microRNA hsa-miR-425-5p and hsa-miR-4523 expressions as biomarkers of active pulmonary tuberculosis, latent tuberculosis infection, and lymph node tuberculosis.

Background: Studies on miRNA highlight its significance as an immunomarker for several diseases, including tuberculosis. This study aimed to determine the difference between miR-425-5p and miR-4523 expressions in patients with active pulmonary TB (PTB), latent TB infection (LTBI), and lymph node TB (LNTB), whose diagnosis remains challenging. Methods: This case-control study was performed on blood samples obtained from 23 patients with PTB, 21 with LTBI, 21 with LNTB, and 25 healthy controls (HC). miRNA hsa-miR-425-5p and hsa-miR-4523 expression levels were measured by RT-qPCR. Statistical analyses were performed using SPSS version 25.0. Results: RT-qPCR showed that hsa-mir-425-5p and hsa-mir-4523 expression levels were significantly different among the four groups (PTB, LTBI, LNTB, and HCs). The hsa-mir-425-5p miRNA expression level in LNTB was higher than that in LTBI (p = 0.003). Meanwhile, the hsa-mir-4523 miRNA expression was downregulated in PTB and LNTB than in LTBI (p < 0.0001 and p = 0.015, respectively). The ROC analysis of a single sample showed that only mir-4523 could discriminate LTBI and HCs, with an AUC of 0.829 (p < 0.001). The ROC curve of each miRNA was further analyzed after logistic regression by adjusting for sex and age. The combination of both miRNAs was also analyzed. The model that analyzed the combination of both miRNAs after adjusting for age had the best performance in differentiating LNTB from LTBI, with an AUC of 0.97 (p < 0.001). Conclusion: miRNA hsa-mir-425-5p was upregulated and miRNA hsa-mir-4523 was downregulated in PTB and LNTB than in LTBI.

Serum iron levels in tuberculosis patients and household contacts and its association with natural resistance-associated macrophage protein 1 polymorphism and expression.

BACKGROUND: Iron deficiency can impair immune function, increasing tuberculosis (TB) susceptibility and severity. The research aimed to investigate iron deficiency anemia in TB patients and household contacts and its association with natural resistance-associated macrophage protein 1 (NRAMP1) polymorphism and expression. METHODS: The levels of iron, ferritin, and transferrin were measured in the serum by ELISA (Enzyme-Linked Immunosorbent Assay). NRAMP1 polymorphisms were determined by polymerase chain reaction (PCR) and sequencing. NRAMP1 gene expression was measured by real-time PCR. Interferon-gamma release assay (IGRA) checked on household contacts to screen household contacts with positive IGRA as the control. RESULTS: This study involved 35 TB cases and 35 TB contacts. The results showed that the serum Fe levels were found to be lower in the TB case group (median 149.6 µmol/L) than in the positive IGRA household contacts group (median 628.53 µmol/L) with a p-value <0.001. Meanwhile, ferritin levels in TB cases tended to be higher, in contrast to transferrin, which was found to tend to be lower in TB cases than household contacts but did not show a significant difference. This study found no association between the polymorphism of exon 15 D543 and active TB. However, NRAMP1 gene expression was lower in TB cases than in positive IGRA household contacts (p = 0.011). Besides, there was a positive correlation between NRAMP1 gene expression and serum Fe levels (r = 0.367, p = 0.006). TB was associated with decreased NRAMP1 gene expression (OR 0.086 95% CI 0.02-0.366, p = 0.001). Besides, TB was associated with low Fe levels (OR 0.533 95% CI 0.453-0.629, p < 0.001). CONCLUSION: Comparing the TB case to the household contacts group, decreased serum Fe levels were discovered in the TB case group. This study also shows a correlation of NRAMP1 gene expression to Fe levels in TB patients and household contacts and describes that TB may lead to decreased Fe levels by downregulating NRAMP1 expression.

Sequence analysis of SARS-CoV-2 Delta variant isolated from Makassar, South Sulawesi, Indonesia.

