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Background Human malaria is a major threat in rural communities of central Vietnam. Anopheles dirus and Anopheles minimus species are critical malaria vectors in Vietnam, which transmit Plasmodium parasites. However, the entomological aspects of malaria transmission in some of the central provinces of Vietnam remain unexplored. Hence, a cross-sectional entomological survey was carried out to identify the malaria vector species and the transmission of Plasmodium parasites in seven endemic provinces of Vietnam. Methods Mosquitoes were collected from seven provinces, Gia Lai, Khanh Hoa, Phu Yen, Ninh Thuan, Binh Thuan, Dong Nai, and Binh Phuoc. The collection was conducted for four to eight consecutive nights using three established methods, indoor and outdoor human landing catches and light trap method. Nested-PCR analysis was performed to detect the Plasmodium species in the separated thorax and the abdomen of the individual mosquitoes. Results A total of 2278 mosquitoes belonging to one of the four species of anopheline mosquitoes, An. dirus, An. maculatus, An. aconitus, and An. minimus were collected. Among the collected mosquitoes, 1398 were analysed using nested-PCR, of which, 40 mosquitoes were positive for Plasmodium parasites. Most of these parasites were detected in the samples from the thorax region, followed by the abdominal portion. The parasites were detected in both the thorax and abdomen of An. dirus. Seven species of Plasmodium parasites were detected during the analysis, of which, Plasmodium inui was the most common species, followed by Plasmodium falciparum, Plasmodium vivax, Plasmodium cynomolgi, Plasmodium coatneyi, Plasmodium knowlesi, and Plasmodium fieldi. Out of the 49 positive samples, 12 showed mixed infections. Co-infection of P. inui with human and other non-human primate Plasmodium species was common. Conclusions This study demonstrated the presence of human and non-human primate Plasmodium infection in An. dirus, a predominant malarial vector. Further, we showed that An. maculatus and An. minimus species also take part in malarial transmission. This might potentially lead to an alarming situation conducive for the emergence of novel zoonotic malaria.

Between July 2009 and June 2014, a total of 1,546 fecal specimens were collected from children

Background Human malaria is a major threat in rural communities of central Vietnam. Anopheles dirus and Anopheles minimus species are critical malaria vectors in Vietnam, which transmit Plasmodium parasites. However, the entomological aspects of malaria transmission in some of the central provinces of Vietnam remain unexplored. Hence, a cross-sectional entomological survey was carried out to identify the malaria vector species and the transmission of Plasmodium parasites in seven endemic provinces of Vietnam. Methods Mosquitoes were collected from seven provinces, Gia Lai, Khanh Hoa, Phu Yen, Ninh Thuan, Binh Thuan, Dong Nai, and Binh Phuoc. The collection was conducted for four to eight consecutive nights using three established methods, indoor and outdoor human landing catches and light trap method. Nested-PCR analysis was performed to detect the Plasmodium species in the separated thorax and the abdomen of the individual mosquitoes. Results A total of 2278 mosquitoes belonging to one of the four species of anopheline mosquitoes, An. dirus, An. maculatus, An. aconitus, and An. minimus were collected. Among the collected mosquitoes, 1398 were analysed using nested-PCR, of which, 40 mosquitoes were positive for Plasmodium parasites. Most of these parasites were detected in the samples from the thorax region, followed by the abdominal portion. The parasites were detected in both the thorax and abdomen of An. dirus. Seven species of Plasmodium parasites were detected during the analysis, of which, Plasmodium inui was the most common species, followed by Plasmodium falciparum, Plasmodium vivax, Plasmodium cynomolgi, Plasmodium coatneyi, Plasmodium knowlesi, and Plasmodium fieldi. Out of the 49 positive samples, 12 showed mixed infections. Co-infection of P. inui with human and other non-human primate Plasmodium species was common. Conclusions This study demonstrated the presence of human and non-human primate Plasmodium infection in An. dirus, a predominant malarial vector. Further, we showed that An. maculatus and An. minimus species also take part in malarial transmission. This might potentially lead to an alarming situation conducive for the emergence of novel zoonotic malaria. Electronic supplementary material The online version of this article (10.1186/s41182-019-0139-8) contains supplementary material, which is available to authorized users.

