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Malaria remains a public health problem in Thailand, especially along its borders where highly mobile populations can contribute to persistent transmission. This study aimed to determine resistant genotypes and phenotypes of 112 Plasmodium falciparum isolates from patients along the Thai-Cambodia border during 2013-2015. The majority of parasites harbored pfmdr1-Y184F mutation. A single pfmdr1 copy number, had CVIET haplotype of amino acids 72-76 of pfcrt and no pfcytb mutations. All isolates had a pfk13 point mutation (R539T, R539I, and C580Y), and increased ring-stage survival assay (except R539I). Multiple copies of pfpm2 and pfcrt-F145I were first detected in 2014 (12.8%) and increased to 30.4% in 2015. Parasites containing either multiple pfpm2 copies with and without pfcrt-F145I or a single pfpm2 copy with pfcrt-F145I exhibited the elevated IC90 values of piperaquine. Antimalarials were detected in 21 samples (18.8%) by mass spectrometer, and 12 samples (10.7%) by bioassay. Collectively, the emergence of these resistance patterns mirrored the reports of dihydroartemisinin-piperaquine treatment failures in the adjacent province of Cambodia, Oddar Meanchey, suggesting a migration of parasites across the border. As malaria elimination efforts ramp up in Southeast Asia, host nations militaries and other groups in border regions must be a focus for interventions

Background While intensive Plasmodium falciparum multidrug resistance surveillance continues in Cambodia, relatively little is known about Plasmodium vivax drug resistance in Cambodia or elsewhere. To investigate P. vivax anti-malarial susceptibility in Cambodia, 76 fresh P. vivax isolates collected from Oddar Meanchey (northern Cambodia) in 2013–2015 were assessed for ex vivo drug susceptibility using the microscopy-based schizont maturation test (SMT) and a Plasmodium pan-species lactate dehydrogenase (pLDH) ELISA. P. vivax multidrug resistance gene 1 (pvmdr1) mutations, and copy number were analysed in a subset of isolates. Results Ex vivo testing was interpretable in 80% of isolates using the pLDH-ELISA, but only 25% with the SMT. Plasmodium vivax drug susceptibility by pLDH-ELISA was directly compared with 58 P. falciparum isolates collected from the same locations in 2013–4, tested by histidine-rich protein-2 ELISA. Median pLDH-ELISA IC50 of P. vivax isolates was significantly lower for dihydroartemisinin (3.4 vs 6.3 nM), artesunate (3.2 vs 5.7 nM), and chloroquine (22.1 vs 103.8 nM) than P. falciparum but higher for mefloquine (92 vs 66 nM). There were not significant differences for lumefantrine or doxycycline. Both P. vivax and P. falciparum had comparable median piperaquine IC50 (106.5 vs 123.8 nM), but some P. falciparum isolates were able to grow in much higher concentrations above the normal standard range used, attaining up to 100-fold greater IC50s than P. vivax. A high percentage of P. vivax isolates had pvmdr1 Y976F (78%) and F1076L (83%) mutations but none had pvmdr1 amplification. Conclusion The findings of high P. vivax IC50 to mefloquine and piperaquine, but not chloroquine, suggest significant drug pressure from drugs used to treat multidrug resistant P. falciparum in Cambodia. Plasmodium vivax isolates are frequently exposed to mefloquine and piperaquine due to mixed infections and the long elimination half-life of these drugs. Difficulty distinguishing infection due to relapsing hypnozoites versus blood-stage recrudescence complicates clinical detection of P. vivax resistance, while well-validated molecular markers of chloroquine resistance remain elusive. The pLDH assay may be a useful adjunctive tool for monitoring for emerging drug resistance, though more thorough validation is needed. Given high grade clinical chloroquine resistance observed recently in neighbouring countries, low chloroquine IC50 values seen here should not be interpreted as susceptibility in the absence of clinical data. Incorporating pLDH monitoring with therapeutic efficacy studies for individuals with P. vivax will help to further validate this field-expedient method. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-2034-2) contains supplementary material, which is available to authorized users.

We studied cross-reactive antibodies against avian influenza H5N1 and 2009 pandemic (p) H1N1 in 200 serum samples from US military personnel collected before the H1N1 pandemic. Assays used to measure antibodies against viral proteins involved in protection included a hemagglutination inhibition (HI) assay and a neuraminidase inhibition (NI) assay. Viral neutralization by antibodies against avian influenza H5N1 and 2009 pH1N1 was assessed by influenza (H5) pseudotyped lentiviral particle-based and H1N1 microneutralization assays. Some US military personnel had cross-neutralizing antibodies against H5N1 (14%) and 2009 pH1N1 (16.5%). The odds of having cross-neutralizing antibodies against 2009 pH1N1 were 4.4 times higher in subjects receiving more than five inactivated whole influenza virus vaccinations than those subjects with no record of vaccination. Although unclear if the result of prior vaccination or disease exposure, these pre-existing antibodies may prevent or reduce disease severity.

Adenovirus (ADV) types 4 (ADV-4) and 7 (ADV-7) are presently the major cause of febrile acute respiratory disease (ARD) in U.S. military recruits. We conducted a multi-center, randomized, double-blind, placebo-controlled phase 3 study of the new vaccine to assess its safety and efficacy. Healthy adults at two basic training sites were randomly assigned to receive either vaccine (two enteric-coated tablets consisting of no less than 4.5 log10 TCID50 of live ADV-4 or ADV-7) or placebo in a 3:1 ratio. Volunteers were observed throughout the approximate eight weeks of their basic training and also returned for four scheduled visits. The primary endpoints were prevention of febrile ARD due to ADV-4 and seroconversion of neutralizing serum antibodies to ADV-7, which was not expected to circulate in the study population during the course of the trial. A total of 4151 volunteers were enrolled and 4040 (97%) were randomized and included in the primary analysis (110 were removed prior to randomization and one was removed after randomization due to inability to swallow tablets). A total of 49 ADV-4 febrile ARD cases were identified with 48 in the placebo group and 1 in the vaccine group (attack rates of 4.76% and 0.03%, respectively). Vaccine efficacy was 99.3% (95% CI, 96.0-99.9; P

