Commercial Enzyme-Linked Immunosorbent Assay Kit Is Useful for Detection of Recombinant and Secretory Nonstructural-1 Protein Antigen of Dengue Virus.

Background: Dengue is a mosquito-borne tropical disease, caused by the Dengue virus (DENV). It has become a severe problem and is a rising threat to public health. In this study, we have evaluated commercial Merilisa i Dengue NS1 Antigen kit (Meril LifeSciences India Pvt. Ltd.) to detect recombinant dengue virus 2 NS1 antigen (rDNS1Ag) and secreted forms of NS1 antigen (sDNS1Ag). Methods: To determine the detection limit of the kit, 100 nanogram (ng) to 0.001 ng rDNS1Ag was tested. The sensitivity and specificity of the kit was determined using recombinant NS1 antigens of all serotypes of DENV and other flaviviruses. For testing sDNS1Ag, the culture supernatant of the Vero cell lines infected with DENV-2 was tested. Further, a spiking experiment was carried out to check the sensitivity of the kit to detect rDNS1Ag in the pools of Aedes aegypti mosquitoes. Results: It was observed that the kit can detect the rDNS1Ag at 1 ng concentration. The kit was sensitive to detect NS1 antigen of DENV-1, DENV-2 and DENV-3 serotypes and specific for detection of only DNS1Ag as it did not cross-react with NS1 antigen of flaviviruses. The kit was sensitive to detect rDNS1Ag in the mosquito pools as well. In addition, the kit was able to detect the sDNS1Ag in Vero cell culture supernatant. Conclusions: Overall, we observed that the Merilisa i Dengue NS1 Ag kit is sensitive and specific for the detection of DNS1Ag both in recombinant and secretory forms.

Detection of Wuchereria bancrofti infection in mosquitoes in areas co-endemic with Brugia malayi in Balasore district, Odisha, India.

Lymphatic filariasis (LF) is a crippling and disfiguring parasitic condition. India accounts for 55% of the world's LF burden. The filarial parasite Wuchereria bancrofti is known to cause 99.4% of the cases while, Brugia malayi accounts for 0.6% of the issue occurring mainly in some pockets of Odisha and Kerala states. The Balasore (Baleswar) district of Odisha has been a known focus of B. malayi transmission. We employed molecular xenomonitoring to detect filarial parasite DNA in vectors. In six selected villages, Gravid traps were used to collect Culex mosquitoes and hand catch method using aspirators was followed for collection of mansonioides. A total of 2903 mosquitoes comprising of Cx. quinquefasciatus (n = 2611; 89.94%), Cx. tritaeniorhynchus (n = 100; 3.44%), Mansonia annuliferea (n = 139; 4.78%) and Mansonia uniformis (n = 53; 1.82%) were collected from six endemic villages. The species wise mosquitoes were made into 118 pools, each with a maximum of 25 mosquitoes, dried and transported to the laboratory at VCRC, Puducherry. The mosquito pools were subjected to parasite DNA extraction, followed by Real-time PCR using LDR and HhaI probes to detect W. bancrofti and B. malayi infections, respectively. Seven pools (6.66%) of Cx. quinquefasciatus, showed infection with only W. bancrofti while none of the pools of other mosquito species showed infection with either W. bancrofti or B. malayi. Although the study area is endemic to B. malayi, none of the vectors of B. malayi was found with parasite infection. This study highlights the ongoing transmission of bancroftian filariasis in the study villages of Balasore district of Odisha and its implications for evaluating LF elimination programme.

Detection of Wuchereria bancrofti DNA in wild caught vector and non-vector mosquitoes: implications for elimination of lymphatic filariasis.

