Abstract Tuberculosis (TB) and type 2 diabetes mellitus (DM), a major TB risk factor, are both accompanied by marked alterations in metabolic processes. Dissecting the specific metabolic changes induced by disease through metabolomics has shown potential to improve our understanding of relevant pathophysiological mechanisms of disease, which could lead to improved treatment. Targeted tandem liquid chromatography–mass spectrometry (LC-MS/MS) was used to compare amine and acylcarnitine levels in plasma samples of patients with TB or TB-DM from Indonesia at time of diagnosis and during antibiotic treatment. Partial least squares discrimination analysis (PLS-DA) showed good separation of patient groups. Amine levels were strongly altered in both disease groups compared to healthy controls, including low concentrations of citrulline and ornithine. Several amino acid ratios discriminated TB from controls (phenylalanine/histidine; citrulline/arginine; kynurenine/tryptophan), possibly reflecting changes in indoleamine-pyrrole 2,3-dioxygenase (IDO) and nitric oxide synthase (NOS) activity. Choline, glycine, serine, threonine and homoserine levels were lower in TB-DM compared to TB, and, in contrast to other analytes, did not normalize to healthy control levels during antibiotic treatment. Our results not only provide important validation of previous studies but also identify novel biomarkers, and significantly enhance our understanding of metabolic changes in human TB and TB-DM.
Introduction: Isoniazid (INH) is an anti-tuberculosis drug used widely. It's efficacy in killing Mycobacterium tuberculosis has made this drug as both chemoprophylaxis and first-line therapy in combating tuberculosis (TB). However, irrational dosage of INH may potentiates to isoniazid-induced hepatitis. NA T2 gene is responsible to metabolize INH and change it to the inactive form. In the geneitself, single nucleotide polymorphisms (SNPs) mayappeared, creating changes on itsgenotype, phenotype, andfunctionalactivity on itskinematics. Genotypevariationshave created three phenotypes, namely rapid, intermediate, and slow acetylators. As TB shares comorbiditywith diabetes mellitus (DM), observing this genemight be useful to control progression and potential side effect from TB-DM treatment. This study aimed to analyze the NAT2acetylator status in TB and TB-DM patientsinKupang, Indonesia. Methods: 5 TBand 5 TB-DM patients in Kupang were surveyed. Vein puncture was performed prior to DNA isolation and sequencingto observethe NAT2 sequence. Results: The genotypes of TB patients are NAT2*4/*4 (rapid acetylator), NAT2*4/*6A (intermediate acetylator), and NAT2*13A/*6J (intermediate acetylator). TB-DM patients have NAT2*4/*4, NAT2*4/*5G (intermediate acetylator), NAT2*13A/*6J (intermediate acetylator), NAT2*6A/*6A (slow acetylator) and NAT2*7B/*7B (slow acetylator). Conclusion: NAT2 rapid acetylators may have risk on therapyfailure; slow acetylators may gain risk of INH-induced hepatitis. TBDM patients should be monitored fordouble burden potencies regarding their DM therapy and isoniazid-induced hepatitis. Thus, gene-guided regimen therapy might benefit the patients for more personalized and saferlNH therapy.
Background: Human papillomavirus (HPV)-45 genotype circulates in high percentage in Bandung area - Indonesia, after HPV-16 and HPV-18. The aim of this study was to analyse variations of major capsid (L1) HPV-45 and its phylogeny. Furthermore in silico protein structure and epitope prediction was explored. Methods: L1 gene of HPV-45 was amplified, sequenced and aligned. Phylogenetic tree had been built and compared with a complete L1 HPV-45 sequence. Structure and epitope prediction of L1 protein were then developed in silico. Results: Of 5 L1 HPV-45 sequences collected, we have detected one variant of sub lineage A2 which was considered as a new variant, and two variants of B2. Superimposition of structure of these two variants with reference showed very similar structure. Furthermore, seven amino acid substitutions were found within these L1 variants of which two substitutions might change the polarity of corresponding amino acid I329T and S383G. The S383G occurred in surface loop (HI-Loop) of new L1 HPV-45 variant. Conclusion: Similar structure of Indonesian variants indicates that amino acids variations do not affect the L1 structure. However, one substitution with altered amino acid polarity found within the area of surface loop suggests a potential impact in antibody recognition and neutralization.