Introduction: This study aimed to perform mutation and phylogenetic analyses of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Delta variants and analyze the characteristic signs and symptoms of patients infected with SARS-CoV-2 Delta variant originated from Makassar during the Delta outbreak.Methods: We collected samples from patients who were infected with coronavirus disease 2019 (COVID-19) between June and October 2021. We selected the Quantitative Reverse Transcription-Polymerase Chain Reaction (PCR)-positive samples with a cycle threshold value of <30 for whole genome sequencing. Total viral ribonucleic acid (RNA) was isolated from 34 PCR-positive nasopharyngeal swab samples, and whole genome sequencing was performed using the Oxford Nanopore GridlON sequencer. Phylogenetic and maximum clade credibility analyses were performed using the Bayesian Markov chain Monte Carlo method. Results: It was found that 33 patients were infected with the SARS-CoV-2 Delta variant in this cohort study, among whom 63.6% (21) patients were female. According to the clinical data, 24 (72.7%), 7 (21.2%), and 2 (6.1%) patients had mild, moderate, and severe COVID-19 infections. Phylogenetic analysis based on the spike and RNA-dependent RNA polymerase (RdRp) genes showed that the collected samples were clustered in the main lineage of B.1.617.2 (Delta variant). The Delta variants had a high frequency of distinct mutations in the spike protein region, including T19R (94.12%), L452R (88.23%), T478K (91.17%), D614G (97%), P681R (97%), and D950 N (97%). Other unique mutations found in a smaller frequency in our samples were present in the N-terminal domain, including A27T (2.94%) and A222V (14.70%), and in the receptor-binding domain, including Q414K (5.88%), G446V (2.94%), and T470 N (2.94%). Conclusion: This study revealed the unique mutations in the S protein region of Delta variants. T19R, L452R, T478K/T478R, D614G, P681R, and D950 N were the most common substitutions in Makassar's Delta variant.

Plasma Macrophage Migration Inhibitory Factor Concentration at Each Spectrum of Tuberculosis.

Macrophage migration inhibitory factor (MIF) is an inflammatory mediator in several diseases, including tuberculosis (TB). However, the role of MIF in each stage of TB remains to be further elucidated. Thus, this study aimed to analyze the differences in plasma MIF protein levels in patients with active pulmonary TB, positive and negative interferon-gamma release assay (IGRA) household contacts (HHCs), and healthy controls (HCs). Plasma MIF concentration was significantly higher in patients with active-new pulmonary tuberculosis (ATB) and HHCs compared with HCs (mean ± standard deviation: 17.32 ± 16.85, 16.29 ± 14.21, and 7.29 ± 5.39 ng/mL, respectively; P = 0.002). The plasma MIF concentration was not statistically different when compared between patients with ATB, IGRA-positive HHCs (17.44 ± 16.6 ng/mL), and IGRA-negative HHCs (14.34 ± 8.7 ng/mL) (P = 0.897). In conclusion, ATB patients, IGRA-positive HHCs, and IGRA-negative HHCs have a higher MIF concentration than HCs. This shows the involvement of MIF in each stage of TB, starting from TB exposure and infection, but not symptomatic, to the active stage.

Expression of miRNA-29a-3p and IFN-γ as biomarkers in active and latent pulmonary tuberculosis.