BACKGROUND: Transmission Assessment Survey (TAS) is the WHO recommended method used for decision-making to stop or continue the MDA in lymphatic filariasis (LF) elimination programme. The WHO has also recommended Molecular Xenomonitoring (MX) of LF infection in vectors as an adjunct tool in settings under post-MDA or validation period. Screening of non-vectors by MX in post-MDA / validation settings could be useful to prevent a resurgence of LF infection, as there might be low abundance of vectors, especially in some seasons. In this study, we investigated the presence of LF infection in non-vectors in an area endemic for LF and has undergone many rounds of annual MDA with two drugs (Diethylcarbamazine and Albendazole, DA) and two rounds of triple drug regimens (Ivermectin + DA). METHODS AND RESULTS: Mosquitoes were collected from selected villages of Yadgir district in Karnataka state, India, during 2019. A total of 680 female mosquitoes were collected, identified morphologically by species and separated as pools. The female mosquitoes belonging to 3 species viz., Anopheles subpictus, Culex gelidus and Culex quinquefaciatus were separated, pooled, and the DNA extracted using less expensive method and followed by LDR based real-time PCR assay for detecting Wuchereria bancrofti infection in vector as well as non-vector mosquitoes. One pool out of 6 pools of An. subpictus, 2 pools out of 6 pools of Cx. gelidus, and 4 pools out of 8 pools of Cx. quinquefaciatus were found to be positive for W. bancrofti infection by RT-PCR. The infection rate in vectors and non-vectors was found to be 1.8% (95% CI: 0.5-4.2%) and 0.9% (95% CI: 0.2-2.3%), respectively. CONCLUSIONS: Our study showed that non-vectors also harbour W. bancrofti, thus opening an opportunity of using these mosquitoes as surrogate vectors for assessing risk of transmission to humans in LF endemic and post MDA areas.

Detection of recombinant dengue virus 2 NS1 protein in Aedes aegypti mosquitoes using commercial Dengue NS1 ELISA kit.

Dengue, a vector-borne disease remains as one of the most serious public health problems globally. Incidence of this disease is on an increasing trend and currently over a billion people in tropical and subtropical regions are at risk. In the absence of an operational vaccine, prevention of dengue virus (DENV) is primarily focused upon controlling mosquito vectors. Mosquito vector surveillance programmes require simple and rapid tools to detect mosquitoes infected with DENV. Here, we tested the commercially available DENV Detect™ NS1 ELISA kit (InBios International, Inc.) for detection of recombinant DENV-NS1 protein in Aedes mosquito samples. The kit was evaluated to find out the minimum detection limit of recombinant DENV-2 NS1 protein following the manufacturer's instructions. Initially, the NS1 protein detection threshold of the kit was determined and later the assay was standardized for detection of NS1 protein in Aedes aegypti mosquito pools containing 5, 10 and 25 mosquitoes. The ELISA kit displayed high sensitivity towards detection of recombinant dengue virus-2 NS1 protein in mosquito pools (up to 25 mosquitoes per pool) at 25 pico gram concentration. Since the commercial NS1 ELISA is highly sensitive and follows a very simple procedure, it could be employed for DENV surveillance in Aedes aegypti mosquitoes, after carrying out laboratory and field bioassays with DENV infected specimens.

Dengue NS1 antigen kit shows high sensitivity for detection of recombinant dengue virus-2 NS1 antigen spiked with Aedes aegypti mosquitoes.

Dengue, caused by the dengue virus (DENV) is a significant vector-borne disease. In absence of a specific treatment and vaccine, dengue is becoming a rising threat to public health. Currently, control of dengue mainly focuses on the surveillance of the mosquito vectors. Improved surveillance methods for DENV in mosquito populations would be highly beneficial to the public health. However, current methods of DENV detection in mosquitoes requires specialized equipment and expensive reagents and highly trained personnel. As an alternative, commercially available dengue NS1 antigen ELISA kits could be used for detection of DENV infection in Aedes aegypti mosquitoes. In this study, we explored the utility of commercially available Dengue NS1 antigen kit (J. Mitra & Co. Pvt. Ltd) for the detection of recombinant dengue virus-2 (rDENV-2) NS1 protein and serum of dengue infected patient spiked with Ae. aegypti mosquito pools. The kit was found to be highly sensitive and specific towards detection of all serotypes of DENV. Further, it could detect as low as 750 femto gram rDENV-2 NS1 protein. It was also observed that rDENV-2 NS1 antigen spiked with blood-fed and unfed mosquito pools could be detected. In addition, the kit also detected dengue infected patient serum spiked with Ae. aegypti mosquito pools. Overall, the Dengue NS1 antigen kit displayed high sensitivity towards detection of recombinant as well as serum NS1 protein spiked with Ae. aegypti mosquito pools and could be considered for the dengue virus surveillance after a field evaluation in Ae. aegypti mosquitoes.

Aptamers: An Emerging Tool for Diagnosis and Therapeutics in Tuberculosis.

