Plasmodium falciparum Clearance Is Rapid and Pitting Independent in Immune Malian Children Treated With Artesunate for Malaria

Background. In Plasmodium falciparum-infected patients treated with artemisinins, parasitemia declines through so-called pitting, an innate splenic process that transforms infected red blood cells (iRBCs) into onceinfected RBCs (O-iRBCs). Methods. We measured pitting in 83 French travelers and 42 Malian children treated for malaria with artesunate. Results. In travelers, O-iRBCs peaked at 107.7% initial parasitemia. In Malian children aged 1.5-4 years, OiRBCs peaked at higher concentrations than in children aged 9-13 years (91.60% vs 31.95%; P = .0097). The parasite clearance time in older children was shorter than in younger children (P = .0001), and the decline in parasitemia in children aged 1.5-4 years often started 6 hours after treatment initiation, a lag phase generally absent in infants and older children. A 6-hour lag phase in artificial pitting of artesunate-exposed iRBCs was also observed in vitro. The proportion of iRBCs recognized by autologous immunoglobulin G (IgG) correlated with the parasite clearance time (r = −0.501; P = .0006) and peak O-iRBC concentration (r = −0.420; P = .0033). Conclusions. Antimalarial immunity correlates with fast artemisinin-induced parasite clearance and low pitting rates. In nonimmune populations, artemisinin-induced P. falciparum clearance is related to pitting and starts after a 6-hour lag phase. In immune populations, passively and naturally acquired immune mechanisms operating faster than pitting may exist. This mechanism may mitigate the emergence of artemisinin-resistant P. falciparum in Africa

Intestinal parasites infections in hospitalized AIDS patients in Kinshasa, Democratic Republic of Congo

To determine the prevalence and the species spectrum of intestinal parasites (IP) involved in hospitalized AIDS patients, a prospective observational and cross-sectional study was carried out in the four main hospitals in Kinshasa, Democratic Republic of the Congo. From November 2006 through September 2007, a single stool sample was collected from 175 hospitalized AIDS patients older than 15 years. Parasites were detected by light microscopy, including Ziehl-Neelsen, Fungi-Fluor, modified trichrome stains, and by immunofluorescence antibody tests and PCR for species diagnosis of microsporidia. At baseline, 19 patients (10.8 %) were under antiretroviral therapy and 156 (89.2 %) were eligible for ART. The main diagnosis for justifying hospitalization was intestinal infection associated with diarrhea in 87 out of 175 (49.7 %). 47 out of 175 (26.9 %) were found to harbor an IP, and 27 out of 175 (15.4 %) were infected with at least one opportunistic IP (OIP). Prevalence rate for OIP were 9.7 %, 5.1 %, 1.7 % and 0.6 % for Cryptosporidium sp., Enterocytozoon bieneusi, Isospora belli and Encephalitozoon intestinalis respectively. Considering patients with diarrhea only, prevalence rate were 12.6 %, 4.6 %, 3.4 % and 1.1 % respectively. The other IP observed were Entamoeba histolytica/Entamoeba dispar in nine cases (5.1 %), Ascaris lumbricoïdes in seven cases (4.0 %), Giardia intestinalis in three cases (1.7 %), hookworm in two cases (1.1 %) and Trichiuris trichiura, Enterobius vermicularis, Schistosoma mansoni in one patient each (0.6 %). No significant relationship was established between any individual IP and diarrhea. These results underline the importance of OIP in symptomatic AIDS patients regardless of diarrhea at the time of the hospitalisation, and showed that routine microscopic examination using stains designed for Cryptosporidium spp. or the microsporidia should be considered due to the absence of clinical markers

Specific PCR assay for direct detection of intestinal microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal specimens from human immunodeficiency virus-infected patients.

A routine assay based on the PCR was developed for the detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal samples. Two oligonucleotide primer pairs from a conserved region in the small-subunit rRNA genes of E. bieneusi (primer pair V1 and EB450) and E. intestinalis (primer pair V1 and SI500) were used to amplify microsporidian DNA. We achieved specific amplification of a 382-bp DNA fragment in E. intestinalis and a 353-bp DNA fragment in E. bieneusi. Boiling of the samples appeared to be most effective for DNA extraction. Fecal samples containing fewer than 10 microsporidia gave a positive result in the PCR assay. Fecal specimens from 30 human immunodeficiency virus-infected patients with microsporidiosis and fecal specimens from 42 patients suspected of having microsporidiosis were investigated by the PCR assay. The PCR assay was validated against standard staining methods (the Uvitex 2B and Chromotrope 2R staining methods) and immunofluorescence assay specific for E. intestinalis. This comparative study has shown that PCR improved species determination and can thus be considered a fast and reliable method for the detection and identification of each intestinal species