Background The recent dramatic decline in dihydroartemisinin-piperaquine (DHA-PPQ) efficacy in northwestern Cambodia has raised concerns about the rapid spread of piperaquine resistance just as DHA-PPQ is being introduced as first-line therapy in neighbouring countries. Methods Ex vivo parasite susceptibilities were tracked to determine the rate of progression of DHA, PPQ and mefloquine (MQ) resistance from sentinel sites on the Thai–Cambodian and Thai–Myanmar borders from 2010 to 2015. Immediate ex vivo (IEV) histidine-rich protein 2 (HRP-2) assays were used on fresh patient Plasmodium falciparum isolates to determine drug susceptibility profiles. Results IEV HRP-2 assays detected the precipitous emergence of PPQ resistance in Cambodia beginning in 2013 when 40 % of isolates had an IC90 greater than the upper limit of prior years, and this rate doubled to 80 % by 2015. In contrast, Thai–Myanmar isolates from 2013 to 14 remained PPQ-sensitive, while northeastern Thai isolates appeared to have an intermediate resistance profile. The opposite trend was observed for MQ where Cambodian isolates appeared to have a modest increase in overall sensitivity during the same period, with IC50 declining to median levels comparable to those found in Thailand. A significant association between increased PPQ IC50 and IC90 among Cambodian isolates with DHA-PPQ treatment failure was observed. Nearly all Cambodian and Thai isolates were deemed artemisinin resistant with a >1 % survival rate for DHA in the ring-stage assay (RSA), though there was no correlation among isolates to indicate cross-resistance between PPQ and artemisinins. Conclusions Clinical DHA-PPQ failures appear to be associated with declines in the long-acting partner drug PPQ, though sensitivity appears to remain largely intact for now in western Thailand. Rapid progression of PPQ resistance associated with DHA-PPQ treatment failures in northern Cambodia limits drugs of choice in this region, and urgently requires alternative therapy. The temporary re-introduction of artesunate AS-MQ is the current response to PPQ resistance in this area, due to inverse MQ and PPQ resistance patterns. This will require careful monitoring for re-emergence of MQ resistance, and possible simultaneous resistance to all three drugs (AS, MQ and PPQ). Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1569-y) contains supplementary material, which is available to authorized users.

In addition to evidence for a protective role of antibodies to the malaria blood stage antigen merozoite surface protein 1 (MSP1), MSP1 antibodies are also considered as a marker of past malaria exposure in sero-epidemiological studies. In order to better assess the potential use of MSP1 serology in malaria chemoprophylaxis trials in endemic areas, an analysis for the prevalence of antibodies to both Plasmodium falciparum and Plasmodium vivax MSP142 in healthy Cambodian adults was conducted at two sites as part of an active, observational cohort evaluating the efficacy of dihydroartemisinin-piperaquine (DP) for uncomplicated malaria ( ClinicalTrials.gov identifier NCT01280162). Rates of baseline sero-positivity were high (59 and 73 % for PfMSP142 and PvMSP142, respectively), and titers higher in those who lived in a higher transmission area, although there was little correlation in titers between the two species. Those volunteers who subsequently went on to develop malaria had higher baseline MSP142 titers than those who did not for both species. Titers to both antigens remained largely stable over the course of the 4–6 month study, except in those infected with P. falciparum who had multiple recurrences. These findings illuminate the difficulties in using MSP142 serology as either a screening criterion and/or biomarker of exposure in chemoprophylaxis studies. Further work remains to identify useful markers of malarial infection and/or immunity.

The dichloromethane/methanol (1:1) extracts of the roots of Pentas longiflora and Pentas lanceolata showed low micromolar (IC(50) = 0.9-3 µg/mL) IN VITRO antiplasmodial activity against chloroquine-resistant (W2) and chloroquine-sensitive (D6) strains of PLASMODIUM FALCIPARUM. Chromatographic separation of the extract of PENTAS LONGIFLORA led to the isolation of the pyranonaphthoquinones pentalongin (1) and psychorubrin (2) with IC(50) values below 1 µg/mL and the naphthalene derivative mollugin (3), which showed marginal activity. Similar treatment of Pentas lanceolata led to the isolation of eight anthraquinones ( 4-11, IC(50) = 5-31 µg/mL) of which one is new (5,6-dihydroxydamnacanthol, 11), while three--nordamnacanthal (7), lucidin-ω-methyl ether (9), and damnacanthol (10)--are reported here for the first time from the genus Pentas. The compounds were identified by NMR and mass spectroscopic techniques.

In vitro drug sensitivity and molecular analyses of Plasmodium falciparum track drug resistance. DNA-binding fluorescent dyes like SYBR Green I may allow field laboratories, proximal to P. falciparum collection sites, to conduct drug assays. In 2007-2008, we assayed 121 P. falciparum field isolates from western Kenya for 50% inhibitory concentrations (IC(50)) against 6 antimalarial drugs using a SYBR Green I in vitro assay: 91 immediate ex vivo (IEV) and 30 culture-adapted, along with P. falciparum reference clones D6 (chloroquine [CQ] sensitive) and W2 (CQ resistant). We also assessed P. falciparum mdr1 (Pfmdr1) copy number and single nucleotide polymorphisms (SNPs) at four codons. The IC(50)s for IEV and culture-adapted P. falciparum isolates were similar, and approximated historical IC(50)s. For Pfmdr1, mean copy number was 1, with SNPs common at codons 86 and 184. The SYBR Green I assay adapted well to our field-based laboratory, for both IEV and culture-adapted P. falciparum, warranting continued use.

From April 2005 to April 2006, a phase 2 malaria vaccine trial in Kenya enrolled 400 children aged 12–47 months. Each received mixed supervised and unsupervised artemether-lumefantrine for uncomplicated malaria, using a standard six-dose regimen, by weight. Children were followed for detection of parasitemia and clinical malaria. A median of two negative malaria blood films occurred during every recurrent parasitemia (RP) episode, suggesting reinfection over late recrudescence. Median time to RP after starting artemether-lumefantrine was 37 days (36–38). Of 2,020 evaluable artemether-lumefantrine treatments, there were no RPs in 99% by day 14, 71% by day 28, and 41% by day 42. By World Health Organization standards, 71% of treatment courses had adequate responses. Although recrudescence in some cannot be ruled out, our cohort had a shorter median time to RP compared with other artemether-lumefantrine treatment studies. This underscores patient counseling on completing all treatment doses for optimal protection from RP.

Plasmodium falciparum Liver Stage Antigen 1 (LSA-1) is a pre-erythrocytic stage antigen. Our LSA-1 vaccine candidate is a recombinant protein with full-length C- and N-terminal flanking domains and two of the 17 amino acid repeats from the central repeat region termed "LSA-NRC." We describe the first Phase I/II study of this recombinant LSA-NRC protein formulated with either the AS01 or AS02 adjuvant system. We conducted an open-label Phase I/II study. Thirty-six healthy malaria-naïve adults received one of four formulations by intra-deltoid injection on a 0 and 1 month schedule; low dose (LD) LSA-NRC/AS01:10microg LSA-NRC/0.5ml AS01 (n=5), high dose (HD) LSA-NRC/AS01: 50microg LSA-NRC/0.5ml AS01 (n=13); LD LSA-NRC/AS02: 10microg LSA-NRC/0.5ml AS02 (n=5) and HD LSA-NRC/AS02: 50microg LSA-NRC/0.5ml AS02 (n=13). Two weeks post-second immunization, the high dose vaccinees and 6 non-immunized infectivity controls underwent experimental malaria sporozoite challenge. The vaccines showed a reassuring safety profile but were moderately reactogenic. There were no serious adverse events. All subjects seroconverted after the first immunization. Following the second immunization, LSA-1-specific CD4+ T cells producing two cytokines (IL-2 and IFN-gamma) were found by intra-cellular staining in all subjects in the LD LSA-NRC/AS01B group and in 3 of 5 subjects in the LD LSA-NRC/AS02 group. In contrast, the HD LSA-NRC/AS01 and HD LSA-NRC/AS02 group subjects had fewer LSA-1-specific CD4+ T cells, and minimal to no IFN-gamma responses. There was no increase in LSA-1-specific CD8+ T cells found in any group. Per protocol, 22 high dose vaccinees, but no low dose vaccinees, underwent P. falciparum homologous malaria challenge (3D7 clone). All vaccinees became parasitemic and there was no delay in their pre-patent period versus controls (p=0.95). LSA-NRC/AS01 and LSA-NRC/AS02 elicited antigen-specific antibody and CD4+ T cell responses, but elicited no protective immunity. Although the optimal antigen dose of LSA-NRC may not have been selected for the challenge portion of the protocol, further vaccine development based upon LSA-1 should not be excluded and should include alternative vaccine platforms able to elicit additional effector mechanisms such as CD8+ T cells.

