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Pneumocystis pneumonia-colonisation versus infection with qPCR in HIV positive & negative patients

February 23 2016

Pneumocystis pneumonia (PCP) caused by P. jirovecii is a serious opportunistic infection frequently seen in HIV positive patients. Despite highly active antiretroviral therapy (HAART), it is still a serious life threatening opportunistic infection. Quantitative PCR on respiratory samples is the most sensitive and specific laboratory test, but the actual cut-off to define a case has been a matter for debate.

Louis et al 2015 (1) analysed retrospectively PCP cases in Paris, to investigate qPCR cut-offs. They established composite diagnoses of PCP from clinical radiological, microscopy and qPCR performed on BAL fluids, over 3 years to 2013. PCP was identified in 56 HIV-positive and in 49 HIV-negative patients indicating that PCP is a significant occurrence in immunocompromised groups other than HIV. The direct fluorescence assay – regarded as the gold standard for PCP diagnosis, showed a 92.8 % sensitivity in HIV positive patients but only a 55.1% sensitivity in HIV negative patients; a lower fungal burden is seen in HIV negative patients (2).
 Typical example of moderately severe PCP in AIDS, with bilateral fluffy mid and lower lobe shadows:PCP in AIDS

A single cut-off at 1.5x104 copies/ml allowed the differentiation between colonized and infected HIV-positive patients with 100% sensitivity and specificity. In HIV-negative patients, cut-off values of 2.9x104 and 3.4x103 copies/ml resulted in 100% specificity and sensitivity, respectively.

Several PCR based methods (Morris 2008; Damiani 2013) have been developed allowing enhancement of sensitivity and they also led to the discovery of colonisation with PCP - essentially the detection of P. jirovecii DNA in bronchopulmonary samples without any clinical and radiological signs of PCP.  The first multicentre evaluation of a real-time PCR (MycAssay, Myconostica Ltd) published in 2011 (3) Hauser et al) showed very good correlation with direct fluorescence diagnosis and also showed much stronger PCR signals in HIV patients compared with non-HIV patients. However, the lower signal strengths overlapped with PCP cases, preventing a simple cut-off to be determined (Fig 1, ref 3). Additional studies determining PCP cut-off in HIV and non-HIV patients with other Pneumocystis PCR assays are required. This is especially the case for HIV negative patients as PCP has a higher mortality in these patients - rates of ~50%.

 

The concordance between four real-time PCR assays - one in house PCR assay and three commercial kits – was evaluated recently (4) and showed a wide dispersion of the Ct values for the four assays. A lack of standardisation between both samples and variation in the standard curves of each assay did not allow direct comparison of cut-offs, but a good concordance or positive/negative for each test was seen between the in house assay, AmpliSens® and the MycAssay® for Pneumocystis.

Diagnosis of PCP with clinical and radiological data is distinctive in some patients, although usually only if far advanced, and laboratory confirmation currently relies on identification of cysts & trophic forms of P. jirovecii directly from respiratory samples such as BAL. Standard staining (Giemsa or toluidine blue), or Calcofluor® white allows visualisation of fungal elements but use of a direct immunofluorescence assay greatly enhances detection.

Asymptomatic colonisation represents a real drawback to PCP diagnosis, especially using PCR. Positive samples need to be evaluation in the context of the patient, and possibly in conjunction with the use of beta 1,3 glucan detection in serum.

REFS:

1. Louis et al 2015, J Clin Microbiol  Dec, 53 (12), 3870

2. Maillet M, 2014. Pneumocystis jirovecii (Pj) quantitative PCR to differentiate Pj pneumonia from Pj colonization in immunocompromised patients. Eur J Clin Microbiol Infect Dis 33:331–336.

3. Hauser et al  2011, J. Clin. Microbiol, May 2011, p. 1872–1878

4 Sasso et al 2016, J Clin Microbiol Feb, 54 (2);  doi:10.1128/JCM.02876-15

More info on PCP