Comparative performances of seven quantitative Reverse - Transcription Polymerase Chain Reaction assays (RT-qPCR) for detecting SARS-CoV-2 infection in samples from individuals suspected of COVID-19 in São Paulo, Brazil

Introduction Brazil is the second largest country with COVID-19 positive cases worldwide. Due to the potent spread of the virus and the scarcity of kits and supplies, the Brazilian Ministry of Health has granted authorization for the use of kits available during this emergency, without an accurate evaluation of their performance. This study compared the performance and cost-effectiveness of seven molecular assays/kits available in São Paulo, Brazil, for SARS-CoV-2 diagnosis Materials and methods A total of 205 nasopharyngeal/oropharyngeal samples from suspected cases of COVID-19, were tested using the following assays: (i) GeneFinder COVID-19 plus RealAmp kit; (ii) 2019-nCoV RNA PCR-Fluorescence Probing, Da An Gene Co.; (iii) in-house RT-qPCR SARS-CoV-2 IAL; (iv) 2019-nCoV kit, IDT; (v) molecular SARS-CoV-2 (E) kit, Bio-Manguinhos; (vi) Allplex 2019-nCoV modified Assay, Seegene Inc, and (vii) Biomol one-step COVID-19 kit, IBMP. The criteria for determining a SARS-CoV-2 true positive result included the cycle threshold cut-off values, the characteristics of exponential/linear curves, the gene target diversity, and a positive result in at least two assays Results The overall sensitivity of the assays listed were GeneFinder 83.6%, Da An Gene 100.0%, IAL 90.4%, IDT 94.6%, Bio-Manguinhos 87.7%, Allplex 97.3%, and IBMP 87.7%. The minor sensitive gene target was RdRP. Although all assays had a Cohen's Kappa index ≥0.893, the best tests used multiplex assays identifying N-gene and/or E-gene targets Conclusion All assays tested accurate for diagnosis, but considering cost-effectiveness (cost, time consumption, number of samples tested, and performance), the in-house IAL assay was ideal for COVID-19 diagnosis in São Paulo, Brazil.

COVID-19 laboratory diagnosis: comparative analysis of different RNA extraction methods for SARS-CoV-2 detection by two amplification protocols

The gold standard for the laboratory diagnosis of COVID-19 is the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) assay, which searches for SARS­CoV-2 target genes in nasopharyngeal/oropharyngeal (NP/OP) samples, and its performance depends on the quantity and quality of the RNA input. This study compared the performance and cost-effectiveness of three different kits/reagents for RNA extraction used in COVID-19 diagnosis in Sao Paulo, Brazil. A total of 300 NP/OP samples belonging to suspected cases of COVID-19 stored in a biorepository were randomly selected, and RNA was extracted using (i) automated extraction (Loccus, Extracta Kit FAST), (ii) manual extraction (BioGene Kit, Bioclin, Quibasa), and (iii) quick extraction methods (Lucigen, Quick DNA Extract Kit). Next, the samples were tested using RT-qPCR for SARS-CoV-2 with the Allplex 2019-nCoV modified assay and the Charité-Berlin protocol. All assays/kits were used according to the manufacturer's instructions. For the Allplex kit, the sensitivity in detecting SARS-CoV-2 with previously extracted RNA by different procedures was 100.0% for Loccus, 100.0% for BioGene and 91.9% for Quick. Using the Charité-Berlin protocol, the sensitivities were 81.4% for Loccus, 81.2% for BioGene and 60.7% for Quick. The least sensitive target gene and the gene most affected by RNA extraction procedures was the RNA-dependent RNA polymerase gene (Charité-Berlin protocol). No false-positive SARS-CoV-2 results were detected using RNA obtained from any of the different protocols. In conclusion, Loccus and BioGene RNA extractions were efficient for RT-qPCR assays, and although the BioGene procedure is less expensive, Loccus is the best choice because it allows the rapid handling of hundreds or thousands of samples, a desirable feature during pandemics. Although less sensitive, the Quick extraction is useful during outbreaks coupled with the Allplex amplification kit for SARS-CoV-2 diagnosis (κ = 0.925).

