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Analyses of cytochrome b mutations in Plasmodium falciparum isolates in Thai-Myanmar border, 2002–2005
ARTICLE IN PRESS 408
Abstracts
P14
P13 Analyses of cytochrome b mutations in Plasmodium falciparum isolates in Thai-Myanmar border, 2002–2005 a,b
a
a
Y. Naoshima-Ishibashi , M. Iwagami , T. Mitamura , S. Looareesuwanc, S. Kanoa,b, a
International Medical Center of Japan, Japan b University of Tsukuba, Japan c Mahidol University, Thailand The combination of atovaquone and proguanil (MalaroneTM, GlaxoSmithKline) has been established as an effective drug of choice not only for treatment but also for prophylaxis of multi-drug resistant Plasmodium falciparum malaria in travellers. However, several cases of resistance against MalaroneTM have already been reported in some parts of Africa, and many of the cases are associated with mutations at codon 268 of cytochrome b gene in mitochondrial genome of P. falciparum. On the contrary, in Asian countries, only limited numbers of clinical failures with the drug has so far been reported, and no mutations could be found at the codon 268. To maintain the usefulness of MalaroneTM in travel medicine, monitoring of the effectiveness of the drug is needed. In this study, mutations of atovaquone binding site on the cytochrome b were investigated by PCR amplification and sequencing of the gene (1131 bp length) in the parasites from patients at the Hospital for Tropical Diseases Mahidol University in Bangkok, Thailand, in 2002 through 2005. Twenty-five blood samples were from complicated falciparum malaria patients and another 25 were from non-complicated falciparum patients who were infected in multi-drug resistant areas in Thai-Myanmar border. Forty-seven out of 50 samples were successfully analyzed, and no mutation was observed in putative ubiquinol (Q0) binding site 1 and 2. In fact, they showed identically conserved nucleotide sequences, as was reported by our previous study conducted in the same area in 1999. We may conclude from these findings that MalaroneTM is still effective and recommended for treatment and prophylaxis for travellers to Mekong region. KEYWORDS Plasmodium falciparum; Malarone; Atovaquone; Cytochrome b
Further reading [1]. Naoshima-Ishibashi Y, et al. Analyses of cytochrome b mutations in Plasmodium falciparum isolates in Thai-Myanmar border. Travel Med Infect Dis 2007;5(2):132–4. [2]. Krudsood S, et al. Efficacy of atovaquone-proguanil for treatment of acute multidrug-resistant Plasmodium falciparum malaria in Thailand. Am J Trop Med Hyg 2007;76(4):655–8. 10.1016/j.tmaid.2007.09.027
Optimization of a flow cytometry protocol for detection and viability assessment of Giardia lamblia
J. Barbosaa,b,, S. Costa-de-Oliveiraa, A.G. Rodriguesa,c, C. Pina-Vaza,d a
Department of Microbiology, School of Medicine, Portugal b Jean Piaget Superior School of Health, Portugal c Department of Plastic and Reconstructive Surgery, Hospital S. Joa˜o, Portugal d Department of Microbiology, Hospital S. Joa˜o, Portugal Giardia lamblia is one of the most important cause of diarrhoea in humans around the world. Transmission routes are complex including fecal—oral as well as waterborne and foodborne transmission. Detection methods in water and stools involve cyst concentration procedures, followed by conventional microscopy which is time-consuming. Higher sensibility was described with antigen detection but showing unpredictable specificity. Molecular genetic studies are complex and difficult for routine analysis. We have optimised a specific Flow Cytometric (FC) protocol for detection of G. lamblia, established its detection limit and assayed the possibility of cross-reaction with other microorganisms. Viability studies, important to evaluate the infectious risk and for treatment monitorization, were also performed by FC. A known concentration of G. lamblia cysts (for Waterborne Inc, USA and from a Giardia culture) were stained with serial concentrations of a fluorescein-labelled (FL) mouse monoclonal antibody (Giardia–a–Glo, Waterborne) and analysed by in a FACS Calibur cytometer (Becton Dickinson, Canada) at FL1 (525 nm-green). Several dilutions of the stained cysts (from 2 105 to 1 102 cysts/ml) were analysed by FC for assessment of the detection limit. Crossreactions were investigated using both prokaryotic (Escherichia coli, Staphylococcus aureus) and eukaryotic microorganisms (Candida albicans, Cryptosporidium parvum oocysts). Suspensions with dead and viable cysts were stained with 5 mg/ml of propidium iodide (PI, Sigma), associated with the specific fluorescent antibody and analysed at FL3 (670 nm-red). The optimal specific-antibody concentration was 1.5 mg/ml, yielding a clearly separated histogram from autofluorescence. A threshold of 2 102 cysts/ml was established. No crossreaction occurred with bacteria, fungi or parasites. When using the two fluorescent probes, specific-antibody and PI (a marker of dead), viable cysts were distinguished from dead. A new detection method using FC is now available for detection of Giardia and for evaluation of its viability. KEYWORDS Flow Cytometry protocol; Giardia lamblia; Detection limit; Viability
