Efficacy of mefloquine treatment and genetic profiles in uncomplicated Plasmodium falciparum malaria in southern Lao PDR.

We conducted a 28-day follow-up of 17 Laotian patients diagnosed with uncomplicated Plasmodium falciparum malaria treated with mefloquine (Mephaquine, MQ) alone to determine the efficacy. All patients were completely cured with MQ, without reappearance of asexual stage parasitemia at follow-up. Of the 7 isolates tested for genotypic analysis, one isolate was a Y86 mutant type of the pfmdr1 gene, the others were N86 wild. These findings suggest no MQ resistance in the study area possibly because the drug is rarely used in southern Lao PDR.

Pfcrt genotypes and related microsatellite DNA polymorphisms on Plasmodium falciparum differed among populations in the Greater Mekong Subregion.

Malaria morbidity and mortality have decreased gradually in the Greater Mekong Subregion (GMS). Presently, WHO sets a goal to eliminate malaria by 2030 in the GMS. However, drug-resistant malaria has been reported from several endemic areas. To achieve the goal of elimination, the status of the emergence and spread of drug resistance should be monitored. In this study, the genotype of the Plasmodium falciparum chloroquine (CQ) resistance transporter gene (pfcrt) and 6 microsatellite DNA loci flanking the gene were examined. P. falciparum isolates (n = 136) was collected from malaria patients in Thailand (n = 50, 2002-2005), Vietnam (n = 39, 2004), Laos (n = 15, 2007) and Cambodia (n = 32, 2009). Amino acid sequences at codons 72-76 on the gene were determined. All of the isolates from Thailand were CQ-resistant (CVIET), as were all of the isolates from Cambodia (CVIET, CVIDT). Thirteen of the 15 isolates (87%) from Laos were CQ-resistant (CVIET, CVIDT), whereas the other 2 (13%) were CQ-susceptible (CVMNK). In contrast, 27 of the 39 isolates (69%) from Vietnam were CQ-susceptible (CVMNK), whereas the other 12 (31%) were CQ-resistant (CVIET, CVIDT, CVMDT) or mixed (CVMNK/CVIDT). The mean of expected heterozygosity of the microsatellite loci was 0.444 in the Thai population, 0.482 in the Cambodian population, and 0.734 in the Vietnamese population. Genetic diversity in the Thai population was significantly lower than that in the Vietnamese population. These results suggested that chloroquine selective pressure on P. falciparum populations is heterogeneous in the GMS. Therefore, further examination to understand the mechanisms behind the emergence and spread of drug-resistant malaria are needed.

A Unique Subset of γδ T Cells Expands and Produces IL-10 in Patients with Naturally Acquired Immunity against Falciparum Malaria.

Although expansions in γδ T cell populations are known to occur in the peripheral blood of patients infected with Plasmodium falciparum, the role of these cells in people with naturally acquired immunity against P. falciparum who live in malaria-endemic areas is poorly understood. We used a cross-sectional survey to investigate the role of peripheral blood γδ T cells in people living in Lao People's Democratic Republic, a malaria-endemic area. We found that the proportion of non-Vγ9 γδ T cells was higher in non-hospitalized uncomplicated falciparum malaria patients (UMPs) from this region. Notably, we found that the non-Vγ9 γδ T cells in the peripheral blood of UMPs and negative controls from this region had the potential to expand and produce IL-10 and interferon-γ when cultured in the presence of IL-2 and/or crude P. falciparum antigens for 10 days. Furthermore, these cells were associated with plasma interleukin 10 (IL-10), which was elevated in UMPs. This is the first report demonstrating that, in UMPs living in a malaria-endemic area, a γδ T cell subset, the non-Vγ9 γδT cells, expands and produces IL-10. These results contribute to understanding of the mechanisms of naturally acquired immunity against P. falciparum.

Involvement of IL-2/IL-2R system activation by parasite antigen in polyclonal expansion of CD4(+)25(+) HTLV-1-infected T-cells in human carriers of both HTLV-1 and S. stercoralis.

