Automation for clinical CD4 T-cell enumeration, a desirable tool in the hands of skilled operators.

BACKGROUND: Automation in HIV clinical flow cytometry when appropriately applied brings considerable standardisation benefits. The Canadian Immunology Quality Assessment Program (CIQAP) detected situations where operators did not manually override automated software in the event of improper output on the Epics XL and FC500 CD4 immunophenotyping platforms. The automated gating algorithm identifies lymphocytes using a double gate strategy based on CD45 × side scatter (SS) gating and a light scatter FS × SS gate known to fail with sub optimal specimens. METHOD: To generate correct interpretation and results CIQAP introduced a simple protocol modification, bypassing the light scatter gate to include all cells characterized by the CD45 gate. Seventeen problem cases were reanalysed for both absolute and relative T-cell subsets accuracy and compared to the CIQAP group mean values. Results were found to be associated with the percentage of lymphocytes excluded by the automated light scatter gate. RESULTS: The modified manual protocol resolved poor performance in 14 instances out of 17 problem cases. It was found to improve accuracy when the light scatter gate excluded greater than 5% of the cells. The remaining three cases had a lymphocyte recovery of greater than 94.6% in the original automated analysis. CONCLUSION: There is a risk in relying solely on automated gating procedures when using the Epics XL and FC500 CD4 immunophenotyping platforms. Laboratory managers have the responsibility to intervene when required. EQA providers are equally responsible to alert the clinical laboratories of the need to update operator training to deal with stressed specimens. © 2016 International Clinical Cytometry Society.

Efficacy comparison between anti-malarial drugs in Africans presenting with mild malaria in the Central Republic of Africa: a preliminary study.

Drug resistance to Plasmodium falciparum contributes to major health problems in central Africa and, as a consequence, poverty. We have analyzed the efficacy of three currently available antimalarial drugs to treat symptomatic, uncomplicated P. falciparum malaria in semi-immune adults living in Bangui, Central Republic of Africa. 210 consecutive individuals were enrolled in the survey, of which 45 were excluded. Those having received dihydroartemisin proved significantly less parasitemic than those having received quinine per os or sulfadoxin-pyrimethamin (chi2 = 16.93; p < 0.05), and 75% recovered in two days compared to 57 and 44%, respectively. The 25% who did not recover benefited from a second cure with dihydroartemisin, which proved 100% efficient. The most accurate protocol remains to be established by analyzing clinical and parasitological data and taking into account the economics of the country.

Schistosomiasis co-infection in humans influences inflammatory markers in uncomplicated Plasmodium falciparum malaria.

Malaria and schistosomiasis are the two major parasite diseases present in developing countries. The epidemiological co-infection with schistosomiasis could influence the development of the physiological reaction associated with Plasmodium falciparum infection in human. Most studies have demonstrated the association of circulating levels of interferon-gamma (IFN-gamma), tumour necrosis factor-a (TNF-alpha), interleukin-10 (IL-10), transforming growth factor (TGF-beta) and soluble Tumour Necrosis Factor Receptors (sTNF-RI and sTNF-RII) with the morbidity of malaria. In the present study, we showed that Schistosoma haematobium co-infection influences, in an age-dependent manner, the unbalance between pro- and anti-inflammatory circulating cytokines that play a key role during malaria infection. Indeed, children co-infected by S. haematobium have higher levels of IFN-gamma and sTNF-RII than children infected only by P. falciparum. In contrast, co-infected adults presented a significant increase of IFN-gamma, IL-10, TGF-beta, sTNF-RI and sTNF-RII rates and IL-10/TNF-alpha ratio. Taken together, this study indicates that schistosomiasis co-infection can unbalance the regulation of inflammatory factors in uncomplicated P. falciparum malaria. The possible consequences of the schistosomiasis co-infection for age-dependent malaria morbidity are discussed.

Malaria co-infection in children influences antibody response to schistosome antigens and inflammatory markers associated with morbidity.

The epidemiological coexistence of schistosomiasis and malaria is frequently observed in developing countries. Co-infection with malaria in children could influence the development of acquired immunity associated with the resistance or the pathology of schistosomiasis. In the present study, performed during May to June 1996 in Senegal, the humoral immune response to Schistosoma haematobium 28 kDa glutathione S-transferase (Sh28GST) vaccinal antigen and to soluble egg antigens (SEA) has been evaluated in individuals infected by S. haematobium. Specific immunoglobulin G3 (IgG3) and IgE responses were significantly higher in co-infected children with Plasmodium falciparum compared with children infected with S. haematobium only. In addition, circulating levels of interferon-gamma (IFN-gamma), interleukin-10 (IL-10), and soluble tumor necrosis factor receptor II (sTNF-RII), 3 parameters associated with schistosomiasis morbidity, were significantly increased in co-infected children. Taken together, this study indicated that malaria co-infection can both influence the acquired specific immune response to schistosome antigens and unbalance the regulation of inflammatory factors closely involved in schistosomiasis pathology.

Short report: IgG1/IgG3 antibody responses to various analogs of recombinant ypfmsp119--a study in immune adults living in areas of Plasmodium falciparum transmission.

