Confirmation of Plasmodium Falciparum Treatment Failure Cases by Polymerase Change Reaction Genotyping

Background: Treatment failure in clinical trial, may be caused by P. falciparum resistant to antimalarialdrug. This study aimed to confirm the treatment failure cases of P. falciparum whcther caused byrecrudescence / resistant or new infection of different strain parasite.Methods: This study was a part of the activity in antimalarial drug efficacy trials (artemisinin-naphthoquine/AN versus dihydroartemisinin – piperaquine/DHP). P. falciparum infections on failure cases weregenotyped for allelic variation in those 3 markers by comparing before (D0) and after treatment (DF) ifparasites recurrent with nested polymerase change reaction (PCR).Results: Thirteen of 19 P. falciparum failure cases showed PCR genotyping completely successful 100%for MSP1 (D0 & DF), MSP2 (DF) and GLURP (D0) and the lowest (76,9% ) for GLURP (DF). Whenall 3 genes were combined, the amplification result showed 69.2%. Identification allele for each locusgenes shown that MSP1 had just one (D0 or DF). Conversely, for MSP2 and GLURP, there were someadditional alleles either at D0 and DF. By comparing the pattern of genotype (alleles) P. falciparum atD0 and DF each locus genes, the confirmation of P. falciparum resistant from new infection could bedetermined The proportion of recrudescence and new infection almost the same, 8 of 13 failed cases werefrom artemisinin-naphthoquine (AN) group.Conclusion: The confirmation of P. falciparum by comparing genotype at D0 and DF could determine parasiteresistant and new infection from treatment failure cases. Recrudescence occurred within 17 days after treatmentand new infection occurred >28 days after treatment. (Health Journal of Indonesia. 2015;6:29-37

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

Epidemologi Molekuler Genotipe Human Immunodeficiency Virus -1 (Hiv-l) Pada Orang Dengan Hiv/aids (Acquired Immunodeficiency Syndrome) Atau Odha Di Jawa Timur Dan DKI Jakarta

Human Immunodeficiency Virus is retrovirus that can decrease immune system in human body. There are two types of HIV which could be genetically specified as HIV-I and HIV-2. HIV-I consists of three groups: M, N, and 0. The 'M' group is further classified as nine subtypes i. e, A, B, C, D, E, F, G, H, and I subtypes. In certain condition, two viruses from different subtypes can mix and form a hybrid virus, called Circulating Recombinant Forms (CRFs). The aim of this research is to map the distribution of HIV subtypes in all of Indonesia's provinces. Sera from HIV patients from hospitals in DKI Jakarta and East Java were taken and examined for genetic analysis. As we may already aware that DKI Jakarta and East Java are provinces that have high prevalence of HIV. All of the specimens were tested and analyzed using RT-PCR technique followed by PCR Nested and sequences confirmation. In East Java Province, HIV-I AE subtypes was the most dominant with 74%followed by Band E subtypes, each of them I3%. In DKI Jakarta province, HIV-I E subtype was the most dominant 60%, AE and B subtypes were 35%, and 5% respectivel

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

Deteksi dan Spesiasi Parasit Malaria Sampel Monitoring Pengobatan Dihydroartemisinin-piperaquine di Kalimantan dan Sulawesi: Mikroskopis Vs Polymerase Chain Reaction

In monitoring the treatment of malaria with Dihydroartemisinin-piperaquine (DHP), microscopic cross check and Polymerase Chain Reaction (PCR) performed to validate the results of laboratory examinations in the field. This study used finger prick samples from subjects with a diagnosis of malaria in monitoring the treatment of malaria with DHP in Kalimantan and Sulawesi. Samples taken at day 0, blood smears made on slides for microscopic and blood spot on filter paper for PCR examination. The PCR method used is a single-round multiplex polymerase chain reaction that has been modified, the examination of each species carried out in different tubes to distinguish the species P. falciparum or P. Vivax. Target of DNA amplification is a species-specific gene sequences in the small-subunit ribosomal RNA (SSUrRNA), 300 bp for P. falciparum and 276 bp for P.vivax. P. falciparum and P.vivax identified in 229 samples of blood smears and blood spots. Microscopic and PCR gave the same results, positive 93.4% and negative 6.6% with a sensitivity of 99% and specificity 93.3%. P.falciparum sensitivity and specificity of 92% and 99%, P.vivax 97% and 94%, PCR as a gold standard. There are differences in the results of examination of 5 samples, ie with microscopic examination identified as P.vivax while the PCR as P. falciparum. In this study, identification of the microscopic parasite similar to the results of identification by PCR, but differ in determining the types of parasites. In general, the ability to microscopic diagnosis of malaria is very good, but confirmation by PCR is still needed

A study of the TNF/LTA/LTB locus and susceptibility to severe malaria in highland papuan children and adults

Background: Severe malaria (SM) syndromes caused by Plasmodium falciparum infection result in major morbidity and mortality each year. However, only a fraction of P. falciparum infections develop into SM, implicating host genetic factors as important determinants of disease outcome. Previous studies indicate that tumour necrosis factor (TNF) and lymphotoxin alpha (LT alpha) may be important for the development of cerebral malaria (CM) and other SM syndromes

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

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

IDENTIFIKASI TIPE HLA KELAS II DENGAN TEKNIK PCR

HLA (Human Leukocyte Antigen) contains a set of genes located together on the short arm of chromosome 6. These genes control immune responses, graft acceptance or rejection and tumor surveillance. These abilities have close relationship with genetic variation (occur in "many forms" or alleles) that bind and present antigens to T lymphocytes. Using advanced technology and molecular biology approaches (PCR technique) detection of genetic variation in the HLA region (or HLA typing) has been performed based on DNA.. PCR is an in vitro technique to amplify the DNA sequence enzymatically. "Sequence Specific Primers" (SSP) are designed for this PCR to obtain amplification of specific alleles or groups of alleles. The PCR products are visualized through agarose gel electrophoresis stained with ethidium bromide. The PCR technique requires small amount of whole blood (0.5 - 1 ml), gives rapid, accurate and complete result. This paper discuss identification of HLA class II typing using PCR-SSP technique and show the examples of the results.   Key words: HLA (Human Leukocyte Antigen) class II, PCR (Polymerase Chain Reaction