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Diagnosis of Pneumocystis jirovecii pneumonia

Diagnosis of Pneumocystis jirovecii pneumonia (PCP) is conventionally based on direct staining and visualization. Challenges in obtaining alveolar samples have stimulated interest in techniques for detection of Pneumocystis DNA in non-invasive samples, which can give good sensitivity and specificity. Robust diagnosis is key to ensuring appropriate therapy.

by Dr Farnaz Dave, Dr Ashley Horsley, Dr Thomas Whitfield
and Dr Clare van Halsema

Pneumocystis jirovecii (previously Pneumocystis carinii) is a pathogen capable of causing life threatening Pneumocystis pneumonia (PCP) in the immunocompromised with case fatality rates among those hospitalized of around 10% [1]. PCP typically occurs in individuals with hematological malignancies on chemotherapy or with other causes of acquired cellular immunodeficiency or, most frequently, in human immunodeficiency virus (HIV)-positive individuals with CD4 T-cell counts <200 cells/µL or <14% of total white cell count [2, 3]. First-line treatment is co-trimoxazole, a combination of the antibiotics sulfamethoxazole and trimethoprim, at high dose for 3 weeks, which has the clinically significant potential side effects of bone marrow suppression, rash and bronchial hypersensitivity. Unfortunately the classical clinical presentation of PCP of progressive dry cough, dyspnoea and malaise is non-specific and chest examination and radiographs are often normal or near normal [4]. Oxygen desaturation on exercise is a helpful clinical sign in the right patient population [5]. Furthermore, many individuals with PCP do not produce sputum, so laboratory confirmation can be challenging. Given the serious nature of the illness and the possible side effects of treatment, accurate diagnosis is key to making an informed treatment decision. Incidence of PCP among HIV-positive individuals has declined since the widespread availability of antiretroviral therapy (ART) and has also declined as a cause of hospitalization of HIV-positive individuals [6]. In a study in a large HIV centre in London, mortality for all hospitalizations was around 10% during the first 10 years of availability of effective ART [1]. In our unit a decrease in mortality among those admitted to intensive care with PCP was seen between the periods of 1986–1995 and 1996–2004. This improvement is thought to be due to advances in critical care rather than treatment of PCP itself [7]. Reaching a diagnosis
Traditional methods

P. jirovecii is a fungus that cannot be cultured in vitro and so the organism is identified using histochemical staining techniques of fluid samples. Grocott-Gomori methenamine silver nitrate or direct immunofluorescence monoclonal antibody (IFA) stains on deep respiratory samples are generally regarded as the gold standard in diagnosis [8]. The life cycle of P. jirovecii is demonstrated by trophic, pre-cystic and cystic forms by morphological criteria. Diagnosis through microscopy of the cystic stage requires significant technical expertise and can still lead to false-negative results. In florid disease, P. jirovecii is present throughout the bronchial tree, from the upper respiratory tract down to the alveolar surface. Induced sputum samples are recommended by most guidelines, if routinely available, as spontaneously expectorated sputum is not considered an adequate alveolar sample and microscopy could be falsely negative. If the results of testing on induced sputum are not conclusive then a bronchoalveolar lavage (BAL) is recommended (sensitivity 86–98%) [9, 10]. In clinical practice, induced sputum is often not readily available, and BAL may not be possible due to hypoxia or may lead to a delay in sampling of several days; therefore, in clinical practice spontaneously expectorated samples are often processed. The disadvantages of this are twofold: a lower yield of cystic forms for visualization and potential for false-positive results due to colonization. Only in rare cases would a lung biopsy be appropriate (sensitivity 95–98%) in circumstances of poor response to empirical treatment and negative initial testing [9].

Molecular techniques
Molecular testing of lower respiratory tract secretions and blood is an alternative and operator-independent method for confirming the presence of P. jirovecii. Nucleic acid amplification techniques (NAAT) can be used with a number of primers targeting different substrates – most commonly the major surface glycoprotein (MSG), mitochondrial large subunit (MTLSU) rRNA and internal transcribed spacer (ITS) region genes [11]. One potential pitfall with these techniques is that the detection of specific nucleic acid sequence does not distinguish between colonization and disease or between viable and non-viable organisms [12]. P. jirovecii RNA is less stable and rapidly degraded after cell death so is a more reliable marker of viable organisms. Modification of standard PCR protocols with quantitative methods (e.g. quantitative touch-down PCR) may help to differentiate between colonization and infection through the selection of thresholds to maximize sensitivity [13]. An added advantage of these molecular techniques is they may provide information on molecular epidemiology and resistance-associated mutations in the gene encoding dihydropteroate synthase (DHPS), the target of sulfamethoxazole, though the benefit of this is controversial [14]. One caveat to this is the potential for point mutations in DNA paired with primer sequences and risk of false negatives as a result.

