Detection and Significance of Anti-Mycobacterium tuberculosis-specific Immunoglobulin G Antibody Response for the Diagnosis of Pulmonary Tuberculosis Using Enzyme-linked Immunosorbent Assay.

Rapid diagnosis of pulmonary tuberculosis(TB) infection is critical in clinical practice. To establish an effective serological diagnostic technique, we generated the several Mycobacterium tuberculosis(MTB)-specific immunogenic antigens and evaluated the clinical benefits of detection of immunoglobulinG (IgG) and IgM antibodies raised against these target antigens for the diagnosis of patients with active TB. The genes encoding the MTB-specific antigens 6-kDa early secretory antigenic target of MTB (ESAT-6), 10-kDa culture filtrate protein (CFP-10), ESX-1 substrate proteinC (ESPC), 14KD/38KD and ESAT-6/14KD/38KD, were amplified from the MTB genome by PCR. Prokaryotic vectors were constructed for the expression of the individual MTB antigens. The target recombinant protein was expressed in Escherichiacoli (BL21/DE3) and purified using immobilized metal affinity chromatography (IMAC). An ELISA based immunoassay was set up using these target antigens for the diagnosis of active TB. The detection samples included 98patients with active TB and 102healthy control volunteers. The cutoff OD value for IgG and IgM antibodies was selected according to a receiver operating characteristic (ROC) analysis. The sensitivity, specificity and positive likelihood ratio were also determined. We successfully cloned, expressed and purified the ESAT-6, CFP-10, ESPC, 14KD/38KD and ESAT‑6/14KD/38KD recombinant antigens of MTB. The mean levels of IgG antibodies were significantly higher in patients with pulmonary TB compared with control groups. The target MTB-specific antigens can distinguish a TB infection from a non-TB infection, showing significant difference in statistics (P<0.001). The sensitivity of the IgG test ranged from 69.4% (ESAT-6) to 77.6% (ESAT-6/14KD/38KD) in the active TB patients; the specificity of assays varied from 78.4% (CFP-10) to 90.2% (14KD+38KD) in the healthy control groups. The IgM antibody test can not distinguish a TB infection from a non-TB healthy control. In conclusion, clinical use of the ESAT-6, CFP-10, ESPC, 14KD/38KD and ESAT-6/14KD/38KD antigens based on serodiagnostic IgG assay is of significant value for the rapid diagnosis of TB and for the discrimination between active TB patients and healthy controls.

The immunoglobulin G (IgG) and IgA antibody responses to different Schistosoma mansoni antigens have been determined in chronically infected mice as well as in unisexually infected animals. With a panel of enzyme-linked immunosorbent assays (ELISAs), soluble antigens from furcocercariae, adult worms, and eggs were probed with sera collected at 3-week intervals. Bisexually infected animals developed significant IgG and IgA antibody responses to the antigens tested, which increased after egg deposition. In unisexual infections no significant differences were recorded in the IgG antibody profile for furocercaria and adult worm antigens, whereas the IgA antibody response was impaired. Both the IgA and IgG antibody responses toward egg antigens were reduced compared with those in a bisexual infection. Furthermore, a specific mucosal IgA antibody response was observed only in the bisexually infected animals. Histological analysis performed on bisexually infected mice led to the observation of eggs and granulomatous lesions within the Peyer's patch follicles, which are essential sites for the induction of mucosal immunity in the intestine. These data suggest a relationship between egg deposition and the induction of the IgA antibody response toward schistosomes.

