Polymorphisms of the pfmdr1 but not the pfnhe-1 gene is associated with in vitro quinine sensitivity in Thai isolates of Plasmodium falciparum.

BACKGROUND: The emergence of Plasmodium falciparum resistance to most currently used anti-malarial drugs is a major problem in malaria control along the Thai-Myanmar and Thai-Cambodia borders. Quinine (QN) with tetracycline/doxycycline has been used as the second-line treatment for uncomplicated falciparum malaria. In addition, QN monotherapy has been the first-line treatment for falciparum malaria in pregnant women. However, reduced in vitro and in vivo responses to QN have been reported. To date, a few genetic markers for QN resistance have been proposed including Plasmodium falciparum chloroquine resistance transporter (pfcrt), P. falciparum multidrug resistance 1 (pfmdr1), and P. falciparum Na+/H+ exchanger (pfnhe-1). This study was to investigate the role of the pfmdr1 and pfnhe-1 gene on in vitro QN sensitivity in Thai isolates of P. falciparum. METHODS: Eighty-five Thai isolates of P. falciparum from the Thai-Myanmar and Thai-Cambodia borders from 2003-2008 were determined for in vitro QN sensitivity using radioisotopic assay. Polymorphisms of the pfmdr1 and pfnhe-1 gene were determined by PCR-RFLP and sequence analysis. Associations between the in vitro QN sensitivity and the polymorphisms of the pfmdr1 and pfnhe-1 gene were evaluated. RESULTS: The mean QN IC50 was 202.8 nM (range 25.7-654.4 nM). Only four isolates were QN resistant when the IC50 of >500 nM was used as the cut-off point. Significant associations were found between the pfmdr1 mutations at codons N86Y and N1042D and in vitro QN sensitivity. However, no associations with the number of DNNND, DDNNNDNHNDD, and NHNDNHNNDDD repeats in the microsatellite ms4760 of the pfnhe-1 gene were identified. CONCLUSION: Data from the present study put doubt regarding the pfnhe-1 gene as to whether it could be used as the suitable marker for QN resistance in Thailand. In contrast, it confirms the influence of the pfmdr1 gene on in vitro QN sensitivity.

Application of QuantiFERON ELISA for Detection of Interferon-Gamma Autoantibodies in Adult-Onset Immunodeficiency Syndrome.

OBJECTIVE: Patients who develop interferon-gamma autoantibodies (IFN-ɤ autoAbs) in adult-onset immunodeficiency (AOID) syndrome are more likely to develop opportunistic and recurrent intracellular infections. The assay to detect IFN-ɤ autoAbs is essential for the diagnosis and therapeutic monitoring of AOID syndrome. Therefore, this study applied the QuantiFERON assay for the detection of IFN-ɤ autoAbs. METHODS: Serum from patients with AOID syndrome (n = 19) and serum from healthy patients (n = 20) was collected and applied using 2 neutralizing platforms of enzyme-linked immunosorbent assay (ELISA) kits (the BD ELISA and the QuantiFERON ELISA) for IFN-ɤ autoAbs detection. RESULTS: The pooled serum from patients with AOID syndrome showed >50% inhibition at 1:5000 dilution (positive), whereas the pooled serum from healthy patients showed <50% inhibition at 1:5000 dilution (negative) according to the neutralizing QuantiFERON ELISA. Each specimen showed the same result according to both the neutralizing BD ELISA and the neutralizing QuantiFERON ELISA. Moreover, the patient serum showed a variation in titer ranging from 1:5000 to >1:5,000,000 according to the neutralizing QuantiFERON ELISA. CONCLUSION: The QuantiFERON ELISA kit could be applied for the detection of IFN-ɤ autoAbs for the diagnosis and therapeutic monitoring of AOID syndrome.

In vitro atovaquone/proguanil susceptibility and characterization of the cytochrome b gene of Plasmodium falciparum from different endemic regions of Thailand.

