Molecular typing of Pneumocystis jirovecii found in formalin-fixed paraffin-embedded lung tissue sections from sudden infant death victims

Abstract

Previous studies have provided histological evidence of an association between primary Pneumocystis infection and sudden infant death syndrome (SIDS). The aim of this work was to determine the species of clustered Pneumocystis organisms found in formalin-fixed paraffin-embedded (FFPE) lung tissue sections from Chilean sudden infant death (SID) victims. This approach needed first to optimize a DNA extraction method from such histological sections. For that purpose, the QIAamp DNA Isolation from Paraffin-Embedded Tissue method (Qiagen) was first tested on FFPE lung tissue sections of immunosuppressed Wistar rats inoculated with rat-derived Pneumocystis. Successful DNA extraction was assessed by the amplification of a 346 bp fragment of the mitochondrial large subunit rRNA gene of the Pneumocystis species using a previously described PCR assay. PCR products were analysed by direct sequencing and sequences corresponding to Pneumocystis carinii were found in all the samples. This method was then applied to FFPE lung tissue sections from Chilean SID victims. Pneumocystis jirovecii was successfully identified in the three tested samples. In conclusion, an efficient protocol for isolating PCR-ready DNA from FFPE lung tissue sections was developed. It established that the Pneumocystis species found in the lungs of Chilean SID victims was P. jirovecii.

Near-Universal Prevalence of Pneumocystis and Associated Increase in Mucus in the Lungs of Infants With Sudden Unexpected Death

“…This high prevalence is consistent with previous evidence that Pneumocystis is common in infant lungs [9, 10] including a study documenting Pneumocystis DNA by nPCR in all of 58 infants of undisclosed age [25]. Furthermore, the structural forms of the fungus were all recognized using Giemsa and GMS stains, suggesting active replication [26]. …”

SYTO-13, a Viability Marker as a New Tool to Monitor In Vitro Pharmacodynamic Parameters of Anti-Pneumocystis Drugs

“…In HIV-negative patients submitted to immunosuppressive treatments for malignancies, auto-immune diseases or organ transplantation, PcP is the cause of pneumonia in 10%-40% of these patients, with high mortality rates [ 3 , 4 ]. In addition, Pneumocystis organisms were detected relatively frequently in neonates or small children [ 5 , 6 ], pregnant women [ 7 ] and patients with chronic underlying diseases [ 8 – 10 ]. Indeed, in the latter population, Pneumocystis organisms have been considered as co-morbidity factors worsening the prognosis [ 3 , 5 , 11 , 12 ].…”

“…In addition, Pneumocystis organisms were detected relatively frequently in neonates or small children [ 5 , 6 ], pregnant women [ 7 ] and patients with chronic underlying diseases [ 8 – 10 ]. Indeed, in the latter population, Pneumocystis organisms have been considered as co-morbidity factors worsening the prognosis [ 3 , 5 , 11 , 12 ]. Particularly, in patients with chronic obstructive pulmonary disease (COPD), a colonization of the lungs by P .…”

Comparison of Single and Touchdown PCR Protocols for Detecting Pneumocystis jirovecii DNA in Paraffin‐Embedded Lung Tissue Samples

“…These techniques have proved more effective in detecting P. jirovecii DNA than conventional methods (Helweg‐Larsen, Lundgren, and Lundgren 2001). Pneumocystis DNA has been isolated from paraffin‐embedded pig (Ramos Vara et al 1998) and human (Chabe et al 2004; Varela et al 1998; Vargas et al 1999) lung tissues. In those previous studies only the single PCR method was used (Chabe et al 2004; Vargas et al 1999).…”

“…Pneumocystis DNA has been isolated from paraffin‐embedded pig (Ramos Vara et al 1998) and human (Chabe et al 2004; Varela et al 1998; Vargas et al 1999) lung tissues. In those previous studies only the single PCR method was used (Chabe et al 2004; Vargas et al 1999). Because there are now several different techniques available for amplifying DNA, in the present study we compared single and touchdown PCR procedures to verify the ability to amplify P. jirovecii in paraffin‐embedded lung specimens and to determine which method is most effective for this purpose.…”