The association between PTPN22 C1858T gene polymorphism and type 1 diabetes mellitus: an Indonesian study.

BACKGROUND: Type 1 diabetes mellitus (T1DM) is disease caused by the destruction of ß pancreatic cells. The activation of T-lymphocyte and proliferation inhibitor are induced by protein tyrosine phosphatase non-receptor type 22 (PTPN22). However, the link between PTPN22 C1858T gene polymorphism and T1DM is still controversy. This study aimed to analyse the C1858T gene polymorphism in Indonesian children with T1DM. MATERIALS AND METHODS: This case-control study was conducted from March 2021 to May 2022 in the Endocrinology Outpatient Clinic at Dr. Soetomo Hospital and Tropical Disease Center Universitas Airlangga. Patients with controlled T1DM during the study period were included. The PTPN22 analysis used polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: Sixty-two children voluntarily participated in this study, and were equally divided into the T1DM and control groups. Most of the patients (94%, 58/62) are Javanese. This study revealed a more frequent CC genotype (9.4%) and allele-C (54.6%) polymorphism in the T1DM group, while more frequent CT genotype (100%) and allele-T (50%) polymorphism were in the control group. The C- and T-allele frequency was 54.6% and 45.4% in the T1DM group, respectively. The T1DM and control groups did not significantly differ (p= .2381). CONCLUSIONS: PTPN22 homozygous genotype-CC and allele-C polymorphisms are more frequent in patients with T1DM. However, the PTPN22 C1858T gene polymorphism did not significantly correlate to T1DM children in this study.Key Messages:The PTPN22 C1858T gene polymorphism does not significantly affect the susceptibility of T1DM in Indonesian children.PTPN22 homozygous genotype-CC polymorphism was more observed in the T1DM group; thus, this genotype may play as a risk factor for T1DM children in the Indonesian population.

Cytotoxic T-lymphocyte-associated protein 4 +49A/G polymorphism in Down syndrome children with Hashimoto's thyroiditis.

Thyroid dysfunction is the most common endocrine disorder in Down syndrome (DS) children. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is one of the immune regulatory genes that correlates with Hashimoto's thyroiditis (HT). However, studies on CTLA-4 +49A/G in DS children with HT are still limited. We aimed to evaluate CTLA-4 +49A/G gene polymorphism in DS children with HT. This case-control study, conducted from February 2020 to February 2022 at Dr. Soetomo General Hospital, Surabaya, enrolled 40 DS children with HT and 50 healthy children. The DNA sequencing was performed to identify the polymorphism (Sanger sequencing). Thyroid peroxidase antibodies (TPOAb), thyroglobulin antibodies (TgAb), thyroid-stimulating hormone (TSH), and free thyroxine (FT4) levels were analyzed by enzyme-linked immunosorbent assay (ELISA). The mean age of DS children with HT was 1.78 years. Males predominated in the study population. Subjects with GG genotype were diagnosed earliest with hypothyroidism (8 months) compared with other studies. The most common thyroid dysfunction was central hypothyroidism, with TgAb positivity present in all patients. The AA genotype (odds ratio [OR] 0.265, 95% confidence interval [CI] 0.094-0.746; P = 0.012) and A allele (OR 0.472, 95% CI 0.309-0.721; P = 0.0002) were significantly more frequent in the control group. The AG genotype (OR 2.65, 95% CI 0.094-0.746; P = 0.003) and G allele (OR 2.116, 95% CI 1.386-3.23; P = 0.003) were more frequent in the DS with HT group. The age of the subjects in this study was younger than in previous studies. The AG genotype and the G allele were more prevalent in the DS with HT group and may be a risk factor in HT development in DS children. Furthermore, the AA genotype may act as a protective factor against HT in DS children.

Protein tyrosine phosphatase non-receptor type 22 C1858T gene polymorphism in children with down syndrome and autoimmune thyroid diseases.