10.1016/j.tmaid.2007.09.028
Abstracts
P14
P13 Analyses of cytochrome b mutations in Plasmodium falciparum isolates in Thai-Myanmar border, 2002–2005 a,b
a
a
Y. Naoshima-Ishibashi , M. Iwagami , T. Mitamura , S. Looareesuwanc, S. Kanoa,b, a
International Medical Center of Japan, Japan b University of Tsukuba, Japan c Mahidol University, Thailand The combination of atovaquone and proguanil (MalaroneTM, GlaxoSmithKline) has been established as an effective drug of choice not only for treatment but also for prophylaxis of multi-drug resistant Plasmodium falciparum malaria in travellers. However, several cases of resistance against MalaroneTM have already been reported in some parts of Africa, and many of the cases are associated with mutations at codon 268 of cytochrome b gene in mitochondrial genome of P. falciparum. On the contrary, in Asian countries, only limited numbers of clinical failures with the drug has so far been reported, and no mutations could be found at the codon 268. To maintain the usefulness of MalaroneTM in travel medicine, monitoring of the effectiveness of the drug is needed. In this study, mutations of atovaquone binding site on the cytochrome b were investigated by PCR amplification and sequencing of the gene (1131 bp length) in the parasites from patients at the Hospital for Tropical Diseases Mahidol University in Bangkok, Thailand, in 2002 through 2005. Twenty-five blood samples were from complicated falciparum malaria patients and another 25 were from non-complicated falciparum patients who were infected in multi-drug resistant areas in Thai-Myanmar border. Forty-seven out of 50 samples were successfully analyzed, and no mutation was observed in putative ubiquinol (Q0) binding site 1 and 2. In fact, they showed identically conserved nucleotide sequences, as was reported by our previous study conducted in the same area in 1999. We may conclude from these findings that MalaroneTM is still effective and recommended for treatment and prophylaxis for travellers to Mekong region. KEYWORDS Plasmodium falciparum; Malarone; Atovaquone; Cytochrome b
Further reading [1]. Naoshima-Ishibashi Y, et al. Analyses of cytochrome b mutations in Plasmodium falciparum isolates in Thai-Myanmar border. Travel Med Infect Dis 2007;5(2):132–4. [2]. Krudsood S, et al. Efficacy of atovaquone-proguanil for treatment of acute multidrug-resistant Plasmodium falciparum malaria in Thailand. Am J Trop Med Hyg 2007;76(4):655–8. 10.1016/j.tmaid.2007.09.027
Optimization of a flow cytometry protocol for detection and viability assessment of Giardia lamblia
J. Barbosaa,b,, S. Costa-de-Oliveiraa, A.G. Rodriguesa,c, C. Pina-Vaza,d a
Department of Microbiology, School of Medicine, Portugal b Jean Piaget Superior School of Health, Portugal c Department of Plastic and Reconstructive Surgery, Hospital S. Joa˜o, Portugal d Department of Microbiology, Hospital S. Joa˜o, Portugal Giardia lamblia is one of the most important cause of diarrhoea in humans around the world. Transmission routes are complex including fecal—oral as well as waterborne and foodborne transmission. Detection methods in water and stools involve cyst concentration procedures, followed by conventional microscopy which is time-consuming. Higher sensibility was described with antigen detection but showing unpredictable specificity. Molecular genetic studies are complex and difficult for routine analysis. We have optimised a specific Flow Cytometric (FC) protocol for detection of G. lamblia, established its detection limit and assayed the possibility of cross-reaction with other microorganisms. Viability studies, important to evaluate the infectious risk and for treatment monitorization, were also performed by FC. A known concentration of G. lamblia cysts (for Waterborne Inc, USA and from a Giardia culture) were stained with serial concentrations of a fluorescein-labelled (FL) mouse monoclonal antibody (Giardia–a–Glo, Waterborne) and analysed by in a FACS Calibur cytometer (Becton Dickinson, Canada) at FL1 (525 nm-green). Several dilutions of the stained cysts (from 2 105 to 1 102 cysts/ml) were analysed by FC for assessment of the detection limit. Crossreactions were investigated using both prokaryotic (Escherichia coli, Staphylococcus aureus) and eukaryotic microorganisms (Candida albicans, Cryptosporidium parvum oocysts). Suspensions with dead and viable cysts were stained with 5 mg/ml of propidium iodide (PI, Sigma), associated with the specific fluorescent antibody and analysed at FL3 (670 nm-red). The optimal specific-antibody concentration was 1.5 mg/ml, yielding a clearly separated histogram from autofluorescence. A threshold of 2 102 cysts/ml was established. No crossreaction occurred with bacteria, fungi or parasites. When using the two fluorescent probes, specific-antibody and PI (a marker of dead), viable cysts were distinguished from dead. A new detection method using FC is now available for detection of Giardia and for evaluation of its viability. KEYWORDS Flow Cytometry protocol; Giardia lamblia; Detection limit; Viability
10.1016/j.tmaid.2007.09.028