Background Border malaria in the Greater Mekong region of Southeast Asia poses a serious threat to the health of the ethnic minority populations of the region. Traditionally thought to be caused primarily by the malaria parasites Plasmodium falciparum and Plasmodium vivax, recently a zoonotic parasite, Plasmodium knowlesi, has been identified in some countries of the region. The presence of this parasite poses a challenge to malaria control programmes, as it is maintained in a zoonotic reservoir of forest-dwelling macaque monkeys. Methods A cross-sectional malaria parasite species prevalence survey was conducted along the Laos-Vietnam border in the central part of the two countries. Human blood samples were collected from Savannakhet in Laos and Quang Tri in Vietnam between August and October 2010 and assayed for the presence of human malaria parasite species and P. knowlesi. A PCR targeting the 18S small subunit ribosomal RNA gene and circumsporozoite protein gene was used for Plasmodium species identification. Results Nine cases of P. knowlesi were detected by PCR in blood samples from the Laos side and three from the Vietnam side. All P. knowlesi infections were found in co-infection with P. vivax, with some triple infections of P. knowlesi, P. vivax and P. falciparum detected in Laos. Phylogenetic analysis of these parasites suggests that P. knowlesi is circulating in the Laos-Vietnam border region. Conclusion This report shows that P. knowlesi is transmited on both sides of the Vietnam-Laos border. Continued monitoring of the range and prevalence of P. knowlesi on both the sides of Laos-Vietnam border is of importance to the National Malaria Control Programmes of both countries.

Border malaria in the Greater Mekong region of Southeast Asia poses a serious threat to the health of the ethnic minority populations of the region. Traditionally thought to be caused primarily by the malaria parasitesA cross-sectional malaria parasite species prevalence survey was conducted along the Laos-Vietnam border in the central part of the two countries. Human blood samples were collected from Savannakhet in Laos and Quang Tri in Vietnam between August and October 2010 and assayed for the presence of human malaria parasite species andNine cases ofThis report shows that

Background Plasmodium falciparum has developed resistance against artemisinin in Southeast Asia. Mutations in the P. falciparum Kelch-13 (Pfk13) gene are associated with artemisinin resistance in vitro and in vivo. We investigated the prevalence of mutations in PfK13 from sporozoite-stage parasites isolated from the salivary glands of Anopheles dirus mosquitoes. Methods Mosquitoes were caught by human-landing catches at two locations within the Khanh Phu commune, South-Central Vietnam. Identification of Anopheles species was performed based on morphological features and nucleotide sequence analysis. Sporozoite-infected salivary glands were stored on filter paper and at 4–6 °C. A nested-PCR targeting the small subunit ribosomal RNA gene was used for Plasmodium species identification. Pfk13 was amplified by nested PCR, and subjected to nucleotide sequencing. Results Five of 33 P. falciparum sporozoite samples carried the P553L mutation at the PfK13 locus. This mutation has been recorded previously in Vietnam, but not in Khanh Hoa province, were surveys of K13 polymorphism have not previously been carried out. Conclusion These results demonstrate the utility of mosquito-stage malaria parasite samples for studies on the molecular epidemiology of drug resistance.

G12 rotaviruses are globally emerging rotavirus strains causing severe childhood diarrhea. However, the whole genomes of only a few G12 strains have been fully sequenced and analyzed, of which only one G12P[4] and one G12P[6] are from Africa. In this study, we sequenced and characterized the complete genomes of three G12 strains (RVA/Human-tc/KEN/KDH633/2010/G12P[6], RVA/Human-tc/KEN/KDH651/2010/G12P[8], and RVA/Human-tc/KEN/KDH684/2010/G12P[6]) identified in three stool specimens from children with acute diarrhea in Kenya, Africa. On whole genomic analysis, all three Kenyan G12 strains were found to have a Wa-like genetic backbone: G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 (strains KDH633 and KDH684) and G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 (strain KDH651). Phylogenetic analysis showed that most genes of the three strains examined in this study were genetically related to globally circulating human G1, G9, and G12 strains. Of note is that the NSP4 genes of strains KDH633 and KDH684 appeared to be of porcine origin, suggesting the occurrence of reassortment between human and porcine strains. Furthermore, strains KDH633 and KDH684 were very closely related to each other in all the 11 gene segments, indicating derivation of the two strains from a common origin. On the other hand, strain KDH651 consistently formed distinct clusters of 10 of the 11 gene segments (VP1-2, VP4, VP6-7, and NSP1-5), indicating a distinct origin of strain KDH651 from that of strains KDH633 and KDH684. To our knowledge, this is the first report on whole genome-based characterization of G12 strains that have emerged in Kenya. Our observations will provide important insights into the evolutionary dynamics of emerging G12 rotaviruses in Africa.

Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be "sub-microscopic" blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.

SUMMARYFour species of malaria parasite, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium knowlesi infect humans living in the Khanh Phu commune, Khanh Hoa Province, Vietnam. The latter species also infects wild macaque monkeys in this region. In order to understand the transmission dynamics of the three species, we attempted to detect gametocytes of the three species in the blood of infected individuals, and sporozoites in the salivary glands of mosquitoes from the same region. For the detection of gametocyte-specific mRNA, we targeted region 3 of pfg377, pvs25, pmg and pks25 as indicators of the presence of P. falciparum, P. vivax, P. malariae and P. knowlesi gametocytes, respectively. Gametocyte-specific mRNA was present in 37, 61, 0 and 47% of people infected with P. falciparum (n = 95), P. vivax (n = 69), P. malariae (n = 6) or P. knowlesi (n = 32), respectively. We found that 70% of mosquitoes that had P. knowlesi in their salivary glands also carried human malaria parasites, suggesting that mosquitoes are infected with P. knowlesi from human infections.

Understanding why some malaria-infected individuals are infective to mosquitoes while others are not, is of great importance when considering interventions to stop malaria transmission. Whether gametocytes are produced in every individual infected with Plasmodium falciparum remains unclear. Using a highly sensitive reverse transcription (RT)-PCR assay, we attempted to detect gametocyte-specific mRNA transcripts in isolates from Thai patients which newly adapted to continuous in vitro culture. We then compared the allelic types of the pfg377 gene between patient blood and culture-adapted parasites in order to determine whether the same parasite lines were producing gametocytes in vivo and in vitro. Transcripts of pfg377 were detected in all parasite isolates and in the corresponding cultured isolates, revealing that all patients had gametocytes circulating in their blood at the time of sampling. For isolates in continuous in vitro culture, there was a match between pfg377 allelic types detected by PCR from genomic DNA (and thus indicative of the dominant allelic type of asexual parasites) and those detected by RT-PCR of mRNA (gametocyte-specific), whereas in freshly isolated patient blood there were some differences between the asexual parasite allelic type and that of the gametocytes in the same infection. Seven isolates contained asexual stage parasites harbouring pfg377 alleles that were not detectable in gametocytes from the same infections, suggesting that some clones were not producing gametocytes at the time of sampling, or that they were below the level of detection., International Journal for Parasitology, 41(3-4), pp.317-323; 2011

The feasibility of identifying parasite DNA and specific mRNAs from wild-caught Anopheles dirus mosquitoes was assessed using dried mosquito salivary glands preserved on filter paper. We were able to detect Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium knowlesi DNA by conventional PCR and, furthermore, detected P. falciparum gametocyte-specific genes, pfg377 and pfs16 mRNA, P. knowlesi circumsporozoite protein (CSP) and sporozoite surface protein 2 (SSP2) mRNA by reverse transcription-PCR. Using this technique, we were able to confirm the presence of P. vivax, P. falciparum and P. knowlesi in one particular wild-caught mosquito. These results indicate that P. knowlesi may be transmitted by the primary human malaria vector in forested areas in Vietnam. This study also shows that the preservation of mosquito salivary glands on filter paper, and the down-stream extraction of parasite DNA and RNA from those, offers a powerful resource for molecular epidemiological studies on malaria., International journal for parasitology, 39(14), pp.1533-1537; 2009

Background Recent studies have described natural human infections of the non-human primate parasites Plasmodium knowlesi and Plasmodium cynomolgi. In Southeast Asia, mosquitoes of the Anopheles leucosphyrus group bite both humans and monkeys in the forest and thus offer a possible route for Plasmodium species to bridge the species barrier. In this study we analysed the species composition of malarial sporozoites infecting the salivary glands of Anopheles dirus in order to determine their potential role as bridge vectors of Plasmodium parasites from monkeys to humans. Methods Mosquitoes were collected in the forest and forest fringe area of Khanh Phu commune by human-baited landing collection. Anopheles species were determined on the basis of morphologic features. Sporozoite-infected salivary glands were applied to filter paper and dried in an ambient atmosphere, before storage in closed vials at 4–6 °C. Detection and identification of Plasmodium species in salivary glands were carried out by nested-PCR of the small subunit ribosomal RNA gene. Results Six species of Plasmodium parasites were detected by PCR, of which P. vivax was the most common, followed by P. knowlesi, P. inui, P. cynomolgi, P. coatneyi and P. falciparum. Twenty-six of the 79 sporozoite infected mosquitoes showed multiple infections, most of which were a combination of P. vivax with one or more of the non-human primate Plasmodium species. Conclusions These results suggest that humans overnighting in this forest are frequently inoculated with both human and non-human primate malaria parasites, leading to a situation conducive for the emergence of novel zoonotic malaria. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-0995-y) contains supplementary material, which is available to authorized users.