Autoimmune Thyroid Disease (AIT) is a frequent comorbidity in Down Syndrome (DS). Protein Tyrosine Phosphatase Non- Receptor Type 22 C1858T (PTPN-22 C1858T) gene polymorphisms have a role in the progression of AIT. The study on PTPN- 22 C1858T gene polymorphism as the risk factor of AIT in DS children is still limited. This study aims to evaluate PTPN-22 C1858T polymorphism in Indonesian DS children. A cross-sectional study involving 31 DS children with hypothyroidism (19 boys/12 girls) was conducted for ten months from February to November 2020 at Dr. Soetomo General Hospital Surabaya. The PTPN-22 C1858T gene polymorphism was analyzed using Polymerase Chain Reaction-Restriction-Fragment-Length Polymorphism (PCR-RFLP). Anti-Thyroid Peroxidase (Anti- TPO) and Anti-Thyroglobulin (Anti-TG), FT4, T3, and TSH levels were analyzed using Enzyme-Linked-Immunosorbent-Assay (ELISA). The mean age of the subjects was 19.45±17.3 months. The CT variant of PTPN-22 C1858T was observed in all subjects. The mean level of T3, FT4, and TSH were 1.59±0.45 ng/mL, 0.81±0.57 ng/mL, 0.22±0.21 µU/mL, respectively. Around 83.9% of patients suffered from central hypothyroidism, 12.9% from primary hypothyroidism, and 3.2% from subclinical hypothyroidism. The positive anti-TG and anti-TPO were observed in 96.8% and 58.1%, respectively. CT variant was observed in Indonesian DS children who suffered from hypothyroidism.

Mono-Parasitic and Poly-Parasitic Intestinal Infections among Children Aged 36-45 Months in East Nusa Tenggara, Indonesia.

The prevalence of intestinal parasitic infection remains high in developing countries, especially because of geographic and socio-demographic factors. This study aimed to evaluate intestinal parasitic infection, as well as its risk factors, among children aged 36-45 months in a rural area (North Kodi) and an urban area (Kupang) of East Nusa Tenggara, Indonesia. Anthropometry, socio-demographic factors and personal hygiene practices were assessed. A total of 214 children participated in the study, and 200 stool samples were collected for intestinal parasite examination. Approximately 30.5% (61/200) of the children were infected with one or more intestinal parasites (67.2%; 41/61 being mono-parasitic infections and 32.8%; 20/61 being poly-parasitic infections). A total of 85 intestinal parasites were detected, consisting of 35.3% (30/85) protozoa and 64.7% (55/85) helminths. The predominant protozoa were Giardia lamblia (43%; 13/30) and Blastocystis spp. (33.3%; 10/30), whereas the predominant helminths were Trichuris trichiura (50.9%; 28/55) and Ascaris lumbricoides (43.6%; 24/55). Moreover, intestinal parasitic infection was associated with rural area (OR 4.5; 95%CI 2.3-8.6); the absence of treatment with deworming drugs (OR 2.56; 95%CI 1.3-5.0); sanitation facilities without a septic tank (OR 4.3; 95%CI 2.1-8.5); unclean water as a source of drinking water (OR 4.67; 95%CI 2.4-9.4); no handwashing practice after defecation (OR 3.2; 95%CI 1.4-7.3); and stunted children (OR 4.4; 95%CI 2.3-8.3). In conclusion, poly-parasitic infections were common in this study. Poor personal hygiene practice and sanitation factors contributed to the high prevalence of intestinal parasitic infection in 36-45-month-old children in East Nusa Tenggara, Indonesia.

CTLA-4 CT-60 A/G and CTLA-4 1822 C/T Gene Polymorphisms in Indonesians with Type 1 Diabetes Mellitus.

Introduction: CTLA-4 gene polymorphism plays an important role in children with type 1 diabetes mellitus (T1DM). However, data on this subject vary among different races and ethnics. Purpose: To analyze CTLA-4 CT-60 A/G and CTLA-4 1822 C/T gene polymorphism among children with T1DM compared to control. Patients and Methods: The CTLA-4 CT-60 A/G and CTLA-4 1822 C/T gene polymorphism in children with T1DM using polymerase chain reaction-restriction fragment length polymorphism in 25 T1DM and 25 controls. The inclusion criteria were patients regularly controlled at the Pediatric Endocrine Outpatient Clinic of Dr. Soetomo Hospital, aged 4-18 years and willing to join this study and the exclusion criteria were T1DM patients hospitalized in the pediatric intensive care unit. In the control group, the inclusion criteria were healthy children, aged 4-18 years and willing to join this study. The exclusion criteria included children with ongoing infection, history of other autoimmune diseases, allergies, or malignancy. Results: The mean age was 12.48 years old, and the mean of T1DM onset was 9.28 years old. The CTLA-4 1822 T allele observed in 62% T1DM and 56% in control (p = 0.388, OR = 0.78, 95% CI = 0.44-1.37) and CTLA-4 CT-60 G allele observed in 52% T1DM and 58% in control (p = 0.393, OR = 1.27, 95% CI = 0.73-2.22). The C/T genotypes was significantly higher in control group (p = 0.045, OR = 3.27, 95% CI = 1.00-10.62). The A/G genotypes was commonly found in control group (p = 0.765, OR = 1.20, 95% CI = 0.37-3.86). The Javanese was the dominant ethnic group in our study. Conclusion: The frequency of CTLA-4 CT-60 A/G polymorphism almost equivalent in T1DM and control group. However, CTLA-4 1822 C/T polymorphism was more prevalent in the control group; thus, this genotype may have a protective effect against T1DM.

