Onchocerca volvulus-specific antibody and cytokine responses in onchocerciasis patients after 16 years of repeated ivermectin therapy.

The recommended control option against onchocerciasis is repeated ivermectin treatment, which will need to be implemented for decades, and it remains unknown how repeated ivermectin therapy might affect immunity against Onchocerca volvulus in the long term. O. volvulus-specific antibody reactivity and cellular cytokine production were investigated in onchocerciasis patients receiving ivermectin (150 microg/kg) annually for 16 years. In treated patients, the T helper type 2 (Th2) cytokine interleukin (IL)-5 and T regulatory IL-10 in response to O. volvulus antigen (OvAg) and bacteria-derived Streptolysin O (SL-O) diminished to levels found in infection-free endemic controls; also, cellular release of Th1-type interferon (IFN)-gamma at 16 years post initial ivermectin treatment (p.i.t.) approached control levels. In ivermectin-treated onchocerciasis patients, IL-5 production in responses to the mitogen phytohaemagglutinin (PHA) decreased, but IL-10 in response PHA increased, and neither attained the cytokine production levels of endemic controls. At 16 years p.i.t., O. volvulus-specific IgG1 and IgG4 subclass reactivity still persisted at higher levels in onchocerciasis patients than in O. volvulus exposed but microfilariae-free endemic controls. In addition, cytokine responses remained depressed in onchocerciasis patients infected concurrently with Mansonella perstans and Necator americanus or Entamoeba histolytica/dispar. Thus, long-term ivermectin therapy of onchocerciasis may not suffice to re-establish fully a balanced Th1 and Th2 immune responsiveness in O. volvulus microfilariae-negative individuals. Such deficient reconstitution of immune competence may be due to an as yet continuing and uncontrolled reinfection with O. volvulus, but parasite co-infections can also bias and may prevent the development of such immunity.

Chemokines in onchocerciasis patients after a single dose of ivermectin.

Ivermectin treatment will effectively diminish microfilariae (Mf) of Onchocerca volvulus in the skin of patients, but therapy is associated with adverse host inflammatory responses. To investigate the association of proinflammatory chemokines with the intensity of infection and clinical adverse reactions, chemokine serum levels were measured in patients following ivermectin treatment (100 microg/kg, 150 microg/kg or 200 microg/kg) or placebo. The density of O. volvulus Mf per mg skin decreased by 85%, 97%, 97% and 90% at day 3, at month 3, month 6 and at 1 year post-ivermectin. The cutaneous T cell-attracting chemokine (CTACK/CCL27) was found highly elevated in onchocerciasis patients compared to infection-free European controls (P = 0.0004) and it did not change following ivermectin or placebo to 1 year post-therapy. The chemokine RANTES/CCL5 (regulated on activated and normally T cell-expressed) was similarly high in onchocerciasis patients and infection-free European controls; the RANTES/CCL5 levels did not change following treatment until 6 months post-therapy but were slightly elevated at 1 year post-therapy (P < 0.02). In contrast, the Th2-type chemoattractants, thymus and activation regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), were activated at 3 days post-ivermectin (P < 0.0001) to return to pretreatment or lower levels thereafter. The Th1-type chemoattractants, macrophage inflammatory protein (MIP)-1alpha/CCL3 and MIP-1beta/CCL4 were low before ivermectin treatment, but following clearance of microfilariae of O. volvulus their levels increased from 6 months post-therapy onwards (for both at 12 months post-therapy, P < 0.0001). The adverse reaction scores (RS) in treated patients increased significantly on day 3 (P < 0.02) while it remained unchanged in those who received placebo (P = 0.22); RS interacted with the microfilarial density (P = 0.01), but not with the dose of ivermectin or with the serum levels of MIP-1alpha/CCL3, MIP-1beta/CCL4, TARC/CCL17, MDC/CCL22 and CTACK/CCL27. Our observations suggest that following ivermectin, macrophages as well as memory Th2-type lymphocytes and B cells, attracted and activated by MDC/CCL22, TARC/CCL17 and CTACK/CCL27, may contribute to dermal immune responses and O. volvulus Mf killing and clearance. The transient changes of TARC/CCL17 and MDC/CCL22 were not associated with clinical adverse responses, and the later rise of MIP-1alpha/CCL3 and MIP-1beta/CCL4 showed a reactivation of Type 1 immune responses associated with persistent low levels of O. volvulus microfilariae and an expiring O. volvulus infection.

Diagnostic value of molecular markers in chloroquine-resistant falciparum malaria in Southern Mauritania.