Tuberculosis (TB) has been plaguing human civilization for centuries, and currently around one-third of the global population is affected with TB. Development of novel intervention tools for early diagnosis and therapeutics against Mycobacterium tuberculosis (M.tb) is the main thrust area in today's scenario. In this direction global efforts were made to use aptamers, the chemical antibodies as tool for TB diagnostics and therapeutics. This review describes the various aptamers introduced for targeting M.tb and highlights the need for development of novel aptamers to selectively target virulent proteins of M.tb for vaccine and anti-TB drugs. The objective of this review is to highlight the diagnostic and therapeutic application of aptamers used for tuberculosis. The discovery of aptamers, SELEX technology, different types of SELEX development processes, DNA and RNA aptamers reported for diseases and pathogenic agents as well have also been described in detail. But the emphasis of this review is on the development of aptamers which can block the function of virulent mycobacterial components for developing newer TB vaccine candidates and/or drug targets. Aptamers designed to target M.tb cell wall proteins, virulent factors, secretory proteins, or combination could orchestrate advanced diagnosis and therapeutic measures for tuberculosis.

Coronavirus disease 2019 in children: Clinical & epidemiological implications.

Despite the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there are limited data emerging in children. This review provides an update on clinical features, diagnosis, epidemiology, management and prevention of coronavirus disease 2019 (COVID-19) in children. Specific characteristics noted in children and their implications in disease management as well as transmission control are highlighted. Besides respiratory symptoms, gastrointestinal and atypical features such as chilblains, neurological symptoms and multisystem inflammation are also reported. Younger infants and those with comorbidity were found to be at risk of severe illness. Infected pregnant women and neonates were reported to have good prognosis. It is possible to manage the children with mild disease at home, with strict infection prevention control measures; severely affected require respiratory support and intensive care management. There are anecdotal reports of using antiviral and immunomodulatory drugs, benefit of which needs to be confirmed in clinical trials. A significant percentage of asymptomatic infection in children has epidemiological implication as these may act as links in transmission chain in the community. There is a need for systematic data on extra-pulmonary manifestations and atypical features, risk factors of severity, role of imaging and biomarkers, testing and management strategies and trials with antivirals and immunomodulatory drugs in children. The psychosocial effects of quarantine, closure of schools, lack of play activities and impact of lockdown need to be addressed. Understanding the biological basis for the profound age-dependent differential outcome of COVID-19 infection is important. Elucidating the protective mechanisms in children may aid in developing novel treatment strategies.

PPE17 (Rv1168c) protein of Mycobacterium tuberculosis detects individuals with latent TB infection.

Latent tuberculosis infection (LTBI) is a clinically distinct category of Mycobacterium tuberculosis (Mtb) infection that needs to be diagnosed at the initial stage. We have reported earlier that one of the Mtb proline-proline-glutamic acid (PPE) proteins, PPE17 (Rv1168c) is associated with stronger B-cell and T-cell responses and could be used to diagnose different clinical categories of active TB patients with higher specificity and sensitivity than PPD and ESAT-6. Based on these observations we further tested the potential of PPE17 for the diagnosis of LTBI. We tested 198 sera samples collected from LTBI individuals (n = 61), QFT-negative (n = 58) and active TB patients (n = 79). Individuals were defined as LTBI by QuantiFERON-TB Gold In-Tube test (QFT-GIT) positive results, while active TB patients were confirmed based on the guidelines of the Revised National TB Control Programme of India. The antibody responses against PPE17, ESAT-6:CFP-10 and PPD were compared in these subjects by enzyme-linked immunosorbent assay. We observed that LTBI individuals show a higher sero-reactivity to PPE17 as compared to currently used latent TB diagnostic antigens like ESAT-6, CFP-10 and PPD. The LTBI and active TB patients display almost similar sensitivity. Interestingly, PPE17 could discriminate LTBI positive subjects from the QFT-negative subjects (P < 0.001). Our study hints that PPE17 may be used as a novel serodiagnostic marker to screen the latently infected subjects and may also be used as a complimentary tool to the QFT-GIT.

The N-terminal domain of Mycobacterium tuberculosis PPE17 (Rv1168c) protein plays a dominant role in inducing antibody responses in active TB patients.