Determination of Cassiarin A Level of Cassia siamea Leaf Obtained from Various Regions in Indonesia Using the TLC-Densitometry Method.

Cassia siamea leaf has been proven in vitro and in vivo to have a strong antimalarial activity with Cassiarin A as its active compound. To obtain a source of C. siamea medicinal plant with high level of active antimalarial compound (Cassiarin A), a valid method for determining Cassiarin A level is needed. For this reason, this research conducts the validation of the Cassiarin A content with determination method using thin-layer chromatography (TLC) densitometry which includes the determination of selectivity (Rs), linearity (r), accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ). Cassiarin A was chromatographed on silica gel 60 F254 TLC plate using chloroform : ethanol (85 : 15 v/v) as a mobile phase. Cassiarin A was quantified by densitometric analysis at 368 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r = 0.9995. The method was validated for precision, recovery, repeatability. The minimum detectable amount was found to be 0.0027 µg/spot, whereas the limit of quantitation was found to be 0.008 µg/spot. The results of this validation are then used to determine the Cassiarin A level of C. siamea leaf from various regions in Indonesia. Based on the results of the study, it can be concluded that the TLC-densitometry method can be used to determine level of the Cassiarin A compound with the advantages of being fast, easy, accurate, and inexpensive. In addition, it showed that C. siamea leaves from Pacitan have the highest level of Cassiarin A compared to other areas studied.

Origins and spread of novel genetic variants of sulfadoxine-pyrimethamine resistance in Plasmodium falciparum isolates in Indonesia.

BACKGROUND: While malaria incidence in Indonesia has decreased threefold in the last decade, more than 200,000 cases were reported in 2016. Different endemicity of Plasmodium falciparum malaria among several islands in Indonesia has been recognized and two unique mutations of P. falciparum dihydropteroate synthase (pfdhps) affecting sulfadoxine-pyrimethamine (SP) resistance were detected from the research of SP efficiency and genotype analysis in South Kalimantan. In this study, geographical distribution and origin of these pfdhps K540T and I588F mutations were analysed. METHODS: Malaria parasites DNA from several endemic areas in Indonesia; Sumatera, Java, Kalimantan, Lombok, Sumbawa, Timor, Sulawesi, and Papua islands; in two periods, 2004-2006 and 2009-2012 were subjected for pfdhfr and pfdhps sequence analysis. RESULTS: Different genotype polymorphisms of pfdhfr and pfdhps were observed in the parasites from various regions in Indonesia and relatively more divergent genotypes were determined from Kalimantan isolates in both 2004-2006 and 2009-2012. The parasites containing K540T mutation were identified in 2004-2006 isolates from East Kalimantan, East Java and Sumbawa as an SGTGA haplotype. The other I588F mutation was also determined in 2004-2006 parasites, isolated from Lombok and Sumbawa islands as an SGEAA(588F) haplotype. The parasites with pfdhfr/pfdhps quintuple or sextuple mutation, a genotype marker of SP resistance, were determined mostly in Kalimantan in both 2004-2006 and 2009-2012. CONCLUSION: Analysis of the prevalence and pfdhfr/pfdhps combined genotypes of K540T or I588F mutations suggested that K540T might be origin in Kalimantan Island and I588F in Sumbawa Island and then these were spread to other areas along with people movement. This research indicates regular monitoring of drug efficacy and parasite genotype analysis is important to keep efficiency and prevent the spread of resistance. It is also essential for the latest anti-malarial drug artemisinin-based combination therapy.

