Development of a reverse transcription loop-mediated isothermal amplification assay with novel quantitative pH biosensor readout method for SARS-CoV-2 detection.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a molecular amplification method that can detect SARS-CoV-2 in a shorter time than the current gold-standard molecular diagnostic reverse transcription-polymerase chain reaction (RT-PCR). However, previously developed RT-LAMP assays have mostly relied on highly subjective visual colorimetric interpretation. In this study, an RT-LAMP assay was developed with quantitative measurement of reaction pH using a novel portable pH biosensor compared to qualitative colorimetric interpretation and gel electrophoresis, with 57 clinical COVID-19 samples used for validation of the test. The LoD of the assay is 103 copies/µL. The highest sensitivity was found in the qualitative methods (93.75%), while the highest specificity and likelihood ratio was found in the pH sensor (87.5% and 6.72). On the sensor measurement, a significant difference (p < 0.0001) was observed between the average pH of the RT-PCR (+) COVID-19 (6.15 ± 0.27), while the average pH of the RT-PCR (-) samples (6.72 ± 0.22). Correlation analysis revealed a strong correlation (r = 0.78, p < 0.0001) between the Ct values obtained from RT-PCR with the biosensor pH readout. RT-LAMP with the quantitative pH sensor readout method has the potential to be further developed as an objective molecular assay for rapid and simple detection of SARS-CoV-2.

A potential mechanism of miana (Coleus scutellariodes) and quercetin via NF-κB in Salmonella typhi infection.

Purpose: To prove the effect of Miana (M), Quercetin (Q), and the combination as an anti-inflammatory agent and Cefixime (C) as an antibiotic in Balb/c mice infected with Salmonella enterica serovar Typhi (S. Typhi) and related to the dynamics of NF-κB mRNA expression and NF-κB protein levels. Methods: A cohort study on male Balb/c mice with subjects consisted of 8 groups with 5 each group by administration of M, Q, M + Q, M + C, Q + C, M + Q + C, C only and sterile distilled water group as negative control. The statistical significance of the difference group was defined as P values less than 0.05. Results: Decreased mRNA expression of NF-κB, NF-κB protein levels, and bacterial load after administration of M + C, Q + C, or M + Q + C showed significant differences when compared to the negative control. The decline in NF-κB was stronger when M + Q + C was given compared to M, Q, M + Q, or C only. Conclusion: The effects of NF-κB suppression appear to be the same between the administration of M, Q and the M + Q when C added. However, the suppression of NF-κB was not significant without adding C.

Decreasing mRNA HMGB1 expression in Klebsiella pneumoniae infection treated by Miana (Coleus scutellarioides (L.) Benth): a cohort experimental study.

Pneumonia is one of the most common infections caused by the bacterium Klebsiella pneumoniae. During the initiation of an infection, the immune system recognizes the pathogen through the release of high mobility group box 1 (HMGB1), thereby triggering the inflammation process. Miana has demonstrated potent inhibitory effects on the inflammatory process during infection in animal models. The aim of this study was to determine the effect of Miana leaf extract on mRNA HMGB1 expression in Balb/c mice infected with K. pneumoniae. Methods: This study comprised a cohort experiment using 20 Balb/c mice divided into four groups. Balb/c mice in each group were intraperitoneally injected with K. pneumoniae. Group 1 was given a placebo; Group 2 was given Miana; Group 3 was given levofloxacin; and Group 4 was given both levofloxacin and Miana. The levels of mRNA HMGB1 expression were measured using real-time PCR before, during, and after the infection as well as after the treatments. Results: The initial examination results showed that the average level of mRNA HMGB1 expression was 5.51 fc. The mRNA HMGB1 expression in mice after being challenged with K. pneumoniae was 9.64 fc. Group 1 that was given a placebo had a mean mRNA HMGB1 expression level of 14.99 fc. Group 2 that was given Miana had a mean mRNA HMGB1 expression level of 13.95 fc. Group 3 that was given levofloxacin had an average mRNA HMGB1 expression level of 6.45 fc, and Group 4 that was given levofloxacin and Miana together had an average mRNA HMGB1 expression level of 5.59 fc. Conclusion: Miana (Coleus scutellarioides (L.) Benth) increased mRNA HMGB1 expression at the initial administration via regulation of the immune system. Administration of Miana following K. pneumoniae infection inhibited the increase in mRNA HMGB1 expression. Treatment with levofloxacin reduced the level of mRNA HMGB1 expression, and the effect was optimized by the administration of Miana leaf extract as a supplement.

