Evaluation of analyte-specific reagents for the direct detection of Pneumocystis jirovecii

ABSTRACT

Pneumocystis jirovecii pneumonia (PJP) is a serious and sometimes fatal infection occurring in immunocompromised individuals. High-risk patients include those with low CD4 counts due to human immunodeficiency virus infection and transplant recipients. The incidence of PJP is increasing, and rapid detection of PJP is needed to effectively target treatment and improve patient outcomes. A common method used is an immunofluorescent assay (IFA), which has limitations, including labor costs, low sensitivity, and requirement for expert interpretation. This study evaluates the performance of the DiaSorin Molecular Pneumocystis jirovecii analyte-specific reagent (ASR) in a laboratory-developed test (LDT) for the direct detection of P. jirovecii DNA without prior nucleic acid extraction. Respiratory samples (n = 135) previously tested by IFA from 111 patients were included. Using a composite standard of in-house IFA and reference lab PJP PCR, the percent positive agreement for the LDT using the DiaSorin ASR was 97.8% (90/92). The negative percent agreement was 97.7% (42/43). The lower limit of detection of the assay was determined to be 1,200 copies/mL in bronchoalveolar lavage fluid. Analytical specificity was assessed using cultures of oropharyngeal flora and common respiratory bacterial and fungal pathogens. No cross-reactivity was observed. Our study suggests that the DiaSorin Pneumocystis ASR accurately detects P. jirovecii DNA and demonstrates improved sensitivity compared to the IFA method.

IMPORTANCE

Our study is unique compared to other previously published studies on the DiaSorin analyte-specific reagent (ASR) because we focused on microbiological diagnostic methods commonly used (immunofluorescent assay) as opposed to pathology findings or reference PCR. In addition, in our materials and methods, we describe the protocol for the use of the DiaSorin ASR as a singleplex assay, which will allow other users to evaluate the ASR for clinical use in their lab.

INTRODUCTION

Pneumocystis jirovecii (previously Pneumocystis carnii) is a nonculturable, unicellular organism lacking the phenotypic features of typical fungi. It contains more morphological characteristics similar to protozoa (1). Anti-fungal drugs are ineffective in treating Pneumocystis, while drugs used to treat protozoans are effective for treatment. Pneumocystis is thought to be spread by airborne transmission and can be acquired early in life (2). The clinical presentation of Pneumocystis pneumonia consists of nonspecific symptoms, including fever, cough, difficulty breathing, chest pain, chills, and fatigue. It mainly causes disease in the alveolar spaces of immunocompromised patients with advanced human immunodeficiency virus (HIV) disease, solid organ or hematopoietic transplant recipients, and other immunocompromised patients.

P. jirovecii lacks ergosterol, a typical component of fungal cell walls, and doesn’t grow in culture, making detecting it challenging. Diagnosis of Pneumocystis jirovecii pneumonia (PJP) relies on adequate sampling, especially for microscopic diagnostic techniques such as immunofluorescent assay (IFA). Lower respiratory tract specimens are the specimens of choice for diagnostics, including bronchoalveolar lavage fluid (BALF), sputum, biopsy, or tracheal aspirate (TA). BALF and bronchial washing (BW) are the preferred specimens as they reach the lung alveoli where the organism resides (3, 4). There are challenges with bronchoscopically obtained specimens in severely ill patient populations with low platelet counts or other contraindications for bronchoscopy. Sputum is an inferior sample for PJP diagnosis. Most of the supporting data for sputum testing are primarily from patients living with HIV, and sensitivity is highly dependent on the organism burden (4).

Other methods of diagnosis include serum β-D-glucan levels, which have been shown to be positive, especially with high levels of P. jirovecii organism burden (5, 6). Polymerase chain reaction (PCR) is an alternative method of PJP diagnosis. Our study evaluated the performance of the DiaSorin Molecular analyte-specific reagent (ASR) for the detection of P. jirovecii DNA using a composite reference method (IFA and reference lab PCR) to assess performance.

MATERIALS AND METHODS

Specimens and patients

Respiratory samples (n = 135) previously tested by IFA from 111 patients were included (positive, n = 84; negative n = 45; indeterminate, n = 6). Specimens were collected between 2012 and 2022 (2012, n = 1; 2013, n = 9; 2014, n = 14; 2015, n = 9; 2016, n = 3; 2017, n = 14; 2018, n = 14; 2019, n = 7; 2020, n = 13; 2021, n = 3; and 2022, n = 48) and stored at −70°C. Specific specimen types are depicted in Fig. 1A. Patients had a variety of immunocompromised statuses, including HIV, cancer, and organ transplant, as seen in Fig. 1B.

Fig 1.

(A) Different specimen types included by number of specimens, and (B) immunocompromised status of patients tested by percentage of patients. BALF: bronchoalveolar lavage fluid, BW: bronchial washing, TA: tracheal aspirate, ES: expectorated sputum, IS: induced sputum.

