Undifferentiated febrile illnesses in South Sudan: a case series from Operation TRENTON from June to August 2017.

Undifferentiated febrile illnesses present diagnostic and treatment challenges in the Firm Base, let alone in the deployed austere environment. We report a series of 14 cases from Operation TRENTON in South Sudan in 2017 that coincided with the rainy season, increased insect numbers and a Relief in Place. The majority of patients had headaches, myalgia, arthralgia and back pain, as well as leucopenia and thrombocytopenia. No diagnoses could be made in theatre, despite a sophisticated deployed laboratory being available, and further testing in the UK, including next-generation sequencing, was unable to establish an aetiology. Such illnesses are very likely to present in tropical environments, where increasing numbers of military personnel are being deployed, and clinicians must be aware of the non-specific presentation and treatment, as well as the availability of Military Infection Reachback services to assist in the management of these cases.

Undifferentiated febrile illnesses amongst British troops in Helmand, Afghanistan.

OBJECTIVES: Undifferentiated febrile illnesses have been a threat to British expeditionary forces ever since the Crusades. The infections responsible were identified during the Colonial Era, both World Wars and smaller conflicts since, but nearly all remain a significant threat today. Undiagnosed febrile illnesses have occurred amongst British troops in Helmand, Afghanistan since 2006 and so a fever study was performed to identify them. METHODS: From May to October 2008, all undifferentiated fever cases seen at the British field hospital in Helmand, Afghanistan were assessed using a standard protocol. Demographic details, clinical features and laboratory results were recorded and paired serum samples were sent for testing at the UK Special Pathogens Reference Unit (SPRU). RESULTS: Over 6 months, there were 26 cases of"Helmand Fever" assessed and 23 diagnoses were made of which 12 (52%) were sandfly fever, 6 (26%) were acute Qfever and 5 (22%) were rickettsial infections. Four cases had co-infections and 7 cases were not diagnosed (mostly due to inadequate samples). The clinical features and laboratory results available at the British field hospital did not allow these diseases to be distinguished from each other. The exact type of rickettsial infection could not be identified at SPRU. CONCLUSIONS: These cases probably represent the "tip of an iceberg" for British and Allied forces. More resources for diagnostic facilities and follow-up of patients are required to improve the management and surveillance of "Helmand Fever" cases; until then doxycycline 100 mg twice daily for 2 weeks should be given to all troops who present with an undifferentiated febrile illness in Helmand, Afghanistan. Patients with acute Q fever should be followed-up for at least 2 years to exclude chronic Q fever. Prevention of these diseases requires a better understanding of their epidemiology, but prophylaxis with doxycycline and possibly Q fever vaccine should be considered.

Effect of hepatitis C infection on tacrolimus doses and blood levels in liver transplantation recipients.

BACKGROUND: In our cohort of patients with hepatitis-C virus, which is the most common indication for liver transplantation, we have noted higher relative blood levels of tacrolimus compared to patients without hepatitis-C virus. AIM: To verify this observation and determine its clinical significance, we performed a comparison of doses and blood levels of tacrolimus in hepatitis-C virus and non- hepatitis-C virus liver transplantation recipients. METHODS: Tacrolimus dose and trough level, as well as mean alanine aminotransferase, for all patients transplanted at our center with a deceased donor between 1/1995 and 12/1999 with hepatitis-C virus were recorded at monthly intervals during the first 24 months following transplantation and compared to patients without hepatitis-C virus. RESULTS: The tacrolimus levels for hepatitis-C virus and non-hepatitis-C virus patients were not significantly different at any of the monthly intervals, except month 9. In addition, the overall mean tacrolimus levels for hepatitis-C virus and non-hepatitis-C virus patients were not significantly different (P = ns). However, the mean tacrolimus dose (mg/kg) was significantly higher for hepatitis-C virus patients at 12, 15, 18, 21 and 24 months, P < 0.01. The total mean tacrolimus dose in hepatitis-C virus patients was lower during year one by 39% (P = 0.018) and by 73% (P = 0.001) during year two. The total difference in cost of tacrolimus (for year one and two) administered to hepatitis-C virus patients was $4920, P = 0.03. The serum alanine aminotransferase was significantly higher in hepatitis-C virus patients at each monthly interval except month 1, P < or = 0.01. CONCLUSIONS: Liver transplant recipients with hepatitis-C virus require significantly lower oral doses of tacrolimus to achieve the same blood levels compared to non-hepatitis-C virus patients. This difference may result in a significant reduction in the cost of tacrolimus in hepatitis-C virus patients. The most likely explanation for these findings is decreased hepatic clearance of tacrolimus caused by mild hepatic injury from recurrent hepatitis-C virus.

