RT-qPCR Test Research Articles

A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

Literature review on virus and host response proteins in COVID-19: pathobiology, management, diagnosis and treatment.

Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome caused by a novel strain of coronavirus (SARS-CoV-2) which was declared by WHO as a cause of global pandemic. By human-to-human transmission it caused severe damage to mankind with increased mortality rate worldwide. Coronavirus is a spherical enveloped virus with single stranded positive-sense RNA with a size of ~30 kilobases encoding various structural, non-structural and accessory proteins. The entry of coronavirus into the host cells is mediated by spike proteins. SARS-CoV-2 efficiently replicates in host cell and by evading immune surveillance, like innate and adaptive immune responses, in the host cells ultimately leads to increased virulence and disease outcome. In the current review, we highlighted the molecular insights of SARS-CoV-2 and its infection mechanism in the host cell via host-viral protein interactions based on currently available data up to 16thMay 2020 using various research literature databases. The diagnostics of SARS-CoV-2 is mainly done by RT-qPCR and serological tests. There is no effective treatment for COVID-19, however, few methods like plasma therapy and remdesivir treatment are reported to show promising results in improving patient's health and decreasing mortality rate. Keywords: SARS-CoV; spike protein; nucleocapsid; COVID-19; interferon.

Influenza Vaccination and Risk of SARS-CoV-2 Infection in a Cohort of Health Workers.

Vaccines may induce positive non-specific immune responses to other pathogens. This study aims to evaluate if influenza vaccination in the 2019–2020 season had any effect on the risk of SARS-CoV-2 confirmed infection in a cohort of health workers. During the first SARS-CoV-2 epidemic wave in Spain, between March and May 2020, a cohort of 11,201 health workers was highly tested by RT-qPCR and/or rapid antibody test when the infection was suspected. Later in June, 8665 of them were tested for total antibodies in serum. A total of 890 (7.9%) health workers were laboratory-confirmed for SARS-CoV-2 infection by any type of test, while no case of influenza was detected. The adjusted odds ratio between 2019–2020 influenza vaccination and SARS-CoV-2 confirmed infection was the same (1.07; 95% CI, 0.92–1.24) in both comparisons of positive testers with all others (cohort design) and with negative testers (test-negative design). Among symptomatic patients tested by RT-qPCR, the comparison of positive cases and negative controls showed an adjusted odds ratio of 0.86 (95% CI, 0.68–1.08). These results suggest that influenza vaccination does not significantly modify the risk of SARS-CoV-2 infection. The development of specific vaccines against SARS-CoV-2 is urgent.

Pooling Samples to Increase SARS-CoV-2 Testing.

As SARS-CoV-2 continues to propagate around the world, it is becoming increasingly important to scale up testing. This is necessary both at the individual level, to inform diagnosis, treatment and contract tracing, as well as at the population level to inform policies to control spread of the infection. The gold-standard RT-qPCR test for the virus is relatively expensive and takes time, so combining multiple samples into “pools” that are tested together has emerged as a useful way to test many individuals with less than one test per person. Here, we describe the basic idea behind pooling of samples and different methods for reconstructing the result for each individual from the test of pooled samples. The methods range from simple pooling, where each pool is disjoint from the other, to more complex combinatorial pooling where each sample is split into multiple pools and each pool has a specified combination of samples. We describe efforts to validate these testing methods clinically and the potential advantages of the combinatorial pooling method named Tapestry Pooling that relies on compressed sensing techniques.

Impact of Anticoagulation and Sample Processing on the Quantification of Human Blood-Derived microRNA Signatures.

