Cancer Stem Cells (CD44+/CD24-), RAD6, DDB2 Immunohistochemistry Expression and IHC-UNEDO Scoring System As Predictor of Ovarian Cancer Chemoresistance.

Backgrounds: Ovarian cancer is a deadly women cancer with many chemoresistance after standard treatment. Ovarian cancer tissues' CD44+/CD24- (CSCs), RAD6 overexpression and DDB2 underexpression are associated with chemoresistance, recurrence, and poor prognosis of the disease because of the existence of cancer stem cells (CSCs). We tried to analyze the expression of those three proteins while building a predictor scoring system to predict the ovarian cancer chemoresistance from the ovarian cancer tissue immunohistochemistry. Materials and Methods: We conducted a cohort study of 64 patients divided into two groups (32 patients in each group) at the Cipto Mangunkusumo, Tarakan, Dharmais, and Fatmawati Hospital which are located in Jakarta city, Indonesia. The patients underwent cytoreductive debulking and histopathological examination continued by six series of chemotherapy followed by six months of observation. We divided the groups into chemoresistant and chemosensitive by using Response Criteria in Solid Tumors (RECIST) criteria. Ovarian cancer tissue immunohistochemistry tests were then performed to count the CSCs, RAD6 and DDB2 expressions. Results: We found relationship between increased CSCs, RAD6 and reduced DDB2 (p < 0.05) expression in ovarian cancer tissue with the chemoresistance. A possible predictor scoring system named IHC-UNEDO scoring was built to aid the ovarian cancer chemoresistance prediction. Conclusions: The conclusion is that CSCs, RAD6 and DDB2 expressions are significantly associated with ovarian cancer chemoresistance, and IHC-UNEDO scoring should be considered as a tool to predict ovarian cancer chemoresistance.

Effect of cholecalciferol supplementation on hand grip strength, walking speed, and expression of vitamin D receptor, interleukin-6, and insulin-like growth factor-1 in monocyte in pre-frail older adults: A randomized double-blind placebo-controlled trial.

AIM: To investigate the effect of cholecalciferol supplementation on hand grip strength, walking speed, and expression of vitamin D receptor (VDR), interleukine-6 (IL-6) and insulin-like growth factor-1 (IGF-1) in monocyte in pre-frail older adults. METHODS: We conducted a randomized double-blinded placebo-controlled clinical trial for 12 weeks, involving 120 pre-frail older adults who were randomized to the cholecalciferol group (cholecalciferol 4000 IU/day) or the placebo group. All subjects were given calcium lactate 500 mg/day. Hand grip strength and walking speed, as primary outcomes, were analyzed using intention-to-treat analysis. The expression of VDR, IGF-1 and IL-6 in monocytes, as secondary outcomes, were analyzed using per-protocol analysis. RESULTS: After a 12-week intervention, there was a significant increase in serum 25(OH)D levels in both groups, with the increase being higher in the cholecalciferol group than in the placebo group (49.05 vs. 24.01 ng/mL; P < 0.001). No statistically significant differences were observed in hand grip strength (P = 0.228) and walking speed (P = 0.734) between the groups. There were no differences in the expression of VDR (P = 0.513), IL-6 (P = 0.509), and IGF-1 (P = 0.503) monocytes between the groups. CONCLUSIONS: Cholecalciferol supplementation for 12 weeks increased serum 25(OH)D levels among pre-frail older adults. However, it did not improve hand grip strength and walking speed, and nor did it change the expression of VDR, IL-6, and IGF-1 in monocytes. Geriatr Gerontol Int 2024; 24: 554-562.

TNFα induces Caspase-3 activity in hematopoietic progenitor cells CD34+, CD33+, and CD41 + of myelodysplastic syndromes.

BACKGROUND: Cytopenia is the primary feature of Myelodysplastic Syndrome, even in the presence of hypercellular bone marrow. TNFα is recognized as both a proinflammatory, and proapoptotic cytokine with a well established role in promoting apoptosis in MDS. Therefore, TNFα has the potential to be a valuable biomarker for predicting the progression of cytopenia in MDS. This study aims to establish the role of TNFα exposure in triggering apoptosis through caspase-3 activity in CD34+, CD33+, and CD41 + cells in MDS. METHODS: This study is an in vitro comparative experimental research. Bone marrow mononuclear cells were isolated as the source of hematopoietic progenitor cells. Subsequently, CD34+, CD33+, and CD41 + cells were exposed to rhTNFα, and the caspase-3 activity was measured using flowcytometry. RESULTS: In MDS CD33 + and CD41 + caspase-3 activity of rhTNFα exposed cells was significantly higher than without exposed cells. The opposite result was found in CD34 + cells, where the caspase-3 activity without rhTNFα exposed cells was significantly higher than rhTNFα exposed cells. CONCLUSION: rhTNFα exposure led to an elevation in caspase-3 activity in MDS progenitor cells, especially in those that had differentiated into myeloid cell CD33 + and megakaryocyte cell CD41+, as opposed to the early progenitor cells CD34+.

