A unified approach to molecular epidemiology investigations: tools and patterns in California as a case study for endemic shigellosis

<p>Abstract</p> <p>Background</p> <p>Shigellosis causes diarrheal disease in humans from both developed and developing countries, and multi-drug resistance is an emerging problem. The objective of this study is to present a unified approach that can be used to characterize endemic and outbreak patterns of shigellosis using use a suite of epidemiologic and molecular techniques. The approach is applied to a California case study example of endemic shigellosis at the population level.</p> <p>Methods</p> <p>Epidemiologic patterns were evaluated with respect to demographics, multi-drug resistance, antimicrobial resistance genes, plasmid profiles, and pulsed-field gel electrophoresis (PFGE) fingerprints for the 43 <it>Shigella </it>isolates obtained by the Monterey region health departments over the two year period from 2004-2005.</p> <p>Results</p> <p>The traditional epidemiologic as well as molecular epidemiologic findings were consistent with endemic as compared to outbreak shigellosis in this population. A steady low level of cases was observed throughout the study period and high diversity was observed among strains. In contrast to most studies in developed countries, the predominant species was <it>Shigella flexneri </it>(51%) followed closely by <it>S. sonnei </it>(49%). Over 95% of <it>Shigella </it>isolates were fully resistant to three or more antimicrobial drug subclasses, and 38% of isolates were resistant to five or more subclasses. More than half of <it>Shigella </it>strains tested carried the <it>tetB</it>, <it>catA</it>, or <it>bla</it>_TEMgenes for antimicrobial resistance to tetracycline, chloramphenicol, and ampicillin, respectively.</p> <p>Conclusion</p> <p>This study shows how epidemiologic patterns at the host and bacterial population levels can be used to investigate endemic as compared to outbreak patterns of shigellosis in a community. Information gathered as part of such investigations will be instrumental in identifying emerging antimicrobial resistance, for developing treatment guidelines appropriate for that community, and to provide baseline data with which to compare outbreak strains in the future.</p

Acute childhood diarrhoea in northern Ghana: epidemiological, clinical and microbiological characteristics

<p>Abstract</p> <p>Background</p> <p>Acute diarrhoea is a major cause of childhood morbidity and mortality in sub-Saharan Africa. Its microbiological causes and clinico-epidemiological aspects were examined during the dry season 2005/6 in Tamale, urban northern Ghana.</p> <p>Methods</p> <p>Stool specimens of 243 children with acute diarrhoea and of 124 control children were collected. Patients were clinically examined, and malaria and anaemia were assessed. Rota-, astro-, noro- and adenoviruses were identified by (RT-) PCR assays. Intestinal parasites were diagnosed by microscopy, stool antigen assays and PCR, and bacteria by culturing methods.</p> <p>Results</p> <p>Watery stools, fever, weakness, and sunken eyes were the most common symptoms in patients (mean age, 10 months). Malaria occurred in 15% and anaemia in 91%; underweight (22%) and wasting (19%) were frequent. Intestinal micro-organisms were isolated from 77% of patients and 53% of controls (<it>P </it>< 0.0001). The most common pathogens in patients were rotavirus (55%), adenovirus (28%) and norovirus (10%); intestinal parasites (5%) and bacteria (5%) were rare. Rotavirus was the only pathogen found significantly more frequently in patients than in controls (odds ratio 7.7; 95%CI, 4.2–14.2), and was associated with young age, fever and watery stools. Patients without an identified cause of diarrhoea more frequently had symptomatic malaria (25%) than those with diagnosed intestinal pathogens (12%, <it>P </it>= 0.02).</p> <p>Conclusion</p> <p>Rotavirus-infection is the predominant cause of acute childhood diarrhoea in urban northern Ghana. The abundance of putative enteropathogens among controls may indicate prolonged excretion or limited pathogenicity. In this population with a high burden of diarrhoeal and other diseases, sanitation, health education, and rotavirus-vaccination can be expected to have substantial impact on childhood morbidity.</p

Specific Depletion of Ly6C^hi Inflammatory Monocytes Prevents Immunopathology in Experimental Cerebral Malaria

