Keragaman Genetik Petanda P. Falciparum dari Specimen Subyek Penelitian Monitoring Dihidroartemisinin-piperakuin di Kalimantan dan Sulawesi

Treatment failure in falciparum malaria may be caused by parasite resistant to antimalarial drug or new infection. Polymorphism genetic marker of P. falciparum namely MSP1, MSP2 and GLURP locus genes in the population should be identified as a baseline to distinguish the cause of treatment failure. A nested Polymerase Chain Reaction (PCR) method was applied to each locus gene separately. A total 121 dried blood spot specimens from subjects infected with P. falciparum in monitoring Dihydroartemisinin-Piperaquine treatment in Kalimantan and Sulawesi Islands were analyzed. Locus genes of MSP1, MSP2 and GLURP were successful identified 82.6%, 96.7% and 81.0% respectively. However, the three (MSP1, MSP2 and GLURP) locus genes were only found in 71.9% (87 of 121) samples. All of MSP1 locus gene had just one allele, two alleles on most of MSP2 (67.5%) and few of GLURP (14.3%). Multi genotype infection was likely dominant than a single genotype infection (65.5% vs. 34.5%). Based on allele length classification, MSP2 locus gene shows more variety of allele class (12 alleles) than GLURP (9 alleles) and MSP1 (7 alleles), with an allele length mostly for MSP1: 440 - 479 bp, MSP2: 480–519 bp and GLURP: 580–639 bp. In this study, falciparum malaria cases were commonly as multi-genotype infection, and MSP2 was a dominant and polymorphic genetic marker of P.falciparum. Keywords: P. falciparum, PCR, MSP1, MSP2, GLURP, allele Abstrak Gagal pengobatan pada malaria falsiparum dapat disebabkan oleh parasit yang resisten terhadap obat antimalaria atau oleh infeksi baru. Keragaman genetik petanda Plasmodium falciparum yaitu lokus gen MSP1, MSP2 dan GLURP dalam suatu populasi perlu diidentifikasi sebagai dasar untuk membedakan penyebab gagal pengobatan. Metode pemeriksaan yang digunakan adalah nested Polymerase Chain Reaction (PCR) terhadap masing-masing lokus gen secara terpisah. Telah dianalisis 121 spesimen resapan darah kering pada kertas filter dari subyek terinfeksi P.falciparum pada studi monitoring pengobatan dengan Dihidroartemisinin-Piperakuin di Kalimantan dan Sulawesi. Masing-masing lokus gen MSP1, MSP2 dan GLURP yang dapat diidentifikasi sebanyak 82,6%, 96,7% and 81,0%. Sedangkan ketiga lokus gen tersebut ditemukan hanya pada 71,9% (87/121) sampel. Lokus gen MSP1 semuanya mempunyai 1 alel, sedangkan dua alel ditemukan pada sebagian besar MSP2 (67,5%) dan sebagian kecil GLURP (14,3%). Infeksi multi-genotip oleh dua atau lebih genotip P.falciparum ditemukan pada 65,5% sampel dan infeksi tunggal hanya 34,5% sampel. Keragaman klas alel paling banyak ditemukan pada lokus gen MSP2 sebanyak 12 klas alel, GLURP sebanyak 9 klas alel, dan MSP1 sebanyak 7 klas alel. Alel pada lokus gen MSP1 sebagian besar pada kisaran 440 - 479 bp, MSP2: 480 – 519 bp, dan GLURP: 580–639 bp. Pada penelitian ini kasus malaria falsiparum umumnya merupakan infeksi multi-geotip, dan MSP2 merupakan petanda gen P.falciparum yang dominan dan beragam. Kata kunci : P. falciparum, PCR, MSP1, MSP2, GLURP, ale

Infantile acute megakaryoblastic leukaemia with T(1:22) in a non-down syndrome child.

