jirovecii n n

part of

ISSN 1746-0913



Matos & Esteves

polymorphism, single strand conformation polymorphism or type-specific oligonucleotide hybridization [20,30–34] . With these techniques, several polymorphic loci have been identified and characterized (see Table 1) [28,29,35–52] . Genetic diversity

Sequence heterogeneity has been observed in several P. jirovecii genes. Two loci have been most widely studied with the purpose to determine the genetic diversity of this organism, the mtLSUrRNA and the ITS regions of the nuclear rRNA operon. Nevertheless, other variable regions of the P. jirovecii genome have also received the attention of researchers, the nuclear rRNA genes encoding the 5.8S and 26S, b-TUB, TS, CYB, SOD, TRR1, DHPS and DHFR. Several multilocus typing studies have been performed [14,18,24,32,33,53–58] , allowing the investigation of genetic diversity of isolates from PcP patients, from different countries (Table 1) . The results obtained demonstrate that patients are infected with a single genotype, while others are coinfected by more than one P. jirovecii genotype [24,32,54–57,59] . New P. jirovecii genotypes have been identified in several loci [18,58] . In two studies involving PcP patients from the USA and Italy, significant differences in the distribution of some ITS genotypes between DHPS wild‑type and mutant sequences were observed, suggesting that some ITS genotypes (i.e., Eg, Ne, Kf or Ee) are more likely to contain DHPS mutations [14,60] . Data showed that a linkage between the ITS genotypes and DHPS mutations may exist. However, the correlation between ITS and DHPS genotypes is in contrast to other studies, involving PcP patients from Portugal [33,59,61] . The differences observed in those studies may be due to the geographical variation in the prevalence of P. jirovecii ITS genotypes and DHPS mutations, possibly caused by intrinsic epidemiological factors that influence the circulation and transmission of different genetic organisms. In addition, Portuguese specimens usually showed a higher prevalence of the wild‑type sequence and a lower frequency of mutations than in the USA, perhaps because of differing use of sulfa or sulfone drugs for PcP prophylaxis [22,61–63] . The low frequencies of DHPS mutants observed in the Portuguese population may contribute to the impairment of the statistical ana­lysis between those genotypes and the ITS genotypes. In another study conducted in a P. jirovecii-infected Portuguese population the results of the combinatorial test d1 demonstrated that one P. jirovecii multilocus genotype E (MLG E) occurred at a 1258

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higher frequency than would be expected, suggesting linkage disequilibrium and clonal propagation. This test takes a panmitic situation as a null hypothesis, and explores the criteria of clonality. The presence of a particular MLG in great excess is often the most robust and significant evidence of clonal reproduction, indicating that the MLG is replicating as a unit without the gene shuffling attributable to sexual recombination. The authors suggested that the persistence of this specific MLG could be a consequence of clonal reproduction of this successful genotype in a geographically isolated P. jirovecii population with epidemic structure [58] . This type of population arrangement is characterized by frequent recombination within all members of an organism population; however, occasionally a highly successful individual arises and increases rapidly in frequency to produce an epidemic clone [64] . Temporal diversity

Investigation of the temporal diversity of P. jirovecii isolates has been achieved by using multilocus genotyping (Table 1) . However, the two first studies were based on single gene typing. They examined paired P. jirovecii isolates from HIVpositive patients who had two episodes of PcP and it was observed that genetically distinct isolates were associated with each episode [65,66] . Also, in a third study involving three genetic markers (ITS1, ITS2 and mtLSU rRNA loci), change in the sequence of the ITS, which is located in the nucleus, was associated with changes in the mitochondrial gene, excluding mutation as the cause of the genetic differences between P. jirovecii strains isolated during different episodes of PcP [67] . More recent multilocus genotyping studies have demonstrated the temporal stability of the genes analyzed. A study showed that isolates of P. jirovecii from non-HIV immunocompromised patients from before the AIDS pandemic, were genetically very similar to those currently found in HIV-positive patients [68] . In another study involving 10 European hospitals over a nine year period, the stability and ubiquitous nature of the genetic markers (26S rRNA, mtLSU rRNA, b-TUB and ITS1 loci) was suggested by the observation of the same types in different hospitals several years apart, as well as in pairs of specimens collected from the same patients at an interval of up to eight weeks [24] . These more recent observations indicate an apparent temporal stability instead of diversity of the P. jirovecii genotypes observed at different times. This would be expected if P. jirovecii organisms were latent in human lungs. future science group