DiaSorin molecular real-time PCR-based assay

DiaSorin Molecular P. jirovecii ASR primers target the mtLSU gene for the detection and amplification of Pneumocystis DNA. Nucleic acid extraction was not performed. BALF and BW samples were run straight, while sputa were diluted 1:1 in universal transport media for ease of pipetting the sample. Amplification of Pneumocystis DNA was carried out on the DiaSorin Molecular 96-well Universal Disc using the LIAISON MDX instrument. All reactions used 2 µL of the non-extracted or diluted sample with 8 µL of master mix that contained 0.2 µL of Pneumocystis primer pair, 4 µL TA master mix, 0.2 µL Simplexa Extraction and Amplification Control primer pair, 0.2 µL Simplexa Extraction and Amplification Control DNA, and 3.4 µL nuclease-free water. The following cycling conditions were used: 1 cycle at 97°C for 120 s, followed by 40 cycles at 97°C for 10 s with a ramp speed of 2 °C/s, and 60°C for 30 s with a ramp speed of 2 °C/s with capture mode on. Target and internal control fluorescence thresholds were set at 20,000 and 10,000, respectively. Data collection and analysis were performed with LIAISON MDX Studio software. The cycle number (CN) value for positive samples was defined as the cycle number at which the fluorescence generated within a reaction crossed the fluorescence threshold. Samples that had a failed internal control on the initial run were repeated. If a sample failed a second time, it was tested at a 1:4 dilution.

Analytical sensitivity and specificity

The limit of detection (LoD) was determined using a quantified stock of Pneumocystis (Exact Diagnostics, Fort Worth, TX, USA), serially diluted in a negative pooled human BALF matrix. The LoD was determined by Probit analysis as the lowest detectable dilution at which the quantified Pneumocystis stock (copies/mL) resulted positive with a 95% probability of detection. Precision (i.e., coefficient of variation) was determined using replicates of a low concentration (2,400 copies/mL, or 2× LoD) and a high concentration (1,700,000 copies/mL) among multiple days and four instruments. For the specificity study, a 0.5 McFarland suspension was prepared in tryptic soy broth, and a 1:10 dilution was tested for each of the following organisms: Moraxella catarrhalis, Staphylococcus aureus, Cryptococcus neoformans, Candida glabrata, Candida albicans, Candida tropicalis, Candida krusei, Candida parapsilosis, Streptococcus pneumoniae, Haemophilus influenzae, and mixed oropharyngeal flora.

Immunofluorescent assay

The BioRad Monofluo Pneumocystis jirovecii IFA Test Kit (Hercules, CA, USA) was performed according to the manufacturer’s instructions.

Composite reference

All 135 respiratory samples were tested by IFA as the standard diagnostic method used at our institution at the time of collection. There were 13 negative or indeterminate IFA samples that were sent for reference lab PCR by physician request as part of routine patient care. In addition, six IFA/DiaSorin discordant samples were sent for reference lab PCR. When IFA and DiaSorin were discordant, the reference lab PCR was used as the comparator method.

Statistical analysis

Statistical significance was determined using a t test with one-tailed distribution and two-sample unequal variance as the parameters. P values <0.05 were deemed statistically significant.

This study was approved by the Institutional Review Board of the University of North Carolina at Chapel Hill with a waiver of informed consent.

RESULTS

Method comparison

Of the 135 total samples, there were 122 concordant samples (89.6%) between IFA and DiaSorin. Concordantly positive samples (n = 84) included 68 BALF, 13 induced sputa, 2 BW, and 1 TA. Concordantly negative samples (n = 38) included 35 BALF, 2 induced sputa, and 1 BW. Six samples (five BALF, one sputum) had indeterminate IFA results; two were positive by DiaSorin (BALF, sputum), and four BALF were negative by DiaSorin. All six initial IFA indeterminate samples were sent for reference lab PCR, and those results were consistent with DiaSorin results (both positive and negative). There were seven BALF samples with discordant results between IFA and DiaSorin. One sample that was IFA positive and DiaSorin negative had a repeat positive DiaSorin result; a pipetting error was determined to be the cause of the initial false negative DiaSorin. The remaining six IFA/DiaSorin discordant samples were sent to a reference lab for PCR testing, and the results are presented in Table 1. Four samples were IFA negative and DiaSorin positive and confirmed positive by the reference lab PCR. The final two samples were IFA negative, DiaSorin positive, and reference lab PCR negative. Using a composite standard of in-house IFA and reference lab PJP PCR, the positive percent agreement for the DiaSorin ASR was 97.8% (90/92). The negative percent agreement was 97.7% (42/43). Of the 135 specimens, six initially had an internal control failure (4.4%). Upon repeat, three failures resolved, while three required a dilution to obtain a result.

TABLE 1.