BACKGROUND: The emergence of Plasmodium falciparum resistant to most currently used antimalarial drugs is the major problem in malaria control along the Thai-Myanmar and Thai-Cambodia borders. Although artemisinin-based combination therapy has been recommended for the treatment of multidrug-resistant falciparum malaria, these combinations are not available for some people, such as travelers from North America. A fixed-dose combination of atovaquone and proguanil (Malarone) has been proved to be effective for the treatment and prophylaxis of malaria which is already approved by countries in North America and Europe. Determination of the phenotypes and genotypes related to atovaquone/proguanil response in Thai isolates of P. falciparum will be useful for rationale drug use. The main purpose of this study was to explore the in vitro atovaquone/proguanil susceptibility of recently adapted Thai isolates of P. falciparum. Genotypic characterization of the cytb gene of these isolates was also determined since it has been reported that point mutations, particularly codon 268 in the cytochrome b gene (cytb) have been linked to atovaquone/proguanil treatment failure. METHODS: Eighty three P. falciparum isolates collected during 1998 to 2005 from four different multidrug resistance areas of Thailand were determined for the in vitro atovaquone/proguanil susceptibilities using radioisotopic assay. Mutations in the cytb gene were determined by PCR-RFLP and sequence analysis. RESULTS: The mean atovaquone and proguanil IC50 was 3.4 nM and 36.5 muM, respectively. All 83 Thai isolates were atovaquone sensitive. None of the 83 isolates contained the mutations at codon 268 of the cytb gene. DNA sequencing of the cytb gene of 20 parasite isolates showed no other mutations. CONCLUSION: In agreement with a recent efficacy study of atovaquone/proguanil, the present information indicates that atovaquone/proguanil can be one of the drugs of choice for the treatment and prophylaxis of multidrug-resistant falciparum malaria in Thailand.

Influence of the pfmdr1 Gene on In Vitro Sensitivities of Piperaquine in Thai Isolates of Plasmodium falciparum.

Piperaquine combined with dihydroartemisinin is one of the artemisinin derivative combination therapies, which can replace artesunate-mefloquine in treating uncomplicated falciparum malaria in Thailand. The aim of this study was to determine the in vitro sensitivity of Thai Plasmodium falciparum isolates against piperaquine and the influence of the pfmdr1 gene on in vitro response. One hundred and thirty-seven standard laboratory and adapted Thai isolates of P. falciparum were assessed for in vitro piperaquine sensitivity. Polymorphisms of the pfmdr1 gene were determined by polymerase chain reaction methods. The mean and standard deviation of the piperaquine IC50 in Thai isolates of P. falciparum were 16.7 ± 6.3 nM. The parasites exhibiting chloroquine IC50 of ≥ 100 nM were significantly less sensitive to piperaquine compared with the parasite with chloroquine IC50 of < 100 nM. No significant association between the pfmdr1 copy number and piperaquine IC50 values was found. In contrast, the parasites containing the pfmdr1 86Y allele exhibited significantly reduced piperaquine sensitivity. Before nationwide implementation of dihydroartemisinin-piperaquine as the first-line treatment in Thailand, in vitro and in vivo evaluations of this combination should be performed especially in areas where parasites containing the pfmdr1 86Y allele are predominant such as the Thai-Malaysian border.

The elicitin-like glycoprotein, ELI025, is secreted by the pathogenic oomycete Pythium insidiosum and evades host antibody responses.

Pythium insidiosum is a unique oomycete that can infect humans and animals. Patients with a P. insidiosum infection (pythiosis) have high rates of morbidity and mortality. The pathogen resists conventional antifungal drugs. Information on the biology and pathogenesis of P. insidiosum is limited. Many pathogens secrete proteins, known as effectors, which can affect the host response and promote the infection process. Elicitins are secretory proteins and are found only in the oomycetes, primarily in Phytophthora and Pythium species. In plant-pathogenic oomycetes, elicitins function as pathogen-associated molecular pattern molecules, sterol carriers, and plant defense stimulators. Recently, we reported a number of elicitin-encoding genes from the P. insidiosum transcriptome. The function of elicitins during human infections is unknown. One of the P. insidiosum elicitin-encoding genes, ELI025, is highly expressed and up-regulated at body temperature. This study aims to characterize the biochemical, immunological, and genetic properties of the elicitin protein, ELI025. A 12.4-kDa recombinant ELI025 protein (rELI025) was expressed in Escherichia coli. Rabbit anti-rELI025 antibodies reacted strongly with the native ELI025 in P. insidiosum's culture medium. The detected ELI025 had two isoforms: glycosylated and non-glycosylated. ELI025 was not immunoreactive with sera from pythiosis patients. The region near the transcriptional start site of ELI025 contained conserved oomycete core promoter elements. In conclusion, ELI025 is a small, abundant, secreted glycoprotein that evades host antibody responses. ELI025 is a promising candidate for development of diagnostic and therapeutic targets for pythiosis.