Pro- and anti-inflammatory cytokines and their signaling pathways play key roles in protection from and pathogenesis of mycobacterial infection, and their balance and dynamic changes may control or predict clinical outcome. Peripheral blood cells' capacity to produce proinflammatory (tumor necrosis factor alpha [TNF-alpha], interleukin-12/23p40 [IL-12/23p40], and gamma interferon [IFN-gamma]) and anti-inflammatory (IL-10) cytokines in response to Mycobacterium tuberculosis or unrelated stimuli (lipopolysaccharide, phytohemagglutinin) was studied in 93 pulmonary tuberculosis (TB) patients and 127 healthy controls from Indonesia. Their cells' ability to respond to IFN-gamma was examined to investigate whether M. tuberculosis infection can also inhibit IFN-gamma receptor (IFN-gammaR) signaling. Although there was interindividual variability in the observed responses, the overall results revealed that M. tuberculosis-induced TNF-alpha and IFN-gamma levels showed opposite trends. Whereas TNF-alpha production was higher in active-TB patients than in controls, IFN-gamma production was strongly depressed during active TB, correlated inversely with TB disease severity, and increased during therapy. By contrast, mitogen-induced IFN-gamma production, although lower in patients than in controls, did not change during treatment, suggesting an M. tuberculosis-specific and reversible component in the depression of IFN-gamma. Depressed IFN-gamma production was not due to decreased IL-12/IL-23 production. Importantly, IFN-gamma-inducible responses were also significantly depressed during active TB and normalized during treatment, revealing disease activity-related and reversible impairment in IFN-gammaR signaling in TB. Finally, IFN-gamma/IL-10 ratios significantly correlated with TB cure. Taken together, these results show that M. tuberculosis-specific stimulation of IFN-gamma (but not TNF-alpha) production and IFN-gammaR signaling are significantly depressed in active TB, correlate with TB disease severity and activity, and normalize during microbiological TB cure. The depression of both IFN-gamma production and IFN-gammaR signaling may synergize in contributing to defective host control in active TB.
Background<br />Epithelial ovarian cancer (EOC) is one of the most common cancers diagnosed in Indonesian women. A combination of paclitaxel and carboplatin is used to treat EOC as standard chemotherapy which is known to have hematologic toxicities. This study aimed to investigate the effect of combined paclitaxel-carboplatin chemotherapy on hematologic status in EOC patients managed at Dr. Hasan Sadikin General Hospital, Bandung, West Java.<br /><br />Methods<br />All patients with confirmed pathological diagnosis of EOC at Dr. Hasan Sadikin General Hospital in the period of 2013 to 2014 were registered. Only patients with complete hematologic data before and after chemotherapy were collected and compared using the paired non-parametric Wilcoxon and McNemar tests. <br /><br />Results<br />In total there were 147 patients with EOC (median age 46 ± 12 years), with the most dominant pathological diagnosis of mucinous (32.7%) and serous (29.3%) types. Only 33 patients had hematologic data before the initiation of chemotherapy. There was a significant decrease after chemotherapy including hemoglobin level (12.0 vs 10.9 g/dL, p=0.013), erythrocyte count (4.53 vs 3.74 million/mL, p<0.001), leukocyte count (7,700 vs 4,000/mm3 p<0.001) and platelet count (343,000 vs 215,000/mm3, p<0.001). Interestingly, anemia cases after chemotherapy were predominant (87.9%) compared with erythopenia, leukopenia, thrombocytopenia i.e. 39.4%, 57.6%, and 27.3% respectively. <br /><br />Conclusions<br />This study confirmed the hematologic toxicities after paclitaxel-carboplatin chemotherapy in EOC patients treated in Hasan Sadikin General Hospital, West Java. The hemoglobin concentration may serve as prognostic factor. Further studies directed to other factors such as genetic factor for polymorphisms may be encouraged to explore the decrease of the hematologic indices.
