Pre-positioned Outbreak Research: The Joint Medical Emerging Diseases Intervention Clinical Capability Experience in Uganda.

The West Africa Ebola virus disease outbreak of 2014-2016 demonstrated that responses to viral hemorrhagic fever epidemics must go beyond emergency stopgap measures and should incorporate high-quality medical care and clinical research. Optimal patient management is essential to improving outcomes, and it must be implemented regardless of geographical location or patient socioeconomic status. Coupling clinical research with improved care has a significant added benefit: Improved data quality and management can guide the development of more effective supportive care algorithms and can support regulatory approvals of investigational medical countermeasures (MCMs), which can alter the cycle of emergency response to reemerging pathogens. However, executing clinical research during outbreaks of high-consequence pathogens is complicated and comes with ethical and research regulatory challenges. Aggressive care and excellent quality control must be balanced by the requirements of an appropriate infection prevention and control posture for healthcare workers and by overcoming the resource limitations inherent in many outbreak settings. The Joint Mobile Emerging Disease Intervention Clinical Capability was established in 2015 to develop a high-quality clinical trial capability in Uganda to support rigorous evaluation of MCMs targeting high-consequence pathogens like Ebola virus. This capability assembles clinicians, laboratorians, clinical researchers, logisticians, and regulatory professionals trained in infection prevention and control and in good clinical and good clinical laboratory practices. The resulting team is prepared to provide high-quality medical care and clinical research during high-consequence outbreaks.

Molecular epidemiology of Plasmodium vivax in Latin America: polymorphism and evolutionary relationships of the circumsporozoite gene.

BACKGROUND: The origins and dispersal of Plasmodium vivax to its current worldwide distribution remains controversial. Although progress on P. vivax genetics and genomics has been achieved worldwide, information concerning New World parasites remains fragmented and largely incomplete. More information on the genetic diversity in Latin America (LA) is needed to better explain current patterns of parasite dispersion and evolution. METHODS: Plasmodium vivax circumsporozoite protein gene polymorphism was investigated using polymerase chain reaction amplification and restriction fragment length polymorphism (PCR-RFLP), and Sanger sequencing in isolates from the Pacific Ocean coast of Mexico, Nicaragua, and Peru. In conjunction with worldwide sequences retrieved from the Genbank, mismatch distribution analysis of central repeat region (CRR), frequency estimation of unique repeat types and phylogenetic analysis of the 3' terminal region, were performed to obtain an integrative view of the genetic relationships between regional and worldwide isolates. RESULTS: Four RFLP subtypes, vk210a, b, c and d were identified in Southern Mexico and three subtypes vk210a, e and f in Nicaragua. The nucleotide sequences showed that Mexican vk210a and all Nicaraguan isolates were similar to other American parasites. In contrast, vk210b, c and d were less frequent, had a domain ANKKAEDA in their carboxyl end and clustered with Asian isolates. All vk247 isolates from Mexico and Peru had identical RFLP pattern. Their nucleotide sequences showed two copies of GGQAAGGNAANKKAGDAGA at the carboxyl end. Differences in mismatch distribution parameters of the CRR separate vk247 from most vk210 isolates. While vk247 isolates display a homogeneous pattern with no geographical clustering, vk210 isolates display a heterogeneous geographically clustered pattern which clearly separates LA from non-American isolates, except vk210b, c and d from Southern Mexico. CONCLUSIONS: The presence of vk210a in Mexico and vk210e, f and g in Nicaragua are consistent with other previously reported LA isolates and reflect their circulation throughout the continent. The vk210b, c and d are novel genotypes in LA. Their genetic relationships and low variability within these vk210 and/or within the vk247 parasites in Southern Mexico suggest its recent introduction and/or recent expansion to this region. The global analysis of P. vivax csp suggests this parasite introduction to the region and likely LA by different independent events.

Postinfectious gastrointestinal disorders following norovirus outbreaks.

