Study and interest of cellular load in respiratory samples for the optimization of molecular virological diagnosis in clinical practice

Acute respiratory infections (ARI) are a major cause of morbidity and mortality in children under five, the elderly, and vulnerable patients [1]. Upper respiratory tract infection incidence is estimated at 7–8 per year in children under 4, and at 2–4 in adults [2].

Most respiratory viruses belong to five different viral families (Para- and Ortho-myxoviridae, Picornaviridae, Coronaviridae, Adenoviridae), and include 14 viral species, defining what is called the “respiratory panel” [3]. Some of them have a high potential of emergence and can cause pandemics. Although some viruses have been associated with particular diseases (respiratory syncytial virus and bronchiolitis, parainfluenza virus 3 and laryngitis, rhinovirus and common cold, influenza virus and flu syndrome), there is no evidence for a clinical specificity, and only the virological diagnosis provides an accurate identification of the ARI [4, 5].

Detection of respiratory viruses is of little interest in general practice, in that the infection does not present a risk of severity for the patient. However, virological confirmation of ARI is needed in severe clinical presentations, requiring hospitalization in intensive care units and occurring in vulnerable subjects [4, 6]. The goal of early virological diagnosis would be an optimization of patient care, which could lead to reduction in length of hospital stay, a saving of antibiotics, and complementary examinations [7].

Virological tests allow for the establishment of accurate diagnosis of infection, assessment of evolving risks (bacterial infection, acute respiratory distress syndrome), and the establishment of measures to limit its spread (isolation, wearing gloves and masks).

Pandemics of Severe Acute Respiratory Syndrome (SARS, 2002-2003) and influenza A-H1N1 (2009) lead to the development of molecular biological techniques applied to virological diagnosis, mainly based on PCR (polymerase chain reaction). Performances of molecular methods in respiratory virology are so significant that they have replaced conventional techniques (culture, detection of viral antigens) as a reference method [8‐11].

Multiplex PCR techniques are particularly suited to medical diagnosis because they can detect multiple viral targets in the same time, avoiding the virologist a selection of viral targets to search. There are now many commercial kits for the detection of a range of 12 to 15 respiratory viruses and some intracellular bacteria [6, 7, 12, 13]. Molecular techniques (real-time PCR) also make it possible to achieve a semi quantification of the viral molecular material present in the sample, giving additional information about the respiratory viral load (interest in therapeutic monitoring and infection transmission risk) [10]. A normalized viral load can be obtained by adding a cell quantification step.

The primary site for replication of respiratory viruses is the ciliated airway epithelium. The sample must be taken as soon as possible after the onset of symptoms. This is usually a nasal swab or nasopharyngeal aspiration (especially realized in children under 2) [14]. These samples are easily accessible and especially adapted to upper ARI. If a rich cell collection appears to be an important prerequisite for the quality of respiratory viral diagnosis, there is currently no information on a possible cellularity threshold that would validate the result of the viral molecular detection.

The main objective of this work is the study of cellularity in 800 respiratory specimens previously characterized virologically. The results should help to define the concept of “cellular richness” and determine the factors that influence it.

Molecular detection, including multiplex techniques, is currently the gold standard for viral respiratory diagnosis. We have very powerful molecular tools, ensuring a quality respiratory viral diagnosis, available for all clinicians supporting hospitalized patients. One factor limiting this diagnosis is represented by the collected respiratory specimens. The main objectives of this work have been to study the cellularity of these clinical respiratory specimens, to propose a possible definition of what is commonly called “cellular richness,” and to measure the impact of this marker on the molecular viral diagnosis. Very few published studies have been completed in this area. However, a number of facts are commonly accepted within the medical community: respiratory specimen should be “rich” to allow for “good” viral diagnosis, the “good” samples are obtained almost exclusively in infants and children. In total we identified 7 studies published in international journals between 2003 and 2014, whose objective was to compare the various upper respiratory samples, in terms of sampling equipment (flocked swabs versus rayon swab), in terms of sampling site (nasal, oropharyngeal, nasopharyngeal, combining sites), and sampling modality (swab, wash, aspiration) [17‐23].

The number of target cells and/or the number of extracellular viral particles collected could define the respiratory sample quality during sampling. It is not unreasonable to consider that the majority of the viral sequences detected by molecular techniques are mainly located in the intracellular compartment. However, several factors could modulate this distribution: the viral species, the cytopathic effect induced in vivo, the inflammatory responses, and the sampling delay from the onset of the symptoms. This study is retrospective; it was therefore not possible to collect data from the time between sampling and clinical symptoms in a standardized way. This matter is nonetheless very interesting and remains to be explored. For this study, we considered cell quantification, or cellular richness, as the main marker of diagnostic efficacy for a sample.

The 7 studies referenced above used indirect measurement of sample richness through the virus detected in it: 5 used molecular detection methods, and one was an antigen detection using rapid diagnostic test. The study published by Peter Daley et al. in 2006 is a direct immunofluorescence detection method. This method allows, when reading with a fluorescence microscope, to evaluate the number of infected and uninfected cells deposited on the slide [17]. Overall, the results are consistent and show a superiority of nasopharyngeal swab versus oropharyngeal and a superiority of flocked swab versus classical ones [14, 17, 19, 20]. The superiority of the “wash” versus the “swab” is not found by all authors, and comes well counteract the widespread idea that washing is always greater than swabbing [14].