Blood-derived microRNA signatures have emerged as powerful biomarkers for predicting and diagnosing cardiovascular disease, cancer, and metabolic disorders. Platelets and platelet-derived microvesicles are a major source of microRNAs. We have previously shown that the inappropriate anticoagulation and storage of blood samples causes substantial platelet activation that is associated with the release of platelet-stored molecules into the plasma. However, it is currently unclear if circulating microRNA levels are affected by artificial platelet activation due to suboptimal plasma preparation. To address this issue, we used a standardized RT-qPCR test for 12 microRNAs (thrombomiR®, TAmiRNA GmbH, Vienna, Austria) that have been associated with cardiovascular and thrombotic diseases and were detected in platelets and/other hematopoietic cells. Blood was prevented from coagulating with citrate–theophylline–adenosine–dipyridamole (CTAD), sodium citrate, or ethylenediaminetetraacetic acid (EDTA) and stored for different time periods either at room temperature or at 4 °C prior to plasma preparation and the subsequent quantification of microRNAs. We found that five microRNAs (miR-191-5p, miR-320a, miR-21-5p, miR-23a-3p, and miR-451a) were significantly increased in the EDTA plasma. Moreover, we observed a time-dependent increase in plasma microRNAs that was most pronounced in the EDTA blood stored at room temperature for 24 h. Furthermore, significant correlations between microRNA levels and plasma concentrations of platelet-stored molecules pointed towards in vitro platelet activation. Therefore, we strongly recommend to (i) use CTAD as an anticoagulant, (ii) process blood samples as quickly as possible, and (iii) store blood samples at 4 °C whenever immediate plasma preparation is not feasible to generate reliable data on blood-derived microRNA signatures.

Abstract 4261: Development of quantitative multi-platform tests for easy readout of AR, ER, PI3K and MAPK pathway activity to unravel pathophysiology across cancer and tissue types

Abstract Introduction: Currently, the selection of targeted therapy is commonly based on genetic data, such as well-known gene mutations, but these mutations do not provide conclusive evidence for the functional activation of the affected pathway. For this reason, we have developed computational models to assess the molecular phenotype of individual tumors, determining the functional activity of oncogenic signaling pathways based on mRNA expression levels of the direct target genes of the respective pathway transcription factors (Verhaegh et al., Cancer Res 2014; Van Ooijen et al., Am J Pathol 2018; Van de Stolpe et al., Sci Rep 2019). For a broader application of these models, we translated them to RT-qPCR, RNA sequencing and microarray tests, for a quantitative and reproducible assessment of signaling pathway activity. Materials and methods: Focusing on the AR, ER, PI3K-FOXO and MAPK-AP1 pathways, we developed PCR primers and probes to assess mRNA expression levels of the pathways' direct target genes. For model parameter calibration, we performed controlled cell line experiments in which the respective pathways were inactivated and activated, to have calibration samples with known pathway activity status. Per pathway, the same set of calibration samples and the same set of target genes were used to develop RT-qPCR, RNA sequencing and microarray tests, to maximize similarity. The resulting OncoSignal tests can be used for a quantitative assessment of pathway activity on individual samples, giving a score on a 0-100 scale. We verified these tests on independent samples with known pathway status. We ran repeat experiments to assess the reproducibility within each platform, and tested the same samples on all three platforms to assess cross-platform concordance. Results: Using the OncoSignal score, low activity test samples were clearly separated from highly active ones. For example, AR-PCR scores went from 19±3 on androgen-deprived LNCaP cell lines to 70±2 after stimulation with DHT, and went down again to 33±1 and 23±1 when subsequently treated with bicalutamide and enzalutamide, respectively. PI3K-FOXO-microarray scores went from 12±1 on control MCF7 experiments to 50±4 after alpelisib treatment. Measurements on 100+ samples on all three platforms showed Pearson correlations between 0.93 and 0.99 in OncoSignal scores, with absolute differences typically below 10. Conclusion: We developed broadly applicable tests to assess AR, ER, PI3K-FOXO and MAPK-AP1 pathway activity in a quantitative manner on individual samples, with good reproducibility on repeat experiments and good concordance between PCR, RNA sequencing and microarray measurements. These tests can be used to unravel disease pathophysiology in (pre-)clinical samples. The added value of OncoSignal for therapy response prediction is currently being evaluated in different studies. Citation Format: Martijn Akse, Henk van Ooijen, Anke Pierik, Sandra van den Bosch, Hans van Zon, Laurent Holtzer, Anne van Brussel, Márcia Inda, Yvonne Wesseling-Rozendaal, Wim Verhaegh, Eveline den Biezen-Timmermans. Development of quantitative multi-platform tests for easy readout of AR, ER, PI3K and MAPK pathway activity to unravel pathophysiology across cancer and tissue types [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4261.

