Review of Clinical Performance of Serology Based Commercial Diagnostic Assays for Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Antibodies.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, which caused the coronavirus disease 2019 (COVID-19) pandemic as declared by the World Health Organization, has created havoc worldwide. The highly transmissible infection can be contained only by accurate diagnosis, quarantining, and exercising social distancing. Therefore, quick and massive deployment of SARS-CoV-2 testing plays a crucial role in the identification and isolation of infected patients. Reverse transcription-polymerase chain reaction is the gold standard for COVID-19 detection; however, it needs expertise, facilities, and time. Hence, for the ease of population-wide screening, serology-based diagnostic assays were introduced. These can help determine the prevalence of infection, understand the epidemiology of the disease, and assist in suitable public health interventions while being user-friendly and less time consuming. Although serological testing kits in markets soared, their sensitivity and specificity were questioned in reports from different parts of the world. In this article, we have reviewed 40 Food and Drug Administration (FDA) and CE-approved clinically evaluated serological kits (8 enzyme-linked immunosorbent assay [ELISA] kits, 10 chemiluminescent immunoassay [CLIA] kits, and 22 lateral flow immunoassay [LFIA] kits) for their sensitivity and specificity and discussed the apparent reasons behind their performance. We observed appreciable sensitivity in the kits detecting total antibodies compared to the kits targeting single isotype antibodies. Tests that determined antibodies against nucleocapsid protein were found to be more sensitive and those detecting antibodies against spike protein were found to have greater specificity. This study was conducted to help the decision-making while acquiring antibody kits and concurrently to be mindful of their shortcomings.

IgG avidity Western blot using Toxoplasma gondii rGRA-7 cloned from nucleotides 39-711 for serodiagnosis of acute toxoplasmosis.

Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.

IgG AVIDITY WESTERN BLOT USING Toxoplasma gondii rGRA-7 CLONED FROM NUCLEOTIDES 39-711 FOR SERODIAGNOSIS OF ACUTE TOXOPLASMOSIS / Avidez do IgG pelo Western Blot usando o r GRA-7 do Toxoplasma gondii clonado de nucleotídeos 39-711 para sorodiagnóstico de toxoplasmose aguda

Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.

Toxoplasmose é uma causa importante de infecção congênita. O presente estudo foi feito para avaliar o uso do recombinante (r) GRA-7 clonado de nucleotídeos (n) 30-711 para discriminar entre toxoplasmose aguda e crônica. Inicialmente IgM, IgG e ELISA avidez IgG comerciais foram usados para determinar o perfil sorológico do soro. Amostras de soro de 20 pacientes sintomáticos com infecção aguda (IgG avidez baixa, IgM positivo), 10 com infecção crônica (alta avidez IgG, IgM negativo) e 10 com avidez IgG indeterminada (IgM positivo) que foram testados para o status de avidez IgG com um doméstico Western Blot desenvolvendo avidez IgG usando o rGRA-7 antígeno recombinante. Todos os 20 soros de provável infecção aguda mostraram bandas que ou se apagaram completamente ou tiveram a sua intensidade significantemente reduzida após tratamento com uréia 8 M, enquanto as intensidades das bandas das 10 amostras de soros de casos crônicos permaneceram iguais. Dos 10 soros com status indeterminado de avidez de IgG, após tratamento com uréia 8 M a intensidade das bandas em seis soros permaneceram iguais, dois soros tiveram bandas apagadas completamente e dois outros tiveram significante redução da intensidade das bandas. Discriminação entre toxoplasmose aguda e crônica foi feita com sucesso através do IgG avidez Western blot doméstico.

Comparative evaluation of a PCR assay with an in-house ELISA method for diagnosis of Tuberculous meningitis.

BACKGROUND: Despite the availability of many investigational methods, diagnosis of Tuberculous meningitis (TBM) is extremely difficult. Polymerase chain reaction (PCR), using specific primers for Mycobacterium tuberculosis (MTB), shows variable sensitivity and specificity. In this study, we assessed the usefulness of the PCR assay for TBM diagnosis and compared it to our in-house enzyme-linked immunosorbent assay (ELISA) based on antigen 85 complex detection. MATERIAL/METHODS: Cerebrospinal fluid (CSF) samples were obtained from 189 patients in 3 different groups: confirmed TBM (n=13), clinically suspected TBM (n=37), and non-TBM (n=139). A PCR assay was performed using a specific pair of primers designed to amplify the insertion sequence IS6110 in the MTB genome, and it was compared to ELISA, using monoclonal antibodies against the purified Ag 85 complex, to analyze CSF samples and diagnose TBM. RESULTS: The PCR assay yielded sensitivity and specificity values of 80% and 84%, which are slightly less, but comfortable to the values obtained for the ELISA method (84% and 91%). Interestingly, a combinatorial approach using both methods provided sensitivity and specificity of 88% and 93%. CONCLUSIONS: The PCR assay was found to be as sensitive and specific as the well-established in-house ELISA technique, suggesting that it can be used for TBM diagnosis.

