POLYMORPHISMS OF PFCRT AND PFMDR-1 GENES AND CHLOROQUINE RESISTANCE OF P. FALCIPARUM IN WAD MEDANI (CENTRAL SUDAN)

Introduction: Malaria parasite resistant to Chloroquine poses severe and increasing health problems in tropical countries. Monitoring the drug resistance by implementing the molecular markers may be essential to overcome the problem, therefore this study aims to assess the Chloroquine resistance of Plasmodium Falciparum parasite in central Sudan, using molecular markers. Methods: One hundred and seventy six patients were confirmed P. falciparaum positive. Sixty-four were selected and only forty patients completed the follow-up. In vivo sensitivity assay was used accompanied with standard regimen of Chloroquine phosphate. DNA was extracted from blood on filter paper (day 0) and was used to amplify two genes P. Falciparum transporters gene Pfcrt and multi-drug resistant gene-1 Pfmdr-1. Results: Among forty patients, 54% responded to Chloroquine regimen with adequate clinical response (ACR), however, 46% showed treatment failure. All treatment failures were treated with Artemether or Quinine. The amplification of Pfcrt gene (n, 18) and Pfmdr 1 gene (n, 29), had shown that 72% of Pfcrt T76 were mutant allele, 22% were K76 wild-type, however, only 5% were mixed alleles T/K. while Pfmdr 1 gene (n, 29) revealed that 55% were wild genotype N 86, 38% were mutant Y 86, and 7% were mixed alleles Y/ N 86. Conclusion: The high frequency of the mutant Pfcrt 76T gene among P. Falciparum isolates was consistent with in vivo study supports the hypothesis that Pfcrt 76T gene could be used as predictive marker for Chloroquine susceptibility in epidemiological surveys

APPLICATION OF PCR TECHNIQUES FOR TB DIAGNOSIS AT THE INSTITUTE OF NUCLEAR MEDICINE, MOLECULAR BIOLOGY & ONCOLOGY (INMO) - UNIVERSITY OF GEZIRA, SUDAN

Traditional methods for laboratory diagnosis of tuberculosis (TB) may require weeks, and delays can impede treatment and control efforts. Nucleic acid amplification (NAA) tests, such as polymerase chain reaction (PCR) and other methods for amplifying DNA and RNA, may facilitate rapid detection of microorganisms. This study emphasized our three years experience in the diagnosis of TB on clinical samples using the PCR method. Among 531 patients with suspected TB, 112 (21.3%) were positive by PCR, while 419 (78.7 %) were negative. The specimens collected from TB suspected patients were sputum, blood, ascitic fluid, pleural fluid, and gastric wash. Blood samples showed high positive results 15/41 (34%) in comparison with sputum samples which is the most frequent sample 64/ 285 (22.3%). Gastric wash samples reported low rate of positive result 1/30 (3.3%). This rapid and sensitive test compared with the other cultural and microscopic tests have now been incorporated into our laboratory practice allowing the physicians to manage proper diagnosis and drug regimens

Variants of CTGF are associated with hepatic fibrosis in Chinese, Sudanese, and Brazilians infected with Schistosomes

Abnormal fibrosis occurs during chronic hepatic inflammations and is the principal cause of death in hepatitis C virus and schistosome infections. Hepatic fibrosis (HF) may develop either slowly or rapidly in schistosome-infected subjects. This depends, in part, on a major genetic control exerted by genes of chromosome 6q23. A gene (connective tissue growth factor [CTGF]) is located in that region that encodes a strongly fibrogenic molecule. We show that the single nucleotide polymorphism (SNP) rs9402373 that lies close to CTGF is associated with severe HF (P = 2 × 10−6; odds ratio [OR] = 2.01; confidence interval of OR [CI] = 1.51–2.7) in two Chinese samples, in Sudanese, and in Brazilians infected with either Schistosoma japonicum or S. mansoni. Furthermore, SNP rs12526196, also located close to CTGF, is independently associated with severe fibrosis (P = 6 × 10−4; OR = 1.94; CI = 1.32–2.82) in the Chinese and Sudanese subjects. Both variants affect nuclear factor binding and may alter gene transcription or transcript stability. The identified variants may be valuable markers for the prediction of disease progression, and identify a critical step in the development of HF that could be a target for chemotherapy

REGRESSION OF LIVER FIBROSIS IN SCHISTOSOMA MANSONI INFECTED SUDANESE SUBJECTS AFTER PRAZIQUANTEL TREATMENT

Objectives: 1-To evaluate the effect of Praziquantel (PZQ) therapy on the regression of liver fibrosis in an endemic population. 2-To determine the factors controlling the regression of hepatic fibrosis (e.g. gender, age and grade of fibrosis).   Material and methods: An association study of a cohort of one hundred seventy seven Sudanese patients infected with Schistosoma mansoni (82 males 46%, 95 females 54%) was conducted to evaluate the factors controlling the regression of liver fibrosis 39 months after treatment with PZQ using ultrasound evaluation. SPSS (Statistical Package for Social Science) software was used for statistical analysis. Chi- Square was used to compare the two phenotypes (regression and progression) in the study subjects. Results: PPF was regressed in 63 patients (36%) from higher grades of fibrosis to lower ones. While in 24 patients (13 %) the disease progressed to higher grades. In addition, the grade of PPF did not change in 90 patients (51%).  The mean values of portal vein diameter (PVD), splenic vein diameter (SVD), and index liver size (ILS) in subjects in whom PPF regressed after treatment were significantly lower than in subjects in whom the disease was progressed (P < .0001, P = .031, and P = .003 respectively).The progression of hepatic fibrosis in males (n = 15, 18 %) was greater than that of females (n = 9, 9 %). Patients who showed regression of PPF or progression of the disease tend to cluster in certain families

