Stage determination and therapeutic decision in human African trypanosomiasis: value of polymerase chain reaction and immunoglobulin M quantification on the cerebrospinal fluid of sleeping sickness patients in Côte d'Ivoire

In human African trypanosomiasis (HAT), two disease stages are defined: the first, or haemo‐lymphatic stage, and the second, or meningo‐encephalitic stage. Stage determination forms the basis of therapeutic decision and is of prime importance, as the drug used to cure second‐stage patients has considerable side‐effects. However, the tests currently used for stage determination have low sensitivity or specificity. Two new tests for stage determination in the cerebrospinal fluid (CSF) were evaluated on 73 patients diagnosed with HAT in Côte d'Ivoire. The polymerase chain reaction (PCR) detecting trypanosome DNA (PCR/CSF) is an indirect test for trypanosome detection whereas the latex agglutination test detecting immunoglobulin M (LATEX/IgM) is an indicator for neuro‐inflammation. Both tests were compared with classically used tests, double centrifugation and white blood cell count of the CSF. PCR/CSF appeared to be the most sensitive test (96%), and may be of use to improve stage determination. However, its value for therapeutic decision appears limited, as patients whose CSF was positive with PCR were successfully treated with pentamidine. This result confirms those of previous works that showed that some patients with trypanosomes in the CSF could be treated successfully with pentamidine. LATEX/IgM, which depending on the cut‐off, showed lower sensitivity of 76% and 88%, but higher specificity of 83% and 71% for LATEX/IgM 16 and LATEX/IgM 8 respectively, appears more appropriate for therapeutic decision making.     

Invasive Salmonella enterica Serotype Typhimurium Infections,Democratic Republic of the Congo, 2007–2011

Infection with Salmonella enterica serotype Typhimurium sequence type (ST) 313 is associated with high rates of drug resistance, bloodstream infections, and death. To determine whether ST313 is dominant in the Democratic Republic of the Congo, we studied 180 isolates collected during 2007–2011; 96% belonged to CRISPOL type CT28, which is associated with ST313.     

Intermediate Susceptibility to Ciprofloxacin among Salmonella enterica Serovar Typhi Isolates in Lima,Peru

Thirty-three Salmonella enterica serovar Typhi blood isolates from Lima, Peru (2008 to 2012), were fully susceptible to trimethoprim-sulfamethoxazole, chloramphenicol, ceftriaxone, and tetracycline; 8/33 (24.2%) showed intermediate susceptibility to ciprofloxacin carrying mutations in the quinolone resistance-determining region of the gyrA gene (Ser83-Phe and Asp87-Asn) and in the gyrB gene (Ser464-Phe).     

Interleukin (IL)-6, IL-8 and IL-10 in serum and CSF of Trypanosoma brucei gambiense sleeping sickness patients before and after treatment

Serum and cerebrospinal fluid (CSF) concentrations of interleukin (IL)-6, IL-8, IL-10, tumour necrosis factor-alpha and interferon-gamma were determined in 46 Trypanosoma brucei gambiense sleeping sickness patients in DR Congo, before and after treatment. According to their CSF cell number before treatment, patients were classified as early-stage (0-5 cells/microL), intermediate-stage (6-20 cells/microL) or late-stage patients (> 20 cells/microL). In serum, slightly higher IL-8 concentrations were found in early-stage patients compared to intermediate- or late-stage patients. These high IL-8 levels dropped after treatment. Higher IL-10 concentrations were detected in serum of patients in intermediate or late stage compared to early-stage patients. In both intermediate- and late-stage groups, serum IL-10 decreased after treatment. In CSF, elevated concentrations of IL-6, IL-8 and especially of IL-10 were observed in late-stage T. b. gambiense patients. After treatment, these concentrations dropped to levels similar to those of the other patients. Tumour necrosis factor-alpha was detected only in a few serum and CSF samples, which were scattered over the different patient groups. Interferon-gamma was detected in serum of 5 patients and remained undetectable in CSF.     

Evaluation of the micro-CATT,CATT/Trypanosoma brucei gambiense,and LATEX/T b gambiense methods for serodiagnosis and surveillance of human African trypanosomiasis in West and Central Africa

OBJECTIVE: To evaluate the performance of serological tests using dried blood on filter-papers (micro-card agglutination test for trypanosomiasis (micro-CATT)) performed under field and laboratory conditions and using whole blood ((CATT/T.b. gambiense) (wb-CATT) and latex agglutination (LATEX/T.b. gambiense) (wb-LATEX)) for the serodiagnosis and surveillance of human African trypanosomiasis in West and Central Africa. METHODS: We evaluated the micro-CATT, wb-CATT and wb-LATEX methods in Côte d''Ivoire and the Central African Republic by screening 940 people. Sensitivity and specificity were calculated for each serological test; only patients with the confirmed presence of trypanosomes in the blood or lymph aspirate were considered true positives. Positive and negative predictive values were also calculated. FINDINGS: Each of the tests showed a lower sensitivity in the Central African Republic than in Côte d''Ivoire. CONCLUSION: The results confirmed the efficiency of the classic wb-CATT to detect sleeping sickness patients. The micro-CATT method can be used for human African trypanosomiasis surveillance if the test is performed on the same day as the blood collection, or if samples are stored at 4 degrees C. Otherwise, micro-CATT can be used when absolute sensitivity is not required. wb-LATEX should only be used for high-specificity screening.     

