Antibody responses to the BBV152 vaccine in individuals previously infected with SARS-CoV-2: A pilot study.

Background & objectives: Vaccination against SARS-CoV-2 is a recommendation from the World Health Organization as the foremost preference in the current situation to control the COVID-19 pandemic. BBV152 is one of the approved vaccines against SARS-CoV-2 in India. In this study, we determined SARS-CoV-2-specific antibody levels at day 0 (baseline, before vaccination), day 28 ± 2 post-first dose (month 1) and day 56 ± 2 post-first dose (month 2) of BBV152 whole-virion-inactivated SARS-CoV-2 recipients, and compared the antibody responses of individuals with confirmed pre-vaccination SARS-CoV-2 infection to those individuals without prior evidence of infection. Methods: Blood samples were collected from 114 healthcare professionals and frontline workers who received BBV152 vaccine from February to May & June 2021. Prior infection with SARS-CoV-2 was determined at baseline. Serum samples were used to estimate SARS-CoV-2 nucleoprotein-specific IgG [IgG (N)], spike protein-specific IgG [IgG (S)] and neutralizing antibodies (NAb). Results: Participants with previous SARS-CoV-2 infection after a single vaccine dose elicited IgG (N) and IgG (S) antibody levels along with NAb binding inhibition responses levels were similar to infection-naïve vaccinated participants who had taken two doses of vaccine. Interpretation & conclusions: Our preliminary data suggested that a single dose of BBV152-induced humoral immunity in previously infected individuals was equivalent to two doses of the vaccine in infection-naïve individuals. However, these findings need to be confirmed with large sized cohort studies.

Genotype analysis of ofloxacin-resistant multidrug-resistant Mycobacterium tuberculosis isolates in a multicentered study from India.

Background & objectives: Drug resistance surveillance offers useful information on trends of drug resistance and the efficacy of control measures. Studies and reports of drug-resistant mutations and phenotypic assays thus become important. This study was conducted to investigate the molecular characteristics of ofloxacin (OFX)-resistant, multidrug-resistant tuberculosis (MDR-TB) isolates from different geographical regions of India and their association with strains of different genotypes. Further, the nitrate reductase assay (NRA) was tested against Mycobacteria Growth Indicator Tube(MGIT) for the determination of OFX resistance as an alternative and cost-effective method. Methods: A total of 116 Mycobacterium tuberculosis isolates were used to assess the mutations in the gyrA, gyrB genes and resistance levels to OFX. Mutational analysis in gyrA and gyrB genes and genotype analysis of M. tuberculosis isolates was done by gene-specific polymerase chain reaction (PCR) followed by DNA sequencing and spoligotyping, respectively. Results: Three (6.25%), 12 (44.44%) and 12 (29.27%) MDR-TB isolates from western, northern and southern India, respectively, were found to be OFX-resistant MDR-TB isolates. OFX resistance was observed to be significantly higher in MDR-TB cases for all study regions. Beijing genotypes from northern India were observed to be associated with OFX-resistant MDR-TB cases (P <0.05). Among 35 (30.15%) phenotypically OFX-resistant isolates, 22 (62.86%) had mutations in the gyrA gene and two (5.71%) isolates had mutations in the gyrB gene. Interpretation & conclusions: These results caution against the PCR-based prediction of OFX resistance patterns and highlight the need for searching other genetic loci for the detection of mutations conferring resistance to OFX in M. tuberculosis. Our study also showed the usefulness of NRA as an alternative method to detect OFX resistance.

Spoligotyping of Mycobacterium tuberculosis isolates at a tertiary care hospital in India.

OBJECTIVE: Spoligotyping is a valuable genotyping tool to study the genetic diversity and molecular epidemiology of Mycobacterium tuberculosis (M. tb). The aim of this study was to analyse different spoligotype patterns of M. tb strains isolated from patients with tuberculosis from different parts of India. MATERIALS AND METHODS: A total of 163 M. tb isolates were spoligotyped between January 2014 and January 2015. About 47% (n = 77) were from patients with extrapulmonary tuberculosis; of these, 10 were MDR, and seven were Pre-XDR. Of the 86 M. tb isolates from patients with pulmonary tuberculosis, 25 were MDR, and 25 were Pre-XDR. RESULTS: We found 61 spoligo patterns, 128 clusters in the spoligotype data base (spoldb4 data base) with spoligo international type (SIT) number and 35 true unique isolates. The most pre-dominant spoligotype was EAI lineage (56), followed by Beijing (28), CAS (20), T(9), U(7), X(3), H(3), BOVIS_1 BCG(1) and LAM(1). CONCLUSION: Although our study identified EAI, CAS and Beijing strain lineages as pre-dominant, we also found a large number of orphan strains (20%) in our study. Beijing strains were more significantly associated with MDR TB than CAS and EAI lineages. Further studies on large sample sizes would help to clearly describe the epidemiology of M. tb in India.