The PPE (proline-proline-glutamic acid) proteins of Mycobacterium tuberculosis are characterized by a conserved N-terminal domain of approximately 180 amino acids and variable C-terminal domain. Since last decade, these proteins have gained much importance in the serodiagnosis of tuberculosis (TB) as they act as a source of antigenic variation. We have demonstrated earlier that one of the PPE proteins PPE17 (Rv1168c) induces strong B-cell and T-cell responses in active TB disease and also displays a higher antibody titer compared to immunodominant antigens such as ESAT-6, Hsp60 and PPD. However, the immunodominant domain of PPE17 (N-terminal or C-terminal) was not examined in detail. In the present study, we observed that antibody responses elicited in TB patients were directed mostly towards the N-terminal domain of PPE17 (N-PPE17). The antibody generated against N-PPE17 in TB patients did not significantly cross-react with N-terminal domains of other PPE proteins used in this study. Our data suggest that the N-terminal domain of PPE17 protein is immunodominant and could be used as a better serodiagnostic marker than the full-length PPE17 protein.

The Mycobacterium tuberculosis PPE protein Rv1168c induces stronger B cell response than Rv0256c in active TB patients.

Tuberculosis (TB) caused by Mycobacterium tuberculosis is a serious global health problem and is responsible for millions of deaths every year. For effective control of this dreadful disease, it is necessary to diagnose TB cases at the initial stages of infection. The serodiagnosis of disease represents simple, rapid and inexpensive method that can be used at the primary health care levels. In this study we have compared sensitivity of two PPE proteins of M. tuberculosis, i.e., Rv0256c and Rv1168c for their use as serodiagnostic markers in active tuberculosis patients. Employing a standardized enzyme immunoassay with these PPE proteins as candidate antigens we were able to successfully discriminate the TB patients' sera from the BCG-vaccinated healthy controls. Further, we observed that Rv1168c displayed higher sensitivity in detecting extrapulmonary and smear negative pulmonary TB cases which are difficult to diagnose by available diagnostic methods. Overall the study highlights that Rv1168c can be used as a potential serodiagnostic marker for the diagnosis of active tuberculosis disease.

Mycobacterium tuberculosis PPE protein Rv0256c induces strong B cell response in tuberculosis patients.

Tuberculosis (TB) is one of the most important diseases of humans and major public health problem worldwide. Early and accurate diagnosis of TB is necessary for the treatment, prevention, and control of TB. Therefore, it is important to identify suitable antigens that can differentiate active tuberculosis patients from BCG-vaccinated individuals. In the present study, we have used Rv0256c (PPE2) protein of Mycobacterium tuberculosis to screen the sera of infected patients belonging to different clinical TB presentations, and BCG-vaccinated clinically healthy individuals by enzyme immunoassay. Our results demonstrated that Rv0256c displayed stronger and specific immunoreactivity against the sera obtained from clinically active tuberculosis patients compared to PPD and ESAT-6 and could differentiate the TB-patients from the BCG-vaccinated controls. Importantly, Rv0256c was also found to detect even the extrapulmonary and smear-negative pulmonary cases which often are tedious and difficult to detect using conventional diagnostic methods. This study suggests that Rv0256c can be used as a potential marker for the serodiagnosis of tuberculosis patients.

Prevalence of Mycobacterium tuberculosis with multiple copies of IS6110 elements in Gulbarga, South India.

This study was carried out to ascertain the prevalence of Mycobacterium tuberculosis by insertion sequence 6110 (IS6110) based DNA fingerprinting method in Gulbarga district belonging to the southern part of India. Results showed that among the 52 M. tuberculosis isolates studied, 57.7% exhibited more than 5 copies of IS6110 showing the prevalence of M. tuberculosis with multiple copies of IS6110 elements.

A review on peripheral blood CD4+ T lymphocyte counts in healthy adult Indians.

The CD4+ T lymphocytes are the crucial cells in the cascade of events in forming immune response to the foreign antigen and hence monitoring the CD4+ T cell counts to understand the extent of immune deficiency is a common practice. CD4+ T cells are also the primary target cells for human immunodeficiency virus (HIV). Hence CD4+ T lymphocyte count is the most important marker of immune dysfunction in HIV disease progression. The estimation of CD4+ T cell counts is used to decide the initiation of anti retroviral therapy (ART), to monitor the efficacy of ART and to start treatment for opportunistic infections (OIs). To develop the threshold levels of CD4+ T cell counts, data from western countries are being used in India. The CD4+ T cell counts are known to be influenced by race and environmental factors. Hence it is important to establish the reference ranges for the CD4+ T cell counts in the target population to understand the immune dysfunction. The information on the lower limits of the CD4+ T cells count is necessary to decide the initiation and monitoring of ART. The published data on the CD4+ T cells count in healthy Indian adult population have been reviewed, analyzed and discussed in this review article. The requirement of establishment of reference ranges in Indian population is discussed.