Longitudinal Survey of Plasmodium falciparum Infection in Vietnam: Characteristics of Antimalarial Resistance and Their Associated Factors

Plasmodium falciparum is the main cause of human malaria and is one of the important pathogens causing high rates of morbidity and mortality. The total number of malaria patients in Vietnam has gradually decreased over the last decade. However, the spread of pathogens with drug resistance remains a significant problem. Defining the trend in genotypes related to drug resistance is essential for the control of malaria in Vietnam. We undertook a longitudinal survey of Plasmodium falciparum malaria in 2001, 2002, and 2005 to 2007. The pfcrt, pfmdr1, pfdhfr, and pfdhps genes were analyzed by sequencing; and correlations by study year, age, gender, and genotype were identified statistically. The ratio of the chloroquine resistance genotype pfcrt 76T was found to have decreased rapidly after 2002. High numbers of mutations in the pfdhfr and pfdhps genes were observed only in 2001 and 2002, while the emergence of parasites with a new K540Y mutation in the P. falciparum dihydropteroate synthetase (PfDHPS) was observed in 2002. For males and those in younger age brackets, a correlation between vulnerability to P. falciparum infection and strains with pfcrt 76K or strains with decreased numbers of mutations in pfdhfr and pfdhps was demonstrated. The parasites with pfcrt 76T exhibited a greater number of mutations in pfdhfr and pfdhps.Plasmodium falciparum has long been one of the most important pathogens, causing severe illness and large numbers of deaths worldwide. In the 1990s, more than 1 million people living in Vietnam suffered from P. falciparum infections, resulting in thousands of deaths per year. Since then, the National Institute of Malariology, Parasitology, and Entomology (NIMPE) and the government of Vietnam have focused a great deal of time and effort on a malaria control program. As a result, the incidence of malaria reported in 2003 was only 12% of that reported in 1992 (2). However, the spread of drug-resistant isolates, including multidrug-resistant strains, has become a critical problem in Vietnam and has led to the significant failure of treatment. Thus, a further understanding of the incidence of malaria cases with detailed parasite genotype information and the identification of factors relating to the acquisition of drug-resistant isolates may prove important for the determination of effective and economical treatment choices in clinical settings.The pfcrt gene is located on chromosome 7 and encodes the vacuolar membrane transporter protein P. falciparum chloroquine-resistant transport (PfCRT) (21). While several point mutations associated with chloroquine resistance have been determined previously, substitution of K for T in codon 76 has been shown to be specifically related to resistance in vitro (21, 23). The allelic variation of pfcrt-related drug resistance differs among various geographical areas. Variants with the sequences CVIET, CVIDT, and SVMNT at pfcrt positions 72 to 76 are prevalent in the Indochinese Peninsula (13, 21, 24). The pfmdr1 gene is located on chromosome 5 and encodes P-glycoprotein homologue 1 (Pgh1). This protein is localized to the digestive vacuole membrane, where it is thought to function in the import of solutes, including some antimalarial drugs, into the digestive vacuole (21). pfmdr1 mutations in codons 86, 184, 1034, 1042, and 1246 have been reported previously and have been shown to correlate with susceptibility to chloroquine, quinine, and mefloquine (23). Sulfadoxine-pyrimethamine (SP) resistance is thought to be due to specific point mutations in the pfdhfr and the pfdhps genes. The pfdhfr gene encodes dihydrofolate reductase (DHFR), the target enzyme of pyrimethamine and trimethoprim. Conformational changes in this enzyme due to point mutations result in the prevention of adequate drug access. The codon positions in the pfdhfr gene that are related to resistance include 16, 50, 51, 59, 108, 140, and 164 (23). The deduced pathway for the resistant mutants suggested that all multiple mutants emerged through stepwise selection from the single mutant with the S108N mutation (18). The pfdhps gene encodes the enzyme dihydropteroate synthetase (DHPS). Point mutations in this gene also lead to conformational changes in DHPS and result in resistance to sulfadoxine and sulfamethoxazole. The loci responsible for resistance have been identified at positions 436, 437, 540, 581, and 613 (23).In Vietnam, chloroquine-resistant P. falciparum was reported for the first time in the 1960s (12, 14). Ngo et al. reported in 2003 that all of the isolates acquired from 18 adult rubber plantation workers residing in southern Vietnam demonstrated the pfcrt 76T mutation (14). In contrast, Phuc et al. reported that the prevalence of the mutant was only 38.5% when the strains from 39 malaria patients in the Quang Tri Province of central Vietnam were investigated (15). P. falciparum strains resistant to antifolates have also continued to increase in prevalence since the 1980s. Masimirembwa et al. analyzed 40 P. falciparum isolates obtained from malaria patients and reported that 97.5% of the isolates demonstrated a pfdhfr mutation that was related to pyrimethamine resistance, while 95.0% demonstrated a pfdhps mutation that was associated with sulfadoxine resistance (12).In the 1990s, the treatment for malaria in Vietnam mainly involved monotherapy with artemisinin or single-dose combinations of mefloquine with artemisinin or artesunate. However, the rates of recrudescence after the use of these treatment regimens were as high as 25%. As a result, the Vietnamese Ministry of Health introduced CV8 treatment, which consisted of dihydroartemisinin, piperaquine, trimethoprim, and primaquine, as part of the National Malaria Control Program (NMCP) (6, 20).Here we present the results of a longitudinal survey conducted in 2001, 2002, and 2005 to 2007 in the Binh Phuoc Province of Vietnam. The study was undertaken to investigate the incidence of malaria caused by P. falciparum and to document the changes in genotype that were related to drug resistance. From this study, we were able to identify the allelic and haplotype changes that occurred over the study years and deduce the factors associated with drug resistance.     