Use of Whole Genome Sequencing Data for a First in Silico Specificity Evaluation of the RT-qPCR Assays Used for SARS-CoV-2 Detection

The current COronaVIrus Disease 2019 (COVID-19) pandemic started in December 2019. COVID-19 cases are confirmed by the detection of SARS-CoV-2 RNA in biological samples by RT-qPCR. However, limited numbers of SARS-CoV-2 genomes were available when the first RT-qPCR methods were developed in January 2020 for initial in silico specificity evaluation and to verify whether the targeted loci are highly conserved. Now that more whole genome data have become available, we used the bioinformatics tool SCREENED and a total of 4755 publicly available SARS-CoV-2 genomes, downloaded at two different time points, to evaluate the specificity of 12 RT-qPCR tests (consisting of a total of 30 primers and probe sets) used for SARS-CoV-2 detection and the impact of the virus’ genetic evolution on four of them. The exclusivity of these methods was also assessed using the human reference genome and 2624 closely related other respiratory viral genomes. The specificity of the assays was generally good and stable over time. An exception is the first method developed by the China Center for Disease Control and prevention (CDC), which exhibits three primer mismatches present in 358 SARS-CoV-2 genomes sequenced mainly in Europe from February 2020 onwards. The best results were obtained for the assay of Chan et al. (2020) targeting the gene coding for the spiking protein (S). This demonstrates that our user-friendly strategy can be used for a first in silico specificity evaluation of future RT-qPCR tests, as well as verifying that the former methods are still capable of detecting circulating SARS-CoV-2 variants.

A Novel Corona Virus (COVID-19) Pandemic, Pathogenesis, Clinical Features and Management Options, Public Health Measures

Introduction: SARS-CoV-2 is an unknown corona virus causing COVID-19 disease responsible for the ongoing pandemic affecting over 190 countries, with a mortality rate of about 5%. The high mortality rate resulted from its ability to elicit cytokine storm via non-specific immune response with delay in specific immune response, notably worst in the elderly, HIV, immunocompromised and cancer diseased patients. The article therefore provides frontline health care workers, the opportunity to understand and equip themselves with management options, public health measures used in coping with COVID-19 infections and enables personnel to make quick preparation for Symptomatic COVID-19 infection in-order to reduce mortalities in health facilities. It also provides a summarised teaching material for medical and allied health students around the world.
 Methodology: Credible data and information were obtained from the World health organization situation report, Johns Hopkins University Corona Virus resource centre and other notable journal publications. Most information was on public domain.
 Results: Clinical features reported include pneumonia, renal dysfunction, hepatobiliary dysfunction and residual tissue damage, especially in the lungs in those that survive. The literature highlights the blood work-up picture (Leucopenia, increased cytokines IL-6, ferritin, Serum creatinine, urea, AST, GGT, ALT and Viremia) and radiological features of the disease. ELISA and RT-qPCR test are required for diagnosis using sputum, or pharyngeal swab, blood, serum, urine and faecal samples which lead to faeco-oral transmission. ELISA/RT-PCR is also required for disease exclusions in co-existing epidemics such as MERS-CoV, SARS-COV and other viral diseases. Treatment modalities employed thus far are trials that have produce results including targeted therapy, anti-retroviral, Favipiravir, Remdesivir, antibody-serum and antimalaria drugs such as chloroquine and quinine which are found among some protocols in African setting and other developing countries. Currently, ongoing preventive measures (face mask, social distancing and hand hygiene, community testing, isolation of confirmed cases and tracking of the exposed, remain the key corner-stone in the management of COVID-19 pandemic.
 Conclusion: COVID -19 disease still remain unclear to many scientists and its pandemic still ongoing. There are many research and clinical trials ongoing while some drugs are used off-label in-order to mitigate the damage caused by the virus in Human body. Cytokine storm needs immunomodulators and viral targeted therapy as discussed in-order to reduce tissue damage, and eventually morbidity and mortalities from COVID-19 infections.

Usefulness of PCR Screening in the Initial Triage of Trauma Patients During COVID-19 Pandemic.