Isolation of Kaeng Khoi virus from dead Chaerephon plicata bats in Cambodia.

A virus isolated from dead Chaerephon plicata bats collected near Kampot, Cambodia, was identified as a member of the family Bunyaviridae by electron microscopy. The only bunyavirus previously isolated from Chaerephon species bats in South-East Asia is Kaeng Khoi (KK) virus (genus Orthobunyavirus), detected in Thailand over 30 years earlier and implicated as a public health problem. Using RT-PCR, nucleotide sequences from the M RNA segment of several virus isolates from the Cambodian C. plicata bats were found to be almost identical and to differ from those of the prototype KK virus by only 2.6-3.2 %, despite the temporal and geographic separation of the viruses. These results identify the Cambodian bat viruses as KK virus, extend the known virus geographic range and document the first KK virus isolation in 30 years. These genetic data, together with earlier serologic data, show that KK viruses represent a distinct group within the genus Orthobunyavirus.

RNA binding properties of bunyamwera virus nucleocapsid protein and selective binding to an element in the 5' terminus of the negative-sense S segment.

The genome of Bunyamwera virus (BUN) (family Bunyaviridae, genus Bunyavirus) comprises three negative-sense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (N). Previous studies have suggested that the encapsidation signal may reside within the 5' terminus of each segment. The BUN N protein was expressed as a 6-histidine-tagged fusion protein in Escherichia coli and purified by metal chelate chromatography. An RNA probe containing the 5'-terminal 32 and 3'-terminal 33 bases of the BUN S (small) genome segment was used to investigate binding by the N protein in vitro using gel mobility shift and filter binding assays. On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a degree of cooperativity in N protein binding. RNA-N complexes were resistant to digestion with up to 1 microg of RNase A per ml. Competition assays with a variety of viral and nonviral RNAs identified a region within the 5' terminus of the BUN S segment for which N had a high preference for binding. This site may constitute the signal for initiation of encapsidation by N.

Molecular mass and volume in radiation target theory.

Radiation target analysis is based on the action of ionizing radiation directly on macromolecules. Interactions of this radiation with the molecules leads to considerable structural damage and consequent loss of biological activity. The radiation sensitivity is dependent on the size of the macromolecules. There has been confusion and discrepancy as to whether the molecular mass or the molecular volume was the determinant factor in the sensitivity. Some proteins are known to change their hydrodynamic volume at low pH, and this characteristic can be utilized to compare the radiation sensitivities of these proteins in the two states. The results show that the radiation sensitivity of proteins depends on the mass of the molecule and is independent of the molecular volume/shape.

Functional and physical molecular size of the chicken hepatic lectin determined by radiation inactivation and sedimentation equilibrium analysis.

Radiation inactivation and sedimentation equilibrium analysis were used to determine the functional and physical size of the chicken hepatic membrane receptor that binds N-acetylglucosamine-terminated glycoproteins. Purified plasma membranes from chicken liver were irradiated with high energy electrons and assayed for 125I-agalactoorosomucoid binding. Increasing the dose of ionizing radiation resulted in a monoexponential decay in binding activity due to a progressive loss of binding sites. The molecular mass of the chicken lectin, determined in situ by target analysis, was 69,000 +/- 9,000 Da. When the same irradiated membranes were solubilized in Brij 58 and assayed, the binding protein exhibited a target size of 62,000 +/- 4,000 Da; in Triton X-100, the functional size of the receptor was 85,000 +/- 10,000 Da. Sedimentation equilibrium measurements of the purified binding protein yielded a lower limit molecular weight of 79,000 +/- 7,000. However, the solubilized lectin was detected as a heterogeneous population of oligomers with molecular weights as high as 450,000. Addition of calcium or calcium plus N-acetylglucosamine decreased the higher molecular weight species, but the lower limit molecular weights remained invariant. Similar results were determined when the chicken lectin was solubilized in Brij 58, C12E9, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS). Results from the present study suggest that in the plasma membrane, the functional species of the chicken hepatic lectin exists as a trimer. However, in detergent solution, the purified receptor forms a heterogeneous population of irreversible oligomers that exhibit binding activity proportional to size.