Point-of-Care Saliva Osmolarity Testing for the Screening of Hydration in Older Adults With Hypertension.

OBJECTIVES: Older adults have an elevated risk of dehydration, a state with proven detrimental cognitive and physical effects. Furthermore, the use of diuretics by hypertensive patients further compounds this risk. This prospective study investigated the diagnostic accuracy of point-of-care (POC) salivary osmolarity (SOSM) measurement for the detection of dehydration in hypertensive adults with and without diuretic pharmacotherapy. DESIGN: Prospective diagnostic accuracy study. SETTING: Home visits to patients recruited from 4 community health centers in West Sulawesi, Indonesia. PARTICIPANTS: A total of 148 hypertensive older adults (57 men, 91 women). The mean ages of male and female patients were 69.4 ± 11.4 and 68.1 ± 7.8 years, respectively. METHODS: Hypertensive adults were divided into 2 groups based on the presence of diuretics in their pharmacotherapeutic regimen. First-morning mid-stream urine samples were used to perform urine specific gravity (USG) testing. Same-day SOSM measurements were obtained using a POC saliva testing system. RESULTS: Both USG (P = .0002) and SOSM (P < .0001) were significantly elevated in hypertensive patients with diuretic pharmacotherapy. At a USG threshold of ≥1.030, 86% of diuretic users were classified as dehydrated compared with 55% of non-using participants. A strong correlation was observed between USG and SOSM measurements (r = 0.78, P < .0001). Using a USG threshold of ≥1.030 as a hydration classifier, an SOSM threshold of ≥93 mOsm had a sensitivity of 78.6% and a specificity of 91.1% for detecting dehydration. CONCLUSIONS AND IMPLICATIONS: Hypertensive patients on diuretics have significantly higher first-morning USG and SOSM values, indicating a higher likelihood of dehydration relative to those on other classes of antihypertensive medication. POC SOSM assessment correlates strongly with first-morning USG assessment, and represents a rapid and noninvasive alternative to urinary hydration assessment that may be applicable for routine use in populations with elevated risk of dehydration.

In vivo and in vitro inhibition effect of propolis on Klebsiella pneumoniae: A review.

For centuries, propolis has been used to treat various diseases in traditional medicine due to its biological and pharmacological activities. It remains popular because of its potentially beneficial role in human health due to its well-known broad multispectrum properties, including antiviral, anti-inflammatory, antibacterial, anesthetic, antioxidant, anticancer, antifungal, antiprotozoal, antihepatotoxic, antimutagenic, and antiseptic activity. Numerous studies have examined the antibacterial activity of propolis and its derivatives, which include many natural antimicrobial compounds with broad spectrum activity against different bacterial types. In vitro studies have shown propolis's antibacterial activity against Gram-positive and Gram-negative bacteria. Many studies have examined propolis's effect on inhibiting bacterial growth. Several studies examining propolis's inhibition of Gram-positive and Gram-negative bacteria have shown it to be an effective antimicrobial agent. Klebsiella pneumoniae is a Gram-negative bacterium commonly associated with respiratory infections, particularly in hospital settings. Inappropriate antibiotic use may contribute to the increasing number of bacterial strains resistant to available drugs. This review summarizes the findings of previous studies on propolis and its potential mechanisms in inhibiting K. pneumoniae growth in animals.

Correlation between innate and adaptive immunity response in TB children post BCG vaccination. Is it effective or not?: Cross-sectional study.