Studies assessing the immunoglobulin G (IgG) antibody responses in severe malaria are not readily available. This study was designed to compare the IgG and IgG1-4 antibody responses in severe malaria and its major clinical presentations (cerebral malaria, severe malarial anemia and respiratory distress) in children (≤15 years) in Buea, Cameroon. In a hospital-based cross-sectional comparative study, children presenting for consultation at the outpatient department/Emergency unit of the Buea Regional Hospital were enrolled and assigned into one of three groups: severe malaria, uncomplicated malaria and negative controls. Baseline characteristics were determined; blood glucose level was measured by glucometer, complete blood count was performed using an automated heamatology analyser and participants were screened for malaria parasites by light microscopy and severe malaria was categorized based on WHO criteria. Total IgG and IgG1-4 antibodies were measured using standard ELISA with Plasmodium falciparum 19-KDa C-terminal region of merozoite surface protein 1 (P.fMSP-119) antigen as capture antigen. A total of 236 participants were enrolled comprising: 66 severe malaria, 70 uncomplicated malaria and 100 negative controls. The participants in the different groups were similar with regards to their ages (p=0.06) and gender (p=0.900). Children with severe malaria had significantly higher levels of anti-P.fMSP-119 IgG4 (p&lt;0.0001) antibodies and significantly lower levels of anti-P.fMSP-119 IgG1 (p&lt;0.0001) and IgG3 (p&lt;0.0001) antibodies. There was no significant variation in the IgG antibody responses between the major clinical forms of severe malaria. The study finding of significantly higher levels of the non-cytophilic antibody IgG4 is suggestive of the role the antibody plays in the pathogenesis of severe malaria. Larger studies investigating how these immune effector cells vary in the major phenotypes of severe malaria are recommended.