Pneumocystis jirovecii multilocus gene sequencing: findings & implications


Table 1. Multilocus genotyping studies of Pneumocystis jirovecii and association with important epidemiological and clinical parameters. Study (year)

Country (time period)

Latouche (1997)

France, Italy (NA)

PcP cases (n)

Tested loci

Ma (1999)

5.8S rRNA, mtLSU rRNA, TS, ITS1, ITS2 U.S.A. (NA) 15 mtLSU rRNA, ITS1, ITS2 NA (1994–1995) 11, HIV-positive 26S rRNA, mtLSU rRNA, b-TUB, ITS1 The Netherlands 6, HIV-positive; AROM, mtSSU rRNA, (1968–1981) non-HIV-positive mtLSU rRNA, ITS1, immunocompromised ITS2 Sweden, France 8, (1 without PcP) mtLSU rRNA, ITS1, (1991–1996) and hospital ITS2 air samples France 14 mtLSU rRNA, DHPS, (1993–1998) ITS1, ITS2 USA (1985–1998) 37, (26 HIV-positive) DHFR, DHPS

Beard (2000)

USA (1995–1998) 324, HIV-positive


Hauser (2001)

Belgium, 212 Denmark, France, Germany, Switzerland (1989–1998) Italy (1994–1997) 19, HIV-positive

26S rRNA, mtLSU rRNA, b-TUB, ITS1

Keely and Stringer (1997) Hauser (1997) Tsolaki (1998)

Olsson (1998)

Santos (1999)

Volpe (2001)

20, HIV-positive

26S rRNA, mtLSU rRNA, b-TUB, ITS1, ITS2 26S rRNA, mtLSU rRNA, b-TUB, ITS1

Hauser (2001)

Belgium, 91 Denmark, France, Germany, Switzerland (NA)

Ma (2001)

USA (1986–1999) 22


Meshnick (2001)




Takahashi (2002) Japan (1994–2002)

34, (24 HIV-positive)


Miller (2002)

2, HIV-positive


Wakefield (2003) UK (1990–2001)

16, HIV-positive


Miller (2003)

76, HIV-positive


UK (2000)


Comments No significant genetic differences between P. jirovecii from the two geographic sites Recurrent PcP caused by reinfection rather than by reactivation of latent P. jirovecii Multitarget genotyping allowing investigation of P. jirovecii diversity Pre-AIDS P. jirovecii isolates are genetically very similar to those currently found in HIV-positive patients Concordant P. jirovecii genotypes between PcP patients and air samples support the hypothesis of airborne transmission DHPS mutations were statistically associated with sulfa drugs use Prior sulfa/sulfone prophylaxis was associated with DHPS mutations Mixed infections were associated with primary PcP. Infection is acquired from a relatively common source Broad diversity of genotypes indicates that multiple sources of the pathogen coexist

Greater genetic diversity among the isolates. Evidence of mixed infections in 32% of the PcP patients Failure of anti-P. jirovecii prophylaxis was associated with a specific MLG. Drug resistance may be detected by molecular typing with use of markers that are apparently unrelated with resistance Mutations of DHPS loci occurred independently in multiple genotypes of P. jirovecii. Exposure to sulfa drugs select for those mutations Association between DHPS mutations and specific ITS genotypes The majority of mutant organisms were collected from patients not exposed to sulfa drugs or atovaquone. Patients with DHPS mutations were more likely to fail TMP-SMX treatment The same genotype in samples from mother and infant, suggesting verticle transmission of P. jirovecii Change in MLGs of sequential samples suggests reinfection, rather than latency. Asymptomatic carriers may supply a reservoir for future infections Described a reliable multilocus method to study the epidemiology and potential transmission of P. jirovecii

Ref. [57] [67] [54] [68]


[82] [45] [32]





[60] [84]




MLG: Multilocus genotype; NA: Not available; PcP: Pneumocystis jirovecii pneumonia; SMX: Sulfamethoxazole; TMP: Trimethoprim.