Assessment of immune response to repeat stimulation with BCG vaccine using in vitro PBMC model.

BACKGROUND: Tuberculosis (TB) is one of the most prevalent cause of death due to a single pathogen. Bacillus Calmette Guérin (BCG) is the only vaccine available for clinical use that protects against miliary TB; however, this vaccine has shown variable levels of efficacy against pulmonary TB. In India, a single dose of BCG vaccine is given and there are few countries where repeated doses of BCG are given. The incidence of TB in India is very high inspite of primary vaccination in neonatal period and therefore requires consideration for repeated immunization. METHODS: To improve BCG immunogenicity, we have evaluated specific antimycobacterial immune responses (anti-BCG IgG and IFN-gamma), T cell activity-ADA, CD4 and CD8 T cell count, and CD4/CD8 ratio in a peripheral blood mononuclear cells (PBMC) model using boost immunization protocols with the BCG vaccine. PBMC were induced with a repeat dose of BCG at 24 and 72 hrs of cell culture. RESULTS: At the end of the experimental time, supernatant was collected to estimate anti-BCG IgG titer, interferon gamma, ADA activity, CD 4 and CD8 T cell count, and CD4/CD8 ratio. We demonstrated that PBMC induced with a repeat dose of BCG showed an increased specific anti-mycobacterial immune responses, T cell activity, and ADA activity as compared to PBMC induced with BCG alone or without BCG induction. CONCLUSION: The repeat BCG stimulation of PBMC obtained from BCG vaccinated individuals shows enhanced immune activation with respect to increased anti-BCG titre, IFN-gamma and ADA activity without concomitant increase in CD4 and CD8 cells. This study provides some basic data in future experiments in animal models with respect to repeat BCG vaccination.

Changes in cerebrospinal fluid cytokine expression in tuberculous meningitis patients with treatment.

BACKGROUND: The prevalence of tuberculous meningitis (TBM) is very high in developing areas of the world. Inflammation and cytokine patterns produced by T lymphocytes play an important role in susceptibility to infections. The inflammatory response and production of cytokines in the cerebrospinal fluid (CSF) of patients with TBM are well documented. Conversely, little is known about the role of pro- and anti-inflammatory cytokines in the CSF of TBM patients. The goal of the present study was to estimate the level of proinflammatory cytokine and anti-inflammatory cytokine levels in CSF samples from TBM patients. MATERIALS AND METHODS: To study this, in vivo levels of IL-2 and IFN-gamma (proinflammatory cytokines), and IL-10 (anti-inflammatory cytokine) in the CSF of 60 adult TBM patients and 50 age- and sex-matched non-TBM controls were measured. These cytokines were estimated in the CSF of TBM patients before and after starting treatment. RESULTS: High levels of proinflammatory cytokines as compared to anti-inflammatory cytokines were found in TBM patients before treatment. However, CSF samples from TBM patients after treatment showed elevated levels of anti-inflammatory and low levels of proinflammatory cytokines. CONCLUSIONS: We hypothesize that an increase in anti-inflammatory cytokines during treatment may indicate a favorable response.

Diagnosis of Chikungunya fever in an Indian population by an indirect enzyme-linked immunosorbent assay protocol based on an antigen detection assay: a prospective cohort study.

A Chikungunya virus (CHIKV) outbreak continues in India. Monitoring of the clinical features of CHIKV infection is an important component of assessing the disease process. Diagnosis is usually made by an immunoglobulin M (IgM)/IgG enzyme-linked immunosorbent assay (ELISA). However, these assays have extremely low sensitivities for the detection of infection in the majority of CHIKV patients during the acute stage of infection (during the 1 to 4 days after infection). In our laboratory, a sensitive ELISA protocol for antigen detection has been developed for the detection of CHIKV infection in the acute stage, and in the present study we assessed the usefulness of this ELISA-based system for the detection of CHIKV infection. We performed a prospective, double-blinded study of 205 Indian patients with suspected CHIKV infection in the Nagpur District. All patients underwent a full clinical assessment, and their serum samples were analyzed for the presence of antigens and of IgM and IgG by an ELISA protocol. In patients with CHIKV infection, the sensitivity of antigen detection was 85%, which was significantly higher (P < 0.001) than that of IgM (17%) or IgG (45%) detection. The sensitivity of IgM (20%) or IgG (25%) detection was significantly lower than that of the antigen assay (95%) for patients with acute infections (i.e., from day 1 to day 5 after infection). Antigen detection not only gives a positive confirmatory result in the early phase of the disease, but it is also useful in the prodromal and subclinical stage and may be useful for field applications for the rapid detection of CHIKV infection.