Background:Hospitals worldwide have postponed all nonessential surgery during the COVID-19 pandemic, but non-COVID-19 patients are still in urgent need of care. Uncertainty about a patient's COVID-19 status risks infecting health care workers and non-COVID-19 inpatients. We evaluated the use of quantitative reverse transcription polymerase chain reaction (RT-qPCR) screening for COVID-19 on admission for all patients with fractures.Methods:We conducted a retrospective cohort study of patients older than 18 years admitted with low-energy fractures who were tested by RT-qPCR for SARS-CoV-2 at any time during hospitalization. Two periods based on the applied testing protocol were defined. During the first period, patients were only tested because of epidemiological criteria or clinical suspicion based on fever, respiratory symptoms, or radiological findings. In the second period, all patients admitted for fracture treatment were screened by RT-qPCR.Results:We identified 15 patients in the first period and 42 in the second. In total, 9 (15.8%) patients without clinical or radiological findings tested positive at any moment. Five (33.3%) patients tested positive postoperatively in the first period and 3 (7.1%) in the second period (P = 0.02). For clinically unsuspected patients, postoperative positive detection went from 3 of 15 (20%) during the first period to 2 of 42 (4.8%) in the second (P = 0.11). Clinical symptoms demonstrated high specificity (92.1%) but poor sensitivity (52.6%) for infection detection.Conclusions:Symptom-based screening for COVID-19 has shown to be specific but not sensitive. Negative clinical symptoms do not rule out infection. Protocols and separated areas are necessary to treat infected patients. RT-qPCR testing on admission helps minimize the risk of nosocomial and occupational infection.Level of Evidence:Level IV, retrospective cohort study. See Instructions for Authors for a complete description of levels of evidence.

Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride.

Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated techniques and enabling accurate, high-throughput diagnostics.

Assessment of patients who tested positive for COVID-19 after recovery

Assessment of patients who tested positive for COVID-19 after recovery

Investigation of The Synergistic Effects of Trastuzumab And Gambogic Acid in Her-2 Positive Breast Cancer Cell Line

HER2 positive breast cancer is one of the biggest health problems in the world, causing millions of deaths every year. Drug combination modeling studies are extensively evaluated in treating many diseases. Pharmacological studies over the last half-century have shown that gambogic acid has potent anti-tumor activity against many types of cancer, including breast cancer. In this study, we examined the synergistic anticancer effect of gambogic acid and trastuzumab in HER2 positive breast cancer cell line (MDA-MB-453). In-vitro synergistic and antiproliferative effects of trastuzumab plus gambogic acid studies were determined with XTT method and the combination index (CI) values of the trastuzumab and gambogic acid combination were calculated by CompuSyn software. To determine molecular mechanisms of the trastuzumab and gambogic acid combination, we analyzed HER2, caspase-9 and Bax gene and protein expression levels quantitative reverse transcription-PCR (qRT-PCR) and ELISA techniques. The combination of 50 µg/ml trastuzumab and 5 µM gambogic acid showed the best synergistic effect at 24 h incubation in MDA-MB-453 cells according to the in-vitro cell proliferation, RT-qPCR and ELISA test. Gambogic acid effects on HER2 positive breast cancer cell line shows its potential as natural compound to inhibit breast cancer cell proliferation in combination with trastuzumab.

Diagnostic methods and potential portable biosensors for coronavirus disease 2019

Timely detection and diagnosis are urgently needed to guide epidemiological measures, infection control, antiviral treatment, and vaccine research. In this review, biomarkers/indicators for diagnosis of coronavirus disease 2019 (COVID-19) or detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the environment are summarized and discussed. It is concluded that the detection methods targeting antibodies are not suitable for screening of early and asymptomatic cases since most patients had an antibody response at about 10 days after onset of symptoms. However, antibody detection methods can be combined with quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) to significantly improve the sensitivity and specificity of diagnosis, and boost vaccine research. Fast, sensitive and accurate detection methods targeting antigens need to be developed urgently. Various specimens for diagnosis or detection are compared and analyzed. Among them, deep throat saliva and induced sputum are desired for RT-qPCR test or other early detection technologies. Chest computerized tomography (CT) scan, RT-qPCR, lateral flow immunochromatographic strip (LFICS) for diagnosis of COVID-19 are summarized and compared. Specially, potential electrochemical (EC) biosensor, surface enhanced Raman scattering (SERS)-based biosensor, field-effect transistor (FET)-based biosensor, surface plasmon resonance (SPR)-based biosensor and artificial intelligence (AI) assisted diagnosis of COVID-19 are emphasized. Finally, some commercialized portable detection device, current challenges and future directions are discussed.