Mycobacterium ulcerans infection is an emerging disease that causes indolent, necrotizing skin lesions known as Buruli ulcer (BU). Approximately 10% of patients develop reactive osteitis or osteomyelitis beneath skin lesions or metastatic osteomyelitis from lymphohematogenous spread of M. ulcerans . The most plausible mode of transmission is by skin trauma at sites contaminated by M. ulcerans . Pathogenesis is mediated by a necrotizing, immunosuppressive toxin produced by M. ulcerans called mycolactone. The incidence of BU is highest in children up to 15 years old and is a public health problem in countries of endemicity due to disabling scarring and bone destruction. Today, most BU occurs in West Africa, but the disease has been reported in over 30 countries. Treatment options for BU are antibiotics and surgery. BCG vaccination provides short-term protection against BU and prevents osteomyelitis. HIV seropositivity may increase the risk for BU and render BU osteomyeletis highly aggressive.

From the seedpods of Tephrosia elata , a new β-hydroxydihydrochalcone named ( S )-elatadihydrochalcone was isolated. In addition, the known flavonoids obovatachalcone, obovatin, obovatin methyl ether and deguelin were identified. The structures were determined on the basis of spectroscopic evidence. The crude extract and the flavonoids obtained from the seedpods of this plant showed antiplasmodial activities. The literature NMR data on β-hydroxydihydrochalcones is reviewed and the identity of some of the compounds assigned β-hydroxydihydrochalcone skeleton is questioned.

Mycobacterium ulcerans is an emerging infection that causes indolent, necrotizing skin lesions known as Buruli ulcer (BU). Bone lesions may include reactive osteitis or osteomyelitis beneath skin lesions, or metastatic osteomyelitis from lymphohematogenous spread of M. ulcerans. Pathogenesis is related to a necrotizing and immunosuppressive toxin produced by M. ulcerans, called mycolactone. The incidence of BU is highest in children up to 15 years old, and is a major public health problem in endemic countries due to disabling scarring and destruction of bone. Most patients live in West Africa, but the disease has been confirmed in at least 30 countries. Treatment options for BU are antibiotics and surgery. BCG vaccination provides short-term protection against M. ulcerans infection and prevents osteomyelitis. HIV infection may increase risk for BU, and renders BU highly aggressive. Unlike leprosy and tuberculosis, BU is related to environmental factors and is thus considered non-communicable. The most plausible mode of transmission is by skin trauma at sites contaminated by M. ulcerans. The reemergence of BU around 1980 may be attributable to environmental factors such as deforestation, artificial topographic alterations and increased manual agriculture of wetlands. The first cultivation of M. ulcerans from nature was reported in 2008.

In a Phase 1 trial, we evaluated the safety of AERAS-402, an adenovirus 35-vectored TB vaccine candidate expressing 3 Mycobacterium tuberculosis (Mtb) immunodominant antigens, in subjects with and without latent Mtb infection. HIV-negative, BCG-vaccinated Kenyan adults without evidence of tuberculosis, 10 QuantiFERON®-TB Gold In-Tube test (QFT-G)(−) and 10 QFT-G(+), were randomized 4:1 to receive AERAS-402 or placebo as two doses, on Days 0 and 56, with follow up to Day 182. There were no deaths, serious adverse events or withdrawals. For 1 AERAS-402 QFT-G(−) and 1 AERAS-402 QFT-G(+) subject, there were 3 self-limiting severe AEs of injection site pain: 1 after the first vaccination and 1 after each vaccination, respectively. Two additional severe AEs considered vaccine-related were reported after the first vaccination in AERAS-402 QFT-G(+) subjects: elevated blood creatine phosphokinase and neutropenia, the latter slowly improving but remaining abnormal until study end. AERAS-402 was not detected in urine or throat cultures for any subject. In intracellular cytokine staining studies, curtailed by technical issues, we saw modest CD4+ and CD8+ T cell responses to Mtb Ag85A/b peptide pools among both QFT-G(−) and (+) subjects, with trends in the CD4+ T cells suggestive of boosting after the second vaccine dose, slightly more so in QFT-G(+) subjects. CD4+ and CD8+ responses to Mtb antigen TB10.4 were minimal. Increases in Adenovirus 35 neutralizing antibodies from screening to end of study, seen in 50% of AERAS-402 recipients, were mostly minimal. This small study confirms acceptable safety and tolerability profiles for AERAS-402, in line with other Phase 1 studies of AERAS-402, now to include QFT-G(+) subjects.

Background Studies are underway to identify more immunogenic formulations of the existing anti-falciparum malaria vaccine RTS,S/AS02A. To supplement in vitro immunogenicity assays, cutaneous delayed-type hypersensitivity (DTH) may be a useful indicator of functional, cell-mediated immunogenicity. Methods Adult rhesus monkeys were immunized with saline or one of four RTS,S/adjuvant formulations: RTS,S/AS01B, RTS,S/AS02A-standard (current formulation), RTS,S/AS05 or RTS,S/AS06 at 0, 4, and 12 weeks. An additional cohort received RTS,S/AS02A-accelerated, at 0, 1, and 4 weeks. Six months after completing immunizations, five vaccine-relevant antigens (high and low doses) and two controls were administered intradermally. DTH reactivity (induration) was measured at 48 and 72 h, and selected sites were biopsied for histological confirmation. Results In comparison with RTS,S/AS02A-standard, RTS,S/AS01B and RTS,S/AS05 each had larger mean reactions (induration) at 5 of 10 (p

Cutaneous leishmaniasis, a parasitic skin disease that was rare in American soldiers who served in Gulf War I (1991), has been more common in Gulf War II (Operation Iraqi Freedom began in 2003). This has prompted interest among military and civilian clinicians in becoming reacquainted with the clinical features of cutaneous leishmaniasis, as well as diagnostic and treatment options. In the US Army, well-established diagnostic procedures include the Giemsa-stained lesional skin smear or punch biopsy, as well as polymerase chain reaction. Therapies for cutaneous leishmaniasis used to treat soldiers include local heat application (ThermoMed™), cryotherapy (liquid nitrogen), oral fluconazole or itraconazole, and intravenous sodium stibogluconate (pentavalent antimony, Pentostam®). Paromomycin, a promising antileishmania aminoglycoside topical preparation, already commercially available in some countries, is under investigation by the US Army as a combined paromomycin–gentamicin topical formulation. This comme...