Pooling of sera for human t-cell lymphotropic virus (HTLV) screening in a time of increasing health care expenditure and limited resouces

Identifying the true prevalence of human T-cell lymphotropic virus, mostly type 1 (HTLV-1), and the number of patients with HTLV-1-associated diseases, in addition to introducing HTLV-1/2 serology during the prenatal of pregnant women and in individuals infected with other viruses that share transmission routes with HTLV-1, are actions that could help to recognize the importance of this virus by WHO and national health organizations, and to control its transmission/dissemination. As Brazil is endemic to HTLV and there is an increase in health care expenditure, but resources are limited, any strategy that could reduce the cost of HTLV screening is needed and welcomed. This study aimed to determine whether the strategy of pooling sera for HTLV antibody determination is feasible and reduces the costs. Two enzyme immunoassays (EIA Murex HTLV-I+II, Diasorin, UK, and Gold ELISA HTLV-1+2, REM Ind. Com. Ltda., SP, Brazil), and serum samples that resulted in different levels of HTLV-1/2 antibodies by EIA and of which a volume allowed assay validation were employed for analysis. The diagnostic sensitivity and specificity and Cohen's Kappa value, as well as the accuracy and precision were analyzed. After validating the five-sample pool using the EIA Murex (Cohen's Kappa = 1.0), the technique was employed for individual cost comparison in 2,625 serum samples from populations at risk of HTLV infections (HBV, HCV, and HIV-infected individuals). The results from individual and pooled samples confirmed the diagnostic sensitivity (100%) and specificity (100%) of the pooling and a cost minimization varying from 60.7% to 73.6%. In conclusion, the results of this study suggest the use of pooling sera in sero-epidemiological surveillance studies and possibly in prenatal care screening programs in Brazil.

Loop-mediated isothermal amplification (LAMP) assay for rapid and accurate confirmatory diagnosis of HTLV-1/2 infection

Laboratory diagnosis of human T-lymphotropic viruses (HTLV) 1 and 2 infection is performed by serological screening and further confirmation with serological or molecular assays. Thus, we developed a loop-mediated isothermal nucleic acid amplification (LAMP) assay for the detection of HTLV-1/2 in blood samples. The sensitivity and accuracy of HTLV-1/2 LAMP were defined with DNA samples from individuals infected with HTLV-1 (n = 125), HTLV-2 (n = 19), and coinfected with HIV (n = 82), and compared with real-time polymerase chain reaction (qPCR) and PCR-restriction fragment length polymorphism (RFLP). The overall accuracy of HTLV-1/2 LAMP (95% CI 74.8­85.5%) was slightly superior to qPCR (95% CI 69.5­81.1%) and similar to PCR-RFLP (95% CI 79.5­89.3%). The sensitivity of LAMP was greater for HTLV-1 (95% CI 83.2­93.4%) than for HTLV-2 (95% CI 43.2­70.8%). This was also observed in qPCR and PCR-RFLP, which was associated with the commonly lower HTLV-2 proviral load. All molecular assays tested showed better results with samples from HTLV-1/2 mono-infected individuals compared with HIV-coinfected patients, who present lower CD4 T-cell counts. In conclusion, HTLV-1/2 LAMP had similar to superior performance than PCR-based assays, and therefore may represent an attractive alternative for HTLV-1/2 diagnosis due to reduced working time and costs, and the simple infrastructure needed.