Enhancing antimycobacterial activity of isoniazid and rifampicin incorporated norbornene nanoparticles.

Background: Tuberculosis (TB) has been identified in skeletons over 6000 years old and still remains as the most prevalent infectious disease in the world; thus, there is a need for development of new drugs or tuning of old drugs. Nanotechnology, an advanced technology, plays a vital role in research for the diagnosis and treatment of TB, thus preventing adverse effects and drug resistance. The objective of this study was to enhance the antimycobacterial activity of isoniazid- (INH) and rifampicin (RIF)-incorporated norbornene (NOR) nanoparticles in comparison with plain INH and RIF without nanoparticles. Methods: Norbornene-polyethylene glycol - Isoniazid copolymer (NOR-PEG-INH) and norbornene polyethylene rifampicin Co polymer (NOR-PEG-RIF) were used for this study. The percentage of INH and RIF in NOR nanoparticles was 35% and 74%, respectively. Mycobacterium growth indicator tube containing Middlebrook 7H9 broth, the liquid medium, was used to analyze in vitro activity of the NOR-based drug and the plain drug. Minimum inhibitory concentration (MIC) of the drugs was determined from H37Rv control strain of mycobacterium TB (MTB). Results: The dosage of INH and RIF is minimal in the combination form with the NOR nanoparticles compared to the plain INH and RIF. The results indicate that the minimum concentration of NOR-PEG-INH and NOR-PEG-RIF required inhibiting H37Rv strain of MTB was 0.05 µg/ml and 0.5 µg/ml, respectively. The results were similar to plain INH and RIF MIC. Conclusion: Low dosage of INH and RIF along with NOR nanocarrier has similar activity to that of INH and RIF; thus this is expected to reduce adverse effects and NOR did not alter the functional activity of INH and RIF, thus becoming eligible for the newer drug carrier in TB treatment.

Specific and early detection of IgG, IgA and IgM antibodies to Mycobacterium tuberculosis 38kDa antigen in pulmonary tuberculosis.

The objective was to apply the purified 38kDa protein antigen of Mycobacterium tuberculosis in ELISA to estimate the IgG, IgA and IgM antibody levels in sera and circulating immune complexes of tuberculosis patients. Sera from smear and culture positive tuberculosis patients were positive for anti 38kDa IgG, IgA and IgM antibodies, with a sensitivity of 61%, 30% and 10%, respectively, and with a specificity of 100% for IgG. The sensitivity of the test improved to a level of 68% for IgG+IgA and of 71.4% for IgG+IgA+IgM without significantly compromising the specificity (IgG of 100%, IgG+IgA of 96%, IgG+IgA+IgM of 90%). Among the smear, culture-negative but X-ray-positive cases, 60% were serum positive for IgG antibody, while in smear-negative but culture-positive cases, 54% were positive for IgG antibody. Measurement of 38kDa antibodies showed a greater than 95% sensitivity in smear and culture-positive, and smear-negative and culture-positive patients, through a combination of assays for serum IgG and circulating immune complex antibodies, while the specificity was 100%.

Antibody response to Mycobacterium tuberculosis 30 and 16kDa antigens in pulmonary tuberculosis with human immunodeficiency virus coinfection.

There is urgent need for rapid diagnostic methods to identify tuberculosis among the HIV positive cases, since the mortality is high. We have isolated and evaluated the serodiagnostic potential of the 30kDa secreted antigen and 16kDa cytosolic antigen of M. tuberculosis, among the HIV-TB patients. Antibody response was studied using Enzyme linked immunosorbent assay. In the HIV-TB group, antibody was found to be 65%, 69% and 6% positive for IgG, A and M isotypes, respectively, against 30kDa. The sensitivity increased to 84%, upon combination of the results of 3 isotypes. Anti-16kDa was detected in 15% (G), 50% (A) and 3% (M) of cases. Combination of results improved the positivity to 57%. There was no difference in antibody response among the HIV-TB cases, related to CD4 counts. Thus the 30kDa antigen proved to be of diagnostic utility in HIV-TB.

Improved diagnosis of pulmonary tuberculosis by detection of free and immune complex-bound anti-30 kDa antibodies.