Alsaleh et al. (2014) performed a molecular cell quantification using real time PCR in order to validate viral detection on nasal swabs. The cellularity of these samples was assessed using the quantification of a human endogenous retrovirus (ERV3) known to be present in two copies per diploid cell [24]. The authors report their results by comparing the Ct obtained upon ERV3 detection and not in terms of cellular load [18].

In our study, we made the choice to use a direct detection method for assessing the number of cells in respiratory samples. To the extent that we needed a large number of samples characterized by many markers for statistical analysis (age of the sampled patient, detected viral target, etc.), a prospective study on freshly sampled respiratory specimens could not be performed. We therefore used previously characterized and stored frozen (-80 °C) respiratory samples. The definition of cellular richness is not affected by freezing or thawing samples since it theoretically does not change the amount of HPRT1 nucleic acid, i.e. 2 copy per haploid cell, whether or not lysed. Similarly, no distinction between living and dead cells was made before the freezing process because such a distinction does not affect the quality of molecular detection by PCR (detection of viral genome into infected cells, living or not).

The analysis of 800 samples, divided into 400 positive and 400 negative in viral detection, failed to establish a minimum cellularity threshold to invalidate the negative results in viral molecular detection. The ROC curve shows that the cellularity is not a quantifiable predictor of the outcome of the virus detection.

However, our work has yielded interesting results concerning the factors influencing the cellularity of samples and the impact of cellularity on the result of molecular detection of respiratory viruses. We have clearly demonstrated that nasal aspirates allowed us to collect more cells than with nasal swabs and this only in the age group under 15 years old. This result must be tempered by the fact that 90 % (173/196) of the nasal aspirations of the study were from children under 9 years of age, since the distribution was random at inclusion of samples in the study. This reflects a common practice of sampling methods in clinical departments: nasal aspirates are rarely performed in adults and the elderly over 65 years old. This gesture is considered invasive and unpleasant. Our results support the idea that, de facto, it would not be appropriate to perform this type of sampling in this group.

For all types of samples (swabs and/or nasal aspirates), the age of the sampled patient remains an important marker influencing cellularity, with a maximum average cellularity under the age of 15, a minimum average cellularity in Adults group, and an intermediate average cellularity in the Elderly group. The reasons for this lack of cellularity in respiratory samples from Adults remain obscure.

Regarding the influence of cellularity on the result of viral detection, it should be noted that negative samples present, all age groups combined, an average cellularity lower than that obtained for the positive samples, even though it has not been possible to establish a predictive relationship. This difference was tenuous for the Children group. This observation leads to two possible explanations: either in the Positive group the infection increases the sample cellularity by promoting epithelial desquamation and, to a lesser extent, the mucus capture, containing free viral particles; or, in the Negative group, there are false-negatives in virus detection, induced by a lack of cells in the sample. This result was obtained from all samples (aspirates and swabs). Insofar as aspirates are evenly distributed in both Positive and Negative groups, we think that the result of comparison is not biased given that aspirates are richer samples. Considering viral detection in its entirety, without analysis of the viral species detected, it is interesting to note that the two factors, “cellularity” and “viral co-detection” (detection of 2 or 3 viruses) are associated positively. This suggests that the detection of several viruses is facilitated in the context of a rich sample. In cases of viral co-detection, the question of whether these are the same cells that are infected or not is not resolved, even if it is conventionally accepted that an already infected cell is less permissive to a second viral infection. It should also be noted that viral co-detection can be either a co-infection, or two or more sequential infections.

Such phenomena have already been discussed in the works of Alsaleh et al. which showed that the positive samples in viral detection had, on average, a greater amount of genetic material of human origin than negative ones. Similarly, samples where the gene ERV3 was not detected had lower viral detection. Finally, they also found that the positive samples for several viruses were also those in which cellular loads were highest [18].

The analysis of the potential impact of cellularity on the specific detection of various viruses included in the “respiratory panel” showed results that should be confirmed with larger numbers in each group. Indeed, on the one hand, the highest average cellularity was obtained in the hMPV positive samples, equally detected among Adults and Children groups; on the other hand, the lowest average cellularity is obtained in the RSV positive samples, mostly detected in Infants and Children groups. These results are surprising in that the two viruses, RSV and hMPV, belong to the same virus family (Paramyxoviridae) as well as to the same virus subfamily (Pneumovirinae), and are genetically close. They have many similarities in the circulatory mode and in the pathophysiology of the infection they cause. Yet the cellularity of hMPV positive samples is significantly greater than that of the RSV positive specimens.

The quality of samples dramatically affects the quality of results provided to clinicians. It is important that a better understanding of the sample characteristics goes along with technological developments. This work is uncommon. He tries to give answers to a trivial scientific question: Respiratory specimens should they be « rich in cells » to ensure optimal virological molecular diagnosis? The cellular load is multifactorial and occurs for many in the sensitivity of molecular detection. However, it was not possible to determine a minimum threshold allowing molecular viral detection.

AdV, adenovirus; ARI, acute respiratory infection; CT, cycle threshold; DNA, deoxyribonucleic acid; EC, extraction control; ERV3, human endogenous retrovirus 3; Flu, influenzae virus; HBoV, human bocavirus; HCoV, human coronavirus; hMPV, human metapneumovirus; PCR, polymerase chain reaction; PIV, paraInfluenza virus; RhV/EV, rhino/entero virus; RNA, ribonucleic acid; ROC, receiver operating characteristic; RSV, human respiratory syncytial virus; SARS, severe acute respiratory syndrome; UTM, universal transport medium