Detection of herpes virus (KSHV) DNA sequences in Brazilian patients with AIDS-associated Kaposi's sarcoma

To explore the possible involvement of herpes viris (KSHV) in AIDS-associated Kaposi's sarcoma (KS) in 7 patients in Brazil, we analyzed 7 AIDS-KS lesions. Using PCR, we found KSHV specific sequences in 3 cases and by using nested PCR, we identified sequences in each of 7 cases. Direct sequencing on nested-PCR products showed a certain degree of variability in relation to classic KSHV sequences, and identified alterations similar to those described in some endemic cases from Africa and in AIDS-associated KS specimens from North America. This mixed pattern of KSHV sequences observed in AIDS-associated KS from Brazil may reflect the geographic origin of the samples, consistent with the environmental and epidemiological backgrounds of people in this country. It is apparent that, just as in other countries in the world, Kaposi's sarcoma in HIV patients is related to herpes virus infection.

Prevalence of HTLV-I and HTLV-II infections among HIV-1-infected asymptomatic individuals in Sao Paulo, Brazil

Individuos infectados pelo virus da imunodeficiencia humana (HIV-1) geralmente apresentam infeccoes por multiplos patogenos, dentre eles, os HTLV-I e HTLV-II. Estes foram descritos com frequencia variavel em pacientes com AIDS e portadores assintomaticos do HIV-1, tanto na Europa como no Japao. Este trabalho foi conduzido com o objetivo de determinar a prevalencia de infeccao HTLV-I e -II em portadores assintomaticos do HIV-1 da cidade de Sao Paulo, e comparar os resultados obtidos com os descritos em literatura e os por nos anteriormente publicados. Foi detectada infeccao HTLV em 1,5 por cento dos 266 portadores assintomaticos do HIV-1 e 14 por cento dos 28 casos de AIDS analisados. Com base em dados epidemiologicos foi confirmado como sendo o principal fator de risco para adquirir a coinfeccao HIV/HTLV, o uso de injetaveis

Dificuldades no diagnostico de infeccao primaria atipica por HIV-1: relato de um caso / Difficulties in diagnosing atypical primary HIV-1 infection : case report

Muitos casos de infeccao primaria pelo HIV nao sao identificados devido a dificuldade em seu diagnostico clinico e laboratorial. Este trabalho relata um caso atipico de infeccao primaria pelo HIV em um usuario de drogas intravenosas que apresentou como primeira manifestacao clinica, quadro agudo de hepatite. A pesquisa de anticorpos dirigidos contra varios virus inclusive o HIV, resultou negativa, na primeira analise do soro do paciente. No entanto, o diagnostico de infeccao primaria pelo HIV foi sugerido inicialmente utilizando um novo metodo laboratorial alternativo para a pesquisa destes anticorpos, denominado IVIAP (in vitro induced antibody production)....

Produçäo induzida de anticorpos in vitro: novo método para o diagnóstico de infecçäo perinatal pelo vírus da imunodeficiência humana / Induced production of in vitro antibodies: new method for diagnosis of perinatal infection by human immunodeficiency virus