Efficacy of LL-37 cream in enhancing healing of diabetic foot ulcer: a randomized double-blind controlled trial.

Wound healing in DFU (diabetic foot ulcer) has prolonged inflammation phase and defective granulation tissue formation. LL-37 has antimicrobial property, induces angiogenesis, and keratinocyte migration and proliferation. This study analyzes the efficacy of LL-37 cream in enhancing wound healing rate and decreasing the levels of IL-1α, TNF-α, and the number of aerobic bacteria colonization in DFU with mild infection. This study was conducted from January 2020 to June 2021 in Jakarta. Subjects were instructed to apply either LL-37 cream or placebo cream twice a week for 4 weeks. Wounds were measured on days 7, 14, 21, and 28 and processed with ImageJ. The levels of LL-37, IL-1α, and TNF-α from wound fluid were measured using ELISA. The number of aerobic bacteria colonization was counted from the isolate grown in culture. The levels of LL-37 in DFU at baseline were equally low in both groups which were 1.07 (0.37-4.96) ng/mg protein in the LL-37 group and 1.11 (0.24-2.09) ng/mg protein in the placebo group. The increase in granulation index was consistently greater in the LL-37 group on days 7, 14, 21, and 28 (p = 0.031, 0.009, 0.006, and 0.037, respectively). The levels of IL-1α and TNF-α increased in both groups on days 14 and 21 (p > 0.05). The decrease in the number of aerobic bacteria colonization was greater in the LL-37 group on days 7, 14 and 21, but greater in the placebo group on day 28 (p > 0.05). In conclusion, LL-37 cream enhanced the healing rate of DFU with mild infection, but did not decrease the levels of IL-1α and TNF-α and the number of aerobic bacteria colonization. This trial is registered at ClinicalTrials.gov, number NCT04098562.

Vitamin D Levels in Pre-frail Older Adults and Its Correlation with Hand Grip Strength.

BACKGROUND: Vitamin D deficiency is frequent in older adults and associated with poor musculoskeletal function. The prevalence of pre-frailty is also high in older persons, who may proceed to a frail state. This study aimed to determine the vitamin D levels in pre-frail older adults and its correlation with hand grip strength. METHODS: A cross-sectional study was conducted on older adults (age > 60 years) with a pre-frail condition who were visiting the outpatient geriatric clinic at Cipto Mangunkusumo Hospital in Jakarta, Indonesia. Serum levels of vitamin D, measured as 25(OH)D, were determined by enzyme-linked immunosorbent assay (ELISA), and hand grip strength was measured using a Jamar hydraulic dynamometer. Correlations between vitamin D levels and hand grip strength were evaluated by Spearman's rank correlation coefficient. Multiple linear regression analysis was carried out to assess contribution of variables that influence hand grip strength. RESULTS: Of 95 pre-frail older adults (mean age 70.08 ± 5.35 years), 67.4% were female,  and the median vitamin D level was 17.91 (interquartile range/IQR 13.68-26.36) ng/mL. Overall, 11.6% of the participants had normal vitamin D levels, whereas 34.7% and 53.7% had insufficient and deficient levels, respectively. Females were more likely to have inadequacy of vitamin D than males.  Those with vitamin D deficiency tended to have a higher body mass index (BMI) and lower vitamin D intake than normal levels. A significant correlation between serum vitamin D levels and hand grip strength was observed (r = 0.283; P = 0.006). After adjusting for age, comorbidities, nutritional status, functional status, BMI, protein intake, and sun exposure score, regression analysis between hand grip strength and vitamin D levels gave standard coefficient beta = 0.255 (P = 0.013). CONCLUSION: In this study, pre-frail older adults had a high proportion of deficient and insufficient vitamin D levels, and a significant correlation was found between serum vitamin D levels and hand grip strength.

CD44+/CD24- Expression as predictors of ovarian cancer chemoresistance: immunohistochemistry and flow cytometry study.