Background: Natural resistance associated macrophage protein (NRAMP) is a proton/ divalent cation transporter which play a role in iron trafficking in the phagosomes. Variations in NRAMP1 gene have been reported to be associated with susceptibility to tuberculosis (TB) because Mycobacterium tuberculosis (MTb), the causative agent of TB, compete with its host to uptake iron for its metabolism. The study aimed to describe the polymorphism of NRAMP among TB patients in Kupang, East Nusa Tenggara. Methods: This is a case-control study, cases were pulmonary TB, new patients, aged 15-55 years with sputum smear positive for acid fast bacili. Control were surrounding neighbours without symptoms and history of TB. All demographic information and blood sample were taken for polymorphism. PCR/RFLP method was performed to explore whether single nucleotide polymorphism D543N of NRAMP1 gene is associated with susceptibility to TB. Results: The study involved 64 pulmonary TB patients and 51 healthy controls. We observed a significant different in the distribution of NRAMP1 genotypes frequencies between TB patients and healthy controls (p = 0.014). Moreover, D543N polymorphism gave significant association only in male subjects. Though the numbers of the subjects are limited, D543N NRAMP1 polymorphism in endemic region in Kupang, the eastern part of Indonesia, seems to be associated with the susceptibility to TB. This is in contrary to studies reported in other part of Indonesia: i.e from west part (Jakarta, Bandung) and central part (Makassar). The population from Kupang may similar genetic background as African population, as Mycobacterium infected in population from Kupang is mostly Mycobacterium africanum. Increasing the number of subjects may enhance the power of the study and possibility to meet Hardy-Weinberg Equilibrium to detect the true associations of this polymorphism in susceptibility to TB. Conclusion: There was a significant difference of polymorphism NRAMP1 which more pronounced among male subjects, however this has not yet fulfilled the Hardy-Weinberg equilibrium. (Med J Indones. 2012;21:160-5)
Thalassaemia carrier screening is commonly conducted among direct-related or immediate family members of thalassaemia patients followed by a counselling about the thalassaemia. In countries where prevalence of thalassaemia carrier is high, carrier screening in general population is mandatory for example in pregnant women, in high school students, young adults or even before marriage [1, 2]. This screening may play a major role for carrier identification. A study conducted in Pakistan showed that siblings identified as β-thalassaemia carriers is higher as opposed to carriers in the general population i.e. 62.2% vs 5 to 8 %, respectively [3]. Therefore, it is practical to focus on siblings of identified thalassaemia patients when both resources and budgets are limited. In Indonesia, thalassaemia major is placed as number 6th in the catastrophic list. Although Indonesia harbours 6 to 10% thalassaemia carriers, carrier screening has not been put as mandatory yet. Furthermore, the willingness for carrier screening is still lacking. The purpose of this report was to explore the willingness for carrier screening in an extended family members of thalassaemia carrier individuals. This was an observational and descriptive study, conducted during a cross sectional event for thalassaemia carrier screening during a family gathering for 3 generations. A thalassaemia carrier individual, designated as Index Carrier (IC) aged 20 years old, was identified during a regular medical check-up prior student admission to the university. After counselling, the IC intended to screen the extended family from both father’s and mother’s side. The family was approached and during their family gathering, thalassaemia case story was introduced and shared. A family tree was drawn to identify whether thalassaemia patients presence among the extended family. The carrier screening was initially offered for the unmarried children older than 15 years old who came in the gathering, however, their parents were also encouraged to screen for the carrier status. After verbal informed consent and permission of the parents, blood was drawn in an EDTA tube. Complete blood count was measured and Shine and Lal index (MCV 2 x MCH/100) was calculated. Value of 3.5%. Interestingly, all boys had normal Hb and all girls had moderate anemia (8-10 g/dL) and they all felt healthy and active in sports. One of those carriers was a sibling of IC while the other IC’s sibling was normal. Further examination showed that IC’s mother was a carrier but not the father. The most common β-thalassaemia mutation in West Java is IVS1nt5, followed by IVS1nt1, and HbE (unpublished data), therefore, DNA analysis is important to explore mutation types, needed for the global data. The limitation of this study was that there was no iron measurement and DNA analysis due to limited budget. Simple haematology parameters may help identifying the carriers. Furthermore, the low knowledge and the anxiety of the participants hindered the willingness to screen their thalassaemia carrier status. As Indonesia is a multi-ethnic population country, a holistic approach to education programs in thalassaemia need to be considered for an optimal thalassaemia carrier screening. Campaign and education for community carrier screening, especially among extended family members of identified carrier individuals, need to be increased, towards zero thalassaemia. Further qualitative study needs to be explored to know the reasons for unwillingness for thalassaemia carrier screening.