Background: How far the role of innate immunity and adaptive immunity do in children who have been BCG vaccinated in controlling the course and the severity of the TB disease has not been completely known. Mycobacterium tuberculosis entry to the body will be recognized by Toll-like receptors found on macrophages, neutrophils, and dendritic cells as part of the innate immune response, after which the dendritic cells will then present the antigen to lymphocyte T0 cells and initiate the adaptive immune response (of which CD4 T cells have an important role in). Was one or were both of these immune responses function well or not in a BCG Vaccinated Children with TB? Objective: This study aim to find a better understanding of the role of innate immune response assessed by TLR2/TLR4 mRNA gene expression and serum TLR2/TLR4 levels, while the role of adaptive immune response is assessed by analyzing serum CD4 level in children with TB who have had BCG vaccination. Methods: This cross-sectional study was conducted among children with TB at the outpatient and inpatient wards at Bhakti Medicare and Jakarta Islamic Hospital. Expression of mRNA gene was measured using the Boom method and protein serum levels were measured using the ELISA method. The results were analyzed by using the SPSS v.23 program. Results: Sixty-nine children were recruited as subjects. In this study, 68.1% of whom had BCG scars. TLR4 mRNA gene expression was found to be higher than TLR2 mRNA gene expression. Serum CD4 level was found to be highest out of TLR2 and TLR4 level, but serum TLR2 level was higher than TLR4 level. TLR2/TLR4 mRNA gene expression, serum TLR2/TLR4 levels, and CD4 levels in subjects with BCG scar were also found to be significantly higher than in subjects without BCG scar (p < 0.001). There was a significant positive correlation between TLR2/TLR4 mRNA gene expression and serum TLR2/TLR4 levels (r = 0.860; r = 0.864; p < 0.001) and between serum levels TLR2/TLR4 with serum CD4 levels (r = 0.822; r = 0.832 p < 0.001). Conclusion: As early as possible, BCG vaccine administration is needed in endemic countries, but it must be ensured that scars can be formed. It is also important to control Latent TB Infection (LTBI) to prevent transmission and relapse of disease.

Positive Correlation of Oestradiol Level on Trigger Day with the Secretion Level of Endometrial Kisspeptin and Leukaemia Inhibitory Factor in the Mid-Luteal Stimulated Cycle.

Background: Kisspeptin plays a role in the oestradiol negative-feedback regulation of GnRH as well as gonadotropin. In addition, kisspeptin has been postulated to induce the production of an important cytokine called leukaemia inhibitory factor (LIF). Aims: This study aims to investigate the correlation between varying oestradiol levels measured on trigger day of the ovarian stimulation and the mRNA expression level of endometrial kisspeptin and LIF. Study Setting and Design: Prospective cross-sectional study took place in Morula IVF Jakarta clinic. Materials and Methods: A total of 43 infertile couples underwent an in-vitro fertilization (IVF) program. Subjects were grouped based on oestradiol levels as follows: group A ([≧ 3000 pg/mL, n = 15], group B [2000-2999 pg/mL, n = 14], group C [<2000 pg/mL, n = 14]). Statistical Analysis Used: ANOVA test was utilised to compare the expression of kisspeptin and LIF among study groups while Pearson correlation was used to identify the correlation between variables. Results: A significantly higher mRNA expression of both Kisspeptin and LIF was found in group A than in groups B and C (P < 0.001). The mRNA expression of kisspeptin and LIF correlated positively with the oestradiol level (r = 0.638, P < 0.001 and r = 0.634, P < 0.001, respectively). Moreover, a strong association between Kisspeptin and LIF expression was also detected (r = 0.700, P < 0.001). Conclusions: mRNA expression of kisspeptin and LIF was significantly different according to the oestradiol levels in the study groups. Increased oestradiol level was shown to elevate the expression of endometrial kisspeptin and LIF in women undergoing the IVF programme.

Molecular Patho-mechanisms of cervical cancer (MMP1).