Osteopontin (OPN) knockout mice (OPN-KO mice) died ofPlasmodium chabaudi chabaudiinfection, although wild-type (WT) mice had self-limiting infections. OPN was detected in the WT mice at 2 days postinfection. OPN-KO mice produced significantly smaller amounts of interleukin-12 and gamma interferon than WT mice produced. These results suggested that OPN is involved in Th1-mediated immunity against malaria infection.

We examined the utility of dried blood on filter paper for the source of cytokine messenger RNA (mRNA). Total RNA was isolated from the dried blood of mice infected with Plasmodium yoelii, and cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). As a reference, we extracted total RNA from peripheral blood collected at the same time as the preparation for dried blood. There was no difference in cytokine mRNA expression between the two sources; the dried blood on filter paper and the peripheral blood. Th1 cells, Th2 cells, and monocytes/macrophages derived cytokine mRNAs in the dried blood from infected mice were detected, and the increase of some of the cytokines mRNAs after infection was also observed. These results suggested that the dried blood on filter paper is satisfactory RNA source for immunological examination in field-based studies.

Results: Urinary PkDNA was detected on day 2, but was not amplified using DNA templates extracted from the samples on day 4, day 5 and day 6. Subsequently, urinary PkDNA was detected from day 7 until day 11, and from day 20 until day 30. PkDNA in faeces was detected from day 7 until day 11, and from day 20 until day 37. Moreover, real-time quantitative PCR showed a remarkable increase in the amount of urinary PkDNA following anti-malarial treatment. This might have been due to the release of a large amount of PkDNA from the degraded parasites as a result of the anti-malarial treatment, leading to excretion of PkDNA in the urine. Methods. Urine and faeces were obtained from a Plasmodium knowlesi infected-Japanese macaque (Macaca fuscata) over the course of an experimentally induced infection. P. knowlesi DNA (PkDNA) extracted from urine and faeces were monitored by nested PCR targeting the P. knowlesi specific cytochrome b (cytb) gene. Background: Diagnostic techniques based on PCR for the detection of Plasmodium DNA can be highly sensitive and specific. The vast majority of these techniques rely, however, on the invasive sampling of blood from infected hosts. There is, currently, considerable interest in the possibility of using body fluids other than blood as sources of parasite DNA for PCR diagnosis. Conclusions: The cytb-PCR system using urine and faecal samples is of potential use in molecular epidemiological surveys of malaria. In particular, monkey faecal samples could be useful for the detection of zoonotic primate malaria in its natural hosts., Malaria Journal, 13(1), 373; 2014

Postmortem brain tissues of 21 cerebral malaria cases were obtained in Myanmar and Vietnam. The tissues were examined by light microscopy and by an immunohistochemical method. Brain microvessels (capillaries and venules) were examined for the presence of immunoglobulins IgE and IgG, Plasmodium falciparum antigen, and parasitized erythrocytes (PRBC). Deposition of IgE, IgG, and P. falciparum antigen was observed in the microvessels from all specimens examined. Sequestered PRBC in the microvessels were positive for IgG in all 21 cases and for IgE in six cases. In the latter cases, the percentage of microvessels with sequestered PRBC was > 50%, with the frequency of IgE-positive cells ranging from 42% to 52%. In contrast, in five cases that were only weakly positive for IgE, the percentage of microvessels with sequestered PRBC was remarkably low (< 1%). These data indicate that the degree of deposition of IgE in microvessels and on PRBC from cerebral malaria patients correlated with that of PRBC sequestration. As IgE-containing immune complexes are known to induce local overproduction of tumor necrosis factor-alpha (TNF-alpha), a major pathogenic factor in cerebral malaria, IgE may contribute to the pathogenesis of this severe disease.