To the Editor: The development of effective COVID-19 vaccines has been essential in slowing the spread of SARS-CoV-2. However, unvaccinated populations as well as those who do not respond to vaccination still remain at risk. Very few cancer patients were included in the COVID-19 mRNA vaccine trials and any individuals receiving chemotherapy or immunotherapy within 6 months were excluded.1 Consequently, we have an inadequate knowledge of how well these vaccines work in the cancer patient population. However, by extrapolation from other vaccines, we hypothesized that patients with hematologic malignancies, especially those on immunosuppressive therapy, would produce poor serological responses to a COVID-19 vaccine.2 In this single-center, observational cohort study we assessed antibody responses in lymphoma patients receiving a COVID-19 mRNA vaccine (BNT162b2, BioNTech/Pfizer, Germany/New York, NY; or mRNA-1273, Moderna, Cambridge, MA). All patients provided written informed consent to participate in observational research, and this study was approved by the Weill Cornell Medicine institutional review board (IRB 21-02023288). Serum samples were obtained before (when possible) and after vaccination. Post-vaccination samples were collected within 11–70 days of the second dose (median 24.5 days). In the healthcare worker (HCW) control group, the post-vaccination samples were obtained within 10–68 days of the second dose (median 40 days) (Figure S1). We also include data from a healthy control group of 35 HCWs enrolled in the NYP-WELCOME (WEilL COrnell Medicine Employees) observational trial (IRB 20-04021831). The use of this cohort in an mRNA vaccine study as well as the assay to quantify immunoglobulin G (IgG) antibodies to the SARS-CoV-2 S-protein has been described previously.3 Additionally, we determined whether any patients had serum antibodies to the SARS-CoV-2 nucleocapsid (N) protein, a marker for prior infection. The anti-S protein response to mRNA vaccination was assessed by enzyme-linked immunosorbent assay using sera from 67 patients with lymphoma and 35 healthy HCW controls. The majority of patients in this study were white (74.6%, Table S1). The median age of the study group was 71 (24–90). The most common comorbidities were hypertension (37.3%) and hyperlipidemia (50.7%). All patients were vaccinated with an mRNA vaccine (31 BNT162b2 and 36 mRNA-1273). The patients were categorized as having Hodgkin lymphoma (NHL; n = 4), chronic lymphocytic leukemia (CLL; n = 21), or other non-Hodgkin lymphomas (n = 42). Patients with other non-Hodgkin lymphomas included follicular lymphoma (7), marginal zone lymphoma (10), mantle cell lymphoma (8), diffuse large B-cell lymphoma (8), Waldenstrom macroglobulinemia (7), and other, unclassified lymphomas (2). No SARS-CoV-2 infections were identified during this study (February to April 2021). The vaccine-induced IgG antibody responses to the SARS-CoV-2 S-protein are shown in Figure 1(A). The median and mean endpoint titers in the HCW control group were higher than in the lymphoma patients, although the difference was not significant. There were also no significant differences in mean titers when patients with different lymphomas were compared. However, while all 35 healthy control group members responded to the vaccine, a substantial proportion of the lymphoma patients did not. Thus, the anti-S endpoint titers in nine of the 21 CLL patients and 17 of the 42 other NHL patients were <10 000 (a cut-off level marked on Figure 1), and were often undetectable. By contrast, the four Hodgkin lymphoma patients all responded to the vaccines. When the data were grouped according to whether the participants received the BNT162b2 or mRNA-1273 vaccine, no differences were apparent. In total, eight lymphoma patients were anti-N-positive while all members of the HCW control group were anti-N-negative. For four of the eight anti-N-positive lymphoma patients, there was evidence of COVID-19 prior to the start of this study. Thus, three patients had prior documented positive SARS-CoV-2 polymerase chain reaction (PCR) tests, while the fourth was not PCR-tested but later had a positive commercial antibody test. Seven of these eight anti-N-positive patients responded to vaccination. Taken together, anti-N-positive lymphoma patients had significantly higher mean anti-S protein titers than their anti-N-negative counterparts (p < 0.0001) and the HCW group (p = 0.02). However, when anti-N-positive lymphoma patients were separated by treatment status (i.e., naïve, active therapy) the sample sizes were too small for comparisons to the anti-N-negative group. We studied the CLL and other NHL patients in more detail to understand the implications of their treatment (Figure 1(B)). Every treatment-naïve and remote-therapy (no treatment in over 24 months) CLL patient responded to vaccination, whereas only 40% (6/15) of those currently being treated had anti-S protein titers above the designated cut-off value. A similar pattern was seen for the other NHL patients, although one individual in each of the treatment-naïve and remote-therapy groups failed to respond to the vaccine. Active therapy in this subgroup was again associated with a poor vaccine response, with only 21.4% (3/14) developing anti-S protein titers above the cut-off. The off-therapy subgroup, who had received treatment within 2 years but not at the time of vaccination, also had a lower vaccine response rate of 55.5% (5/9). The four non-responders in this group had all received an anti-CD20 mAb within the previous 2 years; two within 6 months, one within 1 year, and one within 18 months. None of the patients currently on anti-CD20 mAb therapy seroconverted after vaccination. We next studied the relationship between when anti-CD20 mAb therapy ceased and the vaccine response (Figure 1(C)). None of the 11 CLL and other NHL patients receiving this treatment within 6 months of vaccination had anti-S protein titers above the cut-off, but longer intervals were associated with higher titers. Thus, CLL and other NHL patients who were last treated >24 months before vaccination had response rates of 66.7% (6/9) and 71.4% (10/14), respectively. It is notable that 3/3 CLL and 3/4 other NHL non-responders in this subgroup were receiving a different type of active therapy at the time of vaccination (Table S6). We suggest that even when anti-CD20 mAb therapy ceased >24 months before vaccination, other forms of ongoing active therapy can compromise the vaccine response. Thus we demonstrated that commonly used lymphoma therapies can adversely influence the performance of COVID-19 vaccines, with anti-CD20 mAbs having the greatest impact. With regard to anti-CD20 mAbs, our results are consistent with a growing number of reports that patients on active, or with recent anti-CD20 mAb treatment do not respond to vaccination.4-6 Compared with other studies, we report a higher rate of seroconversion in patients on active BTKi monotherapy.4, 5 Here, we found that 66.7% (4/6) of CLL patients and 50% (2/4) of other NHL patients did develop high-titer IgG antibodies after mRNA vaccination. In a study by Herishanu et al.4 only 16% (8/50) of CLL patients treated with a BTKi responded to vaccination with BNT162b2. In our study, CLL responders on BTKi monotherapy were on treatment for a median length of 53.5 (23–74) months prior to the first vaccine dose. In comparison, CLL non-responders on BTKi monotherapy were on treatment for a median of 2 (1–3) months. The CLL responders were described as having a good response to BTKi monotherapy, with two patients in complete remission and two patients with no progression of disease. All CLL responders were compliant with treatment and only one patient had a recent interruption in therapy. This patient was hospitalized for COVID-19 in April 2020 and treatment was held for approximately 3 weeks after which therapy was restarted. In this study, he was found to be anti-N-positive, consistent with pre-existing serologic immunity from prior infection. Finally, we studied the avidity of IgG antibodies to the Receptor Binding Domain in the lymphoma and healthy control patients (Figure S1). The avidity was significantly higher (p < 0.0001) for anti-N-positive lymphoma patients than for anti-N-negative lymphoma patients as well as healthy controls, all of whom were anti-N-negative (Figure S1(A)). These findings suggest COVID-19 convalescent patients (i.e., anti-N positive) have had longer to affinity mature their anti-S antibodies, which are boosted by the mRNA vaccines. We noted that patients currently receiving venetoclax or a BTKi had lower avidity S-protein antibodies than the other groups, although the group sizes were too small for statistical significance (Figure S1(B)). In conclusion, we found that most lymphoma patients respond to vaccination with an mRNA-based COVID-19 vaccine, but a substantial fraction (>40%) do not and therefore may remain at risk of infection and disease. There were no significant differences in the S-protein IgG antibody response rates or titers between the different lymphoma histologic subtypes. Treatment status was, however, a relevant variable. Treatment-naïve lymphoma patients responded to vaccination in a similar manner to the HCW group, as did patients who had not received therapy for at least 2 years. However, this controlled study presents compelling evidence that patients on active therapy for lymphoma may not respond to vaccination. Our results are particularly concerning for patients on anti-CD20 mAb therapy, given that no patients who had received treatment within 6 months responded well to mRNA vaccination. Thus these patients probably remain at risk of infection with SARS-CoV-2. In this patient population, we suggest exploring alternative strategies for protection such as passive immunization with anti-S monoclonal antibody therapy or, if possible, delaying therapy until after vaccination. This study was funded by the WCM Lymphoma Program, John P. Moore, P. J. Klasse, Erik Francomano and Thomas J. Ketas were supported by NIH grants R01 AI36082 and P01 AI110657. PM has consulted for ADCT, AstraZeneca, Bayer, Beigene, BMS, Cellectar, Epizyme, Gilead, Janssen, Karyopharm, Merck, Regeneron, Takeda, Teneobio, and Verastem; and received research funding from Karyopharm. JPL has consulted for Sutro, Miltenyi, AstraZeneca, Epizyme, BMS/Celgene, Regeneron, Bayer, Gilead/Kite, Karyopharm, GenMab, Genentech/Roche, Abbvie, Incyte, and Janssen. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1: Supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Poor performance of the utility of the serological diagnosis of pulmonary tuberculosis in Nigeria