RTS,S/AS02A, a recombinant Plasmodium falciparum vaccine based on the circumsporozoite protein (CSP) repeat and C-terminus regions, elicits strong humoral and Th1 cell-mediated immunity. In field studies, RTS,S/AS02A reduced malaria infection, clinical episodes, and disease severity. Heterologous prime-boost immunization regimens, optimally spaced, might improve the protective immunity of RTS,S/AS02A.DNA plasmid encoding P. falciparum CSP (3D7) was administered to six experimental groups of rhesus monkeys (N = 5) by gene gun (coded as D), followed by a 1/5th human dose of RTS,S/AS02A (coded as R). Immunization regimens, including a numeral to denote weeks between immunizations, were D-4-R, D-16-R, D-4-D-4-R, D-4-D-16-R, D-16-D-4-R and D-16-D-16-R. A control group (N = 5) received a single 1/5th dose of RTS,S/AS02A. Endpoints were antibody (Ab) to homologous CSP repeat and C-terminus regions and delayed-type hypersensitivity (DTH) to CSP peptides.Monkeys immunized twice with DNA, 16 weeks apart (D-16-D-4-R and D-16-D-16-R), developed higher levels of anti-C-terminus Abs than control monkeys (p0.02). No CSP DNA priming regimen increased RTS,S/AS02A-induced Ab to CSP repeats. At 16 months after first immunization, D-R and D-D-R, but not control, monkeys had histologically confirmed DTH reactions against CSP C-terminus, which persisted at repeat testing 12 months later.Two optimally spaced, particle-mediated epidermal deliveries of CSP DNA improved the humoral immunogenicity of a single dose of RTS,S/AS02A. Further, CSP DNA prime followed by one dose of RTS,S/AS02A gave biopsy proven DTH reactions against CSP C-terminus of up to 2 years duration, implying the induction of CD4+ memory T cells. Heterologous prime-boost strategies for malaria involving gene gun delivered DNA or more potent vectors, administered at optimal intervals, warrant further investigation.

Background. Current recommendations direct health care providers to administer smallpox vaccine to the upper outer arm. However, concerns about cosmetically bothersome scarring, accidental contact transmission, interference by body tattoos, and even malignant transformation suggest evaluation of alternate vaccination sites is warranted.Methods. We randomized 20 vaccinia-naive adults to undergo smallpox vaccination on the outer (n = 10) or inner (n = 10) upper arm. Evaluations included major reaction (“take”) rates and vaccination site cultures on postvaccination day 7, determination of serum vaccinia-specific neutralizing antibody titers on days 0 (prevaccination) and 21, and determination of adverse events.Results. On postvaccination day 7, a total of 18 participants (9 per group) had major reactions, 17 of whom had culture evidence of viable vaccinia. The inner and outer arm groups had similar major reaction mean sizes (P = .17), but the amount of erythema (in square centimeters) was smaller in the inner arm group (P = .05). At day 21, all participants had higher titers of vaccinia-specific neutralizing antibodies, compared with at day 0, and the geometric mean titer values of the 2 vaccine groups were similar (P = .45). Adverse event rates were similar.Conclusion. The comparable clinical, immunological, and tolerability outcomes between smallpox vaccine applied to the conventional upper outer arm site versus the upper inner arm, coupled with modestly less vaccine-site erythema on the inner arm, indicate that the inner arm may be a suitable alternate vaccination site.

Background Severe injury is associated with changes in monocytes that may contribute to poor outcomes. Longitudinal characterization of monocyte response patterns after trauma may provide added insight into these immunological alterations. Methods Venous blood obtained seven times during post-injury days 1 through 13 from 61 patients with an injury severity score >20 was assessed by flow cytometry for monocytes (CD14+) expressing HLA-DR or CD71 (transferrin receptor) and for circulating levels of interleukin (IL) 1α, IL-1β, IL-6, soluble CD14 (sCD14), tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and endotoxin. Urine neopterin was measured by high-pressure liquid chromatography, expressed as a neopterin-creatinine ratio. Results Trauma patients had leucocytosis days 1 through 13, monocytosis days 5 through 13, reduced proportions of CD14+HLA-DR+ cells days 2 through 5, and elevated proportions of CD14+CD71+ cells days 1 through 13. Neopterin was elevated all days, peaking on day 10. sCD14 was elevated days 2 through 13, and there were sporadic elevations of IL-1α, IL-1β, IL-6, TNF-α, PGE2, TXB2, and endotoxin. Sepsis syndrome patients (n = 6) had larger and more prolonged reductions in CD14+HLA-DR+ cells and higher neopterin values, in comparison with uneventful patient outcomes. Conclusions Altered proportions of monocytes expressing HLA-DR and CD71 and elevated sCD14 and urine neopterin levels, for up to 2 weeks after severe injury, underscores an extended period of profound immunological effects. Additional studies to more fully assess temporal monocyte response patterns after severe injury, including activation, may be warranted.

Buruli ulcer, a disease caused by Mycobacterium ulcerans, causes ulcerative skin disease likely generated by a toxin that mediates apoptosis. We analyzed paraffin-embedded sections of surgically excised Buruli ulcer lesions (two ulcers and one edematous plaque) and adjacent non-lesional skin samples (n = 9) for apoptosis by an indirect immunofluorescent terminal deoxynucleotide transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. All samples were stained for acid-fast bacilli (AFB) and cultured for mycobacteria, and most were analyzed with an M. ulcerans-specific diagnostic polymerase chain reaction (PCR). TUNEL (+) bodies were numerous in both ulcers and the plaque, and sparse or absent in adjacent non-lesional skin. The AFB tissue stains and cultures for M. ulcerans were positive only in the three lesions. The result of the PCR for M. ulcerans was positive in all three lesions and in four of six non-lesional tissue samples; three contained sparse TUNEL (+) bodies. An abundance of TUNEL (+) bodies in the three AFB stain (+), culture (+), and PCR (+) Buruli ulcer lesional samples, but not in nearby AFB stain (-), culture (-), and PCR (+) non-lesional skin samples, strengthen the evidence that apoptosis is an important tissue destruction mechanism in human lesions closely associated with viable M. ulcerans.