The 30 kDa secreted antigen of Mycobacterium tuberculosis was purified to homogeneity by serial chromatography, and enzyme linked immunosorbent assay (ELISA) was used to evaluate its diagnostic value in patients with pulmonary tuberculosis. The immunoglobulin (Ig) antibodies G, A, and M were estimated in the two groups: patients who were smear- and culture-positive (S+C+) for pulmonary tuberculosis and normal healthy subjects (NHS). Sensitivity of 67.4%, 14.8%, and 14.3%, with the specificity of 99%, 96.7%, and 92% were obtained for the 3 isotypes respectively. Combination of the results of IgG and IgA increased the sensitivity to 71%, with 97% specificity. Polyethylene glycol precipitation of the circulating immune complexes (CIC) in sera was carried out. The CIC bound antibodies offered a sensitivity of 92.5%, 85.4%, and 68.7%, respectively for the S+C+, S-C+, and S-C- patients, while the specificity was 96.6%. Thus CIC-bound antibodies promise to be a better diagnostic tool in the detection of tuberculosis.

Isolation and evaluation of diagnostic value of two major secreted proteins of Mycobacterium tuberculosis.

Two secreted antigens of Mycobacterium tuberculosis, namely the antigen 85 complex (30/31) and 38kDa antigens, were purified from the whole culture filtrate by using two dimensional preparative electrophoresis and anion exchange chromatography, respectively. Individual components of the antigen 85 complex namely, antigen 85A, 85B and 85C, were separated using hydrophobic interaction chromatography. The humoral antibody activity to these antigens in sputum positive cases of active pulmonary tuberculosis and normal healthy volunteers was determined by enzyme linked immunosorbent assay (ELISA) and immunoblot. Recombinant 38kDa and antigen 6 were used as reference antigens for the assay. None of the healthy volunteers reacted with the 38kDa antigen, while 52% of the TB sera reacted with it. Of the three components of the antigen 85 complex, 85B gave the highest positivity of 40 per cent. The results of combination of 38kDa with antigen 6 offered better results with 76% positivity.

Isotype-specific anti-38 and 27 kDa (mpt 51) response in pulmonary tuberculosis with human immunodeficiency virus coinfection.

There is a need for rapid diagnostic methods to identify tuberculosis among human immunodeficiency virus-positive cases (HIV-TB). This study evaluated the serodiagnostic potential of the native 38 kDa and recombinant 27 kDa (mpt 51) antigens of Mycobacterium tuberculosis purified in the laboratory, when applied to HIV-TB patients. The antibody response was studied using enzyme-linked immunosorbent assays (ELISA). In the HIV-TB group, anti-38 kDa antibody of the immunoglobulin G (IgG), IgA and IgM isotypes was found in 38%, 43% and 7%, of patients, respectively. Antibodies to the 27 kDa antigen occurred in 50%, 31%, and 1%, for IgG, IgA and IgM, respectively. The sensitivity increased upon combination of the results of IgG and IgA isotypes for each of the antigens, without compromising specificity. When the results were analysed based on the smear positivity, 71-78% and 54-69% were positive among smear-positive and smear-negative HIV-TB cases, respectively. A higher sensitivity (71% and 69%) was obtained using the 27 kDa antigen. The use of both antigens offered a sensitivity of 82% in smear-positive and 69% in smear-negative cases. There was no difference in antibody response among the HIV-TB cases, related to CD4 counts. Thus, the combination of the 38 and 27 kDa (mpt 51) antigens proved to be of diagnostic utility in HIV-TB, irrespective of the severity of immunosuppression, in smear-positive and smear-negative TB.

Immunoglobulin G, A, and M responses in serum and circulating immune complexes elicited by the 16-kilodalton antigen of Mycobacterium tuberculosis.

The 16-kDa cytosolic antigen of M. tuberculosis was purified to homogeneity by molecular sieving chromatography, and the diagnostic potential of the antigen was evaluated in various categories of patients by enzyme-linked immunosorbent assay (ELISA). The immunoglobulin G (IgG), IgA, and IgM antibody levels to 16-kDa antigen were estimated in the two polar groups, namely, smear- and culture-positive pulmonary tuberculosis (S(+)C(+)) patients and healthy subjects (HS). Sensitivities of 62, 52 and 11% with specificities of 100, 97, and 95% were obtained for the three isotypes, respectively. The total number of positives by a combination of the three isotypes was analyzed in the polar groups, and the sensitivity improved to 83% with a specificity of 93%. Even when a combination of IgG and IgA alone was considered, the sensitivity was 82% with a specificity of 97%. Polyethylene glycol precipitation of the circulating immune complex (CIC) in sera was carried out. The CIC-bound antibodies to 16-kDa antigen were assessed by ELISA in the S(+)C(+), S(-)C(+), and S(-)C(-) categories of patients. Measuring the IgG-IgA-IgM combination positivities of the CIC-bound antibodies gave sensitivities of 97.5, 100, and 45.3%, respectively. The specificity of the assay with these combinations was maintained at 95.4%.