Sodium cholate-induced changes in the conformation and activity of rat pancreatic cholesterol esterase.

Pancreatic cholesterol esterase (CEase) regulates dietary cholesterol absorption and is activated in the presence of trihydroxy bile salts while remaining inactive monohydroxy bile salts. CEase from rat pancreas has been purified by ammonium sulfate precipitation, hydroxylapatite chromatography, and gel filtration on Sephacryl S-200/S-300 columns connected in series, and its homogeneity and Mr (55,418 +/- 288) have been determined by sedimentation equilibrium centrifugation. The effects of tri-, di-, and monohydroxy bile salts on the conformation of the purified enzyme in buffer solution and in an in vitro assay system were studied by circular dichroism spectropolarimetry. The CD spectrum of the enzyme in solution shows a curve shape suggestive of an alpha-helicity, but low mean residue ellipticity (MRE) values may indicate an important beta-turn contribution. Sodium cholate, a trihydroxy bile salt, induces a decrease in the negative MRE values of the enzyme in solution at bile salt concentrations of 70-100 nM, with no further spectral changes at concentrations as high as 1 mM. Sodium cholate concentrations higher than 1 microM also induce an increase in the enzyme's negative MRE values under activity assay conditions, which reverts toward its original value once the reaction reaches equilibrium. These latter changes are interpreted as induced by substrate binding to the enzyme followed by partial substrate depletion after the reaction reaches equilibrium. Sodium deoxycholate, a dihydroxy bile salt, induces unstable transient increases and decreases in the MRE values of CEase in buffer solution and under activity assay conditions. These changes are bile salt concentration-dependent and may reflect self-association of the protein. Sodium taurolithocholate, a monohydroxy bile salt, does not affect the CD spectrum of CEase, and neither the di- or the monohydroxy bile salt activates the enzyme.

The risk of cytomegalovirus transmission by penetrating keratoplasty.

To investigate the risk of cytomegalovirus transmission by corneal transplantation, we quantitated anticytomegalovirus IgG levels of donor, preoperative, and postoperative serum samples. Of 118 patients, 79 (67%) were seropositive preoperatively. Twenty-five patients who were seronegative preoperatively received a graft from a positive donor and two (8%) seroconverted. Eleven patients who were seronegative preoperatively received a graft from a negative donor and one (9%) seroconverted. None of the patients who seroconverted had a febrile illness and all three grafts were clear.

Effects of pH on allosteric and catalytic properties of the guanosine cyclic 3',5'-phosphate stimulated cyclic nucleotide phosphodiesterase from calf liver.

We have investigated effects of pH on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase purified from calf liver. In the "activated" state, i.e., with 0.5 microM [3H]cAMP plus 1 microM cGMP or at saturating substrate concentrations (250 microM [3H]cAMP or [3H]cGMP), hydrolysis was maximal at pH 7.5-8.0 in assays of different pH. Hydrolysis of concentrations of substrate not sufficient to saturate regulatory sites and below the apparent Michaelis constant (Kmapp), i.e., 0.5 microM [3H]cAMP or 0.01 microM [3H]cGMP, was maximal at pH 9.5. Although hydrolysis of 0.5 microM [3H]cAMP increased with pH from 7.5 to 9.5, cGMP stimulation of cAMP hydrolysis decreased. As pH increased or decreased from 7.5, Hill coefficients (napp) and Vmax for cAMP decreased. Thus, assay pH affects both catalytic (Vmax) and allosteric (napp) properties. Enzyme was therefore incubated for 5 min at 30 degrees C in the presence of MgCl2 at various pHs before assay at pH 7.5. Prior exposure to different pHs from pH 6.5 to 10.0 did not alter the Vmax or cGMP-stimulated activity (assayed at pH 7.5). Incubation at high (9.0-10.0) pH did, in assays at pH 7.5, markedly increase hydrolysis of 0.5 microM [3H]cAMP and reduce Kmapp and napp. After incubation at pH 10, hydrolysis of 0.5 microM [3H]cAMP was maximally increased and was similar in the presence or absence of cGMP. Thus, after incubation at high pH, the phosphodiesterase acquires characteristics of the cGMP-stimulated form. Activation at high pH occurs at 30 degrees C but not 5 degrees C, requires MgCl2, and is prevented but not reversed by ethylenediaminetetraacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the kinetics of cyclic AMP hydrolysis by the cyclic GMP-stimulated cyclic nucleotide phosphodiesterase.