Autoimmune disorders, characterized by inflammation and apoptosis of target cells leading to tissue destruction, are mediated in part by autoantibodies against normal cellular components (autoantigens) that may be overexpressed. For example, antibodies against the autoantigens SS-A/Ro and SS-B/La are primary markers for systemic lupus erythematosus and Sjögren's syndrome. Recently, studies in animals demonstrated that green tea consumption may reduce the severity of some autoimmune disorders, but the mechanism is unclear. Herein, we sought to determine whether the most abundant green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), affects autoantigen expression in human cells. Cultures of pooled normal human primary epidermal keratinocytes and of an immortalized human salivary acinar cell line were incubated with 100 microM EGCG (a physiologically achievable level for topical application or oral administration) for various time periods and then analyzed by cDNA microarray analysis, reverse transcription-polymerase chain reaction, and Western blotting for expression of several major autoantigen candidates. EGCG inhibited the transcription and translation of major autoantigens, including SS-B/La, SS-A/Ro, coilin, DNA topoisomerase I, and alpha-fodrin. These findings, taken together with green tea's anti-inflammatory and antiapoptotic effects, suggest that green tea polyphenols could serve as an important component in novel approaches to combat autoimmune disorders in humans.

Background Cutaneous leishmaniasis (CL), rare in the first Gulf War, is common in American troops serving in Operation Iraqi Freedom. Awareness of the clinical features and treatment options of CL would benefit clinicians who may encounter soldiers, as well as civilians, returning from the Middle East with skin lesions. Objective Our purpose was to describe our clinical experience in treating soldiers with CL. Methods From December 2003 through June 2004, approximately 360 of an estimated 20,000 soldiers returning from a yearlong deployment in Iraq with skin lesions suspected of being CL were examined by dermatologists. We summarized CL diagnoses, laboratory evaluations, and treatments, including localized heat therapy (ThermoMed model 1.8; ThermoSurgery Technologies, Inc, Phoenix, Ariz), oral fluconazole, cryotherapy, and itraconazole. Results Among 237 soldiers diagnosed with CL, 181 had one or more laboratory confirmations, most by Giemsa-stained lesion smears and polymerase chain reaction (PCR). PCR was positive for all 122 smear-positive and 26 biopsy-positive lesions and all 34 smear negative and all 3 biopsy-negative cases. Primary outpatient treatments, including ThermoMed (n=26), oral fluconazole (n=15), cryotherapy (n=4), and itraconazole (n=2), were safe and tolerable. Treatment failure occurred in 2 fluconazole recipients and was suspected in 1 ThermoMed and 2 fluconazole recipients. Seventy-two soldiers elected no treatment. Limitation This was a retrospective study. Conclusion Approximately 1% of Ft Campbell troops returning from Iraq were diagnosed with CL, most by laboratory confirmation. PCR appeared to be the most useful diagnostic technique. Among outpatient treatments, ThermoMed and cryotherapy had favorable safety and efficacy profiles.

In a randomized, double-blind, double-dummy controlled study, 22 men with erythema nodosum leprosum (ENL) received six capsules containing either 100 mg (group A, n = 12) or 300 mg (group B, n = 10) of thalidomide daily for one week. A six-week, four capsules per day taper followed, in which group A received 50 mg/day of thalidomide in weeks 2 and 3, then dummy capsules in weeks 4 through 7, while group B had gradual decrements every two weeks. Both regimens caused comparable improvement in 19 patients at day 7 (group A [12 of 12] versus group B [7 of 10]; P = 0.08), but slower tapering in group B showed less re-emergence of ENL through week 7 (P = 0.02, versus group A). Most patients developed new lesions soon after stopping treatment. Slower tapering from a higher initial thalidomide dose may improve clinical ENL responses, but high recurrence rates after discontinuation indicates further assessment is needed to identify better tapering regimens.

Epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea, exerts chemopreventive effects by selectively inducing apoptosis in tumor cells. In contrast, EGCG accelerates terminal differentiation in normal human epidermal keratinocytes (NHEK) mediated partially by up-regulation of p57/KIP2, a cyclin-dependent kinase inhibitor that confers growth arrest and differentiation. However, it is unclear if EGCG modulates caspase 14, a unique regulator of epithelial cell terminal differentiation associated with cornification. Here, we examined the effect of EGCG on caspase 14 expression in NHEK and correlated the protein and mRNA expression of p57/KIP2 with those of caspase 14 in either normal keratinocytes or p57/KIP2-expressing tumor cells (OSC2, an oral squamous cell carcinoma cell line). Additionally, paraffin-embedded normal and untreated psoriatic (aberrant keratinization) skin sections from humans were assessed for caspase 14 by immunohistochemistry. In NHEK, EGCG induced the expression of caspase 14 mRNA and protein levels within a 24-h period. The expression of p57/KIP2 in OSC2 cells was adequate to induce caspase 14 in the absence of EGCG; this induction of caspase 14 was down-regulated by transforming growth factor-beta1. In human psoriatic skin samples, caspase 14 staining in the upper epidermis was reduced, especially in nuclear areas. These results suggest that, in addition to p57/KIP2, EGCG-induced terminal differentiation of epidermal keratinocytes involves up-regulation of caspase 14. Further understanding of how EGCG modulates cellular differentiation may be useful in developing green tea preparations for selected clinical applications.

Background. Tafenoquine is an 8-aminoquinoline developed as a more effective replacement for primaquine. In a previous dose-ranging study in Thailand, 3 tafenoquine regimens with total doses ranging from 500 mg to 3000 mg prevented relapse of Plasmodium vivax malaria in most patients when administered 2 days after receipt of a blood schizonticidal dose of chloroquine. Methods. To improve convenience and to begin comparison of tafenoquine with primaquine, 80 patients with P vivax infection were randomized to receive 1 of the following 5 treatments 1 day after receiving a blood schizonticidal dose of chloroquine: (A) tafenoquine, 300 mg per day for 7 days (n = 18); (B) tafenoquine, 600 mg per day for 3 days (n = 19); (C) tafenoquine, 600 mg as a single dose (n = 18); (D) no further treatment (n = 13); or (E) primaquine base, 15 mg per day for 14 days (n = 12). The minimum duration of protocol follow-up was 8 weeks, with additional follow-up to 24 weeks. Results. Forty-six of 55 tafenoquine recipients, 10 of 13 recipients of chloroquine only, and 12 of 12 recipients of chloroquine plus primaquine completed at least 8 weeks of follow-up (or had relapse). There was 1 relapse among recipients of chloroquine plus tafenoquine, 8 among recipients of chloroquine only, and 3 among recipients of chloroquine plus primaquine. The rate of protective efficacy (determined on the basis of reduction in incidence density) for all recipients of chloroquine plus tafenoquine, compared with recipients of chloroquine plus primaquine, was 92.6% (95% confidence interval, 7.3%-99.9%; P = .042, by Fisher's exact test). Conclusions. Tafenoquine doses as low as a single 600-mg dose may be useful for prevention of relapse of P. vivax malaria in Thailand.