An unusual rotavirus strain, Ryukyu-1120, with G5P[6] genotypes (RVA/Human-wt/JPN/Ryukyu-1120/2011/G5P[6]) was identified in a stool specimen from a hospitalized child aged 4 years who showed diarrhoea and encephalopathy. In this study, we sequenced and characterized the complete genome of strain Ryukyu-1120. On whole genomic analysis, this strain was found to have a unique genotype constellation: G5-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1. The VP6 and NSP1 genotypes I5 and A8 are those commonly found in porcine strains. Furthermore, phylogenetic analysis indicated that each of the 11 genes of strain Ryukyu-1120 appeared to be of porcine origin. Thus, strain Ryukyu-1120 was found to have a porcine rotavirus genetic backbone and is likely to be of porcine origin. To our knowledge, this is the first report of whole-genome-based characterization of the emerging G5P[6] strains in Asian countries. Our observations will provide important insights into the origin of G5P[6] strains and the dynamic interactions between human and porcine rotavirus strains.

Ultrastructural localization of CD36 in human hepatic sinusoidal lining cells, hepatocytes, human hepatoma (HepG2-A16) cells, and C32 amelanotic melanoma cells. Experimental Parasitology 79, 383-390. CD36 is expressed in the endothelial cells of some human organs, but the ultrastructural localization of this molecule in the sinusoidal lining cells of human liver is not well established. We report the ultrastructural localization of CD36 in the liver using a novel murine monoclonal antibody against CD36, namely MO30, as a primary antibody. Immunocytochemistry by the postembedding method showed that CD36 was localized in endothelial cells of sinusoids and in hepatocyte microvilli protruding into the space of Disse. Moreover, in cultured human hepatoma (HepG2-A16) cells and C32 amelanotic melanoma cells, MO30 reacted with microvilli. Hence, CD36 expressed on these cells may be involved in recognition and/or entry of these cells by malaria sporozoites.

The number and distribution of γδ T cells in spleens from patients who died of cerebral malaria and from rhesus monkeys severely infected with Plasmodium coatneyi were examined by immunocytochemistry. γδ T cells were significantly increased in these spleens. In a rodent malaria model using Plasmodium chabaudi adami, an avirulent strain of murine malaria parasites, the degree of parasitemia appears to be modulated by the number of γδ T cells in the spleen. As parasitemia increases, these T cells increase in number. At some critical point, γδ T cells in collaboration with macrophages and αβ T cells apparently start to clear parasitized erythrocytes from the blood, leading to an abatement of the parasitemia, which is followed by a reduction in the number of γδ T cells. This γδ T cell phenomenon may be responsible for the self-limiting infection in mice.

We studied the brains of rhesus monkeys infected with the primate malaria parasite Plasmodium fragile. Electron microscopy showed that, in these animals, erythrocytes infected with P. fragile undergo sequestration and that parasitized red blood cells adhere to endothelial cells in the cerebral microvessels by means of knobs. Cerebral microvessels with sequestered parasitized red blood cells were shown by immunohistochemical analysis to possess the platelet glycoprotein CD36, thrombospondin, and intracellular adhesion molecule-1. The formation of rosettes also was observed in the cerebral microvessels. In a fashion similar to human cerebral malaria, P. fragile produced neurological symptoms in the animals. Thus, rhesus monkeys infected with P. fragile, like those monkeys infected with Plasmodium coatneyi, can be used as a primate model to study human cerebral malaria.

A 2.5-year-old girl died suddenly during the course of rotavirus gastroenteritis. The autopsy showed encephalopathy with rotavirus systemic infection. Here, we provide evidence of rotavirus replication in multiple organs. Our findings clarify that rotavirus infection in children can extend beyond the intestinal tract through viremia.