BACKGROUND: The US Centers for Disease Control and Prevention estimates 20.9 million norovirus infections annually in the United States. Although the acute disease burden is sizeable, emerging data suggest norovirus may be associated with chronic gastrointestinal problems. We identified known outbreaks in US military recruits and used the Defense Medical Encounter Database (DMED) to identify the risk of new onset functional gastrointestinal disorders (FGD) and gastroesophageal reflux disease (GERD). METHODS: Subjects reporting for care of acute gastroenteritis (AGE) at a military treatment clinic during 3 known norovirus outbreaks were identified. Each AGE subject was matched with up to 4 subjects with unrelated medical encounters. Medical encounter data were analyzed for the duration of military service time (or a minimum of 1 year) to assess for incident FGD or GERD. Relative risks were calculated using regression models. RESULTS: We identified 1718 subjects from 3 outbreaks. After controlling for important demographic covariates, the incidence of constipation, dyspepsia, and GERD was approximately 1.5-old higher (P < .01) in AGE-exposed subjects than matched subjects. We also noted variability in outcome incidence across outbreaks. CONCLUSIONS: It appears that the risk of dyspepsia, constipation, and GERD are higher among those who have AGE during a confirmed norovirus outbreak. Although these findings need confirmation, they suggest that dysmotility may result subsequent to these infections. If confirmed, the costs and morbidity associated with the chronic consequences of norovirus should be considered.

The effect of preparation of cebiche on the survival of enterotoxigenic Escherichia coli, Aeromonas hydrophila, and Vibrio parahaemolyticus.

BACKGROUND: Cebiche is a common dish in Latin America, prepared using raw fish mixed with vegetables and marinated with lime juice. The acidity of the lime juice is commonly believed to destroy bacteria and render cebiche as safe to eat. Little data exist concerning rates of cebiche-associated gastroenteritis outbreaks, although these may be high given the popularity of the dish. METHODS: We inoculated raw fish with Aeromonas hydrophila, Vibrio parahaemolyticus, and enterotoxigenic Escherichia coli to determine the effect of the cebiche preparation process on bacterial viability. Raw fish were exposed to a suspension of 1.0 × 10(8) colony-forming units (CFUs) of each organism in a 50-mL solution, prior to the addition of cebiche ingredients. A typical Peruvian cebiche recipe was used combining limes, onions, sweet potatoes, cilantro, and hot peppers marinated together for 30 minutes. A homogenized mixture of the dish was then evaluated for pH and bacterial counts at 0, 10, and 30 minutes. As much as 100 µL of inocula were streaked onto tryptic soy agar (TSA) agar plates and incubated for 24 hours. RESULTS: The initial average pH of the fish was 6.4 prior to adding cebiche ingredients and 5.0 immediately afterwards. The pH at 10- and 30-minute periods was 5.4 and 5.2, respectively. Little reduction in bacterial counts was observed at either the 10- or 30-minute time periods, with counts increasing at 30 minutes. CONCLUSIONS: The putative bactericidal role of lime juice in the preparation process is not sufficient to reduce the microbial population present in cebiche. Pathogens may remain viable after exposure to acidic conditions. The increasing popularity of Peruvian cuisine may also lead to cebiche-associated illness outside of Latin America.

Comparison of culture techniques at different stages of brucellosis.

The lysis centrifugation technique is preferred for culturing Brucella spp. at all stages of brucellosis because it yields 25% more positive results and on average provides results 10 days earlier than the Ruiz-Castaneda method. This lysis method is inexpensive and easier to use and may be used in laboratories with limited expertise or equipment if all safety precautions are taken.

Susceptibility of the Aotus nancymaae owl monkey to eastern equine encephalitis.

Eastern equine encephalitis virus (EEEV) is an arthropod-borne virus associated with life-threatening encephalitis in humans, equines, birds and many other domestic animals. To investigate the suitability of the Aotus nancymaae New World owl monkey as a viable animal model for EEE candidate vaccine testing we used clinical presentation, serology, viral isolation and PCR to evaluate pathogenesis and immunity in infected animals. Monkeys were inoculated subcutaneously (SQ) or intranasally (IN) with 10(4)pfu of virulent EEEV and were initially followed for 45 days. While none of the animals displayed clinical signs of disease, all of the SC inoculated animals (n=6) manifested a viremia averaging 3.2 days (+/-0.8 days). Likewise, serologic responses (IgM, IgG and PRNT) were observed in all SC infected animals. Interestingly, none of the IN inoculated animals (n=6) became viremic or mounted an antibody response and no pathological abnormalities were observed in two animals that were necropsied on day 6 post-infection (p.i.) from each group. To determine if the antibodies produced by the SC inoculated animals were protective against homologous challenge, three animals from the SC group were serologically evaluated on day 253 p.i. and were administered an inoculum identical to initial challenge on day 270 p.i. A positive control group of four naïve animals was also infected as before. All of the naïve positive control animals manifested a similar viremia as observed initially, averaging 2.75 days (+/-0.5 days) while none of the previously challenged animals became viremic. On days 45 and 253 p.i. geometric mean PRNT titers in the SC group were 453 and 101, respectively. This study demonstrates that the Aotus nancymaae can be reproducibly infected with EEE virus and can serve as a suitable model for infection and immunogenicity for the evaluation of candidate vaccines against EEEV.

