Philippe Esterre

Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6

Artemisinin derivatives are an essential component of treatment against multidrug-resistant Plasmodium falciparum malaria. We aimed to investigate in-vitro resistance to artemisinin derivatives in field isolates. In-vitro susceptibility of 530 P falciparum isolates from three countries (Cambodia, French Guiana, and Senegal) with different artemisinin use was assessed with an isotopic microtest. Artemether IC50 up to 117 and 45 nmol/L was seen in French Guiana and Senegal, respectively. DNA sequencing in a subsample of 60 isolates lends support to SERCA-PfATPase6 as the target for artemisinins. The S769N PfATPase6 mutation, noted exclusively in French Guiana, was associated with raised (>30 nmol/L) artemether IC50s (p<0.0001, Mann-Whitney). All resistant isolates came from areas with uncontrolled use of artemisinin derivatives. This rise in resistance indicates the need for increased vigilance and a coordinated and rapid deployment of drug combinations.

The impact of 34 years of massive DEC chemotherapy on Wuchereria bancrofti infection and transmission: the Maupiti cohort.

Semi‐annual mass DEC chemotherapy combined with vector control at the beginning of the programme, has been administered on the remote island of Maupiti (French Polynesia) since 1955 (except two periods in 1960–67 and 1970–74). The results of two surveys in 1985 and 1989, reporting 0% microfilaraemia, led to the hope that the eradication of lymphatic filariasis had been achieved. We combined parasitological criteria (microfilaraemia by membrane filtration), immunological (antigenaemia and serum levels of specific IgG antibodies) and molecular (PCR‐based evaluation of infection in mosquitoes) techniques and found only good control of the parasite: We found residual microfilaraemia in 0.4% of the sample (mean level in carriers: 101.2 mf/ml), antigenaemia in 4.6% (mean level in positive persons: 714.4 units/ml) and specific IgG in 21.6% (including in one very young child). In addition, an infection rate of 1.4% was calculated in the Aedes polynesiensis vector population. These data, obtained in 1997 just before a hurricane, were partially confirmed in 1999 (0.1% of infection rate in the vector). Together with the possibility of some resistance to DEC, various epidemiological factors critical for the eradication of lymphatic filariasis are discussed.

Clinical findings in female genital schistosomiasis in Madagascar.

To assess the morbidity of S. haematobium infection in women of reproductive age (15–49 years) in the western part of Madagascar, the village of Betalatala with a prevalence of urinary schistosomiasis in women of 75.6% (95% confidence limit 69.3 to 81.9%) was compared with a neighbouring village with similar socio‐economic characteristics and a prevalence of 5.0% (95% confidence limit 0 to 11.75%). The women were questioned in Malagasy about obstetrical history and urogynecological symptoms. They were examined gynaecologically, parasitologically and by ultrasonography. Important STDs were excluded by appropiate diagnostics. In Betalatala significantly more women reported a history of spontaneous abortion (P < 0.01), complaints of irregular menstruation (P < 0.001), pelvic pain (<0.05), vaginal discharge (P < 0.0001), dysuria (P < 0.05) and haematuria (P < 0.01) than in the control village. Biopsies were obtained from the cervix of 36 women with macroscopical lesions, and in 12 cases S. haematobium eggs were found by histological sectioning (33.3%). In the control village no eggs were detected in the histological sections of biopsies taken from 14 women. (P < 0.05). Infections with Candida albicans, Trichomonas vaginalis, Gardnerella vaginalis and Treponema pallidum were found in similar frequencies in both villages. In 9.8% of the women in Betalatala abnormalities of the upper reproductive tract were revealed by ultrasonography versus none in the women from the control village (P < 0.05). Echographic abnormalities of the urinary tract were present in 24% and 3% of the women in the study village and in the control village, respectively (P < 0.0001). These findings were accompanied by an elevated frequency of haematuria (55% versus 20%) and proteinuria (70.4% versus 25%) in the study population (P < 0.0001). Our study indicates that S. haematobium infection in women may not only cause symptoms in the urinary tract, but also frequently in the lower and upper reproductive tract.