Cervical cancer mostly caused by Human Papilloma Virus. Staging and therapy have been extensively studied, and highly correlated with the cellular development of oncogenesis. Mutation was caused by E6 and E7 oncoprotein, also inactivation of 2 tumor suppressor factors (pRB and p53). P53 also regulated MMP1, which dysregulation of MMP transcription would promote tumor metastasis, because of its role in extracellular matrix degradation in tumor invasion. Clinical staging of Cervical Cancer was based on Federation International of Gynaecology and Obstetrics (FIGO) classification from 2018. Management was divided into Surgery, Radiotherapy, and Chemotherapy.

Benefits and challenges in the implementation of virtual laboratory simulations (vLABs) for medical biochemistry in Indonesia.

The pandemic caused major shifts in the delivery of education worldwide. In the teaching of medical biochemistry, the greatest impact was towards the delivery of traditional laboratory simulations. In this study, we highlight the benefits and barriers encountered in the use of virtual laboratories (vLABs) to substitute traditional laboratory practicals. The subjects were a class of 271 medical students at the Faculty of Medicine, Hasanuddin University, all freshman undergoing the Biomedicine Block. The study assessed the use of a commercial vLAB on antibodies and blood typing procedures, which were implemented using our four-step model of vLAB implementation. Collected data include the lecturer-assigned pre- and post-test result, built-in vLAB assessment result of the student first and best attempts, a student perception questionnaire based on a 5-point Likert scale, and an open ended questionnaire regarding student perceptions of the advantages and disadvantages of the vLAB. We observed a remarkable increase of lecturer assigned pre- and post-test scores and built-in first and best attempt scores (p < 0.0001, Wilcoxon signed rank test). A majority of students reported increased motivation when using the vLABs, and favored the ability of mastery through repetition. However, technical and language barriers were highlighted by students during the vLAB implementation. We demonstrate a successful implementation of commercial vLABs in a cohort of non-native English speakers using our four-step approach. Implementation requires strong support from faculty to address technical and language barriers that arise during use of vLABs.

microRNA-221 and tamoxifen resistance in luminal-subtype breast cancer patients: A case-control study.

BACKGROUND: Around 70% of breast cancers (BCs) are estrogen receptor-α (ERα)-positive. Adjuvant endocrine therapy is used to reduce estrogen levels and inhibit signal transduction through the ER. The anti-estrogen drugs that are most commonly used in endocrine therapy belong to the selective ER modulator (SERM) class and include tamoxifen. Although it has been used for three decades in cases of early-stage and ERα-positive BC, resistance to tamoxifen is a common problem. microRNAs (miRNAs) have a potential role in demonstrating BC resistance to tamoxifen therapy. Hence, there is a need to investigate the expression of miRNA-221 (miR-221) in luminal-subtype BC patients receiving tamoxifen therapy. METHODS: This case-control study investigated luminal-subtype BC patients who had undergone endocrine therapy for at least 1 year. The case group comprised patients with local or metastatic recurrence, and the control group comprised patients without local or metastatic recurrence. RESULTS: There was a significant difference in miR-221 expression (p = 0.005) between the case and control groups. There were no significant differences between the groups that were positive and negative for the progesterone receptor (PR) (p = 0.25), had high and low marker of proliferation Ki-67 levels (p = 0.60), were positive and negative for lymphovascular invasion (p = 0.14), and had stage 2 and stage 3 cancer (p = 0.25). CONCLUSION: miR-221 expression was higher in tamoxifen-resistant BC cases. miR-221 is a potential biomarker of tamoxifen resistance.

Relation between expression of hMLH1 and p53 mRNA genes, in the feces of patients with colorectal carcinoma. Cross-sectional study.