These molecular tests are said to have increased sensitivity compared with cyst staining techniques but variable specificity depending on the specimens used, the primer chosen and whether treatment has been started [15]. The three most commonly assessed specimen groups are sputa (ideally induced), oropharyngeal washes (OPW) and blood. The clinical relevance of the known detectability of P. jirovecii DNA in whole blood has not been fully established but could represent colonization as well as disease [16]. The use of cycle threshold values has been proposed as a method to distinguish colonization from disease using BAL samples, although further studies are needed to validate cut offs on different samples [17].

Although there has been a widespread adoption of NAATs the current British HIV Association guidance, and that of similar professional bodies, still suggests combining them where available with a traditional visualization technique as described previously and performing them on alveolar specimens where possible to increase sensitivity and specificity [10, 18].

PCP diagnosis by detection of DNA in non-respiratory samples
Due to the variability in sampling methods, the challenges in obtaining ideal samples and the need for prompt diagnosis research has been conducted on the use of NAATs on OPW and blood. Samples are relatively non-invasive, collection is straight-forward and no special equipment or preparation is required. A study in our unit compared NAATs on OPW and blood with sputum, spontaneously expectorated or induced, using primers for the P. jirovecii MTLSU rRNA gene [19].

All patients were consenting adults presenting to a regional infectious disease unit who were being investigated for PCP as part of routine care. A spectrum of patients was included of different pre-test probabilities to allow estimates of sensitivity and specificity. Each participant was asked to provide sputum (spontaneous or induced), OPW and blood for analysis. OPW was obtained by gargling of normal saline for 10 to 30 seconds without any additional preparation.

Forty-five participants were included, 41 male (91%), 38 Caucasian (84%) with a median age of 39 years. One participant was an HIV-negative renal transplant recipient. Forty-four were HIV-positive with a median CD4 count of 64 cells/mL. Thirty-five of the 44 were not on ART with a median HIV RNA of 164 550 copies/mL. Thirty-nine of the 45 started empirical treatment for PCP a median of 2 days before sampling. We compared the sensitivity and specificity of tests on blood and OPW compared with sputum. Sputum PCR was positive in 60% of participants and in this group 47% of OPW and 50% of blood PCRs were positive. None with negative sputum PCR had positive OPW or blood PCR. A diagnosis of PCP could be reached in 14 of 16 patients with positive NAAT on sputum using these non-respiratory specimens.

Among those with P. jirovecii DNA detected in sputum a sensitivity of 47% for OPW was increased to 80% when considering only OPW samples taken within 48 hours of starting treatment. When this was combined with blood sample testing in the same time frame the sensitivity increased to 88%, which is comparable to that quoted in previous similar studies [12, 13, 15]. There were no false positives based on no OPW or blood PCR positives in those with negative PCR on sputum. As the laboratory techniques used were routine, few additional skills or resources were required.

Overall, using molecular tests on non-respiratory samples was of diagnostic benefit and show potential for savings in time and resources. The molecular tests provide excellent specificity and good sensitivity comparable with sputum without proceeding to time-consuming and invasive tests [20]. However, in view of uncertainty regarding the specificity of testing these non-invasive samples at all, results must be interpreted with care and in the right clinical context.

PCP diagnosis remains a combination of clinical suspicion and physical examination, supported by radiological and microbiological investigations. Using a combination of traditional microscopy with staining and NAAT on appropriate specimens, plus interpretation of results in the clinical context a clear diagnosis can be reached in most cases and this may prevent unnecessary treatment. Using non-respiratory specimens taken early to maximize sensitivity could reduce the requirement for invasive testing or diagnostic uncertainty.

Future developments
We expect the use of NAATs to become even more widely available and useful diagnostic aids alongside traditional techniques. With a plethora of protocols the sensitivity, specificity and utility of these will improve further over time. Combination with other laboratory techniques such as β-D-glucan may be similarly useful. Given the inability to culture the organism and so look for in vitro susceptibility to sulfamethoxazole-based treatment, molecular methods for detecting mutations and potential resistance may develop as a routinely used test.

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The authors

Farnaz Dave MBChB, MRCP; Ashley Horsley MBChB, MRCP; Thomas Whitfield MBChB, MSc, MRCP; Clare van Halsema* MBChB, MRCP, MD, DipHIVMed
North West Infectious Diseases Unit, North Manchester
General Hospital, Manchester M8 5RB, UK

*Corresponding author
E-mail: clare.vanhalsema@pat.nhs.uk