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Matos & Esteves

Table 1. Multilocus genotyping studies of Pneumocystis jirovecii and association with important epidemiological and clinical parameters (cont.). Study (year)

Country (time period)

PcP cases (n)

Matos (2003)

Portugal (1994–1997)

42, DHPS, ITS1, ITS2 immunocompromised

Miller (2003)

UK, Zimbabwe (NA)

51, HIV-positive


Montes-Cano (2004)

Spain (2001–2003)

79, (15 HIV-positive)


Nahimana (2004) Switzerland, France (1993–1996) Medrano (2005) Spain (2003)

33, (25 HIV-positive)


50, immunocompetent


Beard (2005)

Höcker (2005)

Tested loci

USA (1996–2000) 58, infants non-HIV- mtLSU rRNA, DHPS positive, deceased of various causes; 384, HIV-positive adults Germany (2002) 4, children (3 renal 26S rRNA, mtLSU transplant recipients) rRNA, b-TUB, ITS1 60, HIV-positive


Costa (2006)

Portugal (2001–2004)

Valerio (2006)

Italy (1996–2006) 261, HIV-positive


Valerio (2007)

Italy (1994–2004) 207, HIV-positive


Esteves (2008)

Portugal, Spain (2001–2004)

105, (61 HIV-positive)


Siripattanapipong Thailand (2008) (1997–2003)

29, HIV-positive


Montes-Cano (2009)

Spain (NA)

van Hal (2009)

Australia (2001–2007)

40, placentas and 20 mtLSU rRNA, DHPS lung tissues from fetuses 68, specimens mtLSU rRNA, ITS1, ITS2

Esteves (2010)

Portugal (1997–2007)

96, HIV-positive




An association was observed between specific ITS genotypes and treatment failure, bad clinical outcome and childhood Evidence of selection pressure from sulfa drug exposure. P. jirovecii infection arose by recent transmission, not reactivation of latent infection A specific genotype was found to be relatively higher in patients with AIDS/PcP than in patients with chronic pulmonary disease. Data suggest a common source of infection between these two groups P. jirovecii populations may evolve under pressure from DHFR inhibitors. DHFR mutations may contribute to drug resistance P. jirovecii DNA can be frequently detected in immunocompetent adults, suggesting that the general population could be a reservoir and a source of infection Genotype distributions at both loci were significantly different in the two populations, suggesting independent transmission cycles

The affected patients had acquired the same two genotypes, suggesting interhuman infection ITS regions demonstrated a high degree of genetic heterogeneity. No significant association between DHFR, DHPS and ITS genotypes was found No correlation was detected between DHPS and ITS genotypes. Association between DHPS mutations and severity of PcP was suggested Absence of correlation between ITS genotypes and clinical data. A trend of association was observed between DHPS mutations and clinical features Difference in ITS genotypes geographic distribution. The prevalent mtLSU rRNA and DHPS genotypes were statistically associated In comparison with previous reports, higher frequency of DHFR mutations and lower prevalence of DHPS mutations was observed Molecular evidence of P. jirovecii transplacental transmission in humans Patients with DHPS mutations were more likely to have severe disease and poor outcome than patients with DHPS wild‑type sequence Specific MLGs were associated with severity of PcP. Data showed that P. jirovecii haplotypes may be related to the clinical outcome of PcP

MLG: Multilocus genotype; NA: Not available; PcP: Pneumocystis jirovecii pneumonia; SMX: Sulfamethoxazole; TMP: Trimethoprim.


Future Microbiol. (2010) 5(8)

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Pneumocystis jirovecii multilocus gene sequencing: findings & implications


Table 1. Multilocus genotyping studies of Pneumocystis jirovecii and association with important epidemiological and clinical parameters (cont.). Study (year)

Country (time period)

PcP cases (n)

Tested loci


Esteves (2010)

Portugal (2001–2007)

67, HIV-positive


Statistical associations between genotypes and patients’ age groups or PcP clinical status. Evidence of epidemic population structure

Ref. [58]

MLG: Multilocus genotype; NA: Not available; PcP: Pneumocystis jirovecii pneumonia; SMX: Sulfamethoxazole; TMP: Trimethoprim.

