Outbreaks of Pneumocystis pneumonia in 2 renal transplant centers linked to a single strain of Pneumocystis: implications for transmission and virulence

Abstract

Background: There have been numerous reports of clustered outbreaks of Pneumocystis pneumonia (PCP) at renal transplant centers over the past 2 decades. It has been unclear whether these outbreaks were linked epidemiologically to 1 or several unique strains, which could have implications for transmission patterns or strain virulence.

Methods: Restriction fragment length polymorphism (RFLP) analysis was used to compare Pneumocystis isolates from 3 outbreaks of PCP in renal transplant patients in Germany, Switzerland, and Japan, as well as nontransplant isolates from both human immunodeficiency virus (HIV)-infected and uninfected patients.

Results: Based on RFLP analysis, a single Pneumocystis strain caused pneumonia in transplant patients in Switzerland (7 patients) and Germany (14 patients). This strain was different from the strain that caused an outbreak in transplant patients in Japan, as well as strains causing sporadic cases of PCP in nontransplant patients with or without HIV infection.

Conclusions: Two geographically distinct clusters of PCP in Europe were due to a single strain of Pneumocystis. This suggests either enhanced virulence of this strain in transplant patients or a common, but unidentified, source of transmission. Outbreaks of PCP can be better understood by enhanced knowledge of transmission patterns and strain variation.

Figure 1.

Restriction fragment length polymorphism (RFLP) analysis of Pneumocystis samples from Zurich, Switzerland. A–D, The RFLP pattern following agarose gel electrophoresis for the 10 samples that could be amplified for analysis. Labels at the top represent the individual samples. A and C, Gels were run following digestion with DraI. B and D, Gels were run following digestion with Hpy188I. Samples 1–6 and 14 are from renal transplant patients, and samples 7, 10, and 12 are from control patients. The letter G denotes a representative sample from the outbreak in Munich, Germany; + is a positive control. With both enzymes, the RFLP patterns of the renal transplant patients are identical to each other and to the German sample, whereas the control patients showed patterns that were different from each other as well as from the transplant patients. E and F, Southern blots of the gels from panels A and B, confirming the results of the gel analysis. Molecular weight markers are indicated on the left.

Figure 2.

Restriction fragment length polymorphism (RFLP) analysis of Pneumocystis samples from Nagoya, Japan. The RFLP pattern following agarose gel electrophoresis (A) (following digestion with DraI on the left and Hpy188I on the right) and following Southern blotting (B), for the 4 samples that could be amplified for analysis. Labels at the top represent the individual samples. All 4 samples (J1, J2, J5, and J8) are from renal transplant patients. The letter G denotes a representative sample from the outbreak in Munich, Germany; the letter S denotes a representative sample from the outbreak in Zurich, Switzerland; + is a positive control. Samples J1, J2, and J5 showed a pattern identical to each other but different from the G and S samples, whereas sample J8 was different from all other samples. Molecular weight markers are indicated on the left.

Figure 3.

Dendrogram derived by BioNumerics software from restriction fragment length polymorphism (RFLP) analysis of 53 samples following agarose gel electrophoresis. All samples were digested with DraI. Thirty-six are samples from endemic cases of Pneumocystis pneumonia that were included in a prior publication [10]. Seventeen samples are from the current study and include 4 representative samples from the outbreaks and the 9 control samples from Switzerland (Sw) and Germany (Ge), as well as the 4 outbreak samples from Japan (Ja). The Dice coefficient was used to calculate similarities, and unweighted pair group method with average linkages was used for cluster analysis. The position tolerance was 1.9%. The percent similarity scale is shown above the dendrogram and indicated by the numbers at the individual nodes. SDs of the branches are indicated by the gray bars. For branches without a bar, the SD was 0. The samples from the outbreaks in Europe and Japan form unique clusters that are boxed. The control samples from Europe and the outbreak sample from Japan that had a different RFLP pattern are indicated by a +. As previously reported, 6 of the paired samples with 100% identity represent samples from the same patient collected at different times [10].

Figure 4.

Sequence analysis and alignment of a region in the intron of the msg expression site that includes tandem repeats, which are underlined. Shown are results for 2 Swiss samples (S1, S5), a German sample (G), and 2 Japanese samples (J9, J10). Samples J1–J8 (not shown) were identical in sequence to sample J9. For comparison are 4 sequences with 2, 3, 4, or 6 tandem repeats (A2–A6) obtained from a single patient from the United States [14]. Although restriction fragment length polymorphism analysis identified differences between the Japanese and European isolates, in this region the sequences from all renal transplant patients from the 3 countries were identical. The isolate from a nontransplant Japanese patient (J10) differed from the transplant isolates at 2 positions indicated by the arrows.