BACKGROUND: The conventional standard treatment for ovarian cancer is not very effective, and the disease is fatal for women. Cancer Stem Cells (CSCs) that express CD44+/CD24- can contribute to chemoresistance and a poor prognosis. We seek to investigate the expression of CSCs (CD44+/CD24-) in ovarian cancer and their predictive significance. METHODS: The ambispective cohort was performed on 64 patients (32 patients in each group) at four hospitals (Cipto Mangunkusumo, Tarakan, Fatmawati, and Dharmais Hospital). Debulking surgery was performed on the patients, followed by histopathological analysis. The patients had six rounds of chemotherapy and were under monitoring for six months. The therapeutic responses were evaluated using the RECIST criteria (Response Criteria in Solid Tumors) and categorized as chemoresistant or chemosensitive. Using immunohistochemistry, we directly assess the CSCs from ovarian cancer tissue and using flow cytometry to assess the CSCs from the blood. RESULTS: High CSCs expression and ovarian cancer chemoresistance were significantly related in both trials (p 0.05). A better outcome was obtained using CD44+/CD24- immunohistochemistry. CONCLUSIONS: We conclude that there is a substantial association between high CSCs expression and chemoresistance in ovarian cancer and that CSCs immunohistochemistry has a higher predictive value.

Expression of CD44+/CD24-, RAD6 and DDB2 on chemotherapy response in ovarian Cancer: A prospective flow cytometry study.

Backgrounds: Ovarian cancer is the 8th deadliest common cancer in women around the world. Almost all ovarian cancer patients would experience chemoresistance, recurrence, and poor prognosis after cytoreductive surgery and platinum-based chemotherapy. Chemoresistant cancer cells have characteristic expressions of cancer stem cell proteins (CSCs) CD44+/CD24-, RAD6 and DDB2. The increased expression of CD44+/CD24-, RAD6, and decreased DDB2 are believed to be associated with chemoresistance, recurrence, and poor prognosis of the disease. Thus, this study's objective is to analyze the correlation between the expression of CD44+/CD24-, RAD6 and DDB2 with ovarian cancer chemoresistance. Materials and methods: This study was conducted with a prospective cohort of 64 patients who is divided into two groups (32 patients in each group) at the Obstetrics-gynecology and pathology department of Cipto Mangunkusumo, Tarakan, Dharmais, and Fatmawati Hospital. All suspected ovarian cancer patients underwent cytoreductive debulking and histopathological examination. Chemotherapy was given for six series followed by six months of observation. After the observation, we determined the therapy's response with the RECIST Criteria (Response Criteria in Solid Tumors) and then classified the results into chemoresistant or chemosensitive groups. Flow cytometry blood tests were then performed to examine the expression of CD44+/CD24-, RAD6 and DDB2. Results: There was a significant relationship between increased levels of CD44+/CD24-, and RAD6 (p < 0.05) levels with the chemoresistance of ovarian cancer. The logistic regression test showed that the CD44+/CD24- was better marker. Conclusions: These results indicate that CD44+/CD24 and RAD6 expressions are significantly associated with ovarian cancer chemoresistance, and CD44+/CD24- is the better marker to predict ovarian cancer chemoresistance.

Clopidogrel Resistance Among Ischemic Stroke Patients and Its Risk Factors in Indonesia.

OBJECTIVE: Clopidogrel is a common antiplatelet used as secondary prevention of ischemic stroke, known to have better efficacy than aspirin, with a equivalent safety profile. However, clopidogrel resistance is not uncommon but has not been widely studied in Asia. This study will further assess clopidogrel resistance and its risk factors. MATERIALS AND METHODS: A cross-sectional study was conducted at Rumah Sakit Universitas, Indonesia, and Rumah Sakit Cipto Mangunkusumo, Indonesia in 2020-2021. All patients had had at least one episode of ischemic stroke. Clopidogrel resistance was assessed using a VerifyNow assay. RESULTS: 57 subjects were enrolled in this study. We found 15.8% of subjects were clopidogrel resistant. Gender was significantly associated with clopidogrel resistance, with males having 80% lower clopidogrel resistance (OR 0.2 (95% CI 0.022 - 0.638); P=0.006). Meanwhile, smoking was not associated with clopidogrel responsiveness (P=0.051). We found no association between haemoglobin, blood glucose, HbA1c, cholesterol, liver enzymes, serum urea concentration or creatinine levels and clopidogrel resistance. CONCLUSION: Clopidogrel remains an effective treatment to prevent recurrent ischemic stroke in Indonesia. Further studies are needed to assess gene polymorphism and clopidogrel resistance, which may explain the findings of this study.