Leveraging Africa's preparedness towards the next phase of the COVID-19 pandemic

Leveraging Africa's preparedness towards the next phase of the COVID-19 pandemic

Evaluation of COVID-19 RT-qPCR Test in Multi sample Pools.

BackgroundThe recent emergence of SARS-CoV-2 lead to a current pandemic of unprecedented scale. Though diagnostic tests are fundamental to the ability to detect and respond, overwhelmed healthcare systems are already experiencing shortages of reagents associated with this test, calling for a lean immediately-applicable protocol.MethodsRNA extracts of positive samples were tested for the presence of SARS-CoV-2 using RT-qPCR, alone or in pools of different sizes (2-, 4-, 8- ,16-, 32- and 64-sample pools) with negative samples. Transport media of additional 3 positive samples were also tested when mixed with transport media of negative samples in pools of 8.ResultsA single positive sample can be detected in pools of up to 32 samples, using the standard kits and protocols, with an estimated false negative rate of 10%. Detection of positive samples diluted in even up to 64 samples may also be attainable, though may require additional amplification cycles. Single positive samples can be detected when pooling either after or prior to RNA extraction.ConclusionsAs it uses the standard protocols, reagents and equipment, this pooling method can be applied immediately in current clinical testing laboratories. We hope that such implementation of a pool test for COVID-19 would allow expanding current screening capacities thereby enabling the expansion of detection in the community, as well as in close organic groups, such as hospital departments, army units, or factory shifts.

Bioactive micropatterning of biomaterials for induction of endothelial progenitor cell differentiation: Acceleration of in situ endothelialization.

Synthetic grafts do not provide an appealing surface for endothelial cells to adhere and colonize the inner surface. To promote in situ endothelialization the following aspect has to be taken into account, endothelial progenitor cells (EPCs) needs to be mobilized on the surface of the graft. The surface of the graft has to be sufficiently biocompatible to create a prone environment for the EPCs to adhere, proliferate and, differentiate to form a layer and subsequently improve graft patency. In this work, two active molecules GRGDS and sitagliptin, were chosen for their abilities to recruit, enhance adhesion and induce differentiation of endothelial progenitor cells. They were grafted on PET surfaces in order to provide restrained cues triggering cell alignment and evaluate the influence of such structuration on EPCs fate. We then analyze cell behavior onto functionalized biomaterials. Their abilities to control EPCs fate were demonstrated via RT-qPCR, immunofluorescence, and enzymatic tests. The GRGDS/sitagliptin 100 × 10 surface enables to reduce the stemness phenotype on EPCs and induce the expression of endothelial lineage markers. These results highlight the importance of spatial patterning cues in guiding EPCs organization and function, which may have clinical relevance in the development of vascular grafts that promote patency.

The Effect of the Environmental Temperature on the Adaptation to Host in the Zoonotic Pathogen Vibrio vulnificus.

Vibrio vulnificus is a zoonotic pathogen that lives in temperate, tropical and subtropical aquatic ecosystems whose geographical distribution is expanding due to global warming. The species is genetically variable and only the strains that belong to the zoonotic clonal-complex can cause vibriosis in both humans and fish (being its main host the eel). Interestingly, the severity of the vibriosis in the eel and the human depends largely on the water temperature (highly virulent at 28°C, avirulent at 20°C or below) and on the iron content in the blood, respectively. The objective of this work was to unravel the role of temperature in the adaptation to the host through a transcriptomic and phenotypic approach. To this end, we obtained the transcriptome of a zoonotic strain grown in a minimum medium (CM9) at 20, 25, 28, and 37°C, and confirmed the transcriptomic results by RT-qPCR and phenotypic tests. In addition, we compared the temperature stimulon with those previously obtained for iron and serum (from eel and human, respectively). Our results suggest that warm temperatures activate adaptive traits that would prepare the bacteria for host colonization (metabolism, motility, chemotaxis, and the protease activity) and fish septicemia (iron-uptake from transferrin and production of O-antigen of high molecular weight) in a generalized manner, while environmental iron controls the expression of a host-adapted virulent phenotype (toxins and the production of a protective envelope). Finally, our results confirm that beyond the effect of temperature on the V. vulnificus distribution in the environment, it also has an effect on the infectious capability of this pathogen that must be taken into account to predict the real risk of V. vulnificus infection caused by global warming.

