Comparing cytomegalovirus diagnostics by cell culture and quantitative nucleic acid testing in broncho‐alveolar lavage fluids

Abstract

Many clinical laboratories have replaced virus isolation in cell‐culture (VIC) for cytomegalovirus (CMV) by quantitative‐nucleic‐acid testing (QNAT), rendering clinically relevant CMV‐replication difficult to distinguish from CMV‐shedding or latent infection. We compared direct VIC in 1109 consecutive bronchoalveolar lavage fluids (BALFs) and a well‐validated CMV‐QNAT (Basel‐CMV‐UL111a‐77bp). In the retrospective Group 1 (N = 694) and Group 2 (N = 303), CMV‐QNAT was performed within 48 h from 2‐fold and 10‐fold concentrated total nucleic acid (TNA) eluates, respectively. In Group 3 (N = 112), 2‐fold and 10‐fold concentrated TNA eluates were prospectively analyzed in parallel to VIC. CMV was detected by VIC in 79 of 694 (11%) and 26 of 303 (9%) of Groups 1 and 2, but in 114 of 694 (16%) and 57 of 303 (17%) by CMV‐QNAT, respectively. Median CMV loads were significantly higher in VIC‐positive than in VIC‐negative BALF. The likelihood for CMV detection by VIC was 85% for BALF CMV‐ loads >4 log10 copies/ml. In the prospective Group 3, CMV was detected by VIC in 10 of 112 (9%), and in 14 of 112 (13%) and 20 of 112 (18%) by CMV‐QNAT, when using 2‐fold and 10‐fold concentrated TNA eluates, respectively. Notably, CMV was undetectable by CMV‐QNAT in 10 VIC‐positive cases of Groups 1 and 2, but in none of Group 3. We conclude that CMV‐QNAT can be adopted to BALF diagnostics but requires several careful steps in validation. CMV‐QNAT loads >10 000 copies/ml in BALF may indicate significant CMV replication as defined by VIC, if short shipment and processing procedures can be guaranteed. Discordance of detecting CMV in time‐matched plasma samples emphasises the role of local pulmonary CMV replication, for which histopathology remains the gold standard of proven CMV pneumonia.

Detection of cytomegalovirus in bronchoalveolar lavage fluid from immunocompromised patients with pneumonitis by viral culture and DNA quantification

“…Three studies published in recent years also compare the results of viral culture and qPCR for HCMV detection in BAL uid. Based on an analysis of 1,109 BAL samples from immunosuppressed patients, Leuzinger et al (2020) determined that >10,000 copies/mL was an indicator of active replication. A limitation compared with the present study is that only shell vial culture was used, and not traditional culture; also, some of the samples (88%) were frozen and processed for qPCR analysis retrospectively, and the qPCR assay used was not fully automated (it required prior DNA extraction), which can reduce the reproducibility of the results.…”

“…However, it should be noted that the absence of HCMV DNA in plasma does not exclude pneumonitis. Recently, two studies found that approximately one third of patients with HCMV pneumonitis had an undetectable plasma VL, demonstrating that local replication of the virus can occur in the lower respiratory tract without systemic involvement [11,14,19].…”

“…Despite being the reference techniques for the diagnosis of viral infections, in most laboratories culture-based methods have been replaced, due to their laboriousness and longer turnaround time, by molecular techniques such as PCR. However, although PCR analysis is more sensitive and rapid than viral culture, it is limited in that it detects viral genetic material without being able to distinguish between clinically relevant HCMV replication (disease) and viral shedding or latent HCMV infection [11]. Due to this lack of speci city, guidelines recommend quanti cation of the viral load (VL) of HCMV in BAL uid as an aid to interpret the positive PCRs [5].…”

“…Again, no unanimous conclusions have been reached. While some reports indicate that it has a high predictive value for lung disease [15][16][17], others found that a considerable percentage of pneumonitis patients have undetectable HCMV in the blood as a consequence of local replication in the lung, concluding that a negative-DNAemia does not exclude pneumonitis [11,18,19]. This lack of consensus in interpreting VL values in both BAL uid and blood renders the diagnosis of pneumonitis a challenge.…”

“…Since the introduction of molecular techniques in the 1990s, several studies have compared the results of HCMV DNA quanti cation in BAL uid with those obtained by culture-based techniques, and have attempted to establish an HCMV load in BAL uid indicative of clinically signi cant virus replication (pneumonitis). Most have concluded that qPCR in BAL uid is more sensitive and has a higher negative predictive value compared to culture but has less speci city and positive predictive value[8,11,[21][22][23][24][25].…”