Microenvironment induced spheroid to sheeting transition of immortalized human keratinocytes (HaCaT) cultured in microbubbles formed in polydimethylsiloxane

The in vivo cellular microenvironment is regulated by a complex interplay of soluble factors and signaling molecules secreted by cells and it plays a critical role in the growth and development of normal and diseased tissues. In vitro systems that can recapitulate the microenvironment at the cellular level are needed to investigate the influence of autocrine signaling and extracellular matrix effects on tissue homeostasis, regeneration, disease development and progression. In this study, we report the use of microbubble technology as a means to culture cells in a controlled microenvironment in which cells can influence their function through autocrine signaling. Microbubbles (MB) are small spherical cavities about 100-300 μm in diameter formed in hydrophobic polydimethylsiloxane (PDMS) with ～60-100 μm circular openings and aspect ratio ～3.0. We demonstrate that the unique architecture of the microbubble compartment is advantaged for cell culture using HaCaT cells, an immortalized keratinocyte cell line. We observe that HaCaT cells, seeded in microbubbles (15-20 cells/MB) and cultured under standard conditions, adopt a compact 3D spheroidal morphology. Within 2-3 days, the cells transition to a sheeting morphology. Through experimentation and simulation we show that this transition in morphology is due to the unique architecture of the microbubble compartment which enables cells to condition their local microenvironment. The small media volume per cell and the development of shallow concentration gradients allow factors secreted by the cells to rise to bioactive levels. The kinetics of the morphology transition depends on the number of cells seeded per microbubble; higher cell seeding induces a more rapid transition. HaCaT cells seeded onto PDMS cured in 96-well plates also form compact spheroids but they do not undergo a transition to a sheeting morphology even after several weeks of culture. The importance of soluble factor accumulation in driving this morphology transition in microbubbles is supported by the observation that spheroids do not form when cells - seeded into microbubbles or onto PDMS cured in 96-well plates - are cultured in media conditioned by HaCaT cells grown in standard tissue culture plate. We observed that the addition of TGF-β1 to the growth media induced cells to proliferate in a sheeting morphology from the onset both on PDMS cured in 96-well plates and in microbubbles. TGF-β1 is a morphogen known to regulate epithelial-to-mesenchymal transition (EMT). Studies of the role of Ca(2+) concentration and changes in E-cadherin expression additionally support an EMT-like HaCaT morphology transition. These findings taken together validate the microbubble compartment as a unique cell culture platform that can potentially transform investigative studies in cell biology and in particular the tumor microenvironment. Targeting the tumor microenvironment is an emerging area of anti-cancer therapy.     

