COVID-19 challenges: From SARS-CoV-2 infection to effective point-of-care diagnosis by electrochemical biosensing platforms.

In January 2020, the World Health Organization (WHO) identified a new zoonotic virus, SARS-CoV-2, responsible for causing the COVID-19 (coronavirus disease 2019). Since then, there has been a collaborative trend between the scientific community and industry. Multidisciplinary research networks try to understand the whole SARS-CoV-2 pathophysiology and its relationship with the different grades of severity presented by COVID-19. The scientific community has gathered all the data in the quickly developed vaccines that offer a protective effect for all variants of the virus and promote new diagnostic alternatives able to have a high standard of efficiency, added to shorter response analysis time and portability. The industry enters in the context of accelerating the path taken by science until obtaining the final product. In this review, we show the principal diagnostic methods developed during the COVID-19 pandemic. However, when we observe the diagnostic tools section of an efficient infection outbreak containment report and the features required for such tools, we could observe a highlight of electrochemical biosensing platforms. Such devices present a high standard of analytical performance, are low-cost tools, easy to handle and interpret, and can be used in the most remote and low-resource regions. Therefore, probably, they are the ideal point-of-care diagnostic tools for pandemic scenarios.

Toward a point-of-care diagnostic for specific detection of Mycobacterium tuberculosis from sputum samples.

This study reports the development of a new PCR-free device, using IS6110 gene as biomarker, for Tuberculosis (TB) diagnosis. An arginine film (ARGFILM) was used to prepare the biosensor platform. MT-probe was immobilized on this biosensor platform to identify IS6110 gene. This gene is an excellent biomarker for Mycobacterium tuberculosis (MT). Electrochemical analyses were carried out using differential pulse voltammetry method (DPV) by methylene blue (MB) reduction signal measurement before and after hybridization either between probe and synthetic target or extracted DNA from clinical sputum samples. The optimization study of MT-probe immobilization on modified-electrode surface showed that the best probe concentration was 15 µM. The analytical analysis of hybridization assays was performed using different concentrations of synthetic MT-target (15-500 nM). The linear response was between 15 and 100 nM and the detection limit was 4.4 nM. The biosensor performance was also investigated with extracted DNA from sputum samples (PCR-free). The results showed that the biosensor was able to detect the MT from samples, exhibiting a high sensitivity and satisfactory selectivity. Thus, these results allow for the possibility of developing a portable detection device for effective diagnosis of TB patients.

Assessment of messenger RNA (mRNA) of Mycobacterium tuberculosis as a marker of cure in patients with pulmonary tuberculosis.

AIMS: To analyse the performance of RT-qPCR using 85B mRNA in the diagnosis of Mycobacterium tuberculosis infection and in the assessment of the response to treatment for pulmonary tuberculosis (TB). METHODS AND RESULTS: Ninety-eight patients with signs of pulmonary TB were selected: 56 were considered infected with Myco. tuberculosis and they had positive cultures or evident clinical response to anti-TB treatment. Patients with pulmonary tuberculosis were evaluated by culture and RT-qPCR for a 30-day specific treatment. It was found that both tests demonstrated a decline in viable bacilli at 15 and 30 days after the beginning of the therapy in most of the patients. The quantification of the 85B mRNA target was performed in 52 patients who had initially shown positive results by RT-qPCR and who were followed on the days 15 and 30 after the specific treatment. Thus 85B mRNA was detectable in sputum samples in 52 patients with a confirmed diagnosis of pulmonary tuberculosis on day 0. During the specific treatment the 85B mRNA was detectable in 13 patients on day 15 and in only three patients on day 30. CONCLUSIONS: Mycobacterium tuberculosis mRNA in the sputum is a useful prognostic marker and its quantification, an early and reliable indicator for monitoring response to treatment, drug resistance, re-infection and relapse. SIGNIFICANCE AND IMPACT OF THE STUDY: RT-qPCR is a tool that can be used in clinical and therapeutic monitoring as an indicator of bacterial resistance and indicator of the period of transmissibility of Myco. tuberculosis in patients with pulmonary TB undergoing treatment.

Evaluation of a IS6110-Taqman real-time PCR assay to detect Mycobacterium tuberculosis in sputum samples of patients with pulmonary TB.

AIM: Evaluate the IS6110-Taqman system performance in sputum samples from patients with pulmonary tuberculosis from health services in north-eastern Brazil as a diagnostic laboratory tool for pulmonary tuberculosis. METHODS AND RESULTS: 165 sputum samples from respiratory symptomatic patients were evaluated in the IS6110-TaqMan assay: 66 patients with pulmonary tuberculosis and 99 without TB. When the IS6110-TaqMan assay was evaluated using culture and/or clinical response to the specific treatment as the gold standard, IS6110-TaqMan assay obtained a sensitivity of 87.9% and specificity of 98%. The performance of IS6110-TaqMan assay was also evaluated with the sputum smear microscopy, resulting in a sensitivity of 79.7% and specificity 94.8%. CONCLUSIONS: The IS6110-TaqMan was rapid, sensitive and specific for the diagnosis of pulmonary TB. SIGNIFICANCE AND IMPACT OF THE STUDY: IS6110-TaqMan assay is a promising auxiliary tool for the diagnosis of pulmonary TB when used in conjunction with routine laboratory tests, clinical and epidemiological criteria of the patient, thus increasing the sensitivity and specificity of diagnosis.