The infectivity of rotavirus (RV) is dependent on an activation process triggered by the proteolytic cleavage of its spike protein VP4. This activation cleavage is performed by exogenous trypsin in the lumen of the intestinesin vivo. Here, we report the generation and characterization of a recombinant RV expressing cDNA-derived VP4 with a modified cleavage site (arginine at position 247) recognized by endogenous furin as well as exogenous trypsin. Unexpectedly, the mutant virus (KU//rVP4-R247Furin) was incapable of plaque formation without an exogenous protease, although the mutant VP4s on virions were efficiently cleaved by endogenous furin. Furthermore, KU//rVP4-R247Furin showed impaired infectivity in MA104 and CV-1 cells even in the presence of trypsin compared with the parental virus carrying authentic VP4 (KU//rVP4). Although the total titre of KU//rVP4-R247Furin was comparable to that of KU//rVP4, the extracellular titre of KU//rVP4-R247Furin was markedly lower than its cell-associated titre in comparison with that of KU//rVP4. In contrast, the two viruses showed similar growth in a furin-defective LoVo cell line. These results suggest that intracellular cleavage of VP4 by furin may be disadvantageous for RV infectivity, possibly due to an inefficient virus release process.

Ultrastructural changes induced in Plasmodium falciparum by artemisinin were studied in vitro. Electron microscopic autoradiography was performed on infected erythrocytes that were exposed in vitro to 3H-dihydroartemisinin and 14C-artemisinin. These drugs consistently were located in food vacuoles and mitochondria. Two hours after administration, changes were observed in parasite mitochondria, rough endoplasmic reticulum, and nuclear envelope. At four hours, in addition to the earlier changes, nuclear membranes and, to a lesser extent, some plasma membranes formed myelin figures. In addition, there was a disappearance of ribosomes, and a destruction of food vacuole membranes. These changes may lead to the total disorganization of the parasites. Approximately 30% of the parasites manifested these alterations.

We studied the role of osteopontin (OPN) in host responses against rotavirus (RV) infection. OPN knockout (OPN-KO) suckling mice were more susceptible to RV (strain EW) infection and showed prolonged diarrhea duration compared to wild-type (WT) suckling mice. OPN in the small intestine of WT mice was expressed after 48 h post-infection. On day 2 postinfection, mRNA levels of interleukin-1β, tumor necrosis factor-α, and interleukin-15 in OPN-KO mice were lower than in WT mice, although mRNA expression of Th-1- and Th-2-related cytokines in the small intestine were nearly the same between OPN-KO and WT mice. These results suggested that OPN is involved in innate responses against RV infection.

The detection of gametocytes in human peripheral blood is one of the most important measures in a malaria survey. We attempted to detect gametocytes of Plasmodium falciparum by reverse transcription polymerase chain reaction (RT-PCR) of dried blood on filter paper. On field samples analysis, the specific RT-PCR products for region 3 of pfg377 mRNA were observed in 67 of 131 falciparum malaria patients. The minimum detection level of RT-PCR-positive samples was 0.03 gametocytes/microl on quantitative real-time RT-PCR. Gametocyte positive rate was not dependent on sex or age. A higher frequency of gametocytes was found in single P. falciparum infection than in mixed species infection (P

We examined the role of osteopontin (OPN) in immunity against Plasmodium falciparum infection. We measured the mRNA levels for OPN and several cytokines in RNA preparations extracted from dried blood on filter paper obtained from falciparum malaria patients in Vietnam. Expression of OPN mRNA was detected in 134 of 161 patients. The expression of both interleukin-12 p40 and interferon-gamma mRNAs in the group positive for OPN mRNA was significantly higher than that in the group negative for OPN mRNA. The level of parasitemia in the OPN mRNA-positive group was much lower than that in the negative one. These results suggest that OPN might suppress multiplication of the parasites through T helper 1 cells-mediated immune responses.

Aichi virus is the type species of a new genus, Kobuvirus , of the family Picornaviridae . In this study, we constructed a full-length cDNA clone of Aichi virus whose in vitro transcripts were infectious to Vero cells. During construction of the infectious cDNA clone, a novel sequence of 32 nucleotides was identified at the 5′ end of the genome. Computer-assisted prediction of the secondary structure of the 5′ end of the genome, including the novel sequence, suggested the formation of a stable stem-loop structure consisting of 42 nucleotides. The function of this stem-loop in virus replication was investigated using various site-directed mutants derived from the infectious cDNA clone. Our data indicated that correct folding of the stem-loop at the 5′ end of the positive strand, but not at the 3′ end of the negative strand, is critical for viral RNA replication. The primary sequence in the lower part of the stem was also suggested to be crucial for RNA replication. In contrast, nucleotide changes in the loop segment did not so severely reduce the efficiency of virus replication. A double mutant, in which both nucleotide stretches of the middle part of the stem were replaced by their complementary nucleotides, had efficient RNA replication and translation abilities but was unable to produce viruses. These results indicate that the stem-loop at the 5′ end of the Aichi virus genome is an element involved in both viral RNA replication and production of infectious virus particles.