Megakaryoblastic leukaemia is the commonest form of leukaemia occuring in Down syndrome infants. However, it’s subtype with translocation t(1;22)(p13;q13)is uncommon comprising <1% of all cases and reported to exclusively occur in infant without Down syndrome. It has a female predominance and carries apoor prognosis. We described this rare form of leukaemia in a 9-month-old girl who presented with bruises, massive hepatosplenomegaly and multiple cervical and inguinal lymphadenopathy. The blood film showed severe anaemia with ovalostomatocytosis, thrombocytopenia and mild leucocytosis. The bone marrow aspirate showed numerous blasts showing high nuclear-cytoplasmic ratio and agranular cytoplasm with cytoplasmic blebs. Peroxidase staining was negative. The immunophenotyping of the blasts showed positive expression of CD117, CD13, CD33 and CD61 which confirmed the diagnosis of acute megakaryoblastic leukaemia. Interestingly, the cytogenetic finding of translocation t(1;22) which is most common in acute megaloblastic leukaemia in infants without Down syndrome was found in this case. She received the AML trial 15 ADE protocol chemotherapy regime and developed severe neutropenic sepsis and respiratory distress requiring ventilatory support and granulocyte colony stimulating factor (G-CSF). She recovered wellmafter the first course of chemotherapy and was discharged. Unfortunately, she was not brought in for follow-up chemotherapy and presented a few months later with relapsed AML. She was re-started on ADE protocol and currently is on oral thioguanine for maintenance therapy

Identifikasi Single Nucleotide Polymorphism (SNP) Gen Pvmdr1 Pada Penderita Malaria Vivaks Di Minahasa Tenggara (Sulawesi Utara)

Parasite resistance to antimalarial drugs is an obstacle to malaria elimination. In Plasmodium vivax, up to now, a marker to distinguish between resistant and susceptible is no available yet. Identify Single Nucleotide Polymorphisms (SNP) in P. vivax for multidrug resistance (pvmdr1)is potential approach due to pvmdr1 gene is orthologous to the pfmdr1 of P.falciparum, have been related to multidrug resistance. The purpose of this study was to identify SNPs/mutations on pvmdr1 gene of malaria vivax patients who came to the Primary Healthe Centers, Touluaan and Tombatu Minahasa Tenggara (North Sulawesi).Blood samples and slide of blood smears were collected from patients who infected with P.vivax or mixed infection of P.vivax and P.falciparum. After the species were cross checked by certified microscopist then confirmed by PCR, SNP identification were performed by sequencing technique. Only 83 of 99 recruited subjects were included inclution criteria. Sequensing result showed that 59 of 83 subjects were analysed to identify the SNP. We found 5 nonsynonymous SNPs, namely at the point G698S, M908L, Y976F, L1076F, and K1261E

Deteksi P.vivax Single Nucleotide Polymorphism (Snp) Y976f dari Sampel Monitoring Pengobatan Dihidroartemisinin-piperakuin di Kalimantan dan Sulawesi

This study was a part of the activity of monitoring Dihydroartemisinin-Piperaquine (DHP) treatment in subjects infected with P.falciparum and P.vivax in Kalimantan and Sulawesi. SNP Y976F had been proved as the mutation in pvmdr1 gene which was related to P. vivax resistance chloroquine in Papua. Data of spreading pvmdr1 SNP Y976F outside Papua is needed for using Dihidroartemisinin-Piperakuin policy in the treatment of vivax malaria in Indonesia. Detection of SNP Y976F was done against 95 day0-samples of subjects confirmed infected with P.vivax or mixed infection of P.vivax and P.falciparum by PCR. The results showed that 88 (93%) of a total 95 samples were positive detected 976F mutant which were distributed in all sentinel sites of West Kalimantan (2of 3), Central Kalimantan (6 of 8), North Sulawesi (63 of 65), and Central Sulawesi (17 of 19). In conclusion, pvmdr1 SNP Y976F has been spreaded in all sentinel sites. Key words: P.vivax, pvmdr1, Single Nucleotide Polymorphism Abstrak Penelitian ini merupakan bagian kegiatan dari monitoring pengobatan Dihidroartemisinin-Piperakuin (DHP) pada subyek yang terinfeksi dengan P.vivax atau infeksi campuran P.falciparum dan P. vivax di Kalimantan dan Sulawesi. SNP Y976F merupakan mutasi pada gen pvmdr1 yang terbukti berhubungan dengan P. vivax resisten klorokuin di Papua. Dalam rangka kebijakan penggunaan Dihidroartemisinin-Piperakuin untuk pengobatan malaria vivaks di seluruh Indonesia, perlu data penyebaran parasit SNP Y976F pada gen pvmdr1 di luar Papua. Deteksi SNP Y976F dilakukan terhadap 95 sampel H0 subyek terinfeksi P. vivax atau infeksi campuran P.vivax dan P.falciparum yang telah dikonfirmasi dengan PCR. Hasil menunjukkan bahwa 88 dari 95 sampel (93%) terdeteksi positif galur mutan 976F yang tersebar di Kalimantan Barat (2 dari 3), Kalimantan Tengah (6 dari 8), Sulawesi Utara (63 dari 65) dan Sulawesi Tengah (17 dari 19). Kesimpulannya bahwa P.vivax galur Y976F sudah tersebar di setiap sentinel penelitian. Kata kunci: P.vivax, pvmdr1, Single Nucleotide Polymorphis