We assessed monthly doses of tafenoquine for preventing Plasmodium vivax and multidrug-resistant P. falciparum malaria. In a randomized, double-blind, placebo-controlled study, 205 Thai soldiers received either a loading dose of tafenoquine 400 mg (base) daily for 3 days, followed by single monthly 400-mg doses (n = 104), or placebo (n = 101), for up to 5 consecutive months. In volunteers completing follow-up (96 tafenoquine and 91 placebo recipients), there were 22 P. vivax, 8 P. falciparum, and 1 mixed infection. All infections except 1 P. vivax occurred in placebo recipients, giving tafenoquine a protective efficacy of 97% for all malaria (95% confidence interval [CI], 82%-99%), 96% for P. vivax malaria (95% CI, 76%-99%), and 100% for P. falciparum malaria (95% CI, 60%-100%). Monthly tafenoquine was safe, well tolerated, and highly effective in preventing P. vivax and multidrug-resistant P. falciparum malaria in Thai soldiers during 6 months of prophylaxis.

We compared the safety and immunogenicity of the recombinant Plasmodium falciparum MSP1(42) antigen formulated with four novel adjuvant systems (AS01B, AS02A, AS05 and AS08) to alum in rhesus monkeys. All five formulations of MSP1(42) were safe and immunogenic. Whereas, all MSP1(42) formulations tested generated high stimulation indices for lymphocyte proliferation (ranging from 27 to 50), the AS02A and AS01B formulations induced the highest levels of specific anti-MSP1(42) antibody. ELISPOT assays showed that the AS02A and AS01B vaccine formulations-induced different cytokine response profiles. Using the ratio of IFN-gamma/IL-5 secreting cells as the metric, the AS01B formulation induced a strong Th1 response, whereas the AS02A formulation induced a balanced Th1/Th2 response. The IFN-gamma response generated by AS02A and AS01B formulations persisted at least 24 weeks after final vaccination. The notable difference in Th1/Th2 polarization induced by the AS02A and AS01B formulations warrants comparative clinical testing.

Some mycobacterial infections, such as tuberculosis, are characterized by apoptosis of infected or by-stander mononuclear immune cells. For localized (paucibacillary, PB) and disseminated (multibacillary, MB) leprosy, characterized by polarized Th1-like vs. Th2-like immune responses, respectively, little is known about lesional apoptosis. We analyzed sections of paraffin-embedded, untreated leprosy lesions from 21 patients by an indirect immunofluorescent terminal deoxynucleotide-transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. Some TUNEL (+) PB sections were then reacted with phycoerythrin-conjugated (red) antibodies against T cells, monocytes, or antigen-presenting (Langerhans) cells. TUNEL (+) bodies were detected in 9 of 16 PB lesions (56%) and in 1 of 5 MB lesions (20%). Some TUNEL (+) bodies in PB disease were CD3+ (T cell), as well as CD4+ (T-helper) or CD8+ (T-cytotoxic). Apoptosis characterizes PB and MB leprosy lesions and may be more frequent in PB disease. In PB disease, some TUNEL (+) bodies may derive from T cells.

Malaria vaccine RTS,S combined with thrombospondin-related anonymous protein (TRAP) and formulated with AS02A (RTS,S+TRAP/AS02A) is safe and immunogenic in adult humans and rhesus monkeys (Macaca mulatta). Here, RTS,S+TRAP/AS02A was administered on a 0-, 1-, and 3-month schedule to three cohorts of infant monkeys, along with adult comparators. Cohort 1 evaluated 1/5, 1/2, and full adult doses, with the first dose administration at one month of age; cohort 2 monkeys received full adult doses, with the first dose administration at one versus three months of age; and, cohort 3 compared infants gestated in mothers with or without previous RTS,S/AS02A immunization. Immunization site reactogenicity was mild. Some infants, including the phosphate-buffered saline only recipient, developed transient iron-deficiency anemia, which is considered a result of repeated phlebotomies. All RTS,S+TRAP/AS02A regimens induced vigorous antibody responses that persisted through 12 weeks after the last vaccine dose. Modest lymphoproliferative and ELISPOT (interferon-gamma and interleukin-5) responses, particularly to TRAP, approximated adult comparators. RTS,S+TRAP/AS02A was safe and well tolerated. Vigorous antibody production and modest, selective cell-mediated immune responses suggest that RTS,S+TRAP/AS02A may be immunogenic in human infants.

Monthly doses of rifampin, ofloxacin, and minocycline (ROM) are expected to be effective treatment for multi-bacillary leprosy. Patients with MB leprosy received ROM (n = 10) or World Health Organization multi-drug therapy (MDT) (n = 11). Treatment with ROM was given as 24 consecutive monthly observed doses of rifampin (600 mg), ofloxacin (400 mg), and minocycline (100 mg). Treatment with MDT was given as 24 consecutive monthly observed doses of rifampin (600 mg) and clofazimine (300 mg), and unobserved daily dapsone (100 mg) and clofazimine (50 mg). Twenty patients completed the 24-month regimens with > 99% compliance. Treatments with ROM and MDT were safe, tolerable, and caused similar improvements in lesions, bacterial indices, and histology. All MDT recipients developed clofazimine-induced pigmentation. Six ROM and nine MDT recipients assessed at five or more years after completion of treatment had no evidence of relapse. Twenty-four months of treatment with ROM is a safe, well-tolerated, and convenient regimen that may provide an alternate therapy to MDT for MB leprosy. Larger trials with sufficient follow-up would better define the role of ROM.

Red blood cells (RBCs) infected with Plasmodium falciparum are protected from complement-mediated lysis by surface membrane glycosyl-phosphatidylinositol (GPI)−anchored proteins, which include decay accelerating factor (DAF or CD55) and CD59. To determine if P. falciparum avoids or replicates less efficiently in GPI protein- deficient cells at a higher risk for complement-mediated lysis, we compared P. falciparum infectivity among control RBCs with those from subjects with paroxysmal nocturnal hemoglobinuria (PNH), a condition in which RBCs express variable levels of DAF (negative and positive) and CD59 (negative (−), intermediate (I), and high (H)). Co-cultures of 19 matched samples of control and PNH RBCs were infected with P. falciparum to directly compare parasitic invasion. Each PNH RBC sample was then assessed for P. falciparum infectivity across the spectrum of GPI protein deficiency. Identification methods included biotin-streptavidin for RBC populations, fluorescein isothiocyanate−labeled antibodies to DAF and CD59, hydroethidine for parasite DNA, and flow cytometry. The mean ± SD parasitemias in co-cultured PNH and control RBCs were 24.7 ± 6.9% versus 21.0 ± 5.9% (P 0.12). For individual PNH samples, parasitemias were significantly higher in DAF (−) cells versus DAF (+) cells (25.0 ± 8.9% versus 19.1 ± 8.7%; P < 0.001) and in CD59 (−) cells versus I/H cells (22.5 ± 6.4% versus 17.6 ± 4.2%; P < 0.0003). Across the CD59 spectrum, mean parasitemias were highest in CD59 (−) cells (24.5 ± 6.4%), followed by CD59-H cells (19.5 ± 5.4%), and CD59-I cells (16.4 ± 4.8%). Expression of DAF in 12 (63%) of 19 infected PNH samples was reduced. Thus, P. falciparum does not selectively avoid RBCs with fewer GPI proteins and parasite replication in PNH cells is at least as robust as in normal RBCs.