Circulating immune complex (CIC) is known to play a role in pathological glomerular alterations in malaria. However, the nature of the antigens comprising the CIC is still not fully understood. We report here the isolation of the antigen in CIC and its localisation in mice infected with Plasmodium berghei NK65. The antigen was successfully isolated from CIC extracted from the blood of mice infected with P. berghei, by using C1q-coated microplates. The molecular mass of the antigen separated from CIC bound to C1q was found to be 78 kDa. Furthermore, localisation of the antigen was examined by the fluorescent antibody technique and immunoelectron microscopy. The antigen was detected in the parasitised erythrocyte and the mesangial matrix by both methods. These results suggest that the 78 kDa protein might be associated with the glomerular alterations in malaria infection.

Schistosomiasis haematobia is a parasitic disease caused by the infection with Schistosoma haematobium (S. haematobium) prevalent mainly in Africa and western Asia1), and is characterized by dysuria, pollakisuria, and terminal hematuria with an insidious onset. Schistosomiasis haematobia is not endemic to Japan. However, with increasing number of Japanese traveling or staying in tropical and subtropical areas where S. haematobium is endemic, a few Japanese patients have been reported in the 1990's2). Trichinellosis is a disease caused by parasites of the genus Trichinella that humans acquire from eating the muscles of several kinds of wild animal or domestic pigs. Trichinellosis is one of the most common parasitic zoonoses in the world, and may be found in the USA, Canada, and eastern Europe3). In Japan, there have been some reports of outbreaks of trichinellosis associated with the ingestion of raw bear meat4) and there is a case report of a patient infected with Trichinella from eating raw pork in Thailand5). The disease is characterized by fever, myositis, gastrointestinal symptoms, eosinophilia, and swollen eyelids. We report here the first Japanese case to be coinfected with S. haematobium and Trichinella sp..

We studied the effects of artesunate on rhesus monkeys infected with Plasmodium coatneyi. Sixteen rhesus monkeys were divided in four groups. Group I consisted of three monkeys that were splenectomized and were treated with three doses (loading dose: 3.3 mg/kg, maintenance doses: 1.7 mg/kg) of artesunate, group II consisted of three monkeys that were treated with three doses of artesunate (same as group I), group III consisted of two monkeys that were treated with one dose (3.3 mg/kg) of artesunate, and group IV consisted of five untreated monkeys. Parasitemias of these groups ranged from 13.3% to 19.5% before treatment. Twenty-four hours after administration, the parasitemia was reduced to 2.2% in group I and to < 0.1% in group II; parasitemia was lowered to 10.6% in group III only 3 hr after drug administration. The rate of sequestration in the cerebral microvessels, which was 29.4% in untreated animals, was < 0.1% in groups I and II (24 hr after treatment), and 2.0% in group III (3 hr after treatment). These data clearly indicate that artesunate not only reduced parasitemia, but also reduced the rate of parasitized red blood cell (PRBC) sequestration in cerebral microvessels. In an immunohistologic study, endothelial-leukocyte adhesion molecule-1 (ELAM-1) was not detected in group I after treatment with artesunate, although the presence of CD36, thrombospondin, intercellular adhesion molecule-1, IgG, and C3 in the cerebral microvessels was not altered. This is the first in vivo study to show that artesunate interferes with continued PRBC sequestration in the cerebral microvessels in cerebral malaria.(ABSTRACT TRUNCATED AT 250 WORDS)

Term placentas from 35 patients infected with Plasmodium falciparum were obtained in Malawi in southeast Africa and six term placentas from patients infected with P. falciparum were obtained in Wewak, Papua New Guinea, Melanesia. The placental tissues were examined by light microscopy and by an immunohistologic method to compare the pathologic changes of placentas in the two malaria-endemic countries. Using the number of parasitized red blood cells (PRBC) in intervillous spaces, pregnant women from Malawi with placental parasitemia were categorized into three groups. In the high PRBC group (> 20%, group I), there was no deposition of IgE in fetal blood vessels. In contrast, IgE was observed in fetal blood vessels of the intermediate PRBC group (1–10%, group II) and low PRBC group (< 1%, group III). In all six placentas from Papua New Guinean women, deposition of immune complexes, including IgE, was observed in the fetal blood vessels. All placentas with deposition of IgE in fetal blood vessels showed no sequestration of malaria parasites in intervillous spaces. Our data indicate that the amount of deposition of IgE in the placenta from women infected with P. falciparum is inversely correlated with the degree of parasitemia at that site.