Transcriptome analysis reveals pathogenicity and evolutionary history of the pathogenic oomycete Pythium insidiosum.

Oomycetes form a unique group of microorganisms that share hyphal morphology with fungi. Most of pathogenic oomycetes infect plants, while some species are capable of infecting animals. Pythium insidiosum is the only oomycete that can infect both humans and animals, and causes a life-threatening infectious disease, called 'pythiosis'. Controlling an infection caused by P. insidiosum is problematic because effective antimicrobial drugs are not available. Information on the biology and pathogenesis of P. insidiosum is limited. We generated a P. insidiosum transcriptome of 26 735 unigenes, using the 454 sequencing platform. As adaptations to increased temperature inside human hosts are required for a successful pathogen, we generated P. insidiosum transcriptomes at 28 °C and 37 °C and identified 625 up-regulated and 449 down-regulated genes at 37 °C. Comparing the proteomes of oomycetes, fungi, and parasites provided clues on the evolutionary history of P. insidiosum. Potential virulence factors of P. insidiosum, including putative effectors, were identified. Pythium insidiosum harbored an extensive repertoire of ∼ 300 elicitin domain-containing proteins. The transcriptome, presented herein, provides an invaluable resource for exploring P. insidiosum's biology, pathogenesis, and evolution.

Performance comparison of immunodiffusion, enzyme-linked immunosorbent assay, immunochromatography and hemagglutination for serodiagnosis of human pythiosis.

Pythiosis is a life-threatening infectious disease caused by the fungus-like organism Pythium insidiosum. Morbidity and mortality rates of pythiosis are high. The treatment of choice for pythiosis is surgical debridement of infected tissue. Early and accurate diagnosis is critical for effective treatment. In-house serodiagnostic tests, including immunodiffusion (ID), enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICT) and hemagglutination (HA) have been developed to detect antibodies against P. insidiosum in sera. This study compares the diagnostic performance of ID, ELISA, ICT, and HA, using sera from 37 pythiosis patients and 248 control subjects. ICT and ELISA showed optimal diagnostic performance (100% sensitivity, specificity, positive predictive value and negative predictive value). ICT was both rapid and user-friendly. ELISA results were readily quantitated. ID is relatively insensitive. HA was rapid, but diagnostic performance was poor. Understanding the advantages offered by each assay facilitates selection of an assay that is circumstance-appropriate. This will promote earlier diagnoses and improved outcomes for patients with pythiosis.

Autochthonous disseminated dermal and visceral leishmaniasis in an AIDS patient, southern Thailand, caused by Leishmania siamensis.

We report the first establishment of in vitro cultivation and genotypic characterization of Leishmania siamensis isolated from an autochthonous disseminated dermal and visceral leishmaniasis in a Thai acquired immunodeficiency syndrome (AIDS) patient. The molecular identification has shown that the parasite was identical to L. siamensis, a recently described Leishmania species reported in the southern provinces of Thailand. The phylogenetic analysis has confirmed L. siamensis as closely related to the zoonotic Leishmania species L. enrietti.

Role of Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes on in vitro chloroquine resistance in isolates of Plasmodium falciparum from Thailand.

Resistance to chloroquine is a public health problem worldwide. Polymorphisms of the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes have been linked to chloroquine resistance. Although the K76T mutation in the pfcrt gene has been shown to be a key determinant in chloroquine resistance, evidence suggests that the pfmdr1 gene could modulate the level of chloroquine resistance. However, few studies of field isolates could identify the interactive role of these two genes in chloroquine resistance. Thus, we evaluated the influence of pfcrt and pfmdr1 polymorphisms on in vitro chloroquine sensitivity in 89 adapted isolates of P. falciparum from Thailand. We found that 87 of 89 isolates contained the CVIET haplotype of the pfcrt gene. Two additional mutations in the pfcrt gene were identified, i.e., K6Q and H97L. For the pfmdr1 polymorphisms, the 184F allele was common in the parasites isolated along the Thailand-Cambodia border, and those isolated along the Thailand-Myanmar border contained higher copy numbers. Our results indicate that the additional mutations, in particular H97L in the pfcrt gene and Y184F in the pfmdr1 gene and its copy number, influence the level of chloroquine resistance.