<div><p><i>Plasmodium berghei ANKA</i> (PbA) infection of C57BL/6 mice leads to experimental cerebral malaria (ECM) that is commonly associated with serious T cell mediated damage. In other parasitic infection models, inflammatory monocytes have been shown to regulate Th1 responses but their role in ECM remains poorly defined, whereas neutrophils are reported to contribute to ECM immune pathology. Making use of the recent development of specific monoclonal antibodies (mAb), we depleted <i>in vivo</i> Ly6C^hi inflammatory monocytes (by anti-CCR2), Ly6G⁺ neutrophils (by anti-Ly6G) or both cell types (by anti-Gr1) during infection with Ovalbumin-transgenic PbA parasites (PbTg). Notably, the application of anti-Gr1 or anti-CCR2 but not anti-Ly6G antibodies into PbTg-infected mice prevented ECM development. In addition, depletion of Ly6C^hi inflammatory monocytes but not neutrophils led to decreased IFNγ levels and IFNγ⁺CD8⁺ T effector cells in the brain. Importantly, anti-CCR2 mAb injection did not prevent the generation of PbTg-specific T cell responses in the periphery, whereas anti-Gr1 mAb injection strongly diminished T cell frequencies and CTL responses. In conclusion, the specific depletion of Ly6C^hi inflammatory monocytes attenuated brain inflammation and immune cell recruitment to the CNS, which prevented ECM following <i>Plasmodium</i> infection, pointing out a substantial role of Ly6C⁺ monocytes in ECM inflammatory processes.</p></div

Impact of mononuclear cell subset depletion on IFNγ producing CD8⁺ T cells (brain infiltrates).

<p>Six days after infection, cellular infiltrates from the brains of individual mice were prepared and analysed by flow cytometry for the frequency of infiltrating CD8⁺ cells which produce IFNγ or not upon phorbol myristate acetate (PMA) /Ionomycin restimulation.</p><p>^a n = 4–5 per group</p><p>^b Significant differences between PbTg infected group and naïve mice, (p<0.05, Mann-Whitney U test)</p><p>^c Significant differences between mAb-depleted versus non-depleted PbTg infected mice, (p<0.05, Mann-Whitney U test)</p><p>Impact of mononuclear cell subset depletion on IFNγ producing CD8⁺ T cells (brain infiltrates).</p

Histological analysis of brains of PbTg infected mice upon early depletion.

<p>C57BL/6 mice were infected i.v. with 5*10⁴ PbTg-iRBC and then subdivided into groups that received either anti-Gr1, anti-Ly6G or anti-CCR2 mAb on d0 p.i. (early depletion). On day 6 p.i., tissue sections from the brain parenchyma of individual mice were assessed for pathological changes. (A) Standard H&E staining is depicted in the upper panels whereas immunohistochemical staining for anti-Mac3 and anti-CD3 are shown in the middle and lower panels, respectively. Representative sections from naïve mice are shown on the left whereas those from PbTg infected mice are displayed on the right. (B, C) Quantification of Mac3⁺ cells (B) and CD3⁺ cells T cells (C) in brain sections from the meninges and frontal cortex of individual mice. Scale bars indicate 100μm in the magnifications. Bars show mean ± SEM from n = 8 mice per group from 1 out of 3 independent depletion-infection experiments counted in 10 defined fields (High power fields, HPF) in the frontal cortex. Statistical analysis was performed using Kruskal-Wallis test and Dunn’s Post test and significant differences are indicated by the stars in brackets between the groups (* <i>p</i><0.05).</p

Early depletion of inflammatory monocytes protects PbTg infected mice against arising ECM.

<p>(A) Effective depletion of inflammatory monocytes (Ly6C^hiLy6G^-) or neutrophils (Ly6C^intLy6G⁺) in the blood of PbTg-infected C57BL/6 mice upon treatment with anti-Gr1 (middle), anti-Ly6G (right), or anti-CCR2 (far right) monoclonal antibodies, on d+1 post depletion. CD11b⁺ leukocytes from the blood (upper row) were gated (squares) and analysed for the presence of Ly6C⁺Ly6G^- monocytes and Ly6^intLy6G⁺neutrophils (lower row) The data of the lower row correspond to data of the upper row. n = 4 mice per group. Flow cytometric analyses show a representative data plot for each group from 1 of 3 independent experiments. (B-G) Groups of PbTg-infected C57BL/6 mice were injected i.p. with anti-Gr1 mAb (B and C), anti-Ly6G mAb (D and E) or anti-CCR2 mAb (F and G) on the first day of infection (d0) or on days 3 and 5 p.i. (d3+5). Scores of cerebral pathology were determined on day 6 p.i. and bar graphs in B, D and F depict the mean and SEM of ECM score in individual mice (n = 8 mice per group). Statistical analysis was performed using Kruskal-Wallis test and Dunn’s Post test (** <i>p</i><0.01). Survival data shown in C, E and G were analyzed with Mantel-Cox log-rank test. <i>p</i><0.05 was considered significant (* <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001). n = 10 mice per group.</p