Colorectal carcinoma (CRC) is one of the main public health problems. The mortality of CRC is about 8%. Early detection of CRC is very important to prevent death because this cancer could be cured through surgery if the diagnosis can be made as early as possible. Therefore screening strategy for early detection of CRC is critical in reducing mortality. Many investigations supporting the detection of CRC have been developed, including the fecal DNA mutation test using advanced cytological techniques. It is capable of assessing colonocytes for the presence of DNA, RNA, and protein as molecular biomarkers of neoplasia in CRC, including p53 and hMLH1. This study implemented observational approach with a cross-sectional study of the feces of patients with CRC regardless of the stage and grade. The purpose of this study was to determine the expression of the hMLH1 and p53 mRNA genes in the feces of 48 patients with CRC from two hospitals in Indonesia, Siloam Hospitals in Cikarang and Dr. Wahidin Sudirohusodo Hospital in Makassar. The results showed that all adenocarcinoma feces samples with various tumor stages and grades had excess mRNA expression (more than twice the normal amount in Fold Change units) for both the hMLH1 and p53 genes. The average expression of the hMLH1 mRNA gene was the highest at stage two and grade one, while the lowest was at stage four and grade three. In contrast, the average p53 mRNA gene expression was the highest at stage four and grade three, while the lowest was at stage two and grade one. The study suggested that there was a relation between and the expression of hMLH1 and p53 mRNA gene. We concluded that while both hMLH1 and p53 genes in patients' feces with CRC were overexpressed, they did not significantly affect the grade of CRC.

Prediction of hereditary nonpolyposis colorectal cancer using mRNA MSH2 quantitative and the correlation with nonmodifiable factor.

BACKGROUND: Hereditary non-polyposis colon cancer is a dominantly inherited syndrome of colorectal cancer (CRC), with heightened risk for younger population. Previous studies link its susceptibility to the DNA sequence polymorphism along with Amsterdam and Bethesda criteria. However, those fail in term of applicability. AIM: To determine a clear cut-off of MSH2 gene expression for CRC heredity grouping factor. Further, the study also aims to examine the association of risk factors to the CRC heredity. METHODS: The cross-sectional study observed 71 respondents from May 2018 to December 2019 in determining the CRC hereditary status through MSH2 mRNA expression using reverse transcription-polymerase chain reaction and the disease's risk factors. Data were analyzed through Chi-Square, Fischer exact, t-test, Mann-Whitney, and multiple logistics. RESULTS: There are significant differences of MSH2 within CRC group among tissue and blood; yet, negative for significance between groups. Through the blood gene expression fifth percentile, the hereditary CRC cut-off is 11059 fc, dividing the 40 CRC respondents to 32.5% with hereditary CRC. Significant risk factors include age, family history, and staging. Nonetheless, after multivariate control, age is just a confounder. Further, the study develops a probability equation with area under the curve 82.2%. CONCLUSION: Numerous factors have significant relations to heredity of CRC patients. However, true important factors are staging and family history, while age and others are confounders. The study also established a definite cut-off point for heredity CRC based on mRNA MSH2 expression, 11059 fc. These findings shall act as concrete foundations on further risk factors and/or genetical CRC future studies.

Comparison TLR2 and TLR4 serum levels in children with pulmonary and extrapulmonary tuberculosis with and without a Bacillus Calmette-Guérin (BCG) scar.

The formation of a scar after Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccination influences the effectiveness of protection against Mycobacterium tuberculosis (MTB) infection. The innate immunity plays a critical role both in the pathophysiology of tuberculosis (TB) and BCG vaccination protection mechanism. Parts of innate immunity: macrophages, dendritic cells, and neutrophils, have microbial recognition surface receptors called Toll-like receptors (TLR) 2 and 4. The objective of this study is to compare the serum levels of TLR2 and TLR4 in BCG-vaccinated pediatric patients with pulmonary and extrapulmonary TB. This cross-sectional study included children aged less than 18 years old with contracted TB disease and had received BCG vaccination. The subjects were recruited by convenience sampling from both outpatient and inpatient care at Bhakti Medicare and Jakarta Islamic Hospital, from November 2018 to December 2019. Serum TLR2 and TLR4 levels measured using ELISA of the two groups of subjects: children with pulmonary TB (PTB) and extrapulmonary TB (EPTB), were then compared. The presence of BCG scars was included in the analysis. Independent T-test, ANOVA test, and Kolmogorov-Smirnov normality tests on the SPSS program were used to statistically analyze the results. Serum TLR2 and TLR4 levels were higher in EPTB group, but the difference was not significant (TLR2 p = 0.758 and TLR4 p = 0.646, respectively). Subjects with BCG scars in both groups have significantly higher serum TLR2 and TLR4 levels than those without BCG scars in the EPTB group (EPTB p = 0.001 and p = 0.004, respectively); (PTB p < 0.001 and p < 0.001, respectively). BCG vaccination and MTB infection stimulate better innate immune response in EPTB than in PTB and serum TLR2 and TLR4 levels in those with BCG scars were higher when compared to those without BCG scars.

