October 2020  |  Recent medical releases

SYNLAB recommendation of samples types to be used in RT-PCR testing of SARS-CoV-2

General context

Novel SARS-CoV-2 has spread worldwide and the scientific community is raging besides the pandemic emergence and the SARS-CoV-2 high pathogenicity 4. This pandemic has become one of the biggest worldwide health threats 5. Consequently, novel, costeffective and fast diagnostic techniques are required to improve the patient clinical management and treatment in order to limit the virus transmission and to improve public policies prioritizing the population safety. Appropriate specimen collection at the correct moment is of extreme importance for the accurate diagnosis of SARS-CoV-2. Currently, sampling is mostly based on upper respiratory tract samples 12. However, there are several other possible matrices for the detection such as lower respiratory tract specimens, serum, blood, urine, stool and saliva 8. Nasopharyngeal (NP) and oropharyngeal (OP) swabs are most commonly used, since they are recommended by the WHO 5. Comparing positive rate peaks of RT-PCR, some groups showed positive rates of 93% for bronchoalveolar lavages, 72% for sputum, 63% for nasal swabs and 32% for pharyngeal swabs 4,8,13. Moreover, the positive rate for NP and OP is only about 60-70% 8,14,15. Sputum is a sensitive matrix but sampling can be complex, because most of the infected patients do not generate sputum and also due to its high viscosity which can cause false negative PCR results. 8,16.

SYNLAB Preferred samples:

The current standard-of-care we recommend is the use of samples:

  • NP and/or OP swabs
  • Sputum

Proper sampling performed by well-trained staff is important to obtain a high-quality sample. Self-sampling is also accepted for NP/OP swabs if performed under the guidance of a healthcare professional.

Other validated sample types

“Mouthwash“ sample – recommendations for mouthwash sample use

Mouthwash (gargle) is a simple alternative and non-invasive method, which reduces patient discomfort and pain, healthcare workers’ exposure to aerosols, and also reduces requirements for PPE. Based on the SARS-CoV-2 cycle of infection, different groups have demonstrated that the oral cavity is a host for the virus 20-23. Mouthwash with gargle has the advantage of sampling the entire oral cavity (oral, buccal and gingival tissue) including posterior oropharyngeal, nasopharynx, tongue, saliva, deep-throat saliva, larynx and oral mucosa. During the gargling procedure with sterile saline solution, these zones are rich sources of epithelial cells liberated by the force of the cricopharyngeal muscle during oscillation over the posterior pharyngeal wall 5. Viral RNA has been detected from deep throat saliva even after treatment, with a high diagnosis rate for SARS-CoV-2 13. It is known that the SARS-CoV-2 virus attaches to human cell surfaces via angiotensin-converting enzyme 2 (ACE2). ACE2 is widely expressed including on epithelial cells lining minor salivary gland ducts, sinonasal cavity, pharynx, larynx, trachea and lungs. Interestingly, ACE2 expression levels are higher in salivary glands than in the lung 21. This highlights the utility of saliva and gargle samples as they include a complete sampling of the oral cavity. We note the importance of buccal epithelial cell recovery with our gargling method, since their expression of furin has been shown to be essential for zoonotic coronavirus infection 21,24-26. 


General recommendations for mouthwash sample use
After an extensive internal validation process, SYNLAB recommends the use of mouthwash samples with these limitations and constraints:

1. Technical recommendations

  • Use of automatic extraction system based in magnetic bead extraction, e.g. Magmax (Thermofisher) or equivalent using the maximum initial sample volume declare by the provider. 
  • Direct PCR or related techniques with no RNA extraction are NOT recommended.
  • Use RT-PCR reagents targeting at least 2 genes of SARS CoV-2 genome
    • (this may have country-specific variations; in some countries the test must include at least one specific gene for SARS CoV-2.  Tests targeting both E and N genes can be used if permitted in the country, because of their high sensitivity)
  • Use RT-PCR reagents with a level of detection (LoD) of ≤ 4 copies/ul (100 copies per reaction) or better.
  • Use RT-PCR reagents with internal control, preferably including an endogenous human mRNA (e.g. human RNase P) to assure the presence of sample and reveal any sampling errors due to self-sampling
  • Use of mucolytics for viscous samples.

2. Medical recommendations

  • The preferred sample types to test in symptomatic patients according to international recommendations and SYNLAB internal data are (in order)
    • NP and/or OP swabs or Sputum
    • Mouthwash
  • NP and/or OP remains the recommended method for screening programs to detect asymptomatic CoV-2 carriers.

Sputum or mouthwash can be used for such programs if NP/OP are not practicable, with the understanding that some low-viral titre (high-CT) individuals may be missed.

Sample Collection Procedure (country/language specific)
 

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