T cells and their cytokines are well known for their important role in the pathogenesis of periodontitis. To date, the role of antigen presenting cells (APCs), which are known to be critical in the regulation of T cell response, has been poorly investigated in periodontitis. In this study, we analyzed the expression of co-stimulatory molecules (CD80 and CD86) and CD83, which is a marker of mature dendritic cells, on gingival cells that were isolated from severe periodontitis tissues, with the use of flow cytometry. Significant upregulation of CD86 and CD83 expression was detected in periodontitis lesions, and most of this occurred on B cells. In vitro peripheral blood mononuclear cell cultures showed that stimulation with different periodontopathic bacteria, that included Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Actinomyces viscosus, upregulated both CD86 and CD83 expression on B cells. Therefore, the presence of plaque bacteria may be responsible for the enhanced expression seen in vivo on gingival B cells. APC function by bacterial-activated B cells was further investigated using allogeneic mixed leukocyte reactions. After 24 h culture with either A. actinomycetemcomitans or P. gingivalis, these activated B cells performed as potent APCs in mixed leukocyte reactions, and they stimulated T cells to produce high levels of gamma interferon and minimal interleukin-5. In conclusion, periodontopathic bacterial-induced B cell activation with upregulation of CD86 and CD83 may be associated with enhanced APC function. The results of this study suggest, therefore, that infiltrated gingival B cells have a possible role as APCs in the regulation and maintenance of local T cell response in periodontitis.

Aims To describe the population pharmacokinetics of tafenoquine in healthy volunteers after receiving tafenoquine for malaria prophylaxis. Methods The population consisted of 135 male Thai soldiers (mean age 28.9 years; weight 60.3 kg). All soldiers were presumptively treated with artesunate for 3 days plus doxycycline for 7 days to remove any pre-existing malaria infections. After the treatment regime, 104 soldiers (drug group) received a loading dose of 400 mg tafenoquine base daily for 3 days followed by 400 mg tafenoquine monthly for 5 consecutive months. In the placebo group, 31 soldiers were infected with malaria during the study period. They were re-treated with artesunate for 3 days plus doxycycline for 7 days followed by a loading dose of 400 mg tafenoquine daily for 3 days and then 400 mg tafenoquine weekly for prophylaxis. Blood samples were randomly collected from each soldier on monthly and weekly prophylaxis. Plasma tafenoquine concentrations were measured by h.p.l.c. Population pharmacokinetic modelling was performed using NONMEM. Results A one-compartment model was found best to describe the pharmacokinetics of tafenoquine after oral administration. Age and weight influenced volume of distribution (V/F), and subjects who contracted malaria had higher clearance (CL/F), but none of these factors was considered to have sufficient impact to warrant change in dosing. The population estimates of the first-order absorption rate constant (Ka), CL/F and V/F were 0.694 h1, 3.20 l h1 and 1820 l, respectively. The intersubject variability in these parameters (coefficient of variation, CV%) was 61.2%, 25.3% and 14.8%, respectively. The absorption and elimination half-lives were 1.0 h and 16.4 days, respectively. The residual (unexplained) variability was 17.9%. Conclusions The population pharmacokinetics of orally administered tafenoquine have been determined in Thai soldiers under field conditions. This information, together with its known potent antimalarial activity, portends well for the application of tafenoquine as a useful prophylactic drug or for short-term radical treatment of vivax malaria.

Recognition of dendritic cells (DCs) as initiators and modulators of immune responses and growing use of rhesus monkeys for the preclinical optimization of vaccine formulations prompted characterization of the phenotype and function of isolated rhesus peripheral blood DCs. We developed a flow cytometric method to directly identify and isolate DCs from rhesus peripheral blood whereby a T cell depleted population negative for CD3, CD14, CD16 and CD20 but positive for CD83 yielded a cell population with surface markers, morphology, and a cytokine profile similar to human myeloid DCs. Rhesus blood DCs were more effective than monocytes and B cells in mixed lymphocyte reactions and in the presentation of recombinant malaria blood stage antigen MSP-1 (42) to autologous T cells. The ability to isolate rhesus blood DC from peripheral blood should be a useful tool for immunological investigations.

Chloroquine-induced itch in black-skinned African malaria patients is common and frequently leads to poor compliance or treatment defaulting. To assess the frequency and severity of chloroquine-induced pruritus in an Asian population, we reviewed case records of 1189 Plasmodium vivax malaria patients treated with chloroquine (25 mg/kg over 3days) at the Bangkok Hospital for Tropical Diseases from 1992 through 1997. The majority of patients were Thais or ethnic Burmese (light brown skin), referred from the western border of Thailand. Overall, there were 23 patients (1.9%) with complaints of pruritus during chloroquine therapy. Of these, 12 (52%) had palm and sole involvement, eight (35%) had generalized pruritus including the palms and soles, and three (13%) had palm itching only. One patient developed pruritus on the palms and soles on two consecutive admissions. The pruritus did not interfere with daily activity, was reduced in intensity by anti-histamine therapy, and did not affect the patient's willingness to complete the chloroquine regimen. Therapeutic responses in the 23 patients with chloroquine itch was similar to those without itch. Among the itch patients, there was no association with gender or level of parasitaemias. Our findings indicate that the frequency of chloroquine-induced pruritus in Asian patients treated with chloroquine for P. vivax malaria is low in comparison with black-skinned Africans. This may be related to pharmacogenetic factors, the infective Plasmodium species, drug metabolism or drug-parasite interactions, or a lower affinity of chloroquine for less pigmented skin.

BackgroundSevere injury causes immunological changes that may contribute to a poor outcome. Longitudinal characterization of lymphocyte response patterns may provide further insight into the basis of these immunological alterations.MethodsVenous blood obtained seven times over 2 weeks from 61 patients with injury severity scores above 20 was assessed for lymphocyte phenotypic and activation markers together with serum levels of interleukin (IL) 2, IL-4, soluble IL-2 receptor (sIL-2R), soluble CD4 (sCD4), soluble CD8 (sCD8) and interferon γ.ResultsSevere injury was associated with profound changes in the phenotypic and activation profile of circulating lymphocytes. Activation was indicated by increased numbers of T cells expressing CD25, CD69 and CD71, and raised serum levels of IL-2, sIL-2R, sCD4 and sCD8. Relatively higher levels of sIL-2R and sCD4 were found in patients with sepsis syndrome.ConclusionPolytrauma is associated with dramatic alterations in the phenotypic and activation profile of circulating lymphocytes which are generally independent of clinical course. In contrast, several lymphocyte soluble factors, including sCD4 and sIL-2R, paralleled the clinical course. These data provide new insight into lymphocyte responses after injury and suggest that further assessment of soluble factors as clinical correlates, including those related to lymphocyte activation or generalized inflammation, may be warranted.