To further characterize protective-type (IgG1/IgG3) antibody responses to Plasmodium falciparum blood stage in putatively immune individuals' plasma, we have tested for various analogs of the 19 kDa C-terminus of the MSP1 antigen obtained as secreted recombinant proteins from Saccharomyces cerevisiae. One of four proteins was then identified on the basis of consistent IgG3, along with less variable IgG1 recognition. This protein has thus been selected for further functional assays of IgG1/IgG3 antibodies.

[Differential changes in IgG1 and IgG3 against a major antigen of blood stage Plasmodium falciparum (MSP1(19)) as a function of the parasite transmission period: study among immune Senegalese adults]. / Evolution différentielle des IgG1 et des IgG3 dirigées contre un antigène majeur des formes sanguines de Plasmodium falciparum (msp1(19)) en fonction de la période de transmission paustre: une étude chez des sujets adultes prémunis au Sénégal.

This study examined the evolution of P. falciparum specific IgG1 and IgG3 antibodies "before" and "after" the highest transmission period in clinically immune Senegalese adults settle in Dielmo, a holoendemic area for P. falciparum transmission. Plasma was tested for antibodies to an antigen (Q-KNG- MSP1(19)) known to react with IgG1 and/or IgG3 in the majority of these individuals. There was a decrease in titers in individuals with low, but not high titer IgG1 whereas specific IgG3 remained unchanged following the highest transmission period. These results raise the question of the differential role of anti-MSP1(19)g-IgG1 antibody fractions in the maintenance of immunity to P. falciparum.

Immune responses to P. falciparum-MSP1 antigen: lack of correlation between antibody responses and the capacity of peripheral cellular immune effectors to respond to this antigen in vitro.

Protective immunity to P. falciparum blood stage infection is thought to be dependent on IgG antibodies, although the mechanisms that underlie such immunity are not clearly understood. One of the antigens thought to be involved in this protective response is MSP1. The present study has examined the levels and distribution of IgG (and IgM) antibodies to the C-terminal 19 kDa fragment of MSP1 in plasma from P. falciparum immune adult Senegalese and the capacity of the peripheral blood mononuclear cells from these patients to either proliferate or secrete IFN-gamma, IL-10 or IL-4 in vitro in response to this antigen. Specific antibodies were found in 74% of individuals' plasma; 44% of mononuclear cells proved capable of proliferating in vitro and IFN-gamma, IL-10 and IL-4 were detected in 37, 23 and 0% of culture supernatants, respectively. No significant association was found between the presence of antibodies and immune cell reactivity under the culture conditions used. This study emphasizes the complexity of the mechanisms responsible for the sustained production of potentially protective antibodies in response to proposed T-cell dependent P. falciparum blood stage antigens.

[Antibodies specific to Plasmodium falciparum antigens in immune individuals: III. Seasonal course of the response to a major antigen of the asexual blood forms in two sites of different malaria exposure]. / Anticorps spécifiques d'antigènes de Plasmodium falciparum chez les sujets immuns: III.-Evolution saisonnière des réponses vis à vis d'un antigène majeur des formes sanguines asexuées dans deux sites d'expositions palustres différentes.

Specific IgG1 and IgG3 antibody responses to a major Plasmodium falciparum blood stage antigen i.e. MSP1 have been measured in plasma obtained from immune individuals living in areas with different endemicities and sampled at different periods corresponding to different levels of parasite transmission. The study shows a significant imbalance between IgG1 and IgG3 antibody responses in Dielmo vs. Ndiop, and a differential regulation of IgG1 and IgG3 responses both in subclasses and in titers (low: 1/200, and high: 1/2,000) depending upon intensity of parasite exposure.

Plasmodium falciparum- and merozoite surface protein 1-specific antibody isotype balance in immune Senegalese adults.

This study shows markedly different isotype distributions of antibodies to asexual blood stages of Plasmodium falciparum and to merozoite surface protein 1 in clinically immune Senegalese adults depending on the study site. The relationships between immunoglobulin M (IgM) and IgG and between IgG3 and IgG1 antibodies differed in settings where transmission is perennial compared to settings where it is seasonal. This suggests a role for antibody class and/or subclass production and utilization in the regulation of protective immunity to such antigens.

[Specific antibodies against Plasmodium falciparum antigens in immune subjects: II. Screening of responses against the merozoite major surface antigen (MSP!)]. / Anticorps spécifiques d'antigènes de Plasmodium falciparum chez les sujets immuns: II. Criblage des réponses vis à vis d'un antigène majeur de la surface des mérozoïtes (MSP1).

Specific immune responses to asexual blood stages of P. falciparum antigens (a lysate of parasitized red blood cells and a characterized vaccine candidate i.e. MSP1 p19) were analyzed in plasma samples from immune adult individuals living in three different areas of Senegal, where malaria transmission is different. Most individuals in the three sites had specific IgG and IgM to total P. falciparum antigens, whereas approximately 50% had either IgG or IgM specific to MSP1 p19. Further, no anti-MSP1 p19 IgG2 and IgG4 antibody was noticed in any individual whereas the distribution of anti-MSP1 p19 IgG1 and IgG3 was different upon the epidemiological context. In addition, no relationship was found between antibody responses and in vitro T cell responses against P. falciparum antigens upon those experimental conditions. These data stress on the relatively elevated distribution of specific antibodies to MSP1 p19 in P. falciparum hyperendemic areas and suggest a differential regulation of isotypes depending on individual parasite exposure.