Inter-alpha-trypsin inhibitor heavy chain 4 is a novel marker of acute ischemic stroke.

BACKGROUND: Inflammation and inflammatory markers play an important role in acute ischemic stroke (AIS). In an earlier study, we discovered a 120-kDa protein that was highly expressed in healthy participants, but either was not expressed or was barely expressed in AIS patients. The purpose of the present work was to isolate, characterize, and evaluate this 120-kDa protein in blood samples of AIS patients. In addition, we investigated how the identified protein compared with protein S-100betabeta, neuron-specific enolase (NSE), and cytokine interleukins IL-2 and IL-10. METHODS: The 120-kDa protein band was analyzed via LC-MS/MS analysis. Then, the 120-kDa protein was evaluated using an enzyme-linked immunosorbant assay in serum samples from AIS patients, which were collected 0, 24, 48, 72 and 144 h after admission. RESULTS: The amino acid sequence of an internal fragment identified the 120-kDa protein as inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4). The ITIH4 protein was completely absent in AIS patients as compared to those in the control group, and serum levels returned to normal in AIS patients as their condition improved. Expression of S-100 betabeta, NSE, IL-2, and IL-10 were highly correlated with ITIH4 expression. CONCLUSIONS: ITIH4 is an anti-inflammatory protein, and suggests that further investigation into its potential use in the diagnosis and prognosis of AIS is warranted.

Evaluation of the IS6110 PCR assay for the rapid diagnosis of tuberculous meningitis.

BACKGROUND: Tuberculous meningitis (TBM) is one of the common clinical manifestations of extra-pulmonary tuberculosis. It is difficult to diagnose due to a lack of rapid, sensitive, and specific tests. Newer methods, which are easy and reliable, are required to diagnose TBM at an early stage. Thus our aim was to evaluate the polymerase chain reaction (PCR) technique, using primers directed against the IS6110 gene, for the detection of Mycobacterium tuberculosis in the CSF, for the diagnosis of TBM patients. METHODS: An in-house IS6110 PCR method using a specific pair of primers designed to amplify the insertion sequence, IS6110, in the M. tuberculosis genome was used to analyze CSF. A total of 80 CSF samples from different groups of patients were studied (confirmed TBM n = 35, clinically suspected TBM n = 16, non-TBM infectious meningitis n = 12, non infectious neurological diseases n = 17). RESULTS: PCR gave a sensitivity of 91.4% and specificity of 75.9% for the diagnosis of TBM in patients with TBM confirmed by culture. In 16 clinically diagnosed, but unconfirmed, TBM cases PCR was positive in 10 (62.5%) cases. There were seven (24.1%) PCR-positive cases among the 29 patients with non-TBM and non-infectious neurological disease. CONCLUSION: We conclude that the performance of an in-house IS6110 PCR assay is valuable in the rapid diagnosis of tuberculous meningitis.

Comparison of an adenosine deaminase assay with ELISA for the diagnosis of tuberculous meningitis infection.

BACKGROUND: Diagnosing tuberculous meningitis (TBM) remains problematic despite many new advanced diagnostic methods. Adenosine deaminase (ADA) assays have emerged as novel alternatives to other costly and time-consuming methods for TBM diagnosis. In the present study the usefulness of the ADA method was assessed for the diagnosis of TBM and compared with an in-house developed ELISA method for detecting the antigen 85 (Ag 85) complex of M. tuberculosis in cerebrospinal fluid (CSF) samples of suspected and culture-confirmed TBM patients. MATERIAL/METHODS: ADA activity in CSF was determined at 37 degrees C according to the method of Guisti and Galanti. ELISA, employing monoclonal antibodies against the purified Ag 85 complex, was used to demonstrate Ag 85 complex in CSF from TBM patients. CSF samples were obtained from 153 patients in three different groups: confirmed TBM (n=27), clinically suspected TBM (n=39), and non-TBM (n=87). RESULTS: The ADA method yielded sensitivity and specificity of 83% and 86%, which are similar to those of the ELISA method (89% and 90%). The correlation between the Ag 85 complex activity in absorbance by ELISA and the ADA activity obtained in units per liter per minute (U/l/min) was also positive and significant. (Pearson's correlation coefficient r=0.3234, 95%CI: 0.1621-0.4679, p<0.001). CONCLUSIONS: This study suggests that the ADA method can be performed for TBM diagnosis in low-income TB-affected regions where more sophisticated facilities are generally not available.