Systemic lidocaine administration influences NF-kß gene expression, NF-kß and TNF- α protein levels on BALB/c mice with musculoskeletal injury.

BACKGROUND: The immune system can produce various inflammatory mediators to protect the body from stress and surgical trauma. However, this excessive inflammatory response will interfere with the body's immune system, causing systemic inflammatory response syndrome and multi-organ failure if allowed to continue. Lidocaine as an anti-inflammatory is used to treat surgical pain and pain arising from the disease process and treat ventricular arrhythmias. This study aims to prove the efficacy of systemic lidocaine injection as an anti-inflammatory drug in BALB/c mice with sterile musculoskeletal injuries. METHODS: This study used a prospective experimental laboratory study on experimental animals of BALB/c mice using a simple randomized design. Sixteen adult white BALB/c mice (male, healthy, 10-12 weeks old, 35-40 g body weight, and no disability) were selected and randomly divided into two groups: the group given lidocaine (2 mg/kg body weight) and a group that was given sterile distilled water. NF-kß and TNF-α protein levels were detected by ELISA, while mRNA expression of NF-kß was analyzed and determined by quantitative real-time PCR. RESULTS: Musculoskeletal injury significantly increased the expression of both mRNA and protein levels of NF-kß and TNF-α protein level. In addition, the NF-kß (protein and mRNA) and TNF-α (protein) levels in rats experiencing inflammation due to musculoskeletal injury were significantly decreased in the lidocaine group (p < 0.001). CONCLUSIONS: The administration of systemic lidocaine injection was able to inhibit the expression of mRNA NF-kß, the protein levels of NF-kß, and protein levels of TNF-α in mice with musculoskeletal injuries.

Relationship between expression mRNA gene Treg, Treg, CD4+, and CD8+ protein levels with TST in tuberculosis children: A nested case-control.

BACKGROUND: The ability of Mycobacterium tuberculosis to survive intracellularly, provides a cellular adaptive immune response played by specific T cells to defend against tuberculosis. The adaptive immune response to Bacillus of Calmette and Guerin (BCG) immunization is responded to by B cells, T Follicular B helper, T regulatory, restriction CD1, CD8+, CD4+, Th1, Th2, and Th17. BCG immunization can cause a tuberculin test reaction to being positive. The tuberculin test is a method for diagnosing TB infection and for screening individuals for latent infection and assessing the rate of TB infection in a given population. METHODS: a nested case-control survey was conducted on patients with a diagnosis of TB and parents 0-18 years of age from 3 hospitals in Indonesia during September-November 2019 with a total sample of 69 people undergoing clinical examinations, supporting and diagnosing subjects, blood sampling 1-2 cc for examination mRNA gene Treg, Treg, CD 4+, and CD 8+, then centrifuged at 3000 rpm for 10 min to support blood cells and serum. RESULTS: There was a significant relationship between expression of mRNA gene Treg with TST (p = 0,000), Treg with TST (p = 0,000), and CD4+ with TST (p = 0,000). Meanwhile, CD8 + was not significantly associated with TST (p = 0.118). CONCLUSIONS: It is necessary to check the expression of mRNA gene Treg, Treg, CD4+, and CD8+ with more samples to find the mean value that shows the protective value of further TB.

Detection of voluntary dehydration in paediatric populations using non-invasive point-of-care saliva and urine testing.