Caries experience of adults attending private and public dental clinics in Australia

Objectives: In Australia, the majority of dental patients attend the private sector, while those with means tested eligibility for government assistance may attend the public sector. The aims of this study were to compare dental caries among persons who last visited private and public clinics, controlling for age, sex, reason for visit, and income. Methods: Data were collected in 2004‐06, using a three‐stage, stratified clustered sample of Australians aged 15+ years, involving a computer‐assisted telephone interview (CATI), oral examination, and mailed questionnaire. Results: A total of 14,123 adults responded to the CATI (49 percent response) of whom 5,505 (44 percent of those interviewed) had an oral epidemiological examination. Multivariate regression analysis controlling for age, sex, reason for visit, and income showed (P < 0.05) that persons attending public clinics had higher levels of decayed (β = 0.33) and missing teeth (β = 0.83), but lower levels of filled teeth (β = ?1.09) compared with the reference category of private clinics. Conclusions: Persons who attend for dental care in the public sector have worse oral health than adults who visit private dental clinics, in addition to an independent effect of socioeconomic disadvantage.     

A longitudinal study of the relative importance of factors related to use of dental services among young adults

Roberts‐Thomson KF, Stewart J, Do LG. A longitudinal study of the relative importance of factors related to use of dental services among young adults. Community Dent Oral Epidemiol 2011; 39: 268–275. © 2010 John Wiley & Sons A/S Abstract – Background: The decline in the appropriate use of dental services from childhood to adulthood is of concern. The relative importance of factors influencing use of dental services in young adulthood should inform strategies to address this issue. Aim: To develop models predictive of inadequate utilisation of dental care in young adults and to determine the relative importance of health behaviours, need for dental care, and socio‐demographic factors. Inadequate utilisation of dental care was determined as lack of visit for dental care during the study period and as problem‐based care‐seeking behavior. Methods: A random sample of Adelaide young adults was selected from the electoral roll. Participants were interviewed and offered a dental examination at baseline. Two and a half years later participants were contacted again and interviewed on the use of dental services and usual reason for visiting and a number of explanatory factors. The population attributable fractions for the explanatory variables which were significant in the model for this cohort of young adults were calculated using the log‐binomial method. Results: There were 819 participants at follow‐up a response rate of 65% of baseline participants. In the two and a half year period between baseline and follow‐up about one quarter of young adults did not make a dental visit and over one third reported that they usually made a dental visit for a problem. The attributable fraction calculation indicated that 30% of infrequent visiting was attributed to reporting no need for dental care, 17% to difficulty paying a $100 dental bill, 17% to being male and 10% to smoking. The attributable fraction calculation indicated that 27% of usually visiting for a problem was attributed to having no tertiary education, 23% to not having dental insurance, 14% to being male, 12% to smoking, 10% to avoiding dental care due to cost and 5% to use of the public sector. Conclusion: Need and affordability factors were more important than general health behavior factors in influencing use of dental care by a cohort of young adults.     

Genotypic characteristics of Staphylococcus aureus isolates from a multinational trial of complicated skin and skin structure infections