The kinetics of cAMP hydrolysis by the purified calf liver cGMP-stimulated cyclic nucleotide phosphodiesterase were analyzed in the absence or presence of a number of competitive inhibitors of the methylxanthine type according to a two-site competitive model for allosteric enzymes. Methylxanthines were also classified by graphical analysis of classical competition kinetics at saturating cAMP. This treatment yielded Km/KI ratios which estimated the relative effectiveness of the binding of substrate and inhibitors to the "high affinity" (ES complex) state without establishing individual equilibrium-binding constants of cAMP and inhibitors for specific enzyme states. Individual binding constants for substrate and inhibitors were estimated directly by fitting primary data to the rate equation for the two-site competitive model. The equilibrium dissociation constants for cAMP to the "high" (KS) and "low affinity" (AKS) states were 2.4 +/- 0.8 and 410 +/- 140 microM, respectively. Dissociation constants for various inhibitors to the high (BKI) and low affinity (KI) states were also estimated. The ratio KS/BKI, which directly compared the equilibrium-binding constants of substrate and inhibitors to the high affinity state (ES complex), was in excellent agreement with Km/KI ratios derived from graphical analysis. Whereas a number of the methylxanthine analogues were more effective or as effective as cAMP in binding to the low affinity or "ligand-free" state, only isobutylmethylxanthine was effective as cAMP in binding to the high affinity state (1-methyl-3-isopropylxanthine, and 1,3-dipropylxanthine were somewhat less effective). These findings suggested that allosteric transitions might alter the topography of specific hydrophobic domains at cyclic nucleotide-binding sites and that structural determinants were more stringent for binding to the high affinity state than to the low affinity state.

Effects of temperature on allosteric and catalytic properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver.

We have investigated effects of temperature on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Vmax for cAMP and cGMP increased as assay temperature increased from 5 to 45 degrees C. At substrate concentrations below Kmapp, however, hydrolysis increased as temperature decreased from 45 to 5 degrees C and was much greater at 5 degrees C than at 45 degrees C. As assay temperature decreased, Kmapp for cAMP and cGMP decreased. Hill coefficients for cAMP and cGMP were approximately 1.9 at 45 degrees C and 1.2-1.0 at 5 degrees C. cGMP stimulated hydrolysis of 0.5 microM [3H]cAMP at all assay temperatures. Although maximal activity stimulated by cGMP, like Vmax, was lowest at 5 degrees C, presumably because of the effect of temperature on catalytic activity, the apparent activation constant (K alpha app) for cGMP stimulation was lower at 5 degrees C than at 45 degrees C. Thus, affinity for both substrate and effector was increased at 5 degrees C, suggesting that low temperature promotes transitions of the cGMP-stimulated phosphodiesterase to a "high affinity" state. That cGMP stimulated cAMP hydrolysis at 5 degrees C suggests that temperature-induced transitions are incomplete and/or readily reversible. In assays at 30 degrees C competitive inhibitors, like substrates, induce allosteric transitions which result in enhanced hydrolysis of low substrate (1.0 microM [3H] cAMP) concentrations. At higher substrate concentrations (50 microM [3H]cAMP), with the enzyme in the "activated" state, inhibitors compete with substrate at catalytic sites and reduce hydrolysis. At 45 degrees C, as at 30 degrees C, 1-methyl-3-isobutylxanthine (IBMX) and papaverine increased hydrolysis of 1.0 microM [3H]cAMP and reduced hydrolysis of 50 microM [3H]cAMP. At 5 degrees C, however, IBMX and papaverine inhibited hydrolysis of both 1.0 and 50 microM [3H]cAMP. Enzyme activity was relatively more sensitive to inhibition by IBMX at 5 degrees C than at 45 degrees C. Taken together, these observations support the notion that low temperature induces incomplete or readily reversible transitions to the high affinity state for substrates, effectors, and inhibitors. These observed effects of temperature also point out that enzyme determinants and topographical features responsible for transitions to the high affinity state and expression of catalytic activity can be regulated independently.

Interhepatic duct: a new biliary anomaly.

True accessory bile ducts occur in only 1% of patients. An accessory bile duct connecting the right and left hepatic ducts at the porta hepatis is described. This anomaly has never been reported previously, and was clinically significant in the presence of partial obstruction of an anomalous right hepatic duct by stones. The embryologic origin of this duct, which we term an "interhepatic duct," is uncertain.