Background: Rapid diagnosis of pulmonary tuberculosis (PTB) is critical for TB control. High-resolution computed tomography (HRCT) has been used in the diagnosis of PTB. Aims and objectives: We investigated the utility of HRCT for the rapid diagnosis of sputum smear and polymerase chain reaction (PCR)-negative PTB. Methods: We reviewed the medical records of sputum smear and PCR-negative PTB suspects who checked HRCT from January 2011 to December 2014 in a tertiary hospital of South Korea. Final diagnosis of PTB was made based on culture, bronchoscopy, biopsy and appropriate response to treatment. We assessed the diagnostic accuracy of HRCT in the diagnosis of PTB. We also assessed HRCT findings associated with PTB, Results: Of 113 sputum smear and PCR negative PTB suspects who checked HRCT, 45 (39.8%) were finally diagnosed to have PTB. Among 46 (40.7%) patients who were suggested to have PTB based on HRCT, 36 (78.3%) were finally diagnosed to have PTB and 10 (21.7%) were finally diagnosed not to have PTB (6 pneumonia, 3 bronchiectasis, 1 nontuberculous mycobacterial lung disease). Among 67 patients suggested not to have PTB based on HRCT, 9 (13.4%) were finally diagnosed to have PTB. The sensitivity and specificity, positive predictive and negative predictive value of HRCT for the diagnosis of PTB were 80.0%, 85.3%, 78.3%, 86.6%, respectively. Among the radiologic characteristics of HRCT, the presence of tree-in-bud appearance was significantly associated with PTB (p=0.02). Conclusions: HRCT is useful for the rapid diagnosis of PTB in sputum smear and PCR-negative PTB suspects. The presence of tree-in-bud appearance was the most characteristic HRCT finding of PTB.