Despite its diminishing efficacy because of increased resistance, chloroquine remains the primary antimalarial agent in many endemic areas. Evidence is mounting that point mutations on the Pfcrt and possibly the Pfmdr1 genes are conferring plasmodial resistance to chloroquine. In 1998, atypically strong rainfalls led to an increased activity of falciparum malaria in Mauritania that affected non-endemic regions bordering the Saharan desert. An in vivo study on chloroqine resistance was combined with studies for molecular markers of drug resistance. Detection of Pfmdr1-76-tyrosine showed an increased odds ratio (2.91) for resistance (P = 0.0195). However, by use of this codon alone, sensitivity for detection of resistance was 60.6%, and specificity was 65.3%. In comparison, detection of the K76T mutation at Pfcrt showed a very high sensitivity (100%) while specificity remained relatively low (65.4%). For the combination of mutations on both genes, the odds ratio for detection of resistance increased to 5.31 (P = 0.0005). Here, sensitivity was again decreased to 60.6% while specificity increased to 76.9%. The results of this study suggest that detection of Pfcrt T76 can be applied for predicting chloroquine resistance in epidemiologic settings with sufficiently high sensitivity to make it an attractive alternative to time- and labor-consuming in vivo trials. Additional testing for Pfmdr Y76 provides increased specificity to this approach.

Prevalence of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum isolates from southern Mauritania.

The increasing resistance of Plasmodium falciparum in the treatment of uncomplicated malaria with pyrimethamine/sulphadoxine has been associated in several studies with the occurrence of point mutations in the genes of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). In this study, the prevalence of these mutations was examined in samples from south-east Mauritania, where atypically strong rainfalls in 1998 and 1999 led to a severe outbreak of falciparum malaria. We analysed 386 samples by polymerase chain reaction (PCR) for infection with P. falciparum, of which 162 (41.97%) were positive. These isolates were examined for point mutations in the genes of DHFR (codons 16, 51, 59, 108 and 164) and DHPS (codons 436, 437, 540, 581 and 613) by nested PCR and subsequent mutation-specific restriction enzyme digest. We found a low overall prevalence of DHFR gene mutations (up to 18.6% of isolates), but a high overall prevalence of DHPS gene mutations (up to 49.1% of isolates). Thus, emerging resistance to antifolate drugs may be expected to develop soon in the investigated area. This study demonstrates the utility of simple, relatively rapid and inexpensive molecular methods and their application in surveillance programmes. Testing for prevalence of point mutations conferring antifolate resistance might help to identify the developing of drug resistance at a very early stage.

Population structure of Plasmodium falciparum isolates during an epidemic in southern Mauritania.

While the population structure of Plasmodium falciparum is well analysed in selected areas with high malaria endemicity in East and West Africa, only limited data are available for low endemicity regions bordering the Saharan desert. This is one of the first studies for the Sahel, where atypically strong rainfalls in 1998 and 1999 led to a severe outbreak of falciparum malaria in south-east Mauritania. During a study on in vivo-drug resistance against chloroquine we collected blood samples of patients with fever in two medical centres located in non-endemic and hypoendemic areas. We analysed 386 samples by polymerase chain reaction for infection with P. falciparum, and 173 (45%) tested positive. The isolates were genotyped for three polymorphic genetic markers: merozoite surface protein 1 (MSP1), MSP2 and glutamate-rich protein (GLURP). Differences between the two regions could be shown in either number of clones per infection or in their distribution on the different allelic groups. While the mean minimal number of clones in the non-endemic region around Aioun was 1.57, blood samples collected in the hypoendemic region around Kobeni showed multiple infections with an average of 2.34 clones (P < 0.001). In addition, clear differences between endemic regions were apparent in three of the investigated allelic groups: RO33 of the MSP1 gene and FC and Indochina of the MSP2 gene.

Ivermectin-facilitated immunity in onchocerciasis; activation of parasite-specific Th1-type responses with subclinical Onchocerca volvulus infection.

The present study examined the quantitative and qualitative changes registered in the parasite-specific antibody response, cellular reactivity and cytokine production profile in onchocerciasis patients repeatedly treated with ivermectin over a period of 8 years. The densities of Onchocerca volvulus microfilariae (mf) in treated patients remained significantly reduced, whereas the number of permanently amicrofilaridermic patients (subclinical infection) increased with repeated treatments. In vitro cellular responses to O. volvulus antigen (OvAg) were highest (P < 0.01) in untreated control individuals exposed to infection, but negative for mf of O. volvulus (endemic normals). Cellular reactivity in repeatedly treated patients was higher at 84 than at 36 months post initial treatment (p.i.t); furthermore, the proliferative responses to OvAg, mycobacterial purified protein derivative (PPD) and streptococcal SL-O were greater (P < 0.05) at 84 months p.i.t. in amicrofilaridermic than in microfilaria-positive onchocerciasis patients. In amicrofilaridermic patients such reactivity approached the magnitude observed in endemic normals. Peripheral blood mononuclear cells (PBMC) from patients and endemic normals produced equivalent amounts of IL-2, IL-4 and interferon-gamma (IFN-gamma) in response to mitogenic stimulation with phytohaemagglutinin (PHA); in response to OvAg, however, significantly more IL-2 and IFN-gamma were produced by PBMC from subclinical amicrofilaridermic patients or endemic normals than by mf-positive patients. OvAg-specific production of IL-4 by PBMC from treated patients was lower at 84 than at 36 months p.i.t. At three months p.i.t. the titres of circulating OvAg-specific IgG1-3 had increased (P < 0.05), but they then continuously declined with repeated treatments. Only IgG1 and IgG4 bound to OvAg of mol. wt 2-12 kD at 1 month p.i.t., while recognition of OvAg of mol. wt 10-200 kD by IgG1, IgG2 and IgG4 reached a maximum intensity at 3-6 months p.i.t., with the overall intensity of binding to OvAg gradually weakening thereafter. These results suggest that onchocerciasis-associated immunosuppression is reversible following ivermectin-induced permanent clearance of microfilariae from the skin; and that a vigorous parasite-specific cellular reactivity and a sustained production of IL-2 and IFN-gamma in amicrofilaridermic individuals may contribute to controlling O. volvulus infection.