Risk of developing cognitive impairment on patients with chronic obstructive pulmonary disease: A systematic review.

BACKGROUND: The increasing number of chronic obstructive pulmonary disease (COPD) incidence has led to a great negative impact on older people's lives. This chronic disease was a critical and independent risk factor for cognitive function impairment in the elderly with mild cognitive impairment as a frequent feature. This systematic review aimed to examine the risk of developing cognitive impairment in COPD. METHODS: A structured search of the literature was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-analyses statement guideline, with a pre-determined search strategy starting from study identification, title and abstract screening, eligibility assessment, and inclusion of relevant study. The search was conducted in PubMed and MEDLINE via EBSCOhost, with restriction to human studies. The studies from inception until January 12, 2021. RESULTS: Five original articles were included. Most studies found that patients with COPD had a higher chance of developing cognitive impairment, especially when patients were followed up for more than 5 years. We discovered that the risk of cognitive impairment seemed to be correlated with the length of time spent following the participants, with the highest risk of cognitive impairment being identified in those who had the longest observation period. It is critical to conduct cognitive screening from the time a diagnosis of COPD is obtained and on a continuing basis in order to recognize and treat these individuals appropriately. CONCLUSION: There is a potential association between COPD and mild cognitive impairment. We encourage more studies to be done with higher sensitivity and specificity cognitive screening tools in the future to build better evidence and qualify to be analyzed quantitatively with meta-analysis.

The role of clopidogrel resistance-related genetic and epigenetic factors in major adverse cardiovascular events among patients with acute coronary syndrome after percutaneous coronary intervention.

Despite patients with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI) and receiving clopidogrel therapy, some patients still experience major adverse cardiovascular events (MACEs). Clopidogrel resistance, which may be regulated by genetic and epigenetic factors, may play a role in MACEs. This study aimed to determine the association between genetic (CYP2C19 and P2Y12 polymorphisms) and epigenetic (DNA methylation of CYP2C19 and P2Y12 and miRNA-26a expression) factors and their effects on MACEs among post-PCI patients. Post-PCI patients who received a standard dosage of clopidogrel at Harapan Kita Hospital between September 2018 and June 2020 were included in this study. MACEs were observed in patients within 1 year after PCI. Platelet aggregation was assessed using light transmission aggregometry (LTA). DNA methylation of CYP2C19 and P2Y12 was assessed using the bisulfite conversion method. CYP2C19 and P2Y12 polymorphisms and miRNA-26a expression were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). Among a total of 201 subjects, 49.8% were clopidogrel-resistant, and 14.9% experienced MACEs within 1 year after PCI (death was 7.5%). Hypomethylation of CYP2C19 (p = 0.037) and miRNA-26a upregulation (p = 0.020) were associated with clopidogrel resistance. CYP2C19*2/*3 polymorphisms (p = 0.047) were associated with MACEs in 1 year. This study demonstrated that hypomethylation of CYP2C19 and miRNA-26a upregulation increased the risk of clopidogrel resistance in post-PCI patients, but there was no correlation between clopidogrel resistance and MACEs. However, CYP2C19*2/*3 polymorphisms were the factors that predicted MACEs within 1 year.

Expression of mRNA TNFα and level of protein TNFα after exposure sCD40L in bone marrow mononuclear cells of myelodysplastic syndromes.

BACKGROUND: Cytopenia is the primary phenomenon in myelodysplastic syndrome (MDS) amidst hypercellular bone marrow. The soluble CD40 ligand (sCD40L) is considered as a cytokine that can trigger synthesis of tumor necrosis factor α (TNFα) that promotes apoptosis. The objective of this study is to prove that recombinant human sCD40L (rh-sCD40L) exposure on bone marrow mononuclear cells (BMMC) MDS increases TNFα expression at mRNA level and at protein level. METHODS: BMMC from MDS patients whom diagnosed and classified using the WHO 2008 criteria, were exposed to rh-sCD40L and antiCD40L. The expressions of TNFα mRNAs were quantified by qRT-PCR, level of TNFα were measured using the ELISA method. RESULTS: Exposure of rh-sCD40L significantly increased the expression of TNFα mRNA. The similar exposure also significantly increased the level of TNFα compared to controls. TNFα mRNA expression on BMMC in MDS samples exposed to rh-sCD40L is 3.32 times compared to TNFα mRNA expression without exposure. level of TNFα in supernatant media exposed to rh-sCD40L in MDS samples was higher than that of control samples which were 44.44 and 4.85 pg/mL, P=0.018. CONCLUSIONS: The sCD40L plays a role in increasing the synthesis of TNFα in mRNA level and protein level in BMMC MDS.