AIM: Voluntary dehydration, or lack of fluid intake despite water availability, is common in otherwise healthy children, and can lead to adverse effects. Most dehydration biomarkers are impractical for routine assessment in paediatric populations. This study aimed to assess two non-invasive hydration assessment tools, urine specific gravity (USG ) and a novel point-of-care (POC) salivary osmolarity (SOSM) sensor, in healthy children. METHODS: Volunteers were tested by colorimetric USG and a handheld SOSM system. Observed values were compared against previous studies to determine hydration status, as was the concordance between parameters. RESULTS: At the common USG threshold of 1.020, 42.4% of the 139 healthy children were dehydrated. The same prevalence was found using the 70-mOSM cut-off value. Comparative analysis of SOSM at varying USG thresholds demonstrated significantly higher SOSM in dehydrated children with a USG  ≥ 1.030 (P = 0.002). CONCLUSION: At the USG threshold of 1.020 and SOSM threshold of 70 mOSM, 42.4% of healthy children were found to be voluntarily dehydrated. Significantly higher SOSM was observed in dehydrated children (USG ≥ 1.030). As the first study on the utility of POC SOSM measurements for detecting dehydration, these results provide a foundation for future POC characterisation of SOSM in other populations and clinical contexts.

The correlation of Foxp3 + gene and regulatory T cells with scar BCG formation among children with Tuberculosis.

Tuberculosis infection causes a complex immunological response, where interactions between the pathogen and the host are unique, making it difficult to treat and control this disease. According to WHO, an estimated 1 million children became ill with TB, and 233,000 children died of TB in 2017. Bacillus Calmette-Guérin (BCG) vaccines continue to be the only vaccines to prevent Tuberculosis (TB). Studies suggesting the association of BCG scar with decreased childhood mortality in developing countries have rekindled the interest in BCG scar. However, the direct effect of the BCG scar remains unknown. We examined 76 cases in this study. All Subjects were diagnosed with Tuberculosis. BCG scars were examined directly when physical examination at the BCG vaccination site was performed. Tuberculin Skin Test was performed with 0.1 ml purified protein derivative (PPD) solution (5TU PPD/0.1 ml) injected intradermally. We examined the FOXP3 gene by real-time PCR and the level of Treg byELISA. The comparison of the mean Treg gene expression and the Treg protein content was higher in the positive scar group than in the negative scar group. It shows that Treg plays a role in the Tuberculosis during its active phase development. Treg protein levels were higher in the combination of positive TST and scar. It shows that BCG scarring is an essential marker of a well-functioning immune system. Cheap and straightforward initiatives like early BCG vaccinations, monitoring BCG scarring, and revaccinating scar-negative children could have an enormous immediate impact on global child survival.

Molecular characterisation of mutations associated with resistance to first- and second-line drugs among Indonesian patients with tuberculosis.

OBJECTIVES: This study aimed to determine molecular characteristics of rpoB, katG, rrs, and gyrA genes in Mycobacterium tuberculosis isolated from a cohort of Indonesian patients with tuberculosis. METHODS: Fifty isolates of M. tuberculosis were analysed by testing (DST) for susceptibility to first- and second-line drugs using the proportional method in a liquid medium. The genomic material was extracted to perform multiplex polymerase chain reaction (PCR) for identification and gene sequencing of rpoB, katG, rrs, and gyrA. RESULTS: Approximately 80% (40/50) of the rpoB mutations that were detected outside the hot-spot region (S450L, H445D, D435V, S441L, I491F, and Q432P) conferred rifampicin-resistance on M. tuberculosis. Approximately 11.42% (4/35) of isolates with S315T mutation in katG led to rifampicin-resistance instead of isoniazid-resistance. The mutation in katG gene was found at various locations (P280P, G279R, E340Q, T271I, E340*stop codon, R373G, and S315N). Streptomycin-resistance was detected in 42% (21/50) of the strains, but only two strains had rrs gene mutations (G878A and/or S514R). Approximately 14% (7/50) of M. tuberculosis isolates were kanamycin- and capreomycin-resistant but did not harbour mutations in the rrs gene, while 80% (40/50) of the strains had mutations in the quinolone-resistance determining region (QRDR) of the gyrA gene (S95T, D94V, A90V, and S91P) including the pan-susceptible strain. CONCLUSIONS: Of the 50 strains analysed, most of the mutations in the rpoB gene associated with rifampicin-resistance were also detected in the katG and gyrA genes. Molecular characterisation using DNA sequencing techniques is a highly sensitive approach for detecting mutations.

