Dried blood spots versus plasma for the quantification of HIV-1 RNA using the manual (PCR-ELISA) amplicor monitor HIV-1 version 1.5 assay in Yaounde, Cameroon.

OBJECTIVES: Considering the recent accrued need for viral load quantification in resource-limited settings, this study evaluated the use of dried blood spots (DBS) compared to plasma as a means of sample collection and storage for HIV-1 RNA quantification using a non-automated assay. METHODS: Venous blood was collected from 60 consenting HIV-1-positive patients, plasma separated within 4 hours, and stored at -20 degrees C. Venous blood, 50 microL, was blotted on 4 designated areas of Whatman filter paper and air-dried at room temperature for 2 hours. RESULTS: There was a strong statistically significant correlation between HIV-1 RNA viral load using plasma and DBS (r = .955, P < .001). On average plasma viral loads were only slightly higher than DBS viral loads (mean difference: 0.06 log(10) copies/mL). CONCLUSION: Even when using an entirely manual HIV-quantification assay, DBS may provide a reliable, cost-effective method for sample collection and storage for HIV-1 RNA quantification in resource-limited settings.

Correlation of high serum levels of tumor necrosis factor-alpha with disease severity in human African trypanosomiasis.

The levels of tumor necrosis factor-alpha (TNF-alpha) in sera from Trypanosoma brucei gambiense-infected patients from the endemic region of Boko Songho (Bouenza focus in Congo) were measured. An increase was observed in sera from patients (geometric mean = 53.75 pg/ml, n = 69) compared with control subjects from the same endemic area (6.72 pg/ml, n = 31). The patients were classified as being in the early (blood lymphatic) stage and late (meningo-encephalitic) stage of disease according to the presence of parasites and cells in cerebrospinal fluid (CSF). An increase in TNF-alpha was noted in late stage patients (68.42 pg/ml, n = 28) compared with early stage patients (43.68 pg/ml, n = 41). Those patients with fever, asthenia, and edema and those with neurologic signs had higher levels of TNF-alpha (89.36 pg/ml, n = 26) than others (38.07 pg/ml, n = 43). No differences in TNF-alpha levels were seen when trypanosomes were detected in one location (blood, lymph nodes, or CSF) or two or three locations. These data show that the levels of TNF-alpha in serum of T. b. gambiense-infected patients were correlated with disease severity (presence of signs of inflammation or presence of major neurologic signs) and indicate that TNF-alpha could be involved in some aspects of human African trypanosomiasis physiopathology.

Circulating antibodies directed against tryptophan-like epitopes in sera of patients with human African trypanosomiasis.

Human African trypanosomiasis is often associated with an intense proliferation of B lymphocytes, leading to polyclonal antibody synthesis. Using a modified enzyme-linked immunosorbent assay method, we have found highly significant levels of circulating anti-conjugated tryptophan-like epitope antibodies in sera of patients with sleeping sickness. These antibodies were immunoglobulins (Ig) of the M isotype. There was no correlation between immunologic binding and the Ig levels found in sera of patients with human African trypanosomiasis. Higher antibody levels in stage II of the disease than in stage I may be related to damage to the central nervous system. The specificity of this immunologic binding was evaluated by 1) comparison with that obtained with other related conjugates and 2) serum titration. Anti-conjugated tryptophan-like epitope antibodies were not found in other neurologic diseases tested. Their involvement in this pathology remains unknown.

Importance of L-tryptophan metabolism in trypanosomiasis.

African trypanosomiasis or sleeping sickness is caused by extracellular trypanosomes. The presence of seric antibodies directed to a tryptophan-like epitope in trypanosome infected patients and animals led us to investigate the roles of tryptophan in trypanosomiasis. These antibodies are directed against a tryptophan-rich conserved sequence inside the major parasite surface glycoprotein. In vitro, a rapid uptake of tryptophan by trypanosomes is measured. Seric tryptophan levels are decreased during trypanosomiasis. This decrease may be linked with an increase in indoleamine 2,3-dioxygenase (IDO) induced by Interferon-gamma. In vivo inhibition of IDO by norharman provokes a dramatic increase in circulating parasite number. All these data show the essential role of tryptophan in parasite growth. Moreover, antibodies against tryptophan, the decreased concentration of the neurotransmitter serotonin in the brain following infection and the tryptophan metabolites (tryptophol) produced by trypanosomes may participate to the pathophysiological mechanisms provoking sleeping sickness.

[Defense mechanisms in trypanosomiasis]. / Mécanismes de défense au cours des trypanosomoses.