BackgroundMucosal antibodies can prevent virus entry and replication in mucosal epithelial cells and hence virus shedding. Parenteral booster injection of a vaccine against a mucosal pathogen promotes stronger mucosal immune responses following prior mucosal infection compared to injections of a parenteral vaccine in a mucosally naive subject. We investigated whether this was also the case for the BNT162b2 COVID-19 mRNA vaccine.MethodsTwenty recovered COVID-19 subjects (RCS) and 23 SARS-CoV-2 naive subjects were vaccinated with respectively one and two doses of the BNT162b2 COVID-19 vaccine. Nasal Epithelial Lining Fluid (NELF) and plasma were collected before and after vaccination and assessed for Immunoglobulin (Ig)G and IgA antibody levels to Spike and for their ability to neutralize binding of Spike to ACE-2 receptor. Blood was analyzed one week after vaccination for the number of Spike-specific Antibody Secreting Cells (ASCs) with a mucosal tropism.ResultsAll RCS had both nasal and blood SARS-CoV-2 specific antibodies at least 90 days after initial diagnosis. In RCS, a single dose of vaccine amplified pre-existing Spike-specific IgG and IgA antibody responses in both NELF and blood against both vaccine homologous and variant strains, including delta. These responses were associated with Spike-specific IgG and IgA ASCs with a mucosal tropism in blood. Nasal IgA and IgG antibody responses were lower in magnitude in SARS-CoV-2 naive subjects after two vaccine doses compared to RCS after one dose.ConclusionMucosal immune response to the SARS-CoV-2 Spike protein is higher in RCS after a single vaccine dose compared to SARS-CoV-2 naive subjects after two doses.

Non-sputum-based biomarker assay is urgently required as per WHO's target product pipeline for diagnosis of tuberculosis. Therefore, the current study was designed to evaluate the utility of previously identified proteins, encoded by in vivo expressed mycobacterial transcripts in pulmonary tuberculosis, as diagnostic targets for a serodiagnostic assay. A total of 300 subjects were recruited including smear+, smear- pulmonary tuberculosis (PTB) patients, sarcoidosis patients, lung cancer patients and healthy controls. Proteins encoded by eight in vivo expressed transcripts selected from previous study including those encoded by two topmost expressed and six RD transcripts (Rv0986, Rv0971, Rv1965, Rv1971, Rv2351c, Rv2657c, Rv2674, Rv3121) were analyzed for B-cell epitopes by peptide arrays/bioinformatics. Enzyme-linked immunosorbent assay was used to evaluate the antibody response against the selected peptides in sera from PTB and controls. Overall 12 peptides were selected for serodiagnosis. All the peptides were initially screened for their antibody response. The peptide with highest sensitivity and specificity was further assessed for its serodiagnostic ability in all the study subjects. The mean absorbance values for antibody response to selected peptide were significantly higher (p<0.001) in PTB patients as compared to healthy controls; however, the sensitivity for diagnosis of PTB was 31% for smear+ and 20% for smear- PTB patients. Thus, the peptides encoded by in vivo expressed transcripts elicited a significant antibody response, but are not suitable candidates for serodiagnosis of PTB.

In children admitted to hospital, rapid, accurate diagnosis of pulmonary tuberculosis with the Xpert MTB/RIF assay is possible, but no paediatric studies have been done in the primary care setting, where most children are given care, and where microbiological diagnosis is rarely available. We assessed the diagnostic accuracy of Xpert MTB/RIF in children in primary care. For this prospective study, we obtained repeat induced sputum and nasopharyngeal aspirate specimens from children (<15 years) with suspected pulmonary tuberculosis at a clinic in Khayeliwtsha, Cape Town, South Africa. We compared the diagnostic accuracy of Xpert MTB/RIF with a reference standard of culture and smear microscopy on induced sputum specimens. For the main analysis, specificity of Xpert MTB/RIF versus liquid culture, we included only children with two interpretable Xpert MTB/RIF and induced sputum culture results. Between Aug 1, 2010, and July 30, 2012, we enrolled 384 children (median age 38·3 months, IQR 21·2-56·5) who had one paired induced sputum and nasopharyngeal specimen, 309 (81%) of whom had two paired specimens. Five children (1%) tested positive for tuberculosis by smear microscopy, 26 (7%) tested positive by Xpert MTB/RIF, and 30 (8%) tested positive by culture. Xpert MTB/RIF on two induced sputum specimens detected 16 of 28 culture-confirmed cases (sensitivity of 57·1%, 95% CI 39·1-73·5) and on two nasopharyngeal aspirates detected 11 of 28 culture-confirmed cases (sensitivity of 39·3, 23·6-57·6; p=0·18). The specificity of Xpert MTB/RIF on induced sputum was 98·9% (95% CI 96·9-99·6) and on nasopharyngeal aspirates was 99·3% (97·4-99·8). Our findings suggest that Xpert MTB/RIF on respiratory secretions is a useful test for rapid diagnosis of paediatric pulmonary tuberculosis in primary care. National Institutes of Health, National Health Laboratory Services Research Trust, the Medical Research Council of South Africa, the National Research Foundation South Africa, the European and Developing Countries Clinical Trials Partnership.