Susceptibility of owl monkeys (Aotus nancymaae) to experimental infection with Bartonella bacilliformis.

Bartonellosis, caused by Bartonella bacilliformis, is a clinically significant disease in parts of South America, where it is characterized by fever and hemolytic anemia during the often-fatal acute stage and warty skin eruptions during chronic disease. In this study, we evaluated owl monkeys (Aotus nancymaae) as a potential model for studying the immunogenicity and pathology of bartonellosis. Two groups of animals (n = 3 per group) received either 9.5 x 10(7) CFU B. bacilliformis by the ID route or 1.1 x 10(6) CFU by the IV route and were followed for 140 d. Animals were evaluated by physical exam, complete blood count or hematocrit (or both); infection was confirmed by Giemsa staining of blood smears, PCR amplification, and blood culture. On days 7 and 21, Giemsa-stained blood smears from both groups contained organisms (1% to 4% of erythrocytes). All blood cultures and PCR tests were negative. Complete blood counts and chemistry panels showed no difference from baseline. Serology revealed a greater than 4-fold increase in the IgM titer (compared with baseline levels) in the 3 animals from the ID group and 1 animal from the IV group. On day 35, a dermal lesion was excised from the inguinal region of 1 monkey from each group, with a second lesion excised on day 84 from the same monkey in the IV group. However the histopathology and immunostaining of these samples were not consistent with B. bacilliformis. The present study shows that owl monkeys can be infected with B. bacilliformis, but additional dosage studies are necessary to evaluate the usefulness of this species as a disease model for human bartonellosis.

Mycobacterial lipid II is composed of a complex mixture of modified muramyl and peptide moieties linked to decaprenyl phosphate.

Structural analysis of compounds identified as lipid I and II from Mycobacterium smegmatis demonstrated that the lipid moiety is decaprenyl phosphate; thus, M. smegmatis is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly N-acetylmuramyl-L-alanine-D-glutamate-meso-diaminopimelic acid-D-alanyl-D-alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as N-acetylmuramic acid, N-glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the meso-diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal D-alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of M. smegmatis are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.

Continued proteomic analysis of Mycobacterium leprae subcellular fractions.

Recently the sequence of the Mycobacterium leprae chromosome, the only known obligate intracellular mycobacterium, was completed. It has a dramatic reduction in functional genes, with a coding capacity of only 49.5%, the lowest one so far observed among bacterial genomes. The leprosy bacillus seems to preserve a minimal set of genes that allows its survival in the host. The identification of genes that are actually expressed by the bacterium is of high significance in the context of mycobacterial pathogenesis. In this current study, a proteomic approach was undertaken to identify the proteins present in the soluble/cytosol and membrane subcellular fractions obtained from armadillo derived M. leprae. Proteins from each fraction were separated by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry. A total of 147 protein spots were identified from 2-DE patterns and shown to comprise products of 44 different genes, twenty eight of them corresponding to new proteins. Additionally, two highly basic proteins (with pI >10.0) were isolated by heparin affinity chromatography and identified by N-terminal sequencing. This study constitutes the first application of proteomics to a host-derived Mycobacterium.

SecA2 functions in the secretion of superoxide dismutase A and in the virulence of Mycobacterium tuberculosis.

Tuberculosis remains a severe worldwide health threat. A thorough understanding of Mycobacterium tuberculosis pathogenesis will facilitate the development of new treatments for tuberculosis. Numerous bacterial pathogens possess specialized protein secretion systems that are dedicated to the export of virulence factors. Mycobacterium tuberculosis is part of a developing group of pathogenic bacteria that share the uncommon property of possessing two secA genes (secA1 and secA2). In mycobacteria, SecA1 is the essential 'housekeeping' SecA protein whereas SecA2 is an accessory secretion factor. Here we demonstrate that SecA2 contributes to the pathogenesis of M. tuberculosis. A deletion of the secA2 gene in M. tuberculosis attenuates the virulence of the organism in mice. By comparing the profile of proteins secreted by wild-type M. tuberculosis and the DeltasecA2 mutant, we identified superoxide dismutase A (SodA) as a protein dependent on SecA2 for secretion. SodA lacks a classical signal sequence for protein export. Our data suggests that SecA2-dependent export is a new type of secretion pathway that is part of a virulence mechanism of M. tuberculosis to elude the oxidative attack of macrophages.