Background
The emergence of several virus infections during the last decades like dengue, West Nile (WN), Severe acute respiratory syndrome (SARS), chikungunya (CHIK), Middle East respiratory syndrome coronavirus (MERS-CoV), Ebola (EBO), Zika (ZIK) and Yellow Fever (YF) raised some questions on quickness and reliability of laboratory diagnostic tests for verification of suspected cases. All important questions arising with an emerging infection like source and route of transmission, infectivity of patients, recommendation of safety measures for health care personal and distribution in an animal reservoir or other vectors require the performance of laboratory diagnosis. To investigate the patient’s infections most often a blood sample is taken not only for the analysis of blood parameters but also for pathogen detection mostly by PCR or for testing the presence of specific antibodies. However, sampling of blood requires medically trained personal and comprises some risks for the patient as well as for the health care personal. In particular the blood sampling of patients with highly infectious haemorrhagic fever like Ebola involve a great risk for the physician and the patient. Therefore, the sampling by non-invasive methods (e.g. saliva and/ or urine) might be a very valuable alternative for investigating a diseased patient. The use of saliva and urine for the diagnosis for many viral infections has been reported, however, it is often not performed in the broad routine diagnostics.
After a viral infection it usually takes some days until the propagation of the virus causes symptoms and a clinical investigation of a patient is indicated. Blood sampling for analysing blood parameters belongs to a routine procedure in developed countries. This is different in developing countries, where extensive analyses of clinical parameters are not performed routinely due to financial and technical constraints. Even though the sampling of swaps from the respiratory tracks is a common procedure for influenza, SARS, MERS-CoV and other respiratory infections, the sampling of blood is the prevalent procedure for all other recently emerged viruses. However, with the introduction of the highly sensitive Nucleic Acid Tests (NAT) for diagnostic also additional samples like saliva and/or urine were included in diagnostic sample collections. The advantage of using non-invasive samples like saliva for dengue serology was successfully proven 18 years ago by Cuzzubbo et al. who discovered that dengue primary and secondary infections can be distinguished by the level of IgG in saliva [
1]. The specificity of the diagnosis of acute flavivirus infections like dengue, WN and YF has been improved by applying the NAT because it is not impeded by the widespread cross-reactivity found for most serological assays [
2]. Since urine, saliva and other non-invasive samples were not part of the routine investigation, the sampling procedures and the samples containment are also very different. These differences complicate the interpretation of diagnostic findings and therefore an optimization for these samplings is required. An improvement of the sampling and stabilization of viral RNA in the diagnostic material will help to increase significantly the diagnostic sensitivity.
To analyse the usefulness of alternative non-invasive samples for diagnostic of infectious viral diseases like SARS, WN, dengue, MERS-CoV, Ebola, Zika and YF, we reviewed the importance of saliva and urine for the diagnosis of recently emerging viruses in comparison to conventional blood sampling. Since the extensive evaluation of different samples for human patients is often not performed, also investigations of animal models or vectors are considered. Therefore, we performed an electronic literature review on PubMed for alternative sampling for SARS, WN, dengue, MERS-CoV, Ebola, Zika and YF.
Conclusions
The analysis of using other non-invasive samples for virus diagnostics clearly shows that the procedures for the optimal sampling strategy of the different viral infections are not seriously investigated table S9. In the following overview it became obvious that for the newly emerging viruses like CHIK, MersCoV and Zika more and broader attempts for sampling strategies were performed and investigated than for well known viruses like YF and dengue.
The application of saliva as a sample of choice for the diagnosis of CHIK fever infections in small children or patients whose blood samples are difficult to obtain is of great significant. However, all recommendations/fact sheets for CHIK from national and international organizations (CDC, PAHO, WHO, ECDC) do not mention saliva or urine samples as suitable material for diagnosis because of the missing of meaningful studies that evaluate saliva and urine as diagnostic samples compared with blood. Hopefully, there will be studies performed answering these questions with a representative number of CHIK patients in the future.
The use of saliva and urine for the diagnosis of acute dengue infections is of great importance and requires more attention. Present studies show the usefulness for these samples for diagnostics and also the monitoring of the severity of infection. In particular for surveillance studies and/or investigation of small children the noninvasive approach using saliva or urine for the laboratory diagnosis of dengue cases should be considered when blood samples are difficult to obtain. Although the application of alternative samples for the diagnostic became more popular in the recent years, further studies and improvements are necessary to establish these samples equally to common plasma or serum samples. Commercial assays as well as appropriate sampling equipment have to be verified for diagnostic applicability in representative cohorts and during the course of the disease. It seems that the increased awareness of alternative samples stimulates such studies for dengue diagnosis as demonstrated in the recent publications mentioned above.