An enzyme-linked immunosorbent assay was developed to measure immunoglobulin G (IgG) and IgM type 3 antipneumococcal capsular polysaccharide antibodies. The use of Fab2 fragments of rabbit antipneumococcal IgG antibody in the antibody-antigen sandwich increased the sensitivity for measuring IgM antibodies and decreased background activity in antigen-free cuvettes. This methodology detected type 3 IgM antibody responses in six of six subjects vaccinated with polyvalent pneumococcal vaccine and detected type 3 IgG antibody responses in three subjects. Results of the enzyme-linked immunosorbent assay and radioimmunoassay procedures were concordant, and postvaccination enzyme-linked immunosorbent assay IgM titers showed a stronger correlation with total radioimmunoassay antibody than did postvaccination ELISA IgG titers.

OBJECTIVES: To evaluate the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) anti-spike Immunoglobulin G (IgG) antibody response after CoronoVac immunization of healthcare workers at Phyathai 3 Hospital. MATERIALS AND METHODS: The descriptive study was performed at Phyathai 3 Hospital between March 2021 and May 2021. Healthcare workers who received two doses (three weeks apart) of the CoronaVac vaccine were included. Blood samples for anti-spike IgG antibodies were taken from each healthcare worker before getting vaccinated and four weeks after completing two doses of the vaccine. RESULT: A total of 88 healthcare workers were enrolled in our study. Fifty-three (60%) of them were female, 84(95%) were physicians, 46(52%) were obese and 33(37.5%) had at least one coexisting condition. The mean age was 45.8 ± 9.3 years. Seven (8%) of participants were older than 60 years of age. All participants did not have IgG antibodies at baseline. Eighty-seven (98.9%) healthcare workers had seroconversion of anti-spike IgG antibodies four weeks after completing two doses of the CoronaVac vaccine. The mean level of anti-spike IgG at four weeks after completing vaccination was 115+/-85 unit/ml (range, 1.77-297.3 unit/ml). Anti-spike IgG levels were significantly higher in females than males (p = 0.02). Normal-weight participants showed higher anti-spike IgG levels than obese individuals (p = 0.01). IgG antibody responses tended to decrease with age. The highest IgG levels were observed in the ages of 30-40 years. CONCLUSION: Two doses of the CoronaVac vaccine could induce a 98.9% rate of seroconversion in healthcare workers. Female and normal-weight participants were significantly associated with a higher level of IgG response. Younger adults had a higher immune response than older adults in our setting.

In this study, the seroprevalence of immunoglobulin G (IgG) antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) in dogs bred in Japan was evaluated. Ninety-two non-clinical samples were obtained from three institutes and fifty-seven clinical samples were obtained from a veterinary hospital in Japan. Serum titers of total IgG, IgG1 and IgG2 isotype antibodies against MAP were measured using an indirect enzyme-linked immunosorbent assay (ELISA). The IgG antibodies against MAP in non-clinical serum obtained from three institutes was observed to be 2.4%, 20% and 9.0%. Similarly, the IgG1 antibodies titers against MAP were observed to be 7%, 20% and 0%. Lastly, the IgG2 antibodies against MAP were observed to be 7%, 20% and 4.4%. No significance differences in these titers were observed among the three institutes. The IgG, IgG1 and IgG2 antibodies in serum obtained from a veterinary hospital were observed to be 55.3%, 42% and 42%, respectively. Significant differences were found between the non-clinical and clinical samples. The titers in the clinical samples showed a high degree of variance, whereas low variance was found in the non-clinical samples. The IgG antibody levels were thought to be induced following exposure to MAP-contaminated feed. The difference in titers between the clinical and non-clinical samples is likely to be related to the amount of MAP antigen contamination in dog foods.