Phe-Gly dipeptidomimetics designed for the di-/tripeptide transporters PEPT1 and PEPT2: synthesis and biological investigations

A series of five Phe-Gly dipeptidomimetics containing different amide bond replacements have been synthesized in a facile way from the readily available unsaturated ketoester 1, and their affinities for the di-/tripeptide transporters hPEPT1 (Caco-2 cells) and rPEPT2 (SKPT cells) were tested. The compounds contained the amide bond isosteres ketomethylene (2a), (R)- and (S)-hydroxyethylidene (3a and 4a), and (R)- and (S)-hydroxyethylene (5a and 6a) to provide information on the conformational and stereochemical requirements for hPEPT1 and rPEPT2 affinity. The affinity studies showed that for rPEPT2 there is no significant difference in affinity between the ketomethylene isostere 2a and the natural substrate Phe-Gly (K(i) values of 18.8 and 14.6 microM, respectively). Also the affinities for hPEPT1 are in the same range (K(i) values of 0.40 and 0.20 mM, respectively). This corroborates earlier findings that the amide bond as such is not essential for binding to PEPTX, but the results also reveal possible differences in the binding of ketomethylene isosteres to hPEPT1 and rPEPT2. The trans-hydroxyethylidene and hydroxyethylene isosteres proved to be poor substrates for PEPTX. These results provide new information about the importance of flexibility and of the stereochemistry at the C(4)-position for this class of compounds. Furthermore, the intracellular uptake of 2a-4a in Caco-2 cells was investigated, showing a 3-fold reduction of the uptake of 2a in the presence of the competetive inhibitor Gly-Pro, indicating contribution from an active transport component. No active uptake of 3a and 4a was observed. Transepithelial transport studies also indicated active transport of 2a across Caco-2 monolayers.     

Diagnosis of sleeping sickness stage: towards a new approach

Diagnosis of the neurological disease stage in Trypanosoma brucei (T.b.) gambiense infection is essential to select an optimal chemotherapy. The actual parameters for stage determination, the cerebrospinal fluid (CSF) cell count, total protein concentration and trypanosome detection, are insufficiently specific and sensitive. In order to identify new parameters for stage determination, we studied the neuro-inflammatory immune response in the central nervous system, notably the intrathecal humoral immune response in sleeping sickness patients. The presence of intrathecal IgM synthesis was identified as an excellent marker of central nervous system involvement. However, intrathecal IgM detection cannot be performed under field conditions. As a consequence of the strong intrathecal IgM synthesis, extremely high concentrations of IgM are found in the CSF of sleeping sickness. We therefore developed a latex agglutination field test (LATEX/IgM) indicative for intrathecal IgM synthesis and CNS involvement in sleeping sickness. Based on our observations on the intrathecal immune response and with LATEX/IgM, we propose a new approach for stage determination in sleeping sickness.     

Up‐regulation of cytokines and chemokines predates the onset of rheumatoid arthritis

Objective

To identify whether cytokines, cytokine‐related factors, and chemokines are up‐regulated prior to the development of rheumatoid arthritis (RA).

Methods

A nested case–control study was performed in 86 individuals who had donated blood samples before experiencing any symptoms of disease (pre‐patients) and 256 matched control subjects (1:3 ratio). In 69 of the pre‐patients, blood samples were also obtained at the time of the diagnosis of RA. The plasma levels of 30 cytokines, related factors, and chemokines were measured using a multiplex system.

Results

The levels of several of the cytokines, cytokine receptors, and chemokines were significantly increased in individuals before disease onset compared with the levels in control subjects; i.e., those representing signs of general immune activation (interleukin‐1β [IL‐1β], IL‐2, IL‐6, IL‐1 receptor antagonist, and tumor necrosis factor), activation of Th1 cells (interferon‐γ, IL‐12), Th2 cells (IL‐4, eotaxin), Treg cells (IL‐10), bone marrow–derived factors (IL‐7, granulocyte–macrophage colony‐stimulating factor, and granulocyte colony‐stimulating factor), as well as chemokines (monocyte chemotactic protein 1 and macrophage inflammatory protein 1α). The levels were particularly increased in anti–cyclic citrullinated peptide antibody– and rheumatoid factor–positive individuals, and the concentration of most of these increased further after disease onset. The concentration of IL‐17 in individuals before disease onset was significantly higher than that in patients after disease onset. Individuals in whom RA subsequently developed were discriminated from control subjects mainly by the presence of Th1 cells, Th2 cells, and Treg cell–related cytokines, while chemokines, stromal cell–derived cytokines, and angiogenic‐related markers separated patients after the development of RA from individuals before the onset of RA.

Conclusion

Individuals in whom RA later developed had significantly increased levels of several cytokines, cytokine‐related factors, and chemokines representing the adaptive immune system (Th1, Th2, and Treg cell–related factors); after disease onset, the involvement and activation of the immune system was more general and widespread.