Tendo em vista a capacidade de células mononucleares (CM) de sangue periférico de indivíduos infectados pelo HIV secretarem anticorpos espontaneamente in vitro, foi desenvolvido um novo método laboratorial de produçäo induzida de anticorpos anti-HIV 1 in vitro (IVIAP) a ser utilizado no diagnóstico de infecçäo perinatal. Empregaram-se, na padronizaçäo da IVIAP, CM de dois grupos: 33 adultos e 30 crianças infectados pelo HIV-1 e 16 adultos e 21 crianças sem dados epidemiológicos, hiv-1-soronegativos. Para estimular a secreçäo de anticorpos in vitro foi usado antígeno bruto do HIV-1 adsorvido a placas de um kit comercial de ELISA e testadas várias concentraçöes de CM e diferentes tempos de cultura. Os anticorpos produzidos pela CM foram detectados na placa, por reaçäo imunoenzimática, com os reagentes do próprio kit e as instruçöes do fabricante. Os resultados obtidos mostraram que näo houve reaçäo cruzada ou falso-positiva quando foram avaliadas CM do grupo controle HIV-1 soronegativo. No grupo de indivíduos infectados foi possível determinar como sendo 10 elavado a quinta CM por orifício da placa e 24h de cultura a condiçäo ideal para resultar uma IVIAP positiva. Para comparar a eficiência da IVIAP em relaçäo a outras técnicas de secreçäo de anticorpos in vitro, foram utilizadas CM de 19 adultos e 13 crianças infectados pelo HIV-1 na IVIAP, IVAP e ELISPOT, demonstrando que a IVIAP foi mais sensível para detectar infecçäo pelo HIV, nos casos pediátricos. De um grupo de 57 crianças, acompanhado clínica e laboratorialmente por um período médio de 18 meses, foi possível comparar os resultados obtidos na IVIAP com os da sorologia para o HIV, antigenemia p24 e reaçäo em cadeia de polimerase (PCR). Observaram-se resultados falso-negativos nos casos de hipogamaglobulinemia. A IVIAP se mostrou mais sensível para detectar infecçäo pelo HIV que a sorologia e a antigenemia p24. Houve concordância entre os resultados obtidos na IVIAP e na PCR. Neste estudo a IVIAP apresentou 91 porcento de sensibilidade e 96 porcento de especificidade. Concluindo: foi possível desenvolver um novo método laboratorial para o diagnóstico de infecçäo perinatal pelo HIV-IVIAP - sendo sensível, específico, rápido, prático e de baixo custo, podendo ser usado com crianças de mais de dois meses de idade.

Rapid in vitro detection of HIV-1-specific antibody secretion by cells-culture with virus antigens

The present report describes an alternative method for in vitro detection of HIV-1 -specific antibody secretion in 24h of culture employing as stimulant of peripheral blood mononuclear cells the disrupted inactivated whole virus adsorbed onto microwells in a commercial ELISA kit plates. The results obtained from this technique have showed high sensitivity and specificity since it was capable of detecting HIV-1 infection early after birth. There were neither false-positivity nor false-negativity when blood samples obtained from HIV-1 seronegative asymptomatic individuals, and HIV-1 seropositive adult patients were analized. This rapid, low cost, simple, highly sensitive and specific assay can be extremely useful for early diagnosis of pediatric HIV infection

Neutropenia in an infant: possible autoimmune etiology

The present study was undertaken to determine if the serum of a child with severe neutropenia contained antibodies against parental neutrophils. The presence of IgG antibodies to granulocytes from both parents was demonstrated using the indirect immunofluorecence technique. These data suggest an auatoimmune etiology for the neutropenia of this patient

Caso em foco: doença granulomatosa crônica da infância / Case in focus: chronic granulomatous disease in childhood

Os autores apresentam um caso de Doença Granulomatosa Crônica da Infância em um menino de 23 meses de idade com sintomatologia a partir de dois meses de idade. É salientada a freqüência da doença (terceira imunodeficiência congênita na experiência dos autores) e analisados os testes de avaliaçäo da funçäo fagocitária, bem como aspectos da fisiopatologia e da terapêutica dessa entidade

Primary immunodeficiency diseases: a presentation of 6 cases

The peripheral blood leukocytes of 6 children with clinical data suggestive of primary cellular immunodeficiencies were studied in an attempt the cellular basis of these disorders. The phenotype and function of T and B cells were investigated. According to the clinical and laboratory fetures, the patients were classified as one case of severe combined immunodeficiency (SCID), two of ataxia-telangiectasia (AT), one of Wiskott-Aldrich syndrome (WAAS), one of /edi%george syndrome (DSG), and one of cellular immunodeficiency (CID). The laboratory investigations together with the clinical manifestations permitted a diagnosis of primary immunodeficiency diseases

Absence of mutagenicity of Potomorphe umbellata and Potomorphe peltata in the Salmonella/mammalian-microsome mutagenicity assay