IntroductionThis study sought to explore the immunogenicity of a booster dose of an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in people living with human immunodeficiency virus (HIV) and identify the factors affecting the magnitude of anti-SARS-CoV-2 antibody levels.Materials and methodsA total of 34 people living with HIV (PLWH) and 34 healthy donors (HD) were administered a booster dose of the same SARS-CoV-2 vaccine. Anti-SARS-CoV-2 antibody and immunoglobulin G (IgG) levels were measured using the SARS-CoV-2 S protein neutralizing antibody Enzyme-Linked Immunosorbent Assay (ELISA) and 2019-nCov IgG Chemiluminescent Immunoassay Microparticles, respectively. Spearman correlation analysis was used to measure the correlation between laboratory markers and neutralizing antibody and IgG levels. Peripheral blood mononuclear cells (PBMCs) were extracted from each subject using density gradient centrifugation and the numbers of memory T and T follicular helper (Tfh) cells were determined using flow cytometry.ResultsPLWH had a marked reduction in CD4 and B cell levels that was accompanied by a lower CD4/CD8 T cell ratio. However, those who received a supplementary dose of inactivated SARS-CoV-2 vaccines exhibited antibody positivity rates that were analogous to levels previously observed. The booster vaccine led to a reduction in IgG and neutralizing antibody levels and the amplitude of this decline was substantially higher in the PLWH than HD group. Correlation analyses revealed a strong correlation between neutralizing antibody levels and the count and proportion of CD4 cells. Anti-SARS-CoV-2 IgG antibody levels followed a similar trend. The expression of memory T and Tfh cells was considerably lower in the PLWH than in the HD group.DiscussionPLWH had an attenuated immune response to a third (booster) administration of an inactivated SARS-CoV-2 vaccine, as shown by lower neutralizing antibody and IgG levels. This could be attributed to the reduced responsiveness of CD4 cells, particularly memory T and cTfh subsets. CD4 and cTfh cells may serve as pivotal markers of enduring and protective antibody levels. Vaccination dose recalibration may be critical for HIV-positive individuals, particularly those with a lower proportion of CD4 and Tfh cells.

The Utility of Polymerase Chain Reaction (PCR) in the Diagnosis of Pulmonary Tuberculosis

Objective To screen differentially expressed microRNAs (miRNAs) in peripheral blood CD14+ monocytes between latent tuberculosis infection (LTBI) and pulmonary tuberculosis patients. Methods Thirty–one patients with pulmonary tuberculosis and 31 patients with LTBI were enrolled from Shenzhen Bao'an Chronic Diseases Prevent and Treatment Hospital and Shenzhen Third People's Hospital during June 2013 and February 2014. Differentially expressed miRNAs were detected by using a miRNA chip in 6 pulmonary tuberculosis patients and 6 LTBI patients (male 3, female 3), and TaqMan qPCR test was performed to verify the differentially expressed miRNAs in two groups (25 for each). Receiver operating characteristic (ROC) curve was used to evaluate the differentially expressed miRNAs in diagnosis of pulmonary tuberculosis. Target genes of differentially expressed miRNAs were forecasted using miRFocus database, and GO term and KEGG pathway annotation were performed. Results There were 40 differentially expressed miRNAs in CD14+ monocytes between LTBI and pulmonary tuberculosis patients, among which 4 had > 2–fold up–regulation and 36 had > 2–fold down–regulation in pulmonary tuberculosis patients. All differentially expressed miRNAs could be divided into two clusters. TaqMan qPCR test showed that the expression of miR–378 (up–regulated miRNA) in pulmonary tuberculosis patients was 4.17±0.25, which was significantly higher than that in LTBI patients (2.31±0.24, t=5.25, P<0.01); the expression of miR–483–5p (down–regulated miRNA) in pulmonary tuberculosis patients was 1.71±0.16, which was significantly lower than that in LTBI patients (2.97±0.15, t=5.45, P<0.01). ROC curve analysis showed that the sensitivities and specificities of miR–378 and miR–483–5p in the diagnosis of pulmonary tuberculosis were 0.76, 0.72 and 0.84, 0.76, respectively. Bioinformatic analysis showed that the target genes of miR–378 and miR–483–5p mainly involved in cell proliferation, apoptosis, antigen presentation and signal transduction. Conclusion There are significant differences in miRNA profiles in CD14+ monocytes between LTBI and pulmonary tuberculosis patients, and the expression of miR–378 and miR–483–5p may be used for the differential diagnosis of pulmonary tuberculosis from LTBI. Key words: Mycobacterium tuberculosis; Tuberculosis, pulmonary; MicroRNAs; MicroRNA–378; MicroRNA–483–5p