Metformin improves FOXP3 mRNA expression through suppression of interferon gamma levels in pristane-induced murine models of lupus.

Background:  A recent study has indicated the potential of metformin therapy for lupus in animal models, but there has been no study evaluating the effect on pristane-induced lupus. This study aims to evaluate the effect of intraperitoneal versus oral metformin on interferon (IFN)-γ levels and FOXP3 mRNA expression on pristane-induced female BALB/c mice. Methods: In total, 31 female BALB/c mice, aged 6 weeks, were intraperitoneally induced with 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane). After 120 days, the mice were grouped and treated with various treatments: normal saline 100 mcl, oral metformin 100mg/kgBW, or intraperitoneal metformin 100mg/kgBW. After 60 days of treatment, all treatment groups were sacrificed, and kidney specimens prepared and stained using hematoxylin and esosin. Results: IFNγ levels of saline controls vs. oral metformin group was 309.39 vs. 292.83 pg/mL (mean difference 16.56 pg/mL; 95% CI 0.74-32.37; p=0.042), and saline control vs. intraperitoneal metformin group was 309.39 vs. 266.90 pg/mL (mean difference 42.49 pg/mL; 95% CI 29.24-55.73 pg/mL; p<0.004). FOXP3 mRNA expression changes in saline controls vs. oral metformin group was 6.90 vs. 7.79-fold change (mean difference -0.89-fold change; 95% CI -1.68-(-0.11); p=0.03)  and in saline controls vs. intraperitoneal metformin group was 6.90 vs. 9.02-fold change (mean difference -2.12-fold change; 95% CI -2.99-(-1.25); p=<0.001). Correlation analysis of FOXP3 mRNA expression and IFNγ level changes revealed a Pearson correlation of -0.785 (p=0.001) and R2 value of 0.616 (p=0.001). Conclusion: Metformin is a potential new therapy to reduce the levels of IFNγ and increase FOXP3 mRNA expression in mice models of systemic lupus erythematosus. Intraperitoneal metformin, i.e intravenous administration in human, could provide a novel route of administration to improve the effect of metformin for lupus patients.

Effects of lime (Citrus aurantifolia) peel to the expression of mRNA toll-like receptors 4 in balb/c mice-infected Salmonella typhi.

Lime peel contains metabolic compounds that have lethal effects of bacterial cells, but its effect as an antibacterial modulate innate immunity pathways, especially toll-like receptor 4 (TLR-4) signaling pathway, is unclear. This study examined the effects of lime peel extract (LPE) on the activity of TLR 4 in Balb/c mice induced by Salmonella typhi. Mice were induced intraperitoneally and then 3 days after induction, LPE was given orally on two doses (510 and 750 mg/kg BW). The number of bacterial colonization was counted using peritoneal fluid samples by the method of plate count agar. Intervention LPE for 5 days can degrade TLR-4 and the number of colonies of S. typhi. On day 3 after was induced S. typhi, TLR-4 gene expression of Balb/c mice is increased. Postintervention LPE for 5 days, the expression of TLR-4 gene decreased, significantly different at a dose of 750 mg/kg BW (P = 0.04). There was a positive correlation between the expression of TLR-4 gene by the number of bacterial colonization, decreasing gene expression of TLR-4, the number of bacterial colonization is also getting smaller (P = 0.013, r = 0.408). LPE can modulate the TLR-4 signaling pathway in host immunity so that the gene TLR-4 is expressed fewer in numbers. This mechanism causes the bacterial colony number to decrease, not even growth.