The intermediate state of HTLV-1 infection, often found in individuals dually infected with Strongyloides stercoralis (S. stercoralis) and HTLV-1, is assumed to be a preleukemic state of adult T-cell leukemia (ATL). To investigate the effects of S. stercoralis superinfection on the natural history of HTLV-1 infection, we characterized peripheral blood samples of these individuals in Okinawa, Japan, an endemic area for both HTLV-1 and S. stercoralis and we studied effects of the parasite antigen on T-cells. The dually infected individuals showed a significantly higher provirus load and an increase in CD4(+)25(+) T cell population, with a significant, positive correlation. This increase was attributable to polyclonal expansion of HTLV-1-infected cells, as demonstrated by inverse-long PCR analysis of the integration sites. S. stercoralis antigen activated the IL-2 promoter in reporter gene assays, induced production of IL-2 by PBMC in vitro, and supported growth of IL-2 dependent cell lines immortalized by HTLV-1 infection or the transduction of Tax. Taken collectively, these results indicate that S. stercoralis infection induces polyclonal expansion of HTLV-1-infected cells by activating the IL-2/IL-2R system in dually infected carriers, an event which may be a precipitating factor for ATL and inflammatory diseases.

Biochemical and physiological analyses of a hemolytic toxin isolated from a sea anemone Actineria villosa.

A species of venomous sea anemone Actineria villosa was recently found inhabiting the coastal areas of Okinawa, Japan. This marine animal produces various proteinous toxins, so that a local health organization was called for medical treatment for those who had accidental contact with this animal. In this study we analyzed the biochemical and physiological properties of hemolytic protein from A. villosa. The toxin purified from the tentacles of the animals was found to be a protein with a molecular weight of approximately 19 kDa. We named this newly found hemolytic toxin of A. villosa, Avt-I. Incubation of the toxin with sphingomyelin inhibited hemolytic activity by up to 85%, showing that Avt-I may target sphingomyelin on the erythrocyte membrane. The hemolytic activity was stably maintained at temperatures below 45 degrees C, however, a sharp linear decrease in heat stability was observed within the range of 45-55 degrees C. Our results provide the first evidence that A. villosa produces a toxin with strong hemolytic activity similar in biochemical and physiological properties to other members of actinoporin family previously isolated from related species of sea anemones.

A molecular epidemiologic study of point mutations for pyrimethamine-sulfadoxine resistance of Plasmodium falciparum isolates from Lao PDR.

To understand the current condition of pyrimethamine-sulfadoxine (PS) resistant falciparum malaria in Lao PDR, the frequency of point mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum were examined in 50 blood samples collected from the patients with P. falciparum infection in Southern Lao PDR. Point mutations in 5 codons of the DHFR gene, which is known to be related to pyrimethamine resistance, were detected in 15 out of the 50 samples (30%). Among the 15 samples, 10 samples showed a double mutation of codons 59 and 108 (Cys59Arg with Ser108Asn). In the remaining 5 samples, an additional mutation was observed in codon 51 (Asn51 lle), providing a triple mutation of codons 51, 59 and 108. On the other hand, point mutations in the 4 codons of DHPS gene related to sulfadoxine resistance were observed only in 2 samples (4.0%), namely in codon 437 (Ala437Gly). Only one sample showed mutations in both DHFR and DHPS genes. From the results, it should be considered that the frequency of PS resistant malaria is still low in Lao PDR. Continuous monitoring for the PS resistant malaria, however, is necessary because of the increasing use of PS in this country.

Pyrimethamine-sulfadoxine treatment of uncomplicated Plasmodium falciparum malaria in Lao PDR.

A 28-day in vivo treatment trial to evaluate the efficacy of pyrimethamine/sulfadoxine (Fansidar, PS) was conducted in 21 Lao patients with uncomplicated Plasmodium falciparum malaria. Sixteen patients (76%) were completely cured with PS without any reappearance of asexual stage parasitemia during the follow-up examination. On the other hand, 5 patients (24%) failed to respond to this trial medication, resulting in recrudescence of asexual stage P. falciparum malaria. PS resistance resulted in higher prevalence of post-treatment gametocytemia, 25% gametocyte carriers among PS sensitive cases versus 75% of the resistant cases. These findings suggest that although the level of PS resistance is still valid for treatment of malaria in the study area of Lao PDR, post-treatment induction of gametocytemia among resistant cases may result an increase in transmission rate of PS resistant falciparum malaria.

Falciparum Malaria Incidentally Pretreated with Azithromycin.

A 65-year-old man, who recently returned from Liberia, visited a clinic complaining of fever, and azithromycin was prescribed. The patient presented to a general hospital 5 days after the onset of symptoms, however, a blood smear examination failed to detect malaria. Contrary to the blood smear result, a rapid antigen test in our hospital was strongly-positive for falciparum malaria, indicating a high level of malarial antigen in the blood. Moreover, laboratory examinations on admission showed a tendency for improvement. We assumed that the administration of azithromycin partially treated malaria, thus complicating the blood smear diagnosis. We should be careful in prescribing azithromycin, which is widely used in clinics, to travelers returning from malaria-endemic countries.

