New Short Tandem Repeat-Based Molecular Typing Method for Pneumocystis jirovecii Reveals Intrahospital Transmission between Patients from Different Wards

Abstract

Pneumocystis pneumonia is a severe opportunistic infection in immunocompromised patients caused by the unusual fungus Pneumocystis jirovecii. Transmission is airborne, with both immunocompromised and immunocompetent individuals acting as a reservoir for the fungus. Numerous reports of outbreaks in renal transplant units demonstrate the need for valid genotyping methods to detect transmission of a given genotype. Here, we developed a short tandem repeat (STR)-based molecular typing method for P. jirovecii. We analyzed the P. jirovecii genome and selected six genomic STR markers located on different contigs of the genome. We then tested these markers in 106 P. jirovecii PCR-positive respiratory samples collected between October 2010 and November 2013 from 91 patients with various underlying medical conditions. Unique (one allele per marker) and multiple (more than one allele per marker) genotypes were observed in 34 (32%) and 72 (68%) samples, respectively. A genotype could be assigned to 55 samples (54 patients) and 61 different genotypes were identified in total with a discriminatory power of 0.992. Analysis of the allelic distribution of the six markers and minimum spanning tree analysis of the 61 genotypes identified a specific genotype (Gt21) in our hospital, which may have been transmitted between 10 patients including six renal transplant recipients. Our STR-based molecular typing method is a quick, cheap and reliable approach to genotype Pneumocystis jirovecii in hospital settings and is sensitive enough to detect minor genotypes, thus enabling the study of the transmission and pathophysiology of Pneumocystis pneumonia.

Genotyping Pneumocystis jirovecii: Impacting Our Understanding of Interhuman Transmission

“…Regarding the ability to determine mixtures of P. jirovecii genotypes in samples, STR typing studies reported 70% (Parobek) and 68% (Gits-Muselli) of genotype mixtures, which was confirmed with a European study that analyzed 249 samples (68% of mixtures) [22,23,87]. The maximal ratio of mixture detection using simulated mixed samples was 1/50 [22].…”

“…The selected methods should also be capable of detecting mixtures, and in particular, have the ability to detect minority alleles. Detection of mixtures is an important aspect in the study of P. jirovecii transmission and epidemiology, considering that up to 92% of patients' respiratory samples present coinfections [17,[21][22][23][24][25].…”

“…The discriminatory power reached in this study was 0.999. Another STR analysis scheme (with one locus in common with Parobek's study) was proposed in 2015 and was based on six STR markers [22]. In studying 106 respiratory specimens (corresponding to 91 patients), the discriminatory power was 0.992 after the exclusion of putative epidemiologically related samples.…”

“…Thus, the range of mixture detection varies according to the method used, from 30% using Sanger DNA sequencing to 70% using SSCPs [24,28,32,45,68,76,91,92] and STRs [23,87] to 90% using pyrosequencing [21]. Recently, implementation of STR analysis methods and next-generation sequencing (NGS) has allowed easy detection of low amounts of coinfecting genotypes, with ratios of about 1:50 and 1:1,000, respectively [22,90]. Mixtures are generally encountered with P. jirovecii infections, and it does not seem to depend on the underlying disease since mixture detection was as high in AIDS patients as in solid organ transplant recipients [22,23].…”

Pneumocystis jirovecii Diversity in Réunion, an Overseas French Island in Indian Ocean

“…Indeed, there is only one report on this topic, which concerned P. jirovecii genotypes in French Guiana (Le Gal et al, 2015) located 7,000 km from metropolitan France whereas there are no data on genomic characteristics of P. jirovecii from Réunion, located 9,300 km from metropolitan France and 12,000 km from French Guiana (Figure 1). Conversely, numerous data on P. jirovecii genotypes are available for metropolitan France (Nevez et al, 2003;Totet et al, 2003;Le Gal et al, 2013;Maitte et al, 2013;Gits-Muselli et al, 2015;Alanio et al, 2016;Desoubeaux et al, 2016;Charpentier et al, 2017;Vindrios et al, 2017). In this context, the main objective of the present study was to identify P. jirovecii multilocus genotypes in patients living in Réunion and to compare these genotypes with those identified using the same method in patients living in metropolitan France or French Guiana.…”