Harnessing Dengue Rapid Diagnostic Tests for the Combined Surveillance of Dengue, Zika, and Chikungunya Viruses in Laos.

.Recent expansions of vector-borne diseases highlight the need for improved surveillance, especially in resource-poor settings. Dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV) share the same vectors as well as similar clinical presentations, suggesting that combined surveillance would be useful. We hypothesized that blood spotted on dengue rapid diagnostic tests (RDTs) could be harnessed for sample collection in remote areas for subsequent detection of DENV, CHIKV, and ZIKV by reverse transcription real-time polymerase chain reaction (RT-qPCR). CHIKV and ZIKV dilutions were spotted on dengue RDTs (SD BIOLINE Dengue DUO, Standard Diagnostics, Gyeonggi-do, Republic of Korea), dried, and extracted. As reference, aliquots of each viral dilution were directly extracted. Using specific RT-qPCR tests, both viruses were successfully detected from RDT extracts. However, the limit of detection was slightly lower in comparison to direct extracts, two logfold for CHIKV and one logfold for ZIKV. For analysis of temperature stability, DENV dilutions were spotted on RDTs and stored for up to 2 months at −80°C, 4°C, or 35°C before testing. Storage of RDTs for 2 months at 35°C did not compromise detection of RNA by RT-qPCR; only minimal degradation was observed. This proof-of-principle study demonstrates the potential of using dengue RDTs for DENV/CHIKV/ZIKV combined surveillance in areas without access to laboratory facilities. Further investigations are needed for evaluation of tri-viral surveillance under field conditions using patient samples. Large-scale implementation of surveillance for these viruses is of crucial public health importance for the early detection of epidemics. This method also has important implications for improving understanding of the molecular epidemiology of the three viruses.

Experience in diagnosing congenital Zika syndrome in Brazilian children born to asymptomatic mothers

IntroductionTypical symptoms of primary Zika virus infection are not specific and share similarities with other arbovirus infections such as dengue fever and chikungunya. As acute infection can be asymptomatic in up to 73% of cases, infants with microcephaly represent a diagnostic challenge for pediatricians. We describe the frequency of congenital Zika syndrome (CZS) in Brazilian children born to asymptomatic pregnant mothers and its differential diagnosis. MethodsThis longitudinal, observational study was conducted on children with suspected CZS whose mothers did not report rash during pregnancy, referred to the reference hospital in a metropolitan area of ​ Rio de Janeiro, Brazil. The diagnosis of suspected CZS was based on Brazilian Ministry of Health protocol. ResultsForty-three (17%) of 246 referred children were born to mothers without rash history during pregnancy. Thirteen (30%) of 43 children met the Brazilian Ministry of Health criteria for CZS, all with microcephaly (two post-natal). The other children included 11 cases with post-natal microcephaly due to hypoxic-ischemic encephalopathy (6), non-progressive encephalopathy of unknown etiology (2), microcephaly under investigation (2) and congenital toxoplasmosis (1); 17 children were misdiagnosed with microcephaly and progressed with normal head circumference during the follow-up period; one child was included because of epidemiological link and one was loss to follow-up. All children who underwent laboratory investigation for ZIKV infection during neonatal period had negative RT-qPCR tests. ConclusionWe emphasize the increasing importance of CZS in differential diagnosis of microcephaly at birth or post-natal period. Detailed clinical investigation assisted by neuroimaging tests may clarify the diagnosis of CZS when laboratory tests are not available during the acute phase of the disease.

Diagnosis of SARS-CoV-2 infection based on CT scan vs RT-PCR: reflecting on experience from MERS-CoV

Diagnosis of SARS-CoV-2 infection based on CT scan vs RT-PCR: reflecting on experience from MERS-CoV