The Differential Effects of Propylthiouracil and Methimazole as Graves' Disease Treatment on Vascular Atherosclerosis Markers: A Randomized Clinical Trial.

Background: Hyperthyroidism is related to vascular atherosclerosis. Propylthiouracil (PTU) and methimazole, other than their antithyroid effects, may have different mechanisms in preventing atherogenesis in Graves' disease. Objective: This study aimed to investigate the effect of antithyroid drugs on markers of vascular atherosclerosis in Graves' hyperthyroidism. Methods: This study was a single-blind, randomized clinical trial conducted on 36 patients with Graves' disease in Cipto Mangunkusumo General Hospital, Jakarta, Indonesia, from June 2019 until July 2020. Graves' disease was diagnosed from clinical manifestation of hyperthyroidism with diffuse goiter and then confirmed by thyroid stimulation hormone (TSH), free T4 (fT4), and TSH-receptor antibody (TRAb) measurements. Participants were randomly assigned to either a PTU or a methimazole treatment group and followed up for 3 months. Markers of vascular atherosclerosis were represented by adhesion molecules [intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin], carotid artery stiffness [pulse wave velocity (PWV)], and thickness [carotid intima media thickness (cIMT)]. Results: By the end of the study, 24 participants reached euthyroid condition (13 from the PTU group and 11 from the methimazole group). After 3 months of follow-up, in the PTU group, we noticed an improvement of ICAM-1 [pretreatment: 204.1 (61.3) vs. posttreatment: 141.6 (58.4) ng/ml; p = 0.001], VCAM-1 [837 (707-977) vs. 510 (402-630) ng/ml; p < 0.001] and E-selectin [32.1 (24.1-42.7) vs. 28.2 (21.6-36.8) ng/ml; p = 0.045] in the PTU group. In the methimazole group, only VCAM-1 improvement [725 (565-904) vs. 472 (367-590); p = 0.001] was observed. Meanwhile, we found no significant changes in PWV or cIMT in either group. Conclusion: Antithyroid treatment in Graves' disease leads to improvement in adhesion molecules, with a lesser effect on methimazole, whereas there were no significant changes in PWV or cIMT. PTU may have a better mechanism compared with methimazole in terms of improving adhesion molecules.

Determining a critical threshold for G6PD activity below which red blood cell response to oxidative stress is poor.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme disorder in the world. Its main function is to generate NADPH that is required for anti-oxidative pathway in the cells especially in red blood cells (RBC). G6PD deficiency is X-linked and thus subject to random X-chromosome inactivation in women giving them mosaic expression of G6PD activities in their individual cells. This phenomenon makes it difficult for diagnosis with the currently available G6PD qualitative diagnostic tests. With the rolling out of newly marketed anti-malarial drug tafenoquine, which has a long half-life, screening for G6PD deficiency becomes a necessity where those with < 70% G6PD activity cannot receive this drug. Thus, evidence for a quantitative cut-off for G6PD activity is needed to ensure safe drug administration. METHODS: RBC models were developed to analyse the effect of oxidant on RBC oxidative markers namely total glutathione (GSH)and malondialdehyde (MDA). G6PD activity was measured using quantitative assay from Trinity Biotech and was correlated with cytofluorometric assay. RBC from two G6PD heterozygous women with different G6PD activities were also analysed for comparison. RESULTS: There was a negative correlation between G6PD activity and CuCl concentration and a strong association between G6PD activities and proportion of G6PD normal RBC in CuCl-treated models and in ex vivo RBC. However, in terms of oxidative stress markers analyses, unlike the hypothesis where the lower G6PD activity, the higher MDA and the lower GSH level, the CuCl RBC model showed that in low G6PD activities (10-30%) cells, the MDA level is lower compared to the rest of the models (p < 0.05). The ex vivo models however were in line with the hypothesis, although the result was not significant (p = 0.5). There was a significant difference between RBC with < 60% and those with > 80% G6PD activities in CuCl RBC model, but not in ex vivo RBC (p = 0.5). Genotyping heterozygous subjects showed G6PDViangchan variant with 2.97 U/gHb (33% activity) and 6.58 U/gHb (74% activity). CONCLUSIONS: The GSH analysis has pointed to the 60% G6PD activity cut-off and this data is supportive of the old World Health Organization threshold for intermediate upper limit of 60% G6PD activity. However, there are significant limitations in using MDA assay with CuCl RBC model because the RBC was already stressed due to the copper treatment and thus present a different result when compared to the ex vivo model.