Regulation of arginine metabolism in Saccharomyces cerevisiae. Association of arginase and ornithine transcarbamoylase.

Association of arginase and ornithine transcarbamoylase (OTCase) has been proposed to play an essential role in the regulation of arginine metabolism in Saccharomyces cerevisiae (Wiame, J.-M. (1971) Curr. Top. Cell. Reg. 4, 1-39). In this report multienzyme complex formation is directly demonstrated in the presence of the active-site ligands for OTCase and arginase. Using equilibrium sedimentation, a dissociation constant for complex formation was determined to be 2.3 X 10(-8) M in the presence of ornithine and agmatine, active-site ligands for OTCase and arginase, respectively. A molecular stoichiometry in the complex of one molecule of OTCase to one molecule of arginase was verified using transmission electron microscopy. The dimensions of the complex were determined by negative staining and rotary and unidirectional shadowing techniques to be 102 A wide by 81 A high. These dimensions are quantitively consistent with dimensions of the individual enzymes (Duong, L. T., Eisenstein, E., Green, S. M., Ornberg, R. L., and Hensley, P. (1986) J. Biol. Chem. 261, 12807-12813). The enzymatic activity of OTCase is virtually completely inhibited when associated with arginase, reflecting the dramatic modulation of enzyme activity as a consequence of the acquisition of quaternary structure in this multienzyme complex.

The effects of metal ions and temperature on the interaction of cobra venom factor and human complement factor B.

An alternative pathway C3 convertase is formed by the equilibrium association of Factor B with cobra venom factor (CVF) followed by the activation step catalyzed by Factor D. However, the association of Factor B with CVF has only occasionally been demonstrated and has not been quantitatively analyzed. Here we show that in the absence of metals the two proteins have significant affinity for each other and reversibly associate in a one-to-one stoichiometry with a dissociation constant of 11.6 microM. Upon the addition of metal ions, the complex is stabilized only 2- to 30-fold in the order Ni2+(Kd = 6.6 microM) less than Mg2+(Kd = 1.1 microM) less than Mn2+(Kd = 0.4 microM). These results suggest that metal ions may be less important in stabilizing the CVF.B complex and more important in promoting the subsequent equilibrium association of CVF.B with Factor D. The stability of the CVF.B complex is variously dependent on temperature in the range studied (14-21 degrees C) depending on the metal ion that is present. The complex formation was demonstrated in the analytical ultracentrifuge at sedimentation equilibrium employing a combination of single- and multiple-independent variable nonlinear least squares analytical techniques. Two different numerical approaches gave very similar results.

Adenosine 5'-diphosphate-dependent subunit dissociation of bovine dopamine beta-hydroxylase.

In a previous study, it was shown that purified soluble bovine dopamine beta-hydroxylase exhibits pH-dependent reversible tetramer-dimer dissociation (Saxena, A., Hensley, P., Osborne, J. C., Jr., and Fleming, P. J. (1985) J. Biol. Chem. 260, 3386-3392). Here we report evidence for the dissociation of this enzyme by magnesium-adenosine diphosphate independent of pH in the pH range 5-7. Quantitative binding of ADP to dopamine beta-hydroxylase revealed that there are two binding sites/dimeric species of hydroxylase and that ADP is tightly bound with a KD less than 10(-8) M. Kinetic data obtained at pH 5.5, the pH inside the chromaffin granule, shows that the apparent Km values for both the substrates tyramine and ascorbate are lowered by the presence of ADP without affecting the Vmax of the enzyme. The ADP-dependent lowering of apparent Km values results from a dissociation of the enzyme to the dimeric species which has inherently lower apparent Km values for substrates.

Oligomerization of pregnancy-specific beta 1-glycoprotein (SP1) at physiologic pH and ionic strength.

Highly purified pregnancy-specific beta 1-glycoprotein (SP1) migrated in gel electrophoresis as a homogeneous species and behaved as a single species in 6 mol/l guanidinium chloride (GdmCl), both in the ultracentrifuge and HPLC. At physiologic pH and ionic strength, in the absence of GdmCl, SP1 existed in the form of oligomers of apparent molecular weights of 40 000 to greater than 300 000. The specific activity of these oligomers varied over a 5-fold range. Electrophoretic mobility also varied among SP1 oligomers, with increasing (alpha-like) mobility shown by oligomers of increasing molecular size. Oligomerization may explain some or all of the reports of SP1 heterogeneity.