Association of a sex-related difference of Strongyloides stercoralis-specific IgG4 antibody titer with the efficacy of treatment of strongyloidiasis.

It is difficult to completely eradicate strongyloidiasis, a human intestinal nematode infection with Strongyloides stercoralis with drugs, especially in males. To find host factors involved in the response to treatment, patients infected with S. stercoralis were examined for S. stercoralis-specific antibody titers and the effect of treatment with albendazole on these titers were determined. The cure rate was slightly but not significantly lower in males than in females (P = 0.108). However, a significantly higher titer of S. stercoralis-specific IgG4 antibody was observed in males than in females (P = 0.0097), and the S. stercoralis-specific IgG4 antibody titer was significantly higher in the male non-cured group than in the cured group (P = 0.035). These results suggest that elevation of the S. stercoralis-specific IgG4 antibody titer is associated with resistance to treatment of S. stercoralis infection, especially in males.

The effectiveness of impregnated bed net in malaria control in Laos.

Impregnated bed net (IBN) were used in 366 villages in the central and southern three provinces of Lao PDR from 1999 to 2000. It was confirmed that 81.0% of 40000 bed nets, which were donated by Japanese Grant Aid, were delivered within 2 years. The strengthening of information network systems in anti-malaria and strong relationship between community and local authorities ensured the success of operation in a short period. The number of patients and the slide positive rate of malaria decreased markedly in public health facilities in three provinces after the use of IBN. An entomological survey was conducted in Boualapha district, where malaria is endemic, to investigate the IBN efficacy on malaria vector. The density and parous rate of Anopeles dirus, which is the main malaria vector in the area, were markedly decreased in the village where IBN was used. This mosquito's behavior, which was baiting mainly humans during the time when the inhabitants sleep in the IBN, was considered to be advantageous in preventing malaria infection using by IBN. The area of distribution of A. dirus is similar to the high endemic area of malaria in Lao PDR. Thus, it is expected that the expansion of the IBN program in the southern provinces will lead to successful malaria control in subsequent years.

Expansion of unconventional T cells with natural killer markers in malaria patients.

Immunological states during human malarial infection were examined. In parallel with parasitemia and anemia, granulocytosis was induced in the blood of patients, especially those infected with Plasmodium (P.) falciparum. At that time, the level of lymphocytes remained unchanged or slightly increased in the blood. However, the distribution of lymphocyte subsets was modulated, showing that the proportion of CD56(+)T cells, CD57(+)T cells, and gammadeltaT cells (i.e. all unconventional T cells) had increased in patients infected with P. falciparum or P. vivax. This phenomenon occurred at the early phase of infection and disappeared in the course of recovery. The data from patients with multiple attacks of P. vivax infection showed that there was no augmentation of these responses. In adult cases, the increase in the proportion of unconventional T cells seemed to closely parallel disease severity. However, all these responses were weak in children, even those infected with P. falciparum. In conjunction with accumulating evidence from mouse malaria experiments, the present results suggest that the immunological state induced by malarial infection might mainly be an event of unconventional T cells and that the immunological memory might not be long-lasting, possibly due to the properties of unconventional T cells.

Intranasal sensitization with Blomia tropicalis antigens induces allergic responses in mice characterized by elevated antigen-specific and non-specific serum IgE and peripheral blood eosinophil counts.

In order to evaluate the potential allergenicity of Blomia tropicalis (Bt) antigen, IgE production of both specific and non-specific for Bt antigen was monitored in BALB/c mice after exposure to the antigen by nasal route. It was evidenced that B. tropicalis contains a functional allergen in its components. The allergenic components, however, when administered intranasally without any adjuvant, did not function to induce IgE response within a short period. On the other hand, intranasal inoculation of Bt antigens augmented serum IgE responses in mice pretreated by a subcutaneous priming injection of the same antigens. Inoculation of Bt antigen without subcutaneous priming injections induced IgE antibody production only when the antigen was continuously administered for a long period of over 24 weeks. Even when the priming injection was absent, the Bt antigen inoculated with cholera toxin (CT) as a mucosal adjuvant also significantly augmented the Bt antigen-specific IgE responses depending on the dose of CT co-administered. The present study also demonstrated that Bt antigen/CT-inoculated mice showed increased non-specific serum IgE level and peripheral blood eosinophil rates without noticeable elevations of the total leukocyte counts. The immunoblot analysis demonstrated 5 main antigenic components reactive to IgE antibodies induced. These components at about 44-64 kDa position were considered to be an important candidate antigen for diagnosis of the mite-related allergy.