Toll-like receptor 8 (TLR-8) is known as part of intracellular signaling transduction for bacterial phagocytosis. Mycobacterium tuberculosis (Mtb) is intracellular pathogenic bacteria that is recognized by this receptor, and genetic variation of TLR-8 might alter susceptibility of the host towards pulmonary tuberculosis (PTB). This study aimed to determine whether TLR-8 gene polymorphisms were associated to PTB in Kupang, Indonesia. This case-control study compared demographic and clinical data between 115 PTB patients and 115 controls, then two TLR-8 single nucleotide polymorphisms (rs3764880 and rs3788935) were explored using the GoldenGate® Genotyping for VeraCode® / BeadXpress Illumina®. There is no significant difference between sex distribution of patient vs control groups. The polymorphisms (rs3764880 and rs3788935) are in Hardy-Weinberg Equilibrium in this population (p > 0.05). The distribution of major vs minor genotypes and alleles of TLR-8 polymorphisms in PTB patients were as followed: rs3764880 (GG vs GA vs AA, 50.0% vs 21.4% vs 28.6% ; G vs A, 60.9% vs 39.1% ) and rs3788935 (GG vs GA vs AA, 53.0% vs 21.7% vs 25.3%; G vs A, 62.9% vs 37.1%). Neither genotypes nor alleles were associated with PTB in this population (P > 0.05). Besides, when the analyses were stratified by gender, none of the alleles of polymorphism in both genders were associated with PTB cases. None of the TLR-8 polymorphisms have associated the risk of developing PTB in Kupang, East Nusa Tenggara population (as opposed to other studies in different ethnic groups). These might reflect the diversity of genetic polymorphisms in eastern Indonesia populations, suggesting different genetic backgrounds with western part of Indonesia.
The tissue sample may have important genetic information in diagnostic, prognostic and counselling issues. Formalin-Fixed-Paraffin-Embedded (FFPE) is a routine method for preserving tissues. However, DNA isolated from FFPE tissue is often difficult to be amplified in PCR due to fragmentation and DNA-protein crosslinks. This study aimed to optimize the DNA isolation method from FFPE tissue and compare the performance of four different PCR ready-to-use kits. Genomic DNA was isolated from FFPE tissue colon of Short-segment Hirschsprung (S-HSCR) patients and prostate cancer tissue using Quick-DNA™ FFPE Kit (Zymo Research) with and without pre-heating treatment in KOH/NOH solution. Primers for Androgen Receptor (AR) gene and four different PCR kits: MyTaq HS Red Mix 2X (BioLine), FastStart Taq DNA Polymerase (Roche), KAPA2G fast PCR Kit 2X (KAPA Biosystem) and KOD FX Neo (Toyobo) were used for amplification. DNA electrophoresis was performed to compare the PCR results. BioLine and Toyobo kits gave better PCR results than those of Roche and KAPA Biosystem. Increasing amount of Taq polymerase and dNTPs of Roche kit by two-fold could increase the quality of PCR results. Toyobo could amplify DNA up to 417 bp, however, none of these PCR kits could amplify DNA above 450 bp. Pre-heated treatment of FFPE tissue in NaOH/KOH did not improve the DNA quality and PCR results. Toyobo PCR ready-to-use kit gave the best result among the other three PCR kits used in this study in amplifying DNA isolated from FFPE tissue. Designing the primers producing amplicon not more than 450 bp is suggested.