Characterising SRD5A2 Gene Variants in 37 Indonesian Patients with 5-Alpha-Reductase Type 2 Deficiency.

The 5-alpha-reductase type 2 deficiency (5ARD2) is an autosomal recessive condition associated with impairment in the conversion of testosterone to dihydrotestosterone. This condition leads to undervirilisation in 46,XY individuals. To date, there have been more than 100 variations identified in the gene responsible for 5ARD2 development (steroid 5-alpha-reductase 2, SRD5A2). However, few studies have examined the molecular characterisation of Indonesian 5ARD2 cases. In the current study, we analysed 37 subjects diagnosed with 46,XY DSD (disorders of sex development) with confirmed variations in the SRD5A2 gene. We examined results from testosterone/dihydrotestosterone (T/DHT) and urinary etiocholanolone/androsterone (Et/An) ratios, as well as from molecular and clinical analyses. Twelve variants in the SRD5A2 gene were identified, and 6 of which were novel, namely, c.34-38delGinsCCAGC, p.Arg50His, p.Tyr136 ∗ , p.Gly191Arg, p.Phe194Ile, and p.Ile253Val variants. Moreover, we determined that 20 individuals contained harmful mutations, while the remaining 17 variants were benign. Those containing harmful mutations exhibited more severe phenotypes with median external genitalia masculinisation scores (EMS) of 3 (1.5-9) and were more likely to be diagnosed at a later age, reared as female, and virilised at pubertal age. In addition, the respective sensitivities for detecting severe 5ARD2 cases using T/DHT (cutoff: 10) and urinary Et/An ratios (cutoff: 0.95) were 85% and 90%, whereas mild cases were only identified with 64.7% and 47.1% sensitivity, respectively. Although we were unable to identify clear correlations between genotypic and phenotypic characteristics in this study, we clearly showed that individuals who were homozygous or compound heterozygous for any of the harmful mutations were more likely to exhibit classic 5ARD2 phenotypes, lower EMS, female assignment at birth, and virilisation during puberty. These results serve to inform the development of improved clinical and molecular 5ARD2 diagnostic approaches, specifically in Indonesian patients.

Accurate light microscopic diagnosis of South-East Asian ovalocytosis.

INTRODUCTION: South-East Asian ovalocytosis (SAO) is a common inherited red blood cell polymorphism in South-East Asian and Melanesian populations, coinciding with areas of malaria endemicity. Validation of light microscopy as a diagnostic alternative to molecular genotyping may allow for its cost-effective use either prospectively or retrospectively by analysis of archived blood smears. METHODS: We assessed light microscopic diagnosis of SAO compared to standard PCR genotyping. Three trained microscopists each assessed the same 971 Giemsa-stained thin blood films for which SAO genotypic confirmation was available by PCR. Generalized mixed modeling was used to estimate the sensitivity, specificity, positive predictive value, and negative predictive value of light microscopy vs "gold standard" PCR. RESULTS: Among red cell morphologic parameters evaluated, knizocytes, rather than ovalocytic morphology, proved the strongest predictor of SAO status (odds ratio [OR] = 19.2; 95% confidence interval [95% CI] = 14.6-25.3; P ≤ 0.0001). The diagnostic performance of a knizocyte-centric microscopic approach was microscopist dependent: two microscopists applied this approach with a sensitivity of 0.89 and a specificity of 0.93. Inter-rater reliability among the microscopists (κ = 0.20) as well as between gold standard and microscopist (κ = 0.36) underperformed due to misclassification of stomatocytes as knizocytes by one microscopist, but improved substantially when excluding the error-prone reader (κ = 0.65 and 0.74, respectively). CONCLUSION: Light microscopic diagnosis of SAO by knizocyte visual cue performed comparable to time-consuming and costlier molecular methods, but requires specific training that includes successful differentiation of knizocytes from stomatocytes.

Loss of DMRT1 gene in a Mos 45,XY,-9[8]/46,XY,r(9)[29]/47,XY,+idic r(9)× 2[1]/46,XY,idic r(9)[1]/46,XY[1] female presenting with short stature.