Malaria parasite developmental analyses by the nested polymerase chain reaction method: an implication for the evaluation of mosquito infection rates in epidemiological studies.

A malaria mosquito vector, Anopheles saperoi, and a non-vector, Aedes albopictus, were allowed to feed on mice infected with murine malaria, Plasmodium yoelii nigeriensis, and were subsequently monitored for the development of parasites by the nested polymerase chain reaction (PCR) method, using Plasmodium genus-specific primer pairs. The mosquitos were divided into two parts, head/thorax and abdomen, for DNA analyses. The parasite DNA and murine DNA for each mosquito were examined in parallel. In both groups of mosquitos, murine DNA was detected up to 4 days post-blood meal in both the head/thorax and abdomen. After 4 days, the murine DNA fell below detectable limits. Murine DNA and parasite DNA remained undigested for the first 4 days post-blood meal. Parasite DNA was detected in the abdomen of 25% (3/12) of Ae. albopictus on day five and 10% (1/10) on day six, after murine DNA had fallen below detectable limits. Parasite DNA was not detected in the head/thorax of Ae. albopictus on those days or afterwards in either the head/thorax or abdomen, demonstrating that the parasite detected on days 5 and 6 in the abdomen degenerated and did not develop into mature oocysts or sporozoites. In the vector An. saperoi, parasite DNA was detected continuously in the head/thorax and abdomen for many days after the murine DNA had fallen below detectable limits. The detection rate of parasite DNA in the head/thorax of An. saperoi increased gradually from day 8 post blood meal until it reached a maximum level of 71.4% (15/21 12 days post-infection. Parasite DNA in abdomen reached its maximum level of 81% (17/21) 10 days post-blood meal. The implications of these results for the design and interpretation of epidemiological surveys is discussed.

Field application and evaluation of a rapid immunochromatographic test for detection of Plasmodium falciparum infection among the inhabitants of Lao PDR.

Field application and evaluation of a rapid immunochromatographic test (ICT) for detection of Plasmodium falciparum infection were performed in 13 villages in a southern province of Lao PDR in 1999. More than 2,000 inhabitants, accounting for 61.8% of the total estimated population, were examined. Malaria infection was confirmed in all villages surveyed by ICT and microscopic diagnosis. The positive rates of P. falciparum malaria by microscopy ranged from 9.7% to 59.2% (mean 27.2%), whereas by ICT they were from 11.6% to 64.5% (mean 29.8%). The positive rates by ICT were generally higher in 8 out of 13 villages. However, a significant difference between the positive rates by microscopy and ICT was not observed in all villages. Plasmodium falciparum infection was actually confirmed by microscopy in 84.1% of specimens that tested positive by ICT. The results by ICT were consistent with those of the microscopic diagnosis, the discrepancy of the results was less than 10% (141/2,066). The ICT was falsely-positive in 4.7% and falsely-negative in 2.1% of the test cases. These results showed the efficacy of ICT not only in the diagnosis of the respective cases, but also in the mass-examination in the field.

[Two cases of mixed infection of malaria diagnosed by PCR method].

We here reported two Japanese cases of mixed infections of plasmodium species, whose DNAs were detected using the PCR test. One case was a 31 year-old male, who presented fever and fatigue, and had a travel history to Kenya, Cameroon and Indonesia. Smear test of his peripheral blood found the presence of Plasmodium vivax, while nested-PCR diagnosis detected the DNAs both P. vivax and Plasmodium malariae. The other was a 54 year-old female suffering from general fatigue. She had been treated with chloroquine for falciparum malaria in Indonesia two weeks before. Malaria antigen test showed positive although no Plasmodium organisms were found in the smear test. The nested PCR detected the DNA of Plasmodium ovale in addition to that of Plasmodium falciparum. In conclusion, the PCR test is helpful and useful for detection of mixed infections of Plasmodium species.

Association between serum zinc concentration and the Plasmodium falciparum antibody titer among rural villagers of Attapeu Province, Lao People's Democratic Republic.