Background New treatments for psoriasis are being developed, but many are associated with limited efficacy, side-effects, or rapid recurrence after discontinuation. Thus, the aim of new agents is to induce longer term remissions with fewer side-effects. Preliminary studies have shown that Mycobacterium vaccae, a nonpathogenic organism prepared as a heat-killed suspension, may induce periods of remission in some psoriasis patients when administered intradermally. Methods To further assess the efficacy and tolerability of M. vaccae in patients with moderate to severe psoriasis (psoriasis area and severity index (PASI) of 12–35), we conducted an open label study whereby 24 patients received two intradermal inoculations of M. vaccae in lesion-free deltoid skin, separated by a period of 3 weeks. Results Twelve weeks after starting treatment, 14 of 24 patients (58%) showed marked improvement in the PASI score (greater than 50% reduction), two had moderate improvement (25–50% reduction), six were unchanged ( 5% increase). By 24 weeks, 11 of 22 patients continued to show greater than 50% improvement. Five patients had complete clearance of skin lesions that lasted for at least 6 months. Conclusions Intradermal administration of heat-killed M. vaccae suspension was well tolerated and induced clinically significant improvement in a majority of psoriasis patients in this cohort. Placebo-controlled testing to further define the efficacy of this treatment is warranted.

Animal models for Mycobacterium ulcerans infections (Buruli ulcer) include guinea pigs, rats, and mice, but each has limitations in replicating the spectrum of human disease. Here, 19 adult nine-banded armadillos were inoculated intradermally with M. ulcerans. Injection sites were examined and skin samples obtained for histologic and microbiology studies. Necropsies were conducted to assess systemic involvement. In group 1 (n = 4), 2 animals developed progressive skin ulcers with undermined borders at the injection sites within 6-10 weeks. Biopsies showed features similar to human disease including extensive necrosis in the deep dermis and subcutaneous fat, mixed cellular infiltrates, and acid-fast bacilli (AFB). In group 2 (n = 15), 5 animals developed progressive skin ulcers, 3 had evanescent papulo-nodules, 3 died shortly after inoculation of unknown causes, and 4 showed no signs of infection. Lesion samples from 3 animals with progressive ulcers were culture positive for AFB. Our findings indicate that nine-banded armadillos are susceptible to M. ulcerans and may develop cutaneous lesions that closely mimic Buruli ulcer.

WR 238605 is an 8-aminoquinoline developed for the radical cure of Plasmodium vivax. Forty-four P. vivax-infected patients were randomly assigned to 1 of 4 treatment regimens: 3 groups received a blood schizonticidal dose of chloroquine followed by WR 238605: group A (n=15) received 300 mg daily for 7 days; group B (n=11), 500 mg daily for 3 days, repeated 1 week after the initial dose; group C (n=9), 1 dose of 500 mg. A fourth group (D; n=9) received chloroquine only. Among patients who completed 2-6 months of follow-up (n=23), there was 1 relapse in group B (day 120) and 1 in group C (day 112). Among patients treated with chloroquine only, there were 4 relapses (days 40, 43, 49, and 84). WR 238605 was safe, well tolerated, and effective in preventing P. vivax relapse.

Certain red blood cell (RBC) disorders, including thalassemia, have been associated with an innate protection against malaria infection. However, many in vitro correlative studies have been inconclusive. To better understand the relationship between human RBCs with thalassemia hemoglobinopathies and susceptibility to in vitro infection, we used an in vitro coculture system that involved biotin labeling and flow cytometry to study the ability of normal and variant RBC populations in supporting the growth of Plasmodium falciparum malaria parasites. Results showed that both normal and thalassemic RBCs were susceptible to P falciparum invasion, but the parasite multiplication rates were significantly reduced in the thalassemic RBC populations. The growth inhibition was especially marked in RBCs from -thalassemia patients (both -thalassemia1/-thalassemia2 and -thalassemia1 heterozygote). Our observations support the contention that thalassemia confers protection against malaria and may explain why it is more prevalent in malaria endemic areas.

Buruli ulcer, caused by Mycobacterium ulcerans, is a destructive infection that most commonly affects the skin. Animal models for Buruli ulcer include guinea pigs, rats, mice, and armadillos, but each is limited in replicating the spectrum of human disease. Here, a cynomolgus monkey was infected with two concentrations of M. ulcerans (1.0 and 2.2 ×1 0 8 ) by intradermal inoculation, 3 months apart. All injection sites developed papules that progressed to ulcers with undermined borders within 2-4 weeks. The rate of progression and size of the ulcers were proportional to the numbers of organisms inoculated. Biopsies from ulcer edges showed ulceration, robust inflammatory cell infiltrates, granuloma- tous-like responses, mild edema, and extracellular acid-fast bacilli. The ulcers healed spontaneously between Weeks 8 and 12, with no signs of systemic infection. This report, the first to describe a non-human primate experimentally infected with M. ulcerans, suggests that cynomolgus monkeys are modestly susceptible and develop some of the clinical and histologic features of Buruli ulcer.

Background: Some basal cell carcinomas (BCCs) contain genetic mutations, suggesting that the lesion is composed of a monoclonal population of cells. Clonality, a distinguishing feature of neoplasia, can be inferred by referencing clonal markers such as the pattern of X chromosome inactivation. The X-linked human androgen receptor gene (HUMARA; GenBank) contains a polymorphic DNA marker that reliably illustrates the pattern of X chromosome inactivation in a tissue. Objective: Our purpose was to determine the clonality of sporadic BCCs by examining patterns of X chromosome inactivation. Methods: The patterns of X chromosome inactivation in paired samples of normal skin and sporadic BCCs from 24 women were compared by means of the HUMARA gene assay. Results: All samples from normal skin displayed random X chromosome inactivation, consistent with lyonization. In 15 of 25 tumor samples (60%), nonrandom X chromosome inactivation was detected, consistent with monoclonality. Conclusion: At least some sporadic BCCs are composed of a monoclonal population of cells, strengthening the contention that a collection of mutations confers a growth advantage to this epithelial lesion. (J Am Acad Dermatol 1998;38:49-55.)

(1997). Primaquine-tolerant vivax malaria in Thailand. Annals of Tropical Medicine & Parasitology: Vol. 91, No. 8, pp. 939-943.

Cerebral malaria is a potentially fatal manifestation of ‘severe’ malaria caused by Plasmodium falciparum. It is an especially important problem for African children because it is a major cause of death due to malaria. The pathophysiology of cerebral disease is characterised by complex host-parasite interactions. Optimal management of cerebral malaria is also complex, requiring accurate diagnosis, prompt treatment with one of the few remaining effective antimalarial drugs, and recognition that cerebral disease is frequently accompanied by other major organ dysfunction requiring additional care.