Analysis of discordant results between IFA and DiaSorin

	IFA result	Reference lab PCR result	DiaSorin result	DiaSorin CN value
1	Negative	Positive	Positive	29.5
2	Negative	Positive	Positive	32.8
3	Negative	Positive	Positive	32.9
4	Negative	Positive	Positive	33.3
5	Negative	Negative	Positive	35.2
6	Negative	Negative	Positive	37.1

Analysis of cycle numbers

The mean CN for IFA positive/DiaSorin positive samples was 24.4 [95% confidence interval (CI), 15.5–33.3], and the mean CN for IFA negative/DiaSorin positive samples was 33.4 (95% CI, 28.8–38.1). The difference in CN values between IFA positive and negative samples that were DiaSorin positive was statistically significant (P < 0.05). There was no statistical difference between the CN values by specimen type, immune status, nor date of collection (data not shown).

Analytical sensitivity and specificity

The lower limit of detection of the DiaSorin ASR was determined to be 1,200 copies/mL in BALF. Precision studies performed at 2× LoD (2,400 copies/mL) demonstrated an overall CV of 1.8%, while precision at a high concentration (1,700,000 copies/mL) was 2.3% CV. Analytical specificity was assessed using cultures of oropharyngeal flora and common respiratory bacterial and fungal pathogens. No cross-reactivity was observed.

DISCUSSION

Our study suggests that the DiaSorin Molecular Pneumocystis ASR provides increased sensitivity when compared to the IFA method. The difference in CN values between IFA negative/DiaSorin positive and IFA positive/DiaSorin positive samples supports the increased analytical sensitivity of DiaSorin. The two samples that were IFA negative, reference lab PCR negative, and DiaSorin positive could be attributed to the lower limit of detection of 1,200 copies/mL for DiaSorin versus 6,000 copies/mL for the reference lab PCR (7). This hypothesis is supported by the high CN values for these two samples (35.2 and 37.1), but we recognize these samples could also represent false positives.

There are outstanding challenges with the clinical specificity of molecular assays for diagnosing PJP. A molecular result should be used in conjunction with clinical assessment since highly sensitive PCR assays can detect colonization. Colonization, carriage, asymptomatic infection, and subclinical infection have all been used to describe the presence of the organism of DNA in the absence of pneumonia. Vera et al. described varying percentages of Pneumocystis colonization as anywhere between 1% and 74% (8). Guegan et al. similarly described a wide range of potential cutoff values for colonization versus active infection diagnosis (9). Currently, there is no consensus on these cutoff values. In our study, although there was a statistical difference in the CN values of IFA positive/DiaSorin positive and IFA negative/DiaSorin positive samples, we are unable to assess whether this represents an appropriate cutoff for determining infection versus colonization, particularly given the lack of clinical correlation of results and overlap in the 95% confidence intervals of the CN values (28.8–33.3). More studies are needed to generate the needed clinical data, as the cutoff applied may be dependent or specific to geographic areas and patient populations. Further, CN values may not be sufficiently precise or reproducible to establish a clinical cutoff, and standardized quantitative molecular tests may be required.

When comparing the workflow of the IFA and the LDT with the DiaSorin ASR, we calculated less hands-on time (15 m vs 40 m) and specimen volume (2 µL vs 3 mL) for the DiaSorin ASR. Time to result was also decreased when using the DiaSorin ASR compared to IFA (1.5 h vs 3 h). The ability to test samples without prior extraction is a benefit of the DiaSorin ASR compared to traditional LDTs, including the reference lab PCR used in this study. The approximate cost (supplies and labor) for the DiaSorin ASR was slightly higher than that of the IFA ($130 vs $100, respectively).

There are a limited number of published studies on the DiaSorin primers evaluated in this study. Kilic et al. evaluated a multiplex Pneumocystis and CMV PCR in BALF samples to investigate coinfection rates in immunosuppressed patient populations with suspected pneumonia. This study showed 100% sensitivity and 87.2% specificity in detecting P. jirovecii, which was a higher sensitivity than using IFA for PJP diagnosis (10). Damhorst et al. evaluated the clinical utility of the DiaSorin ASR for the detection of P. jirovecii with reference to pathology and found high sensitivity and specificity (>93%) (11). Recently, Belanger et al. compared the quantitative RealStar P. jirovecii assay to the DiaSorin ASR and found that while they both performed well on negative BALF samples, the RealStar assay had a slightly higher percent positive agreement with a laboratory-developed PCR test or the RealStar assay performed at a reference laboratory (12).

There are several limitations to consider in our study. We did not assess the clinical interpretation of the test result, which can be challenging in the context of colonization versus disease discussed earlier. Ideally, clinical adjudication would be performed, particularly for the IFA-negative, DiaSorin ASR-positive patients. However, this was beyond the scope of the current study especially since specimens spanned two different electronic medical record systems. We also did not include additional test results, such as β-D-glucan levels, in our analysis because few patients had these results. It is important to consider additional clinical and laboratory data in the context of using a molecular test for PJP diagnosis, as a more sensitive diagnostic tool requires more thoughtful interpretation for the patient. Finally, only a small portion (n = 19) of our total sample set (n = 135) was sent for a reference lab PCR comparison, meaning not all samples had equal data points when drawing final conclusions.