Experimental studies have indicated that low serum zinc levels affect immune responses. However, few studies have evaluated the impact of serum zinc levels on antibody responses in the field in developing countries. We investigated an association between the anti-Plasmodium falciparum (Pf) antibody (immunoglobulin G) titer and serum zinc concentration among villagers in rural areas of the Lao People's Democratic Republic. Blood samples were collected to detect Pf infection. An enzyme-linked immunosorbent assay (ELISA) was used to measure the anti-PfIgG antibody titer. Each serum sample was assayed to measure the concentration of zinc. Pearson's correlation coefficient was applied to the association between zinc concentration and anti-PfIgG antibody titers. Multiple linear regression analysis was used to assess the association between zinc concentration and anti-PfIgG antibody titers, controlling for age and albumin level. Of 71 blood samples, 40 were Pf positive and 31 were Pf negative. The median serum zinc concentrations were 56.0 µg/dl in the Pf-positive group and 62.5 µg/dl in the Pf-negative group. The median anti-Pf titers were 833.4 in the positive group and 1237.2 in the negative group. Unexpectedly, there was a negative correlation between serum zinc and anti-Pf IgG antibody titers; the correlation coefficient were -0.453 and (p=0.003) in the positive group and -0.461 (p=0.009) in the negative group. The results of this study indicated sustained antibody responses among the villagers, who had likely been exposed to malaria periodically throughout their lives. Further studies are necessary to determine the conditions in which zinc could be effective against malaria.

Imported malaria cases in Okinawa Prefecture, Japan.

With the increase in global transportation, imported malaria has become a significant public health concern in Japan. In the present study, we retrospectively analyzed all imported malaria cases in Okinawa Prefecture from 1988 to 2012. In that period, 23 patients with imported malaria were admitted to the University of the Ryukyus Hospital. Malaria types observed included Plasmodium falciparum (14 cases), P. vivax (7 cases), combined P. falciparum and P. ovale (1 case), and combined P. vivax and P. malariae (1 case). All cases were resolved by anti-malarial treatment. The clinical data from these patients highlights the importance of collecting patient travel history and ensuring an adequate supply of both diagnostic test and drug treatments in Okinawa.

Characterization of a novel proteinous toxin from sea anemone Actineria villosa.

The sea anemone Actineria villosa expresses a lethal protein toxin. We isolated a novel 120-kDa protein, Avt120, from partially purified toxin and found it to possess extremely strong lethal activity. The 3,453-bp Avt120 gene translates to a 995-amino acid protein. The 50% lethal dose (LD(50)) of purified Avt120 in mice was 85.17 ng. Among several tested cell lines, Colo205 cells were most sensitive to Avt120: 50% of them were damaged by 38.4 ng/mL Avt120. Avt120 exerted ATP degradation activity (10 µmol ATP h(-1) mg(-1)), which was strongly inhibited by ganglioside GM1 to decrease the cytotoxicity of Avt120.

Molecular characterization on the genome structure of hemolysin toxin isoforms isolated from sea anemone Actineria villosa and Phyllodiscus semoni.

We recently identified the existence of new isoforms of Avt-I (from sea anemone Actineria villosa) and Pstx20 (from sea anemone Phyllodiscus semoni) hemolytic toxins, and named them Avt-II and Pst-I. Avt-II and Pst-I differ in length by 14 and 7 bp, respectively, as compared to their corresponding isoform genes. Both newly found isoform genes have the coding regions with the identical length of 1033 bp. The restriction fragment length polymorphism analysis with endonuclease HphI was able to clearly distinguish between the two Avt isoforms, but not Pstx isoforms, and based on the densitometric analysis of DNA bands, it indicated that relative expression levels of Avt-I and Avt-II genes were 18.3% and 81.7%, respectively. PCR amplification of the two Avt isoform genes using the genomic DNA as template indicated the existence of two introns within each toxin isoform gene. The first intron with the identical 242 bp in length for both Avt isoform was found within the 5'-untranslated region, and the second intron with lengths of 654 bp and 661 bp in Avt-I and Avt-II isoforms, respectively, was found within the signal sequence coding region. This is for the first time to identify the existence of introns within hemolysin genes of sea anemone. Having several unique characteristics that have identified only for a new member of actinoporin family of A. villosa and P. semoni, e.g., strong toxicity and genes with introns, it is plausible to speculate that these toxins have a unique genetic evolutionary linage differed from that for other sea anemone hemolytic toxins.