The development of short term tests for measuring the mutagenic activity of genotoxic chemicals has been helpful in increasing security. Natural products from the flora are largely used in popular medicine. Using the Salmonella/mammalian-microsome assay, we investigated the genetic toxicity of two plant species popularly used in malaria treatment, Pothomorphe umbrellata and Pothomorphe peltata. The data show the absence of genetic toxicity for both plant species

Epidermodisplasia verruciforme. Estudo imunologico de tres casos. / Epidermodysplasia verruciform. Immunological study in 3 cases

Tres pacientes com epidermodisplasia verruciforme foram avaliados imunologicamente atraves de testes in vivo e in vitro. Segundo os parametros de avaliacao de imunidade celular por nos adotados, todos os tres pacientes mostraram-se imunodeprimidos. Um dos pacientes que apresentava carcinomas foi submetido a imunoestimulacao com dinitroclorobenzeno (DNCB) e posteriormente reavaliado

Linfócitos T, B no sangue periférico de doentes com hepatite aguda / Levels of peripheral T and B lymphocytes in acute hepatitis

Foi determinado o número de linfócitos T, B e linfócitos T, formadores de roseta estável, a 37 °e, num grupo de 140 doentes com diagnóstico clínico e Iaboratorial de hepatite aguda, atendidos no ambulatório do Hospital Emílio Ribas, São Paulo-SP, Brasil, no período de outubro de 1981 a maio de 1982. As provas sorológicas para pesquisa de antígenos e/ou de antícorpos para os vírus da hepatite A e B revelaram que 37,8% dos doentes apresentavam hepatite A, 50,7% dos doentes, hepatite B, e 11,5%, sorologia negativa para os vírus da hepatite A e B, tendo sido classificados como portadores de hepatite de causa indeterminada. O número de linfócitos nesses doentes foi determinado no dia do diagnóstico clínico, após 60 e 360 dias. Quanto ao número de linfócitos T, estes se mostraram aumentados na maioria dos doentes, durante todo o estudo, retornando a valores normais após 60 360 dias apenas nos doentes com hepatite de causa indeterminada. Em relação aos linfócitos B, estes estavam normais na maioria dos doentes, independentemente da época e tipo de hepatite. Para os linfócitos T, formadores de roseta estável, a 37°e, não ocorreram variações significativas na maioria dos doentes, durante todo o estudo. A tentativa de se cor rela cio na rem níveis de linfócitos com tipo e forma de hepatite discutida no trabalho (AU).

Reatividade tuberculínica e resposta imunológica celular e humoral "in vitro" em doentes com tuberculose pulmonar / In vitro tuberculin reaction and cellular and humoral immune response in patients with pulmonary tuberculosis

Foi estudada a reatividade tuberculínica e a resposta imunológica celular e humoral "in vitro", em 50 doentes de ambos os sexos, de 20 a 80 anos de idade, com tuberculose pulmonar ativa, internados no Parque Hospitalar do Mandaqui da Secretaria de Estado da Saude, Säo Paulo (Brasil), no período de maio a agosto de 1980. Para o estudo da reatividade tuberculínica foi utilizado o PPD, Rt-23, 2 UT, tendo havido 14,0% de näo-reatores, 12,0% de reatores fracos e 74,0% de reatores fortes. O estudo da imunidade celular e humoral "in vitro" foi realizado pela quantificaçäo de linfócitos T e B, transformaçäo blástica de linfócitos, liberaçäo do fator inibidor da migraçäo de leucócitos (LIF) e reaçäo de hemaglutinaçäo passiva. Os resultados mostraram a validade do cálculo do número absoluto dos linfócitos T e B. A cultura de linfócitos e a técnica do LIF, foram capazes de detectar a sensibilizaçäo dos linfócitos ao PPD, mesmo nos doentes näo reatores, e a reaçäo de hemaglutinaçäo passiva revelou a presença de anticorpos específicos na populaçäo estudada em títulos superiores aos encontrados em pessoas normais, independentemente da reatividade tuberculínica