In Vitro Monitoring of Plasmodium falciparum Drug Resistance in French Guiana: a Synopsis of Continuous Assessment from 1994 to 2005

ABSTRACT Implemented as one arm of the malaria control program in French Guiana in the early 1990s, our laboratory has since established in vitro profiles for parasite drug susceptibility to a panel of eight antimalarials for more than 1,000 Plasmodium falciparum isolates from infected patients. The quinine-doxycycline combination was introduced in 1995 as the first-line drug treatment against uncomplicated P. falciparum malaria, replacing chloroquine, and the first-line drug combination was changed to the artemether-lumefantrine combination in 2002. Resistance to chloroquine declined 5 years after it was dropped in 1995 as the first-line drug, but unlike similar situations in Africa, there was a rapid halt to this decline. Doxycycline susceptibility substantially decreased from 2002 to 2005, suggesting parasite selection under quinine-doxycycline drug pressure. Susceptibility to mefloquine decreased from 1997 onward. Throughout the period from 1994 to 2005, most isolates were sensitive in vitro to quinine, amodiaquine, and atovaquone. Susceptibility to amodiaquine was strongly correlated with that to chloroquine and to a lesser extent with that to mefloquine and halofantrine. Susceptibilities to mefloquine and to halofantrine were also strongly correlated. There were two alerts issued for in vitro artemether resistance in the period from 2002 to 2003 and again in 2005, both of which could be associated with the presence of an S769N polymorphism in the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)-type P. falciparum ATPase6 (PfATPase6) gene. Analysis of susceptibility to lumefantrine, conducted for the first time in 2005, indicates an alarming rate of elevated 50% inhibitory concentrations. In vitro monitoring of parasite drug susceptibility should be pursued to further document the consequences of specific drug policies on the local parasite population and, in particular, to establish profiles of susceptibility to individual components of drug combinations to provide early warning signs of emerging parasite resistance.

Community-based study of genital schistosomiasis in men from Madagascar

Detection of Schistosoma haematoblum eggs in 43% of semen samples with Increased levels of eosinophil cationic protein suggests that the genital organs of men are frequently affected with schistosomiasis.

Resistance to Dihydroartemisinin

The original title of our article was “Lack of Plasmodium falciparum in Vitro and Genomic Resistance to Dihydroartemisinin in Travelers Returning to France from Africa.” EID’s shortening of the title (1) led to the perception that the letter title was misleading, but it was not on purpose. We have recently tested the 50% inhibitory concentration for artemether of the S769N PfATPase6 isolate that we had kept in liquid nitrogen, and it showed susceptibility. We underline that the previously reported clinical or parasitologic failures to some artemisinin-based combination therapies (2,3) were not synonymous with the emergence of resistance to artemisinin compounds. In the study by Grandesso et al., a combination of artesunate plus amodiaquine was given to children <5 years of age who lived in an area in which amodiaquine alone was ineffective to adequately treat uncomplicated falciparum malaria in 1 of 3 cases at day 28 (2). Such a combination (artesunate plus amodiaquine) was nearly equivalent in 1 of 3 cases to a 3-day artesunate monotherapy, which may fail to completely cure children because of the short half-life of artesunate. In the study by Bukirwa et al., no recrudescence occurred in patients treated with artesunate plus amodiaquine and only 2 of 199 patients treated with artemether plus lumefantrine experienced recrudescence at day 28 (3). As Birkiwa et al. themselves acknowledged, artemether plus lumefantrine was not administered with food, and it is known that lumefantrine is absorbed better when it is taken with a small amount of fat. Thus, the clinical failures observed did not necessarily reflect P. falciparum resistance to artemisinin compounds.

Cell populations in the lesion of human cutaneous leishmaniasis: a light microscopical, immunohistochemical and ultrastructural study.

