New real-time PCR detects prolonged norovirus excretion in highly immunosuppressed patients and children

Abstract

Noroviruses (NoV) are a major cause of epidemic nonbacterial gastroenteritis and affect all age groups worldwide. Three of five NoV genogroups, namely, genogroup I (GI), GII, and GIV, are associated with human disease. Unfortunately, these genogroups demonstrate a high degree of sequence diversity, complicating the design of pan-NoV diagnostic PCR tests. To decrease the risk of false-negative test results, we have developed a new one-step real-time TaqMan reverse transcription-PCR protocol. This protocol detects all human NoV genogroups in one reaction with a sensitivity of 400 virus genome equivalents/reaction for both GI and GII. The use of in vitro-transcribed NoV RNA as an external standard allows (semi)quantification of viral loads in samples. In a retrospective analysis of 206 stool samples from 77 patient episodes, the duration of NoV excretion and the amount of virus excreted were determined. Twenty (26.0%) of these episodes lasted longer than 10 days. Univariate risk factor analysis revealed the patient status after organ transplantation (odds ratio [OR], 7.49 [95% confidence interval, 2.06 to 28.32]; P < 0.001), immunosuppression (OR, 9.19 [95% confidence interval, 2.50 to 35.39]; P < 0.001), and age of less than 10 years (OR, 4.58 [95% confidence interval, 1.36 to 15.77]; P = 0.004) as risk factors for a NoV excretion period of more than 10 days. These findings were confirmed by time-dependent Kaplan-Meier analyses, whereas multivariate Cox regression analyses identified immunosuppression as the sole risk factor. Surprisingly, in contrast to the excretion periods, the viral loads in stools did not increase in connection with age or immunosuppressive status. This fact may be one important piece in the pattern of high-level NoV transmissibility and may have an impact on the development of transmission prevention strategies.

FIG. 1.

Excerpt from the Clustal alignment depicting all primers and probes and the most variable sequences. Associated genogroups and accession numbers are indicated to the left. Dots indicate identity to the direct primer/probe above. Positions refer to the reference Norwalk virus sequence (accession number M87661).

FIG. 2.

Phylogenetic dendrogram of NoV GII. The phylogenetic tree is based on a 280-bp fragment within NoV orf1 and was calculated with the neighbor-joining algorithm. Twenty-three Hannoverian sequences from the years 2005 to 2008 were compared to 20 well-classified sequence database entries. The GI.1 Norwalk virus sequence was used as the out-group. Hannoverian sequences are indicated by gray boxes. Bootstrap values indicate percentages of 1,000-fold replicates.

FIG. 3.

Distribution of 77 NoV excretion episodes among patients with respect to age (in years) and immunosuppressive status. Episodes in patients with intense immunosuppression are indicated in black, and those in patients with no or mild immunosuppression are indicated in white.

FIG. 4.

Kaplan-Meier survival plots for age (A) and intense immunosuppression (B). The y axes indicate the probability of NoV excretion x days after the first positive sample was taken. Crosses indicate censored cases. Cum., cumulative. (A) Black continuous line, age greater than 10 years; gray dashed line, age less than 10 years; log rank, P < 0.001. (B) Black continuous line, no/mild immunosuppression; gray dashed line, intense immunosuppression; log rank, P = 0.003.

FIG. 5.

Plots of viral loads against the number of days after the first positive result, stratified by the ages (in years) (A) or immunosuppressive statuses (B) of patients. Due to log scaling, negative samples were excluded and values for samples that were positive but had viral loads below the quantification limit were calculated as half the quantification limit, i.e., 45,500 copies/ml of stool suspension. IS, immunosuppression.