Immunological diagnosis of tuberculosis based on recombinant antigens ESAT-6 and CFP-10 in children from an endemic area in northeast Brazil.

Diagnostic tests for tuberculosis (TB) using interferon gamma (IFN-γ) responses produced by T lymphocytes after stimulation by early secretory antigen target 6 (ESAT-6), culture filtrate protein 10 (CFP-10) or purified protein derivate (PPD) were carried out using ELISA (enzyme-linked immunosorbent assay) in whole blood culture supernatants from children with suspected TB disease (n=21), latent TB infection (LTBI; n=17) and negative controls (NC; n=21) from Recife, Pernambuco, Brazil. The results were analysed using the ROC (receiver operating characteristic) curves and the areas under the curve (AUC) generated varied from 0.5 to 1.0 with higher values indicating increased discriminatory ability. Comparisons of AUCs were made using non-parametric assumptions, and the differences were considered significant if P<0.05. The ROC curve showed a statistical difference (P = 0.015) between the LTBI and NC groups with an AUC of 0.731, TB disease and NC (AUC=0.780; P=0.002) and a group with TB (latent infection+disease, n=38) and NC (AUC=0.758; P = 0.001) when the antigen used was ESAT-6. No statistical difference was found between the groups when CFP-10 or PPD was used. In conclusion, the ESAT-6 test may be the most appropriate for diagnosis of childhood TB, both LTBI and TB disease, when associated with epidemiological and clinical data, especially in endemic areas such as Brazil.

Development and optimization of polymerase chain reaction-based malaria diagnostic methods and their comparison with quantitative buffy coat assay.

Polymerase chain reaction (PCR)-based assays targeting the small-subunit rRNA were developed and evaluated, allowing for the simultaneous diagnosis of Plasmodium falciparum and Plasmodium vivax DNA in human blood samples. The PCR methods and quantitative buffy coat (QBC) were compared in 402 patients. The heminested PCR method showed a sensitivity of 97.4%, which was superior to the sensitivity of the QBC method (91.7%, P < 0.05), to simple PCR (84.6%, P < 0.001), and to PCR with digoxigenin labeling (PCR-DIG) (88.5%, P < 0.001). The PCR-DIG and QBC analyses were more sensitive than simple PCR (P < 0.003 and P < 0.05, respectively). There was no significant difference between the sensitivities of the QBC assay and the PCR-DIG assay. The specificity for the 3 PCR-based methods was 100%, superior to the specificity calculated for the QBC assay (88.95%, P < 0.009). The frequency of a positive result in groups from endemic areas but without detectable parasitemia increased, in order, from simple PCR, QBC test, PCR-DIG, to heminested PCR. An association between a positive PCR result and a history of malaria was also found. Taken together, these data suggest that this technology could be further developed to screen people with oligoparasitemia and to monitor malaria treatment.

Risk factors for the occurrence of bancroftian filariasis infection in children living in endemic areas of northeast of Brazil.

The objective of this study was to identify biological and social risk factors for the occurrence of microfilaraemia in a population of 1464 children of both sexes aged 5-14 years, living in two highly endemic areas of Recife a city in the northeast of Brazil. A survey was performed from December 1990 to July 1991 and the microfilaraemia was examined by the thick-drop technique using 45 microliters of peripheral blood. Information was obtained about use of bednet, length of time living in area and number of occupants per household. Risk was quantified by the crude and adjusted Odds Ratio. The 95 per cent confidence interval, Likelihood Ratio Statistics, and P value were used to test the statistical significance. An association was established between microfilaraemia in children and adolescents, and age, number of individuals per household, the presence of microfilaraemic adults in the household, length of time living in the area, and bednet use. Maternal microfilaraemia was not found to be a risk factor for the occurrence of microfilaraemia in offspring. These results allow the identification of children with a greater risk of microfilaraemia. In addition, these findings highlight the role of the household environment in the transmission process.

Bancroftian filariasis in two urban areas of Recife, Brazil: pre-control observations on infection and disease.

Bancroftian filariasis is a major public health problem in the city of Recife in north-eastern Brazil. In some of its urban areas microfilaraemia prevalence reaches 14%. This study describes epidemiological characteristics, infection and disease, in 2 urban areas, Coque and Mustardinha, before control measures were applied. The parasitological survey was performed by a 'door-to-door' census covering 5563 subjects, aged between 5 and 65 years. Microfilaraemia was detected by the thick drop technique, using 45 microL of peripheral blood collected between 20:00 and 24:00. In both areas the prevalence of microfilaraemia was 10%, and males had higher prevalences of infection and disease than females. The prevalence of microfilaraemia was higher in the 15-24 and 25-34 years age groups in both sexes. Most microfilaria (mf) carriers (72.1% in Coque and 79.7% in Mustadrinha) had mf densities < 100/60 microL of blood. Females of reproductive age had significantly lower mf densities than males. The overall disease prevalence in both areas was 6.3%. Amongst the subjects who presented with chronic disease 15.7% were microfilaraemic. Chronic disease prevalence increased from 1.4% in the 5-14 years age group to 11.3% in the oldest age group. The most frequent clinical manifestation was hydrocele (5.4%), followed by lymphoedema (1.8%). The epidemiological pattern of filariasis in the populations studied was marked by high prevalence of microfilaraemia, low mf density, and relatively low prevalence of filarial disease considering the level of endemicity.