In particular for diagnostic of VHF cases, the investigation of the usefulness of non-invasive taken samples should have high priority since blood sampling from such patients is highly risky and often dangerous for patients with hemorrhagic symptoms while continuous bleeding occurs. The virus load in sever cases of VHF is very high in body fluid and the NAT is the method of choice since the production of specific IgM and/or IgG antibodies takes a few days. In very severe cases, the patient even can pass away before developing a detectable antibody reaction, while a general viremia affecting several organs and is detectable in most body fluids. In less severe cases, the exact moment becomes an important issue since viremia only remains for just a few days (3-4 days) and often patient samples were taken too late for successful NAT thus requiring laborious serology testing with in-house assays and risk of false positive reactivity. Onset of disease is also not a well-defined point of time because the perception of a disease might be different in developing compared to developed countries. This makes the comparison of clinical symptoms and diagnostic findings during the course of the disease even more difficult.
Although the analysis of different body fluids during the recent Ebola virus outbreak expands our knowledge of virus transmissibility via eye fluid and semen tremendously, systematic studies on which and for how long the different body fluids will contain viral RNA and/or infectious viruses are still missing. Recommendations on how non-invasive sampling should be performed, what kind of samples are necessary and how they should be stabilized and transported under African environmental conditions are still missing and require thoroughly investigations.
For WNV infections the use of urine is a suitable alternative diagnostic sample compared to serum and should be listed in fact sheets from national and international organizations. Further studies are needed investigating if urine samples are a useful source for surveillance studies on WNV distribution and for analyzing the severity of the disease in patients with neuro-invasive and/or kidney manifestation. The emergence of Zika virus tremendously stimulated the investigation of using non-invasive samples as saliva and urine for diagnostic of Zika virus infections. From the start of the Zika outbreak in French Polynesia saliva was considered as alternative material for the diagnostics. The investigation of urine and saliva shows a higher virus load compared to blood making these samples a suitable alternative to blood. However, this information has to be forwarded to the physicians as it is already part of the PAHO and WHO recommendations.
For YF diagnostic one can hope that the integration of saliva and urine as alternative samples into the recommendation worked out by the WHO for the recent outbreak will help to implement this method into the medical procedure and create some meaningful results for future outbreak scenarios.
Even although SARS seems to have disappeared from the globe, the experience and findings on which and when the different diagnostic samples should be used for a quick and reliable diagnostic should be carefully evaluated, since respiratory infections are on the rise as we have seen a few years later by the influenza and MERS-CoV outbreaks. The great variety on which, how and when respiratory samples have to be taken is reflected by the list of nomenclature found in the publications: for upper respiratory specimens (nasal swab, exudate nose, oronasal swab, pharyngeal swab, tracheal swab, exudate mouth, saliva and sputum) and lower respiratory specimens (bronchoalveolar fluid, bronchoalveolar lavage). Recommendations for standardized procedures for sampling and handling of the respiratory samples will help to allow a better comparison of the findings and provide a better preparedness for the next respiratory disease outbreak.
Because of a higher virus load, samples from the lower respiratory tract are more suitable for diagnostic. However, the sampling of bronchoalveolar-lavage fluid is also invasive and requires an experienced physician and holds a certain risk of nosocomial infections by aerosols. Other non-invasive samples like urine, nasal, pharyngeal, tracheal and rectal swabs were included in the diagnostic analysis for analyzing the pathogenesis and the kinetic of virus release by the different body fluids. Like for SARS standardized procedures for sampling and handling of the respiratory samples will help to allow a better comparison of the findings and provide a better preparedness for future respiratory disease outbreak.
The present review clearly shows that there is still the need to perform more diligent studies analyzing non-invasive patient samples in comparison with blood and serum samples, which used for routine diagnostic of emerging viral diseases. While for some diseases like the respiratory infections, the sampling of nasal swaps and saliva is part of routine sampling, for other viral infections, blood or serum seems the predominant and only type of sample used for laboratory diagnosis.