BACKGROUND: A 46,XY sex reversal syndrome is characterized by discordant genetic and phenotypic sex, leading to normal external female genitalia, undeveloped gonads and presence of Müllerian structures in an otherwise 46,XY individual. Chromosome 9pter aberrations, such as ring chromosome have been reported to cause 46,XY disorders of sex development (DSD), due to involvement of DMRT1 gene located at the 9p24.3 region. CASE PRESENTATION: This study presents a unique case of a 12-year-old female with mos 46,XY, (r)9[31]/45,XY,-9[9] karyotype, presenting with intellectual disability and short stature, mimicking Turner syndrome. Re-karyotyping was performed using standard GTL-banding technique. Further cytogenetic study using standard metaphase fluorescent in situ hybridization (FISH) technique was applied to cultured lymphocytes from peripheral blood, hybridized using green control probe specific to 9q21 loci, and red DMRT1 probe specific to 9p24.3 loci. Cytogenetics and FISH analysis revealed mos 45,XY,-9[8]/46,XY,r(9)[29]/47,XY,+idic r(9)× 2[1]/46,XY,idic r(9)[1]/46,XY[1] and haploinsufficiency of DMRT1 gene in most cells. CGH array revealed a deletion around 1.25 Mb at 9p24.3 loci [arr 9p24.3(204,193-1,457,665)× 1] and three duplications around 13 Mb [9p24.3p22.3(1,477,660-14,506,754)× 3] near the breakage point that formed the ring chromosome 9. CONCLUSIONS: The clinical presentation of the subject that mimics Turner syndrome highlights the importance of cytogenetic analysis to detect the possibility of ring chromosome 9. Sex reversal due to haploinsufficiency of DMRT1 gene in ring chromosome 9 structures is exceedingly rare with only a handful of cases ever reported. This finding further highlights the importance of DMRT1 gene in sex determination and differentiation in males. More research is required to pinpoint the exact mechanism that underlies sex reversal caused by DMRT1 haploinsufficiency.

Prediction of Wound Healing in Diabetic Foot Ulcers: an Observational Study in Tertiary Hospital in Indonesia.

AIM: to evaluate the role of clinical characteristics, functional markers of vasodilation, inflammatory response, and atherosclerosis in predicting wound healing in diabetic foot ulcer. METHODS: a cohort study (February - October 2010) was conducted from 40 subjects with acute diabetic foot ulcer at clinical ward of Dr. Cipto Mangunkusumo National Central General Hospital, Jakarta, Indonesia. Each subject underwent at least two variable measurements, i.e. during inflammatory phase and proliferation phase. The studied variables were clinical characteristics, complete peripheral blood count (CBC) and differential count, levels of HbA1c, ureum, creatinine, lipid profile, fasting blood glucose (FBG), marker of endothelial dysfunction (asymmetric dimethylarginine/ADMA, endothelin-1/ET-1, and flow-mediated dilation/FMD of brachial artery), and marker of vascular calcification (osteoprotegerin/OPG). RESULTS: median of time achieving 50% granulation tissue in our study was 21 days. There were nine factors that contribute in the development of 50% granulation tissue, i.e. family history of diabetes mellitus (DM), previous history of wound, wound area, duration of existing wound, captopril and simvastatin medications, levels of ADMA, ET-1, and OPG. There were three out of the nine factors that significantly correlated with wound healing, i.e. wound area, OPG levels, and simvastatin medications. CONCLUSION: in acute diabetic foot ulcers, wound area and OPG levels had positive correlation with wound healing, whereas simvastatin medications had negative correlation with wound healing.

Assessment of Point-of-Care Diagnostics for G6PD Deficiency in Malaria Endemic Rural Eastern Indonesia.