Concomitant BCR-ABL and JAK2 V617F in a patient with myeloproliferative neoplasm: a case report

Myeloproliferative neoplasm (MPN) is a clonal proliferation of the haematopoietic stem cells leading to activated tyrosine kinase signaling activity. Myeloproliferative neoplasm is classified into BCR-ABL positive chronic myeloid leukemia (CML) and BCR-ABL negative MPN which harbors JAK2 V617F mutation in most cases. The genetic combination of BCR-ABL and JAK2 V617F mutation is rare with estimated frequency of 0.4% based on recent study. Herein, we reported a case of a man diagnosed with CML detected by fluorescence in-situ hybdridisation (FISH) showing atypical BCR-ABL fusion pattern in 29% nucleated cells (cut-off levels ≥5% for positive signals) in the presence of JAK2 V617F mutation. However, the BCR-ABL transcript was not detected by the conventional reverse transcriptase-polymerase chain reaction (RT-PCR) method which was specific for major fragments. Interestingly, complete hematological remission was not achieved despite initiation of tyrosine kinase inhibitor (Imatinib). In conclusion, it is imperative to scrutinise CML cases for concomitant JAK2 V617F mutation especially patients with atypical clinical or laboratory findings. Therefore, close monitoring with clinical and ancillary technique especially FISH and molecular methods such as DNA sequencing were crucial to help achieve complete hematological, cytogenetic and deep molecular response alongside targeted therapy

Parasite-Dependent Expansion of TNF Receptor II–Positive Regulatory T Cells with Enhanced Suppressive Activity in Adults with Severe Malaria

Severe Plasmodium falciparum malaria is a major cause of global mortality, yet the immunological factors underlying progression to severe disease remain unclear. CD4+CD25+ regulatory T cells (Treg cells) are associated with impaired T cell control of Plasmodium spp infection. We investigated the relationship between Treg cells, parasite biomass, and P. falciparum malaria disease severity in adults living in a malaria-endemic region of Indonesia. CD4+CD25+Foxp3+CD127lo Treg cells were significantly elevated in patients with uncomplicated (UM; n = 17) and severe malaria (SM; n = 16) relative to exposed asymptomatic controls (AC; n = 10). In patients with SM, Treg cell frequency correlated positively with parasitemia (r = 0.79, p = 0.0003) and total parasite biomass (r = 0.87, p<0.001), both major determinants for the development of severe and fatal malaria, and Treg cells were significantly increased in hyperparasitemia. There was a further significant correlation between Treg cell frequency and plasma concentrations of soluble tumor necrosis factor receptor II (TNFRII) in SM. A subset of TNFRII+ Treg cells with high expression of Foxp3 was increased in severe relative to uncomplicated malaria. In vitro, P. falciparum–infected red blood cells dose dependently induced TNFRII+Foxp3hi Treg cells in PBMC from malaria-unexposed donors which showed greater suppressive activity than TNFRII− Treg cells. The selective enrichment of the Treg cell compartment for a maximally suppressive TNFRII+Foxp3hi Treg subset in severe malaria provides a potential link between immune suppression, increased parasite biomass, and malaria disease severity. The findings caution against the induction of TNFRII+Foxp3hi Treg cells when developing effective malaria vaccines

Human T cell recognition of the blood stage antigen Plasmodium hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT) in acute malaria

<p>Abstract</p> <p>Background</p> <p>The <it>Plasmodium </it>purine salvage enzyme, hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT) can protect mice against <it>Plasmodium yoelii </it>pRBC challenge in a T cell-dependent manner and has, therefore, been proposed as a novel vaccine candidate. It is not known whether natural exposure to <it>Plasmodium falciparum </it>stimulates HGXPRT T cell reactivity in humans.</p> <p>Methods</p> <p>PBMC and plasma collected from malaria-exposed Indonesians during infection and 7–28 days after anti-malarial therapy, were assessed for HGXPRT recognition using CFSE proliferation, IFNγ ELISPOT assay and ELISA.</p> <p>Results</p> <p>HGXPRT-specific T cell proliferation was found in 44% of patients during acute infection; in 80% of responders both CD4⁺and CD8⁺T cell subsets proliferated. Antigen-specific T cell proliferation was largely lost within 28 days of parasite clearance. HGXPRT-specific IFN-γ production was more frequent 28 days after treatment than during acute infection. HGXPRT-specific plasma IgG was undetectable even in individuals exposed to malaria for at least two years.</p> <p>Conclusion</p> <p>The prevalence of acute proliferative and convalescent IFNγ responses to HGXPRT demonstrates cellular immunogenicity in humans. Further studies to determine minimal HGXPRT epitopes, the specificity of responses for Plasmodia and associations with protection are required. Frequent and robust T cell proliferation, high sequence conservation among <it>Plasmodium </it>species and absent IgG responses distinguish HGXPRT from other malaria antigens.</p