COMPARISON OF MULTIPLEX SINGLE ROUND PCR AND MICROSCOPY IN DIAGNOSIS OF AMOEBIASIS.

BACKGROUND: Amoebiasis, the cause of dysentery and extra-intestinal abscesses, now becomes second fatal parasitic disease in the world. As routine microscopic diagnosis cannot differentiate causative Entamoeba histolytica from non-pathogenic E. dispar and E. moshkovskii, better diagnosis has to be searched. MATERIALS AND METHODS: Multiplex single round PCR was tested and compared with results of microscopy of wet preparation on 30 samples of diarrheic stools and extra intestinal lesions from amoebiasis suspected patients. RESULTS: Microscopy examination showed that 21 (70%) of the samples were positive for E. histolytica/E. dispar/E. moshkovskii complex and 18 (86%) of them contained hematophagous trophozoites. Multiplex single round PCR showed 12 positive results, from which seven were positive for E. histolytica, two were positive for E. moshkovskii, and three showed mixed of E. histolytica and E. moshkovskii. No samples were positive for E. dispar. High positive rate of microscopy might be related with highly suspected amoebiasis cases, while lower positive PCR might be caused by low parasite density and time-related trophozoite disintegration. CONCLUSION: The study showed that multiplex single-round PCR is a valuable diagnostic tool for species differentiation, but cannot replace microscopy in the diagnosis of amoebiasis because of its low sensitivity and impossibility to discriminate the form of E. histolytica and whether it is in the disease-causing stage, while microscopic examination is capable to demonstrate the presence of hematophagous trophozoites that indicates it is invasive and at the disease-causing stage of E. histolytica.

Two novel mutations of pfdhps K540T and I588F, affecting sulphadoxine-pyrimethamine-resistant response in uncomplicated falciparum malaria at Banjar district, South Kalimantan Province, Indonesia.

BACKGROUND: Mutations in pfdhfr and pfdhps genes have been shown to associate with sulphadoxine-pyrimethamine (SP) resistance of Plasmodium falciparum parasites. However, pfdhfr, pfdhps genotypes and the correlations to SP treatment outcome in Indonesia has not yet been well analysed. METHODS: After obtaining informed consent, 61 uncomplicated falciparum malaria patients were recruited in Banjar district, South Kalimantan Province, Indonesia, from October 2009 to August 2010. They were treated by a single oral dose of SP and its effects on clinical and parasitological status were followed until day 28 after treatment. Occasionally, a thick smear blood film for microscopy observation and blood spot on a filter paper for pfdhfr and pfdhps genotype analysis were collected. RESULTS: Pfdhfr and pfdhps genotypes from 24 P. falciparum-infected patients consisting of adequate clinical parasitological response (ACPR) (n = 6; 25.0%) and early treatment failure (ETF) (n = 10; 41.7%) or late parasitological failure (LPF) (n = 8; 33.3%) were obtained by sequencing. Two novel mutations of pfdhps gene, K540T and I588F, were determined in ten and five isolates, respectively. These mutations were present in the pfdhfr/pfdhps combined haplotypes of ANRNI/SGTGA (n = 6), ANRNL/SGTGA (n = 4), and ANRNI/SGEAA(588F) (n = 5), (mutation codons are bold typed); these haplotypes were mostly belonging to parasitological failure (ETF or LPF). The parasites acquiring five mutations in pfdhfr/pfdhps haplotypes and four mutations with additional I588F did not respond adequately to SP treatment. CONCLUSION: Many of Plasmodium falciparum infected patients in Banjar district, South Kalimantan, Indonesia did not respond adequately to SP treatment and these low ineffectiveness of SP in this area was associated with two novel mutations of pfdhps, K540T and I588F.

Evaluation by villagers of the malaria control project on Lombok and Sumbawa Islands, west Nusa Tenggara Province, Indonesia.

The cooperative malaria control project between Indonesian and Japanese institutions was conducted from 2001 to 2004 at small malaria endemic foci on Lombok and Sumbawa Islands. The aim of this research was to evaluate the effects of the project according to the opinions of the villagers. We conducted a KAP survey of a simple random sample of 300 householders on each island. The conclusion of the study was that the project reduced malaria incidence significantly on Lombok. However, the effects were not as clear on Sumbawa. Poor socio-economic status and lack of school education were important related factors. Therefore, health education, or behavioral change communication, was an essential component of malaria control.