Incidence and risk factors of hookworm infection in a rural community of central Thailand.

A cohort study to identify incidence and risk factors of hookworm infection was conducted in a rural community, central Thailand from November 2005 to February 2007. Stool specimens were examined for hookworm eggs using wet preparation, Kato thick smear, and water-ethyl acetate sedimentation technique. The incidence rate of hookworm infection was 7.5/100 person-years. The independent risk factors for acquiring hookworm infection were barefoot walking (incidence rate ratio [IRR] = 4.2, 95% confidence interval [CI] = 1.2-14.5) and raising buffaloes around the house (IRR = 4.8, 95% CI = 1.9-11.8). Sequencing of internal transcribed spacer 1 (ITS1)-5.8S-ITS2 region of the ribosomal RNA gene were performed for identifying species of hookworm. Necator americanus was the most common hookworm identified in this population. Ancylostoma duodenale and A. ceylanicum were also detected. Our data suggest transmission of both human and animal hookworms in this community. Thus, prevention and control strategies of hookworm infection should cover both human and animal infection.

Expressed sequence tags reveal genetic diversity and putative virulence factors of the pathogenic oomycete Pythium insidiosum.

Oomycetes are unique eukaryotic microorganisms that share a mycelial morphology with fungi. Many oomycetes are pathogenic to plants, and a more limited number are pathogenic to animals. Pythium insidiosum is the only oomycete that is capable of infecting both humans and animals, and causes a life-threatening infectious disease, called "pythiosis". In the majority of pythiosis patients life-long handicaps result from the inevitable radical excision of infected organs, and many die from advanced infection. Better understanding P. insidiosum pathogenesis at molecular levels could lead to new forms of treatment. Genetic and genomic information is lacking for P. insidiosum, so we have undertaken an expressed sequence tag (EST) study, and report on the first dataset of 486 ESTs, assembled into 217 unigenes. Of these, 144 had significant sequence similarity with known genes, including 47 with ribosomal protein homology. Potential virulence factors included genes involved in antioxidation, thermal adaptation, immunomodulation, and iron and sterol binding. Effectors resembling pathogenicity factors of plant-pathogenic oomycetes were also discovered, such as, a CBEL-like protein (possible involvement in host cell adhesion and hemagglutination), a putative RXLR effector (possibly involved in host cell modulation) and elicitin-like (ELL) proteins. Phylogenetic analysis mapped P. insidiosum ELLs to several novel clades of oomycete elicitins (ELIs), and homology modeling predicted that P. insidiosum ELLs should bind sterols. Most of the P. insidiosum ESTs showed homology to sequences in the genome or EST databases of other oomycetes, but one putative gene, with unknown function, was found to be unique to P. insidiosum. The EST dataset reported here represents the first steps in identifying genes of P. insidiosum and beginning transcriptome analysis. This genetic information will facilitate understanding of pathogenic mechanisms of this devastating pathogen.

Association between the pfmdr1 gene and in vitro artemether and lumefantrine sensitivity in Thai isolates of Plasmodium falciparum.

We evaluated the influence of pfmdr1 mutations and copy number on in vitro artemether and lumefantrine sensitivity in 101 laboratory and adapted Thai isolates of Plasmodium falciparum. Approximately one-fourth of these isolates exhibited reduced lumefantrine susceptibility. We found that both mutations and amplification of the pfmdr1 gene influenced in vitro artemether and lumefantrine sensitivity. Using multivariate analysis, 184F or 1042N alleles and a copy number of ≥ 4 were identified as the independent markers for decreased lumefantrine susceptibility. Separate analysis also indicated that parasites from different geographical areas were influenced by different genetic markers.