Respiratory tract samples, viral load, and genome fraction yield in patients with Middle East respiratory syndrome

Abstract

Background: Analysis of clinical samples from patients with new viral infections is critical to confirm the diagnosis, to specify the viral load, and to sequence data necessary for characterizing the viral kinetics, transmission, and evolution. We analyzed samples from 112 patients infected with the recently discovered Middle East respiratory syndrome coronavirus (MERS-CoV).

Methods: Respiratory tract samples from cases of MERS-CoV infection confirmed by polymerase chain reaction (PCR) were investigated to determine the MERS-CoV load and fraction of the MERS-CoV genome. These values were analyzed to determine associations with clinical sample type.

Results: Samples from 112 individuals in which MERS-CoV was detected by PCR were analyzed, of which 13 were sputum samples, 64 were nasopharyngeal swab specimens, 30 were tracheal aspirates, and 3 were bronchoalveolar lavage specimens; 2 samples were of unknown origin. Tracheal aspirates yielded significantly higher MERS-CoV loads, compared with nasopharyngeal swab specimens (P = .005) and sputum specimens (P = .0001). Tracheal aspirates had viral loads similar to those in bronchoalveolar lavage samples (P = .3079). Bronchoalveolar lavage samples and tracheal aspirates had significantly higher genome fraction than nasopharyngeal swab specimens (P = .0095 and P = .0002, respectively) and sputum samples (P = .0009 and P = .0001, respectively). The genome yield from tracheal aspirates and bronchoalveolar lavage samples were similar (P = .1174).

Conclusions: Lower respiratory tract samples yield significantly higher MERS-CoV loads and genome fractions than upper respiratory tract samples.

Keywords: Ct value; MERS-CoV; Middle East; RT-PCR; clinical; coronavirus; diagnosis; genome fraction; molecular; screening; viral load.

Figure 1.

Clinical sample type and Middle East respiratory syndrome coronavirus (MERS-CoV) threshold cycle (Ct) values. The Ct of real-time reverse-transcription polymerase chain reaction, a measurement of MES-CoV viral load (upper panel) or fraction of MERS-CoV genome obtained by deep sequencing (lower panel) were plotted by clinical sample type (tracheal aspirates [TAs], nasopharyngeal swab specimens [NPs], sputum specimens, and bronchoalveolar lavage specimens [BALs]). Box and whisker plots were prepared using the Python/Matplotlib box plot module (http://matplotlib.org/examples/pylab_examples/boxplot_demo.html). Data are for 110 specimens collected through 14 November 2014. Gray boxes indicate the lower to upper quartile values of each subset, blue lines indicate median values, and whiskers indicate ranges, with outlier points falling above or below 1.5 times the interquartile range indicated individually.

Figure 2.

Fraction of the sequenced Middle East respiratory syndrome coronavirus (MERS-CoV) genome obtained as a function of MERS-CoV load. Samples were stratified by clinical sample type and the fraction of MERS-CoV genome obtained by deep sequencing was plotted as a function of the MERS-CoV load (presented in terms of the threshold cycle [Ct] value). Data are for 110 specimens collected through 14 November 2014. Tracheal aspirates (TAs) are indicated by red circles, nasopharyngeal swab samples (NPs) are indicated with black Xs, sputum samples are indicated with gray circles, bronchoalveolar lavage samples (BAL) are indicated with green circles, and samples with an unknown type (n = 2) are indicated with black crosses.