Immunohistochemical study of p53 expression in premalignant and malignant cervical neoplasms

One of the most important cervical cancer risk factors is human papillomavirus (HPV) infection. The p53 gene is one of the most important targets of the HPV E6 gene. E6 protein has the ability to stimulate p53 degradation, inhibits several functions of wild-type p53 and it competes with its function including suppression of malignant growth. The aim of this study is to determine the differences in p53 expressions in pre-malignant and malignant cervical neoplasms. This is a retrospective study on 100 cases of cervical neoplasms. There were 21 cases of CIN 1, 8 cases of CIN 2, 25 cases of CIN 3, 36 cases of squamous cell carcinoma, 7 cases of adenocarcinoma and 3 cases of adenosquamous carcinoma. All cases were evaluated by immunohistochemistry using p53 monoclonal antibody. Thirty six of the 54 pre-malignant cases (66.7%) were positive for p53 protein, in contrast to the malignant cases in which, 40 of the 46 cases (87.0%) were positive. The majority of CIN showed absent to focal staining (29/54, 53.7%). In contrast, 84.8% (39/46) of the invasive carcinoma showed regional to diffuse staining. The expression of p53 is greater in the malignant cervical neoplasms than the pre-malignant cervical lesions, suggesting that p53 overexpression is not an early phenomenon in the pathogenesis of cervical cancer. It is also shown to be slightly higher in percentage in CIN 2 and 3 when compared with CIN 1. However, a number of cases were p53 negative, suggesting that other factors may be involved and further HPV studies are indicate

Anaplastic large cell lymphoma presenting as a soft tissue mass – A case report

Anaplastic large cell lymphoma (ALCL) is a rare tumour, accounting for approximately 3% of adult non-Hodgkin lymphomas.1 Primary systemic ALCL frequently involves both lymph nodes and extranodal sites. A 44-year-old woman presented with a firm, mobile mass in the left iliac fossa region. Ultrasound findings showed a well defined inhomogenous soft tissue mass, measuring 4x4x2.6cm in the deep subcutaneous region. Histopathological examination revealed that the mass was infiltrated by large lymphoid cells with marked nuclear atypia including kidney-shaped nuclei. These neoplastic cells expressed anaplastic lymphoma kinase (ALK) (both nuclear & cytoplasmic staining), CD30 and EMA but not for T-cell (CD45RO and CD3), and B-cell (CD20 & CD79α) markers. Fluorescence in situ hybridization (FISH) analysis showed a t(2;5)(p23;q35) chromosomal translocation. Subsequently the patient developed shortness of the breath and a thoracic computed tomography (CT) scan showed a mass encasing the right upper lobe bronchus. She also had bilateral axillary lymph nodes, measuring 1 cm in diameter (biopsy was not done). The mediastinum and endobronchial region did not show any abnormalities. She received 6 cycles of CHOP chemotherapy and remained disease free 2 years after diagnosis. ALCL, rarely present as a soft tissue tumour and this disease should be included as a differential diagnosis of any soft tissue mas

Detection of BCR/ABL gene in chronic Myeloid Leukaemia: comparison of Fluorescence in situ Hybridisation (FISH), conventional Cytogenetics and Polymerase Chain Reaction (PCR) techniques

The reciprocal translocation t(9;22)(q34;q11) which gives rise to the Philadelphia (Ph1) chromosome and BCR/ABL fusion gene, plays a pivotal role in the diagnosis and pathogenesis of chronic myeloid leukemia (CML). In this study, we evaluated the role of fluorescence in situ hybridisation (FISH) in detecting the BCR/ABL rearrangement in CML patients. The sensitivity, specificity and detection rate of BCR/ABL gene using FISH, PCR and conventional cytogenetics (karyotyping) methods were also compared. 18 bone marrow samples of patients with clinically diagnosed CML and suspected of CML were collected. The sensitivity, specificity and positive predictive values of FISH were altogethe