BACKGROUND: Patients infected by Plasmodium vivax or Plasmodium ovale suffer repeated clinical attacks without primaquine therapy against latent stages in liver. Primaquine causes seriously threatening acute hemolytic anemia in patients having inherited glucose-6-phosphate dehydrogenase (G6PD) deficiency. Access to safe primaquine therapy hinges upon the ability to confirm G6PD normal status. CareStart G6PD, a qualitative G6PD rapid diagnostic test (G6PD RDT) intended for use at point-of-care in impoverished rural settings where most malaria patients live, was evaluated. METHODOLOGY/PRINCIPAL FINDINGS: This device and the standard qualitative fluorescent spot test (FST) were each compared against the quantitative spectrophotometric assay for G6PD activity as the diagnostic gold standard. The assessment occurred at meso-endemic Panenggo Ede in western Sumba Island in eastern Indonesia, where 610 residents provided venous blood. The G6PD RDT and FST qualitative assessments were performed in the field, whereas the quantitative assay was performed in a research laboratory at Jakarta. The median G6PD activity ≥ 5 U/gHb was 9.7 U/gHb and was considered 100% of normal activity. The prevalence of G6PD deficiency by quantitative assessment (<5 U/gHb) was 7.2%. Applying 30% of normal G6PD activity as the cut-off for qualitative testing, the sensitivity, specificity, positive predictive value, and negative predictive value for G6PD RDT versus FST among males were as follows: 100%, 98.7%, 89%, and 100% versus 91.7%, 92%, 55%, and 99%; P = 0.49, 0.001, 0.004, and 0.24, respectively. These values among females were: 83%, 92.7%, 17%, and 99.7% versus 100%, 92%, 18%, and 100%; P = 1.0, 0.89, 1.0 and 1.0, respectively. CONCLUSIONS/SIGNIFICANCE: The overall performance of G6PD RDT, especially 100% negative predictive value, demonstrates suitable safety for G6PD screening prior to administering hemolytic drugs like primaquine and many others. Relatively poor diagnostic performance among females due to mosaic G6PD phenotype is an inherent limitation of any current practical screening methodology.

High Prevalence of Hepatitis B Virus Infection in Young Adults in Ternate, Eastern Indonesia.

The incidence of hepatitis B virus (HBV) infection has been declining thanks to the universal hepatitis B infant immunization program. Nevertheless, young adults born before the program implementation might have acquired HBV in early childhood or remain susceptible to infection. This study aimed to evaluate hepatitis B epidemiology in asymptomatic young adult population in Ternate, eastern Indonesia. Serum samples of 376 subjects (aged 17-25, mean 19.82 ± 1.69 years; male/female 138/238) were screened for HBV parameters serologically (HBV surface antigen [HBsAg]; its antibody [anti-HBs]; anti-core antigen [anti-HBc]), and molecularly (HBV DNA). HBsAg, anti-HBc, anti-HBs, and HBV DNA prevalence were 15.7%, 36.2%, 24.2%, and 27.9%, respectively, with male predominance. Of all subjects, 13.0% were HBsAg negative with detectable HBV DNA (occult hepatitis B [OHB]), and 56.4% showed negativity for all seromarkers. This population showed high hepatitis B prevalence with substantial occurrence of OHB. However, a high percentage of the population were still susceptible and at risk of HBV infection. This study emphasizes the necessity to improve prevention strategies to screen and manage HBV carriers, including the adoption of catch-up or booster vaccination targeted to young adult populations. Investigations on the roles of host-virus interactions associated with OHB and its implications are warranted.

Immune response of thalassemia major patients in Indonesia with and without splenectomy.

AIM: to describe non-spesific and specific immune response profile in Indonesian thalassemia major with and without splenectomy. METHODS: this study was held at Thalassaemia Centre, Cipto Mangunkusumo Hospital Jakarta on September 2013-February 2014. A comparative cross sectional study was conducted in healthy, thalassemia major aged more than 12 year and seronegative HIV. They were matched in age and sex for splenectomised and non-splenectomised groups, analysing the non-spesific immune response (neutrophil count and phagocytosis) and specific immune response (count and function of cellular immunity). Infection episodes were also analized as immune response in vivo parameter. RESULTS: splenectomised thalassemia major showed increased neutrophil count but significantly decreased non-spesific immune response (neutrophil phagocytosis). Spesific immune response of splenectomised group presented significantly higher absolute lymphocyte, lymphocyte T, CD4+ and CD8+ counts compared to non-splenectomised thalassemia major (p<0.05). Ratio CD4+/CD8+ were similar in these groups. Serum marker of activated cellular imunity function (IL-2 and TNF-) were similar among two groups. Mild infection episodes on splenectomised and non-splenectomised group were 2.02 (ranged 0 to 12) times and 0.81 (ranged 0 to 8) times (p=0.004), respectively. Severe infection on splenectomised group were sepsis for 2 weeks and diarrhea for 1 week, whereas on non-splenectomised group was typhoid fever for 4 days. CONCLUSION: there were significant differences on immune response among thalassemia major patients. Splenectomised thalassemia major showed a greater degree of susceptibility to infections than non-splenectomised thalassemia major.