The impact of bacterial genetic characteristics on the outcome of patients with Staphylococcus aureus infections is uncertain. This investigation evaluated potential associations between bacterial genotype and clinical outcome using isolates collected as part of an international phase 2 clinical trial (FAST II) evaluating telavancin for the treatment of complicated skin and skin structure infections (cSSSI). Ninety S. aureus isolates from microbiologically evaluable patients with cSSSI enrolled in the FAST II trial from 11 sites in the United States (56 isolates, or 62%) and 7 sites in South Africa (34 isolates, or 38%) were examined for staphylococcal cassette chromosome mec, agr, and the presence of 31 virulence genes and subjected to pulsed-field gel electrophoresis (PFGE). South African methicillin-susceptible S. aureus (MSSA) isolates were more likely to carry certain virulence genes, including sdrD (P = 0.01), sea (P < 0.01), and pvl (P = 0.01). All 44 (49%) methicillin-resistant S. aureus (MRSA) isolates were from the United States; 37 (84%) were strain USA 300 by PFGE. In the United States, MRSA isolates were more likely than MSSA isolates to carry genes for sdrC (P = 0.03), map/eap (P = 0.05), fnbB (P = 0.11), tst (P = 0.02), sea (P = 0.04), sed (P = 0.04), seg (P = 0.11), sej (P = 0.11), agr (P = 0.09), V8 (P = 0.06), sdrD, sdrE, eta, etb, and see (P < 0.01 for all). MRSA isolates were more often clonal than MSSA isolates by PFGE. Isolates from patients who were cured were significantly more likely to contain the pvl gene than isolates from patients that failed or had indeterminate outcomes (79/84 [94%] versus 3/6 [50%]; P = 0.01). S. aureus strains from different geographic regions have different distributions of virulence genes.     

The serotonin transporter expression in Drosophila melanogaster

The serotonin transporter is an important regulator of serotonergic signaling. In order to analyze where the Drosophila melanogaster ortholog of the mammalian serotonin transporter (dSERT) is expressed in the nervous system, a dSERT antibody serum was used. Ectopic expression studies and loss of function analysis revealed that the dSERT antibody serum specifically recognizes dSERT. It was shown that in the embryonic nervous system dSERT is expressed in a subset of Engrailed-positive neurons. In the larval brain, dSERT is exclusively expressed in serotonergic neurons, all of which express dSERT. dSERT-positive neurons surround almost all brain neuropiles. In the mushroom body of the adult brain, extrinsic serotonergic neurons expressing dSERT engulf the mushroom body lobes. These neurons show regional differences in dSERT and serotonin expression. At the presynaptic terminals, serotonin release is sterically linked to serotonin reuptake. In contrast to this, there are other areas in serotonergic neurons where dSERT expression and/or function are uncoupled from synaptic neurotransmitter recycling and serotonin release. The localization pattern of dSERT can be employed to further understanding and analysis of serotonergic networks.     

A rare case of pleuropulmonary blastoma detected in fetus

Pleuropulmonary blastoma (PPB) is among the rarest malignant tumors diagnosed in children. PPBs can be histopathologically classified into 3 types: cystic tumor (type I), mixed cystic and solid tumor (type II), and pure solid tumor (type III). We describe a case of type III PPB that was detected in a prenatal fetus, confirmed using histopathological methods. To the best of our knowledge, this is the first case describing a type III PPB detected in a fetus. Prenatal ultrasonography is an excellent tool for detecting pulmonary lesions during the diagnostic phase, and the possibility of PPB should be considered when solid tumors are detected. Early detection can allow for the performance of full resection, leading to a better prognosis for this cancerous tumor.     

A rare case of fusiform basilar trunk aneurysm

Aneurysms in the posterior circulation and distal sites are more common among the pediatric population than among adults, with a male predominance. Symptoms of an aneurysm in the posterior circulation can include a stiff neck or severe headache due to a ruptured aneurysm, whereas an unruptured aneurysm can cause mass effects or neurological deficits. However, in children, the complete occlusion of the aneurysm while preserving the flow of the main artery can be difficult to achieve when attempting a stent-assisted coil embolization technique. A 25-month-old girl presented with left hemiparesis and was diagnosed with a basilar artery aneurysm 10 months prior, but she did not receive any specific treatment. No history of trauma and no significant familial history were recorded. Angiography showed a fusiform aneurysm on the basilar artery trunk, which was successfully occluded using stent-assisted coiling following dual antiplatelet therapy with clopidogrel and aspirin. She was discharged with the complete restoration of motor deficits.