Efficacy of ivermectin in the treatment of concomitant Mansonella perstans infections in onchocerciasis patients.

As part of an ivermectin dose-ranging study of onchocerciasis patients in Togo, 55 onchocerciasis patients with concomitant mansonelliasis received single oral doses either of ivermectin (100 to 200 micrograms/kg body weight) or placebo. As expected, Onchocerca volvulus microfilariae in the skin were greatly reduced in number soon after drug treatment, but microfilariae of Mansonella perstans reacted differently. Microfilarial densities of M. perstans were assessed with a filtration technique both before, and 4 times after, treatment. In untreated patients microfilarial densities were stable until the end of the study at 6 months. In patients receiving ivermectin, microfilarial densities dropped on average to less than 60% of the pre-treatment level and remained there until the final post-treatment examination. This partial reduction was probably not caused by a microfilaricidal effect of ivermectin, but rather by an altered distribution of microfilariae in the peripheral blood and in a suspected microfilarial reservoir.

Ivermectin-facilitated immunity in onchocerciasis. Reversal of lymphocytopenia, cellular anergy and deficient cytokine production after single treatment.

A longitudinal investigation has been conducted into the cell-mediated immune responses of onchocerciasis patients after a single-dose treatment with ivermectin. Untreated patients tested for delayed cutaneous hypersensitivity (DCH) to seven recall antigens showed lower responses than infection-free control individuals (P less than 0.01), but 6 and 14 months after treatment DCH reactions increased to similar levels to those seen in the controls. The in vitro cellular reactivity to Onchocerca volvulus-derived antigen (OvAg) was reduced in untreated patients as compared with controls, and the lymphocyte blastogenic responses to OvAg and streptolysin-O clearly improved up to 14 months after treatment. Peripheral blood mononuclear cells (PBMC) from untreated patients produced IL-1 beta, tumour necrosis factor-alpha (TNF-alpha) and IL-6 in response to mitogenic stimulation with phytohaemagglutinin (PHA), only low levels of IL-1 beta, IL-2 and TNF-alpha in response to OvAg, but higher amounts of IL-4 and interferon-gamma (IFN-gamma) in response to OvAg than control individuals. After ivermectin treatment, the OvAg-induced production of IL-1 beta and TNF-alpha increased significantly 1 and 14 months after treatment. The PHA-induced production of IL-2 and IL-4 increased 1 month after treatment and remained significantly elevated until 14 months after treatment, whereas the OvAg-specific secretion of IL-2, IL-4 and IFN-gamma did not change after ivermectin treatment. Flow cytometric analysis of lymphocyte-subsets in the peripheral blood of untreated patients revealed a relative and absolute (P less than 0.01) diminution of CD4+ cells and a significantly smaller CD4+/CD8+ cell ratio as compared with controls. By 4 weeks after treatment and thereafter, CD4+ T cells increased relatively and absolutely (P less than 0.01); likewise there was an absolute increase in T-helper-inducer cells (CD4+CD45RO+) and a temporarily improved CD4+/CD8+ cell ratio (P = 0.001). The expression of the low-affinity receptor for IgE (CD23) on total lymphocytes decreased from 14% to 7% by 14 months after treatment. The CD8+ cells and CD3+TCR gamma delta + cells were higher in patients than in controls and both remained elevated until 14 months after treatment. These results suggest a distinctly improved cellular immunity in human onchocerciasis that was facilitated by ivermectin therapy.

[Ivermectin in the treatment of onchocerciasis: feasibility, compliance and efficacy of a mass treatment]. / Ivermectine dans le traitement de l'onchocercose: faisabilité, compliance et efficacité d'un traitment de masse.

We have carried out a massive treatment by ivermectin on 2,425 people affected with onchocercosis (River Blindness) in six villages where this disease is endemic. The people who were affected with the disease and were to be treated have accepted the treatment. The secondary reactions that the patients had experienced had the highest effects on the second and third day after receiving treatment. These secondary reactions were easily controlled. This study has permitted us to notice that the treatment of onchocercosis by ivermectin on a large scale depends on many factors which are as follows: the very long period of treatment, the importance of becoming aware of the disease, the control of secondary reactions, absenteeism, the high mobility of the population and the ethical problems.