Six rhesus monkeys (Macaca mulatta) infected with Plasmodium coatneyi were studied for parasitized red blood cell (PRBC) sequestration in microvessels of the brain. The degree of PRBC sequestration is different in the cerebral, mid-brain, and cerebellar microvessels, with sequestration occurring preferentially in the cerebellum. This pattern resembles that of PRBC sequestration in cerebral and cerebellar microvessels in human falciparum malaria. The morphologic appearance of sequestered cells under light and electron microscopy as well as the PRBC sequestration pattern bolsters the contention that the rhesus monkey infected with P. coatneyi is an appropriate primate model for the experimental study of human cerebral malaria.

Sequestration of parasitized red blood cells (PRBC) in the microvessels results in impairment of microcirculation with organ dysfunction in complicated human Plasmodium falciparum malaria. In cerebral malaria patients, the percentage of small blood vessels with PRBC sequestration is higher in the brain than in other organs. The clinical severity of cerebral malaria depends on the level of PRBC sequestration in the brain. In our study, postmortem samples from cerebrum and cerebellum of 16 patients who died of P. falciparum malaria were examined and compared using light microscopy. In the cerebellum, the percentage of microvessels with PRBC sequestration was higher than that in the cerebrum. The difference in sequestration rates between cerebrum and cerebellum is statistically significant (P < 0.05). There is a higher degree of vascularity in the cerebellum (7 vessels/mm2) than in the cerebrum (5 vessels/mm2), and the difference is also statistically significant (P < 0.025). Perivascular hemorrhages also occur more frequently in the cerebellum than in the cerebrum. The results of this study, which show that differential sequestration of PRBC occurs in the microvessels of the cerebrum and cerebellum, explain the varied neurologic manifestations that result from cerebral and cerebellar dysfunction in human cerebral malaria. This study also reveals the necessity of postmortem histologic examination of the cerebellum in every suspected case of cerebral malaria.

Systemic Lupus Erythematosus (SLE) is one of the chronic autoimmune diseases with various inflammatory manifestations involving multiple organ systems, accompanied by multiple immunological abnormalities. The Preliminary Criteria for the Classification of SLE by American Rheumatism Association (ARA) had been widely used for the diagnosis of patients with SLE since 1971.The criteria was newly revised by ARA in 1982. The sensitivity and the specificity of the 1982 Revised Criteria was evaluated in our patients with connective tissue diseases. The sensitivity for SLE (78 cases) was 94.9%, and the specificities against PSS (17 cases), PM (10 cases) or RA (19 cases) were all 100%.The sensitivity for the diagnosis at initial visits in patients with SLE was also 78.2% and the 1982 Criteria was considered to be quite useful for early diagnosis of SLE.The results indicated that the 1982 Criteria could be more useful in diagnosis of SLE than the 1971 Criteria.

Collagenase-like(CL) peptidase activity in serum, which was measured using a newly synthesized substrate, (succinyl-Gly-Pro-Leu-Gly-Pro)-4-methylcoumaryl-7-amide, was significantly lower in patients with advanced rheumatoid arthritis or with systemic lupus erythematosus than that in normal controls. Decrease of the serum enzyme activity was more pronounced in systemic lupus erythematosus. No significant change in serum CL-peptidase activity was found in other connective tissue diseases such as mixed connective tissue disease and Sjogren's syndrome.

Pancreozymin-Secretin test (PS test) was conducted for 25 cases of Sjorgen's syndrome (SjS) for the purpose of studying pancreatic lesions in SjS. Pancreatic juice was collected for 60 minutes after intravenous injection of secretion, and concentrations of β2-microglobulin (β2-m) and Lactoferin (LF) in pancreatic juice were determined by Radioimmunoassay.Additionally, continuous determination of urine amylase was made for the same cases, and the usefulness of this method as a screening test for pancreatic lesions was studied.As a result, only one case (4%) showed a definite decline in the pancreatic secretory function in the PS test. However, the β2-m level in pancreatic juice was high in 10 (45%) out of 22 cases and the LF level was likewise high in 8 (36%) out of 22 cases, the difference being significant compared with the normal control group (p