Intestinal parasites infections in hospitalized AIDS patients in Kinshasa, Democratic Republic of Congo

To determine the prevalence and the species spectrum of intestinal parasites (IP) involved in hospitalized AIDS patients, a prospective observational and cross-sectional study was carried out in the four main hospitals in Kinshasa, Democratic Republic of the Congo. From November 2006 through September 2007, a single stool sample was collected from 175 hospitalized AIDS patients older than 15 years. Parasites were detected by light microscopy, including Ziehl-Neelsen, Fungi-Fluor, modified trichrome stains, and by immunofluorescence antibody tests and PCR for species diagnosis of microsporidia. At baseline, 19 patients (10.8 %) were under antiretroviral therapy and 156 (89.2 %) were eligible for ART. The main diagnosis for justifying hospitalization was intestinal infection associated with diarrhea in 87 out of 175 (49.7 %). 47 out of 175 (26.9 %) were found to harbor an IP, and 27 out of 175 (15.4 %) were infected with at least one opportunistic IP (OIP). Prevalence rate for OIP were 9.7 %, 5.1 %, 1.7 % and 0.6 % for Cryptosporidium sp., Enterocytozoon bieneusi, Isospora belli and Encephalitozoon intestinalis respectively. Considering patients with diarrhea only, prevalence rate were 12.6 %, 4.6 %, 3.4 % and 1.1 % respectively. The other IP observed were Entamoeba histolytica/Entamoeba dispar in nine cases (5.1 %), Ascaris lumbricoïdes in seven cases (4.0 %), Giardia intestinalis in three cases (1.7 %), hookworm in two cases (1.1 %) and Trichiuris trichiura, Enterobius vermicularis, Schistosoma mansoni in one patient each (0.6 %). No significant relationship was established between any individual IP and diarrhea. These results underline the importance of OIP in symptomatic AIDS patients regardless of diarrhea at the time of the hospitalisation, and showed that routine microscopic examination using stains designed for Cryptosporidium spp. or the microsporidia should be considered due to the absence of clinical markers

Evaluation of an Immunofluorescent-Antibody Test Using Monoclonal Antibodies Directed against Enterocytozoon bieneusi and Encephalitozoon intestinalis for Diagnosis of Intestinal Microsporidiosis in Bamako (Mali)

A 2-month study was carried out in Mali to evaluate an immunofluorescent-antibody test (IFAT) using monoclonal probes specific for Enterocytozoon bieneusi or Encephalitozoon intestinalis. Sixty-one human immunodeficiency virus (HIV)-seropositive adult patients and 71 immunocompetent children were enrolled. Microsporidia were detected in stools from 8 of 61 patients (13.1%) seropositive for HIV. A single species, E. bieneusi, was identified. All the children were negative for microsporidia. The sensitivity and specificity of IFAT were 100% compared with those of PCR, which was used as the “gold standard.” Moreover, species identification by IFAT was more rapid and less expensive than that by PCR. These results show the suitability of IFAT for detection of microsporidia in developing countries

Measuring the Plasmodium falciparum HRP2 protein in blood from artesunate-treated malaria patients predicts post-artesunate delayed hemolysis

Artesunate, the recommended drug for severe malaria, rapidly clears the malaria parasite from infected patients but frequently induces anemia-called post-artesunate delayed hemolysis (PADH)-for which a simple predictive test is urgently needed. The underlying event in PADH is the expulsion of artesunate-exposed parasites from their host erythrocytes by pitting. We show that the histidine-rich protein 2 (HRP2) of the malaria parasite Plasmodium falciparum persists in the circulation of artesunate-treated malaria patients in Bangladesh and in French travelers who became infected with malaria in Africa. HRP2 persisted in whole blood (not plasma) of artesunate-treated patients with malaria at higher levels compared to quinine-treated patients. Using an optimized membrane permeabilization method, HRP2 was observed by immunofluorescence, Western blotting, and electron microscopy to persist in once-infected red blood cells from artesunate-treated malaria patients. HRP2 was deposited at the membrane of once-infected red blood cells in a pattern similar to that for ring erythrocyte surface antigen (RESA), a parasite invasion marker. On the basis of these observations, we developed a semiquantitative titration method using a widely available HRP2-based rapid diagnostic dipstick test. Positivity on this test using a 1:500 dilution of whole blood from artesunate-treated patients with malaria collected shortly after parasite clearance predicted subsequent PADH with 89% sensitivity and 73% specificity. These results suggest that adapting an existing HRP2-based rapid diagnostic dipstick test may enable prediction of PADH several days before it occurs in artesunate-treated patients with malaria