This is in particular disappointing because non-invasive sampling offers a real alternative avoiding additional risk for the patient and the medical personal. Patients suspected to be infected by VHF usually have a very high virus load. Therefore, the analysis of saliva or urine should be routinely performed in parallel to investigation of blood samples. Based on a solid database it will be possible to determine the most appropriate sample for a viral diagnosis before it gets implemented in the routine diagnostic procedure. Diagnostic laboratories and physicians have to gain knowledge when and how alternative samples have to be considered for analyzing an acute or convalescent viral infection. Even though, we have with NAT a very sensitive diagnostic tool, the best point in time during the course of the disease together with the optimal sampling, sample storage and sample preparation are essential to get optimal results for a laboratory diagnosis. In patients presenting with a moderate or mild disease, the viremia is mostly very short lasting just 1-3 days. For these patients the positive NAT is sometimes difficult to achieve and a negative finding is of no predictive value since the virus titer might be just below the detection limit or sample storage and sample preparation is not optimal in case of non-invasive sampling such as saliva and urine. Although the virus disappeared from the blood, the virus genome will be still detectable in the urine as consequence of the clearance process as we have found in Yellow Fever vaccinees or other for Zika [
97,
104]. In contrast, we find a high viral load in patients developing a severe disease sometimes with a general infection affecting several organs. In particular in those patients, the detection of the pathogenic virus by NAT is not difficult analyzing different types of samples. Very often the viral load also reflects the severity of the disease and can be used as prognostic marker for progression of the disease in the patient. Exactly for these patients the non-invasive sampling is less stressful as frequent bleeding performed with the current procedure. This becomes even more important, if point of care diagnostic allows a more frequent monitoring of infection parameters of the diseased patient in the future. For the recently emerging CHIK and Zika in South America, the analysis of throat swaps, saliva and urine samples were already analyzed for their usability in diagnostics. However, the findings are based on case studies or small studies and require a more systematic approach. Preferably there should be performed a systematic analysis of different invasive samples (blood, bronchoalveolar lavage) and non-invasive samples (saliva, urine, etc.) for their diagnostic value using different diagnostic methods for patients with acute and convalescent disease when new infections emerge. This also implies that the procedure taking samples has to be somehow standardized. This became very obvious by the large number of terms for upper and lower respiratory samples most likely reflecting the great variety how the samples were taken. This makes the comparability of diagnostic findings presented in different studies very difficult. To address this problem a clear recommendation for the sampling procedure would help to overcome this imponderability. Furthermore, the methods for storage of the samples regarding temperature and stabilization needs to be worked out to allow an optimal transport and preparation of the diagnostic sample. For example there are special sampling tubes with stabilizer available for both saliva (DNAgard Saliva by biomatrica, USA) and urine (VACUETTE Urine CCM tubes by Greiner Bio-One, Germany and Urine Monovette by Sarstedt, Germany), which will help preserves the integrity of sample at room temperature. Sample preparation from urine and saliva could be also challenging especially for molecular diagnostics, therefore it is very important to optimize protocols using clinical samples rather than spiked mock-samples. Our personal experience showed that the optimized sample preparation method on real clinical samples was not as efficient as on spiked mock samples.
The increasing number of publications considering alternative sampling for diagnostic of the recent outbreaks MERS-CoV, CHIK, Ebola and Zika are promising. However, systematic studies are rare and for some infectious disease outbreaks like the recent yellow fever in Angola, non-invasive samples for diagnostic are still uncommon and require further investigation. Just for diseases like yellow fever, non-invasive samples like urine or saliva might be a very practical alternative, since the first index patients are detected often very late, while blood will be negative by NAT in patients with mild disease and from patients with a severe disease it is difficult taking blood because of a hemorrhage clinical picture. Non-invasive samples might be also a great alternative for investigations of children or other patients were collection of blood samples is difficult [
18,
30,
88]. Also for large surveillance studies, non-invasive samples like saliva and/or urine could be performed without medical personal for collecting blood. However, the usability of these samples for serology or NAT has to be evaluated in comparison to conventional use of sera. Therefore, it is necessary to introduce non-invasive sampling strategies in recommendations as done for the “Yellow Fever Laboratory Diagnostic Algorithm” worked out for the epidemic and non-epidemic countries in Africa during the YF outbreak in Angola by WHO expert team [
135]. Since these investigations could be only performed during an outbreak situation the preparation of a scheme for evaluation of sampling for respiratory as well as for other infectious diseases including the further processing for laboratory diagnostic is highly recommended. This more structured approach will help to improve the diagnostic of emerging infectious diseases significantly as new diagnostic methods and platforms are developed continuously and require effective evaluation under real conditions.
For confirmation of a clinical diagnosis a correct laboratory diagnosis is essential for all further steps of patient's treatment and handling. In particular for new and/or emerging diseases, the conditions for the best sampling regarding time and source (blood, serum, saliva, urine, etc.) are often not known. Since every disease has its own course of symptoms and affected organs, the optimal sampling schema might change and require different strategies regarding sample preparation and processing. While in the early phase when disease symptoms appear often the viral pathogen could be detected by nucleic acid testing in blood or other body fluids in the later phase only antibodies are detectable in serum.
In the present article, we analyzed over 700 scientific publications on diagnostic approaches for dengue, West Nile, SARS, chikungunya, MERS-CoV, Ebola, Zika and Yellow Fever regarding their relevance for using alternative samples like saliva and urine for their versatility for virus diagnostic. The use of non-invasive samples offers a suitable alternative for improving the diagnostic performance and significantly reduces the risk for medical personal and patients for routine diagnostic and during an outbreak situation. The advantage of using non-invasive samples for the diagnostic has to be seriously analyzed and investigated before implementing in the routine diagnostics.