To characterize the in situ cellular immune response in localized cutaneous leishmaniasis (LCL), the authors studied frozen skin biopsies from 50 patients with LCL due toLeishmania braziliensis guyanensis. A panel of 31 monoclonal antibodies was used, which defined the number and distribution of inflammatory cell subsets. Skin inflammatory infiltrates were composed of T cells (with a local CD4/CD8 ratio of 1.05±0.7 vs 1.48±0.3 in peripheral blood), macrophages and a smaller number of B cells, natural killer cells and granulocytes. Most of the T cells expressed activation markers (interleukin-2 and transferrin receptors, HLA-DR+) and an increase in T-cell-receptorγδ expression was noted. Analysis of the CD4+ subpopulations with newly available reagents showed that helper T cells (CD4+CD45RO+) exceeded the suppressor/inducer subset (CD4+CD45RA+) by 1.4∶1. There were no differences between local immune variables from patients with primary infection (45 patients) and those with recurrence (5). In 7 patients, biopsies were analysed before and 1 month after specific treatment, and did not show significant differences except for a small increase of dermal CD1a+ (Langerhans) cells/mm2. The observed pattern of cellular skin infiltration suggests an immune-mediated tissue injury including T-cell-mediated cytotoxicity and delayed hypersensitivity reactions in addition to direct parasitic action.

Granulomatous reaction and tissue remodelling in the cutaneous lesion of chromomycosis

The cell-mediated immune reaction was studied in the cutaneous lesion of chromomycosis, using monoclonal antibodies against polymorphonuclear neutrophils, macrophage and lymphocyte subsets, endothelial and fibroblast cells. In addition, immunostaining of the main degradative enzymes (neutrophil elastase and interstitial collagenase) and certain important cytokines (transforming growth factor-β, tumour necrosis factor-α and interferon-γ) suggested an explanation for the granulomatous reaction and the associated tissue remodelling. The distribution pattern of neutrophils and macrophage subsets, observed by computer-aided image analysis, suggests that the in situ persistence of fungi is the main pathological factor.

Schistosoma haematobium induced lesions in the female genital tract in a village in Madagascar

Female genital schistosomiasis, FGS, was investigated in a gynaecological study as part of an overall community based morbidity survey, including parasitological and ultrasonographical examination, of a Schistosoma haematobium endemic area in Madagascar. Women (103), of childbearing age (15-49 years), were included for a gynaecological examination and visible lesions of vagina and cervix were biopsied in order to determine the origin of the lesion. Furthermore all women were screened for the presence of schistosome ova using PAP smears from the vagina and the endo/exo cervix. In total 15 women showed schistosome ova in the vagina and/or cervix (median age 24 years and range 15-36 years). Of 36 women with cervical abnormalities, 12 eggs were detected by cervical biopsy (33%). In addition, two of the 12 presented vaginal induration, which contained eggs. Six women had eggs in their PAP smears of which three were egg negative by cervical biopsy. The prevalence of positive S. haematobium egg excretion in the urine among the 103 women was 69% and the geometric mean egg count of positive individuals was 51 eggs/10 ml of urine. Five of the 15 women with confirmed FGS had < or = 1 egg/10 ml of urine. Bladder lesions and congestive changes in the kidneys were demonstrated by ultrasonographic examination in 33 and 9% of the 103 women, respectively. None of the 15 women with confirmed FGS had renal congestion. Our study demonstrates that FGS is a common manifestation of the infection with S. haematobium, even in lightly infected individuals.

Morbidity assessment in urinary schistosomiasis infection through ultrasonography and measurement of eosinophil cationic protein (ECP) in urine.

Summary In a Schistosoma haematobium‐endemic village in western Madagascar we evaluated ultrasonography and Eosinophil Cationic Protein (ECP) in urine as means to detect the associated urinary tract pathology. 192 individuals were matched according to age and sex, and grouped into infected persons with bladder and, if present, kidney pathology (n= 96); infected persons without pathology (n= 48) and noninfected persons without pathology (n= 48). The median urinary egg count was significantly higher in individuals with ultrasonographically detectable urinary tract pathology (115 eggs/10 ml urine) than in infected persons without (45 eggs/10 ml of urine). At 136 ng/ml, the median ECP level was significantly higher in the 144 infected individuals than in the 48 noninfected persons (0.35 ng/ml). Egg excretion correlated positively with ECP level. The median ECP level was significantly higher in the group with ultrasonographically detectable urinary tract pathology than in the group without (183 ng/ml vs. 67 ng/ml). The results suggest that minor degrees of pathology, particularly at an early stage of infection with S. haematobium, might be overlooked by ultrasonography despite the presence of marked inflammation, as indicated by markedly increased urinary ECP levels in infected individuals without ultrasonographically detectable urinary tract pathology. ECP may therefore provide important information on the evolution of S. haematobium‐associated urinary tract morbidity.