Cytokine Regulation of Periportal Fibrosis in Humans Infected with Schistosoma mansoni: IFN-γ is Associated with Protection Against Fibrosis and TNF-α with Aggravation of Disease

International audienceHepatic periportal fibrosis, which affects 5–10% of subjects infected by Schistosoma mansoni, is caused by the T cell-dependent granuloma that develop around schistosome eggs. Experimental models of infection have shown that granuloma and fibrosis are tightly regulated by cytokines. However, it is unknown why advanced periportal fibrosis occurs only in certain subjects. The goal of the present study was to evaluate the cytokine response of S. mansoni-infected subjects with advanced liver disease in an attempt to relate susceptibility to periportal fibrosis with an abnormal production of cytokines that regulate granuloma and fibrosis. Fibrosis was evaluated by ultrasound on 795 inhabitants of a Sudanese village in which S. mansoni is endemic: advanced periportal fibrosis was observed in 12% of the population; 35% of the affected subjects exhibited signs of portal hypertension. Age (odds ratio (OR), 11.5), gender (OR, 4.2), and infection levels (OR, 2.2) were significantly (p < 0.01) associated with hepatic fibrosis. Cytokines produced by egg-stimulated blood mononuclear cells from 99 subjects were measured (75 with no or mild fibrosis; 24 subjects with advanced fibrosis). Multivariate analysis of cytokine levels showed that high IFN-γ levels were associated with a marked reduction of the risk of fibrosis (p = ‫OR, 0.1); in contrast, high TNF-α levels were associated with an increased risk (p = 0.05; OR, 4.6) of periportal fibrosis. Moreover, infection levels were negatively associated with IFN-γ production. These results with observations in experimental models strongly suggest that IFN-γ plays a key role in the protection of S. mansoni-infected patients against periportal fibrosis, whereas TNF-α may aggravate the disease

Exome Sequencing Identifies Two Variants of the Alkylglycerol Monooxygenase Gene as a Cause of Relapses in Visceral Leishmaniasis in Children, in Sudan

International audienceVisceral leishmaniasis (kala-azar, KA) is the most severe form of leishmaniasis, characterized by fever, weight loss, hepatosplenomegaly, and lymphadenopathy. During an outbreak of KA in Babar El Fugara (Sudan), 5.7% of cured patients displayed relapses, with familial clustering in half the cases. We performed whole-exome sequencing on 10 relapsing individuals and 11 controls from 5 nuclear families. Rare homozygous and compound-heterozygous nonsense (c.1213C > T, rs139309795, p.Arg405*) and missense (c.701A > G, rs143439626, p.Lys234Arg) mutations of the alkylglycerol monooxygenase (AGMO) gene were associated with KA relapse in 3 families. Sequencing in additional family members confirmed the segregation of these mutations with relapse and revealed an autosomal dominant mode of transmission. These mutations were detected heterozygous in 2 subjects among 100 unrelated individuals with KA who never relapsed after cure, suggesting incomplete penetrance of AGMO deficiency. AGMO is expressed in hematopoietic cells, and is strongly expressed in the liver. AGMO modulates PAF production by mouse macrophages, suggesting that it may act through the PAF/PAF receptor pathway previously shown to have anti-Leishmania activity. This is the first demonstration that relapses after a first episode of KA are due to differences in human genetic susceptibility and not to modifications of parasite pathogenicity

IFN-gamma polymorphisms (IFN-gamma +2109 and IFN-gamma +3810) are associated with severe hepatic fibrosis in human hepatic schistosomiasis (Schistosoma mansoni).

International audienceSchistosome infection is a major public health concern affecting millions of people living in tropical regions of Africa, Asia, and South America. Schistosomes cause mild clinical symptoms in most subjects, whereas a small proportion of individuals presents severe clinical disease (as periportal fibrosis (PPF)) that may lead to death. Severe PPF results from an abnormal deposition of extracellular matrix proteins in the periportal spaces due to a chronic inflammation triggered by eggs and schistosome Ags. Extracellular matrix protein production is regulated by a number of cytokines, including IFN-gamma. We have now screened putative polymorphic sites within this gene in a population living in an endemic area for Schistosoma mansoni. Two polymorphisms located in the third intron of the IFN-gamma gene are associated with PPF. The IFN-gamma +2109 A/G polymorphism is associated with a higher risk for developing PPF, whereas the IFN-gamma +3810 G/A polymorphism is associated with less PPF. The polymorphisms result in changes in nuclear protein interactions with the intronic regions of the gene, suggesting that they may modify IFN-gamma mRNA expression. These results are consistent with the results of previous studies. Indeed, PPF is controlled by a major locus located on chromosome 6q22-q23, closely linked to the gene encoding the alpha-chain of the IFN-gamma receptor, and low IFN-gamma producers have been shown to have an increased risk of severe PPF. Together, these observations support the view that IFN-gamma expression and subsequent signal transduction play a critical role in the control of PPF in human hepatic schistosome infection (S. mansoni)