Quantitative and functional alterations in macrophages are observed in trypanosomiasis. Two molecules from macrophages exert potent anti-microbial effect: Nitric Oxide (NO) and Tumor Necrosis Factor-alpha (TNF-alpha). The role of NO in trypanosomiasis is investigated at first on a murine parasite. Trypanosoma musculi, at first and then on Trypanosoma brucei gambiense and T. brucei brucei. Macrophages from T. musculi-infected mice synthesize NO and their trypanostatic activity is correlated with NO production. In vitro activation of macrophage NO synthase by IFN-gamma induces a trypanostatic activity and TNF-alpha is involved in NO synthase induction. High serum levels of TNF-alpha are correlated with disease severity in human African trypanosomiasis. TNF-alpha is increased in supernatants of leucocyte and trypanosome cocultures. TNF-alpha exerts a strong anti-trypanosomal effect. Messengers RNA of TNF-alpha are detected in monocytes after 8 hours of coculture with trypanosomes. Macrophage effector molecules participate with other immune effector mechanisms in resistance of host to trypanosomes.

[Immunology and immunopathology of African trypanosomiasis]. / Immunologie et immunopathologie de la trypanosomose Africaine.

Human African trypanosomiasis (HAT) is characterized by a major deregulation of the immune system. Hypergammaglobulinemia, auto-antibodies, and immunodepression are cardinal features. Parasitemia occurs in waves due to the successive appearance of parasites with different variable glycoprotein surface antigens (VGSA). Antigenic variation enables parasites to elude the host's immune defenses. Although high levels of immune complexes have been detected during HAT, it seems unlikely that they play a significant pathophysiological role. Numerous auto-antibodies have been detected. B lymphocyte activation is uncommon. In vitro T lymphocytes do not proliferate normally, but synthesize cytokines, such as interferon-g which enhance parasite development. Macrophages bind and destroy parasites in the presence of antibodies. They also synthesize large quantities of TNF-alpha which promote parasite destruction but also increase the severity of clinical symptoms. Nitric acid synthesized by activated macrophages has an antiparasitic effect but induces immunosuppression. In the meningoencephalitic stage of HAT, a severe inflammatory reaction is observed. This event is preceded by astroglia which could be induced by astrocytes secreting TNF-a and IL-1. Auto-antibodies against the central nervous system (e.g. anti-galactocerebrosides, anti-tryptophan-like auto-antibodies) may also be involved in the development of encephalitis. VGSA play a key role in the immunopathology of HAT (antigenic variation, induction of cytokine and autoantibody production). Successive relapses occur with the appearance of new antigenic variants and production of antibodies. The resulting continuous stimulation of the immune system leads to deregulation of immunoglobulin production and cytokine network.

Prevalence and spread of extended-spectrum ß-lactamase-producing Enterobacteriaceae in Ngaoundere, Cameroon.

During April 2010 and June 2010, 334 Enterobacteriaceae isolates from 590 participants (outpatients, inpatients, inpatient carers, hospital workers and members of their households) were collected from faecal samples. Based on ß-lactamase pattern, origin of strains and the relationship between participants, 44 isolates of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae were selected from 44 participants (in Ngaoundere Protestant Hospital and Ngaoundere Regional Hospital, Cameroon). To determine the relatedness of bacterial strains, these isolates were fingerprinted using the automated, repetitive-sequenced-based PCR-based DiversiLab system. Subsequently, E. coli isolates that had undergone DiversiLab analysis were examined with respect to their phylogenetic group and detection of the ST131 clone to shed light on the epidemiology of these isolates in the Ngaoundere hospitals. The prevalence of faecal carriage of ESBL-producing Enterobacteriaceae among the study participants was 54.06%. According to participant groups, the prevalence of faecal carriage was also high (outpatients 45%; inpatients 67%; inpatient carers 57%; hospital workers 44%; and members of their households 46%). Analysis of the molecular epidemiology of ESBL-producing E. coli and K. pneumoniae showed a close relationship of the isolates between related and non-related individuals. In addition, DiversiLab results of E. coli identified four related isolates (4/22) from cluster III belonging to the epidemiologically important clone ST131. Our results highlight the importance of outpatients, inpatients, their carers, hospital workers and their families as reservoirs of ESBL-producing Enterobacteriaceae.

Identification of a tryptophan-like epitope borne by the variable surface glycoprotein (VSG) of African trypanosomes.

Antibodies (Ab) directed against a tryptophan-like epitope (WE) were previously detected in patients with human African trypanosomiasis (HAT). We investigated whether or not these Ab resulted from immunization against trypanosome antigen(s) expressing a WE. By Western blotting, we identified an antigen having an apparent molecular weight ranging from 60 to 65 kDa, recognized by purified rabbit anti-WE Ab. This antigen, present in trypomastigote forms, was absent in procyclic forms and Trypanosoma cruzi trypomastigotes. Using purified variable surface glycoproteins (VSG) from various trypanosomes, we showed that VSG was the parasite antigen recognized by these rabbit Ab. Anti-WE and anti-VSG Ab were purified from HAT sera by affinity chromatography. Immunoreactivity of purified antibodies eluted from affinity columns and of depleted fractions showed that WE was one of the epitopes borne by VSG. These data underline the existence of an invariant WE in the structure of VSG from several species of African trypanosomes.