Supporting this hypothesis, there are some studies that demonstrate the detection of P. jirovecii DNA, by molecular methods, in noninvasive human samples from healthy infants and pregnant women, suggesting that an asymptomatic infection, or a carrier state, is common in segments of the population that are immunocompetent [69,70] . However, another explanation cannot be excluded. P. jirovecii genotypes are known to change over time due to pressure exerted by chemotherapy. The decreased use of sulfa prophylaxis after the introduction of HAART promoted a decline of DHPS gene mutations frequencies and, consequently, a decline in DHPS gene diversity. Geographical diversity

Several studies using multilocus genotyping methods have been carried out to determine the distribution patterns of P. jirovecii isolates in different geographic locations (Table 1) [24,32,57,60,61,71,72] . Some studies demonstrated no differences in the genotypes identified in diverse geographic populations [24,57,61,71] . However, in one study that examined P. jirovecii organisms obtained from adult HIV-positive PcP patients diagnosed in five US cities, genotype distribution patterns differed at each of the cities where samples were obtained and this variation correlated with the place of diagnosis but not with the place of birth [32] . In another study involving PcP patients from different cities in the USA, the percentage of DHPS mutations differed from place to place and there was an association between those mutations and specific ITS genotypes [60] . In a third study involving non-HIV-positive infants who died from various causes, and HIV-positive PcP patients, from different US cities, significantly different genotype distributions were observed in both populations. However, in this study, the geographic distribution of the genotypes found was not determined [72] . In another study, the ITS genotypes found showed a significant diversity in the different populations examined. Also, a statistically significant association between mtLSU rRNA genotype 1 and ITS type Eg was revealed [61] . future science group

Circulation & transmission

The transmission of P. jirovecii is not fully understood, nor has its natural habitat been identified. A number of studies using multi­locus genotyping methods have been conducted to study this issue (Table 1) [11,20,30,61,72–77] . Concordant P. jirovecii genotypes (mtLSU rRNA genotypes 1, 2 and 3) between PcP patients and hospital air samples from those patients’ rooms support airborne transmission [20] . P. jirovecii isolates from persons without underlying lung disease or immunosuppression and from HIV-positive asymptomatic P. jirovecii carriers, were studied supporting the hypothesis that both populations may play a role in transmission of P. jirovecii and may even represent a reservoir for future infections [11,30] . Comparison of data obtained from patients with chronic pulmonary diseases and HIV-positive patients infected with P. jirovecii revealed a similar pattern of genotypes in ana­lysis of mtLSUrRNA in samples isolated from the same city, providing support for the notion of a common infectious source [8] . The possibility that P. jirovecii can be transmitted from a PcP patient to a susceptible person was suggested by the report of an outbreak of PcP in a pediatric renal transplant unit. Multilocus genotyping of the isolates of the affected patients showed that they had acquired the same genotypes, suggesting interhuman infection [73] . Another study also suggested transmission of a P. jirovecii genotype between an HIV-positive mother to her young infant [74] . A third study provided additional data in support of this transmission route [61] . Most of these studies failed to show unequivocal evidence of person-to-person transmission, especially because of the low numbers of persons involved in the studies, but also because the subjects’ history of contact with PcP patients were usually poorly documented. By contrast, two other reports seem to establish that person-to-person transmission can occur. One study involved 10 PcP cases diagnosed in renal transplant recipients. Nosocomial transmission from HIV-positive patients with PcP was suspected because all these patients shared the same hospital building, were not isolated and were receiving suboptimal or no anti-PcP prophylaxis. Using a multilocus typing



Matos & Esteves

method, involving the 26S rRNA, mtLSU rRNA, b-TUB and ITS1 loci, the authors identified the same P. jirovecii genotype (type 1) in all of the patients (five renal transplant recipients and one HIV-positive patient), in the same period of time [75] . Another detailed study was conducted in a nephrology outpatient clinic due to the increase of the incidence of PcP cases in that group of patients. The pulmonary specimens available from renal allograft recipients were studied using four P. jirovecii genetic markers (26S rRNA, mtLSU rRNA, b-TUB and ITS1 loci), revealing the same P. jirovecii genotype. It was observed that all but one of the 19 PcP-infected transplant recipients had at least one concomitant visit with another PcP infected patient a common waiting area [76] . P. jirovecii has also been detected in placentas and lung tissues of aborted fetuses from immuno­competent women who had miscarriages, supporting transplacental transmission in humans [77] . While this evidence points to person-to-person transmission of P. jirovecii via the respiratory route, alternative explanations cannot be ruled out. The presence of P. jirovecii in the air of hospitals or other institutions’ common areas raises the possibility of a common environmental source. Reinfection versus reactivation