Background: Diagnosis and management of latent tuberculosis (TB) infections are one of the challenges of eradicating pulmonary TB. A critical aspect of controlling pulmonary TB spread is early diagnosis. One TB biological marker type under evaluation is microRNAs (miRNAs). Mycobacterium tuberculosis infection causes epigenetic changes. The upregulation of miRNA-29a-3p suppresses the immune response by post-transcriptionally inhibiting interferon (INF)-γ expression in T cells, increasing susceptibility to pulmonary TB. This study aimed to assess miRNA-29a-3p expression as a biomarker of active and latent pulmonary TB infection. Methods: This case-control study included 50 individuals with active TB, 33 household contacts with a positive IFN-γ release assay (IGRA), and 30 healthy controls. An enzyme-linked immunosorbent assay-based IGRA was used to determine latent pulmonary TB infection in household contacts. Quantitative real-time PCR was used to quantify miRNA-29a-3p expression. Data analysis used analyses of variance and receiver operating characteristic (ROC) curves. Results: miRNA-29a-3p expression differed significantly between active TB, latent TB, and healthy participants (controls; p = <0.001. ROC curve analysis showed that miRNA-29a-3p expression had 86% sensitivity and 73% specificity with an area under the ROC curve (AUC) of 0.763 (95% confidence interval [CI]: 0.668-0.858). The miRNA-29a-3p ROC curve had 84.8% sensitivity and 70% specificity with an AUC of 0.808 (95% CI: 0.698-0.919) for latent TB. Additionally, miRNA-29a-3p expression was significantly correlated with active (p < 0.0001) and latent (p < 0.0001) pulmonary TB. However, miRNA-29a-3p expression was not significantly correlated with INF-γ levels in patients with active (R = 0.005; p = 0.62) and latent (R = 0.010; p = 0.38) pulmonary TB or healthy controls (R = 0.060; p = 0.19). Conclusion: miRNA-29a-3p expression was increased in patients with active and latent pulmonary TB. Therefore, miRNA-29a-3p represents a potential biomarker for latent and active pulmonary TB. However, IFN-γ levels were not correlated with miR-29a-3p expression.

Full-genome sequencing and mutation analysis of SARS-CoV-2 isolated from Makassar, South Sulawesi, Indonesia.

Introduction: A global surge in SARS-CoV-2 cases is occurring due to the emergence of new disease variants, and requires continuous adjustment of public health measures. This study aims to continuously monitor and mitigate the impact of SARS-CoV-2 through genomic surveillance, to determine the emergence of variants and their impact on public health. Methods: Data were collected from 50 full-genome sequences of SARS-CoV-2 isolates from Makassar, South Sulawesi, Indonesia. Mutation and phylogenetic analysis was performed of SARS-CoV-2 from Makassar, South Sulawesi, Indonesia. Results: Phylogenetic analysis showed that two samples (4%) were of the B.1.319 lineage, while the others (96%) were of the B.1.466.2 lineage. Mutation analysis of the spike (S) protein region showed that the most common mutation was D614G (found in 100% of the sequenced isolates), followed by N439K (98%) and P681R (76%). Several mutations were also identified in other genomes with a high frequency, including P323L (nsp12), Q57H (ns3-orf3a), and T205I (nucleoprotein). Conclusion: Our findings highlight the importance of continuous genomic surveillance to identify new viral mutations and variants with possible impacts on public health.

Analysis of real-time PCR Melanocortin 3 (MC3R) gene expression to identify new biomarkers inflammation in tuberculosis.

Background: Diagnosis of tuberculosis (TB) in the era of technological sophistication requires accuracy and speed to provide as much information as possible so that TB treatment can be carried out quickly and precisely. New studies have also begun to be carried out to diagnose TB, one of which is by examining genes, either by looking at polymorphisms, mutations, or expressions. Several previous studies have confirmed the association of MC3R and TB genes with polymorphisms; MC3R is a gene that participates in the regulation of the inflammatory process and is also found in macrophages; therefore, we tried to analyze gene expression in the active TB group, household contacts, and healthy controls for looked at the differences between the three groups and confirmed the correlation of MC3R with TB by seeing which group's gene expression increased the most expression of the three groups so that the results can be considered as a TB diagnostic biomarker in the future. Methods: This study included 122 people, 49 patients with confirmed TB, 46 close relatives of patients, and 27 healthy controls. This study used a real-time PCR technique to analyze MC3R gene expression in the three groups, and all data were analyzed using Bio-Rad CFXTM software version 3.1 and one-way ANOVA using SPSS 21.0. Results: The value of MC3R gene expression in the active TB group increased 3.6-fold in the healthy group (p = 0.143), and that of gene expression in the healthy control group increased 1.09-fold in the healthy group (p = 0.007). Conclusion: There is a relationship between MC3R and TB based on the results of gene expression analysis that increased in the active TB group compared to the household contact group and healthy controls.