For many years it was thought that PcP resulted from reactivation of latent infection. In reality, several epidemiological multilocus studies on P. jirovecii support the hypothesis that some PcP cases can be acquired de novo (Table 1) [11,30,32,67,71] . One study involved HIV-positive patients experiencing multiple PcP episodes. Patients exhibited genotype switching between disease episodes in all of the loci analyzed, strongly suggesting reinfection instead of reactivation of dormant P. jirovecii [67] . Another study was designed and performed to test the latency model of Pneumocystis infection in the human host. Identification of single nucleotide polymorphisms ������������� at four independent loci (mtSSU rRNA, mtLSU rRNA, SOD and DHPS) was used to genotype P. jirovecii isolates from HIV-positive patients with symptomatic and asymptomatic P. jirovecii infections. A change in MLGs of sequential samples suggested reinfection, rather than latency [30] . In HIVpositive patients from Zimbabwe and from the UK, reversal of the mutant-to-wild‑type DHPS genotype ratios was seen when selection pressure was absent, as in Zimbabwe, or was removed, as in the UK, supporting the hypothesis that P. jirovecii infection arises by reinfection [71] . Furthermore, this topic was studied in a general population in whose pulmonary specimens 1262

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P. jirovecii was detected. A total of 6 months later, more than 75% of this population had no P. jirovecii DNA detectable in the specimens analyzed, suggesting the possible transience of the carrier state in healthy persons [11] . All these results suggest that reinfection with P. jirovecii organisms is a common occurrence, rather than reactivation of latent organisms in the human lungs. However, the existence of reactivation of P. jirovecii latent infections cannot be ruled out. Molecular detection techniques have shown that P. jirovecii can be carried in the lungs of asymptomatic persons with mild immunosuppression and even in the lungs of immunocompetent persons [4–11] . It is thought that these groups function as reservoirs for Pneumocystis infections in other susceptible (immunocompromised) persons [30] . Also, population studies have shown that genotype frequency distribution patterns are associated with the place of diagnosis rather than place of birth [32] . This implies that any infection acquired early in life has natural limits [3] . Demographic & clinical parameters

The relationship between P. jirovecii diversity and several demographic and clinical parameters in PcP patients have been investigated by several authors. Some of those utilized multilocus genotyping methodology (Table 1) [14,18,24,27,58,59,78,79]. In a study involving the P. jirovecii 26S rRNA, mtLSU rRNA, b-TUB and the ITS1 loci, using the PCRsingle strand conformation polymorphism typing method, no significant association was found between the 35 P. jirovecii genotypes identified and sex, age or HIV status of the 212 patients with PcP, from 10 European hospitals [24] . However, another study showed statistical associations between P.  jirovecii genotypes and patients’ age groups and PcP clinical status. mtLSU rRNA genotype 4 and DHFR 312 corresponded more frequently in patients 40 years of age or older (p = 0.045 and p = 0.050, respectively); and isolates with SOD 1 corresponded more frequently to patients between 30 and 39 years (p = 0.031). In addition, the generated data showed that DHFR wild‑type was more frequent among patients with confirmed AIDSrelated PcP cases (p = 0.014) and less common among colonized patients (p = 0.047) [58] . In other studies, the genomic characteristics of P. jirovecii isolates from groups of patients with diverse forms of P. jirovecii parasitism (patients colonized by the organism, immunocompetent infants with bronchiolitis developing mild PcP and immunocompromised patients with PcP) and living in the same geographical region were investigated. Shared features of the P. jirovecii genotypes were observed at future science group

Pneumocystis jirovecii multilocus gene sequencing: findings & implications

the genomic regions studied in the three groups of patients, suggesting that patients infected with this organism, whatever the form of infection they present, were part of a common human reservoir for P. jirovecii [78,79] . In other studies, associations of specific ITS genotypes with treatment failure, clinical outcome and childhood [59] and of DHPS mutants and some specific clinical PcP features (higher lactate dehydrogenase and the need for intubation) and worse outcomes [14] , were observed. In another study three P. jirovecii specific MLGs were found to be associated with severity of PcP, showing that P. jirovecii haplotypes may be related to PcP clinical outcome. The genotypes mt85C/SOD215C and SOD110T/SOD215C were detected mainly in PcP cases with favorable follow-up, both being associated with less virulent PcP cases (p 

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