Specific K39 antibody response and its persistence after treatment in patients with imported leishmaniasis.

The sensitivity of a K39 ELISA (Leishmania IgG, Virion/Serion) for the detection of antibodies in patients with imported leishmaniasis was compared with an immunofluorescence assay (IFA), which was applied as "golden standard". The retrospective study comprised 93 IFA-positive or borderline sera from 42 patients with visceral (n = 16) or cutaneous (n = 26) leishmaniasis. Patients had acquired infection predominately in the Mediterranean area or the Middle East. The Leishmania species (Leishmania donovani/infantum, Leishmania tropica, Leishmania major) were identified by real-time PCR. The majority (94%) of first samples from patients with visceral leishmaniasis (VL) tested positive by K39 ELISA. Antibody levels ranged from low to very high (33.19-1990.00 U/ml; median 596.66 U/ml) but did not correlate with the respective IFA titers. High K39 ELISA values correlated with acute infection in immunocompetent individuals. K39 antibodies declined in all individuals after clinically successful therapy, but time to seronegativity varied considerably (51 weeks to >6 years). In patients with cutaneous leishmaniasis (CL), the sensitivity of the K39 ELISA was low (23%) compared to IFA (92% positive). Antibody levels ranged from low to medium (10.85-524.77 U/ml; median 19.77 U/ml). The highest antibody concentrations were seen in L. infantum-infected individuals. Summarizing, a high K39 ELISA value indicates active VL. The assay is, like IFA, not a measure for effective therapy but may support post-treatment monitoring. Low level positivity can indicate subclinical, previous or clinically cured VL or even CL. The K39 ELISA can supplement highly sensitive screening tests in the diagnosis and follow-up of imported leishmaniasis.

In vitro activity of wALADin benzimidazoles against different life cycle stages of Plasmodium parasites.

wALADin1 benzimidazoles are specific inhibitors of δ-aminolevulinic acid dehydratase from Wolbachia endobacteria of filarial nematodes. We report that wALADin1 and two derivatives killed blood stage Plasmodium falciparum in vitro (50% inhibitory concentrations, 39, 7.7, and 12.8 µM, respectively). One of these derivatives inhibited gliding motility of Plasmodium berghei ANKA infectious sporozoites with nanomolar affinity and blocked invasion into hepatocytes but did not affect intrahepatocytic replication. Hence, wALADin1 benzimidazoles are tools to study gliding motility and potential antiplasmodial drug candidates.

Chagas Disease: Detection of Trypanosoma cruzi by a New, High-Specific Real Time PCR.

BACKGROUND: Chagas disease (CD) is a major burden in Latin America, expanding also to non-endemic countries. A gold standard to detect the CD causing pathogen Trypanosoma cruzi is currently not available. Existing real time polymerase chain reactions (RT-PCRs) lack sensitivity and/or specificity. We present a new, highly specific RT-PCR for the diagnosis and monitoring of CD. MATERIAL AND METHODS: We analyzed 352 serum samples from Indigenous people living in high endemic CD areas of Colombia using three leading RT-PCRs (k-DNA-, TCZ-, 18S rRNA-PCR), the newly developed one (NDO-PCR), a Rapid Test/enzyme-linked immuno sorbent assay (ELISA), and immunofluorescence. Eighty-seven PCR-products were verified by sequence analysis after plasmid vector preparation. RESULTS: The NDO-PCR showed the highest sensitivity (92.3%), specificity (100%), and accuracy (94.3%) for T. cruzi detection in the 87 sequenced samples. Sensitivities and specificities of the kDNA-PCR were 89.2%/22.7%, 20.5%/100% for TCZ-PCR, and 1.5%/100% for the 18S rRNA-PCR. The kDNA-PCR revealed a 77.3% false positive rate, mostly due to cross-reactions with T. rangeli (NDO-PCR 0%). TCZ- and 18S rRNA-PCR showed a false negative rate of 79.5% and 98.5% (NDO-PCR 7.7%), respectively. CONCLUSIONS: The NDO-PCR demonstrated the highest specificity, sensitivity, and accuracy compared to leading PCRs. Together with serologic tests, it can be considered as a reliable tool for CD detection and can improve CD management significantly.

Combinations of registered drugs reduce treatment times required to deplete Wolbachia in the Litomosoides sigmodontis mouse model.

Filarial parasites can be targeted by antibiotic treatment due to their unique endosymbiotic relationship with Wolbachia bacteria. This finding has led to successful treatment strategies in both, human onchocerciasis and lymphatic filariasis. A 4-6 week treatment course using doxycycline results in long-term sterility and safe macrofilaricidal activity in humans. However, current treatment times and doxycycline contraindications in children and pregnant women preclude widespread administration of doxycycline in public health control programs; therefore, the search for shorter anti-wolbachial regimens is a focus of ongoing research. We have established an in vivo model for compound screening, using mice infected with Litomosoides sigmodontis. We could show that gold standard doxycycline treatment did not only deplete Wolbachia, it also resulted in a larval arrest. In this model, combinations of registered antibiotics were tested for their anti-wolbachial activity. Administration of rifamycins in combination with doxycycline for 7 days successfully depleted Wolbachia by > 2 log (>99% reduction) and thus resulted in a significant reduction of the treatment duration. Using a triple combination of a tetracycline (doxycycline or minocycline), a rifamycin and a fluoroquinolone (moxifloxacin) led to an even greater shortening of the treatment time. Testing all double combinations that could be derived from the triple combinations revealed that the combination of rifapentine (15mg/kg) and moxifloxacin (2 x 200mg/kg) showed the strongest reduction of treatment time in intraperitoneal and also oral administration routes. The rifapentine plus moxifloxacin combination was equivalent to the triple combination with additional doxycycline (>99% Wolbachia reduction). These investigations suggest that it is possible to shorten anti-wolbachial treatment times with combination treatments in order to achieve the target product profile (TPP) requirements for macrofilaricidal drugs of no more than 7-10 days of treatment.

Comparison of Doxycycline, Minocycline, Doxycycline plus Albendazole and Albendazole Alone in Their Efficacy against Onchocerciasis in a Randomized, Open-Label, Pilot Trial.

The search for new macrofilaricidal drugs against onchocerciasis that can be administered in shorter regimens than required for doxycycline (DOX, 200mg/d given for 4-6 weeks), identified minocycline (MIN) with superior efficacy to DOX. Further reduction in the treatment regimen may be achieved with co-administration with standard anti-filarial drugs. Therefore a randomized, open-label, pilot trial was carried out in an area in Ghana endemic for onchocerciasis, comprising 5 different regimens: the standard regimen DOX 200mg/d for 4 weeks (DOX 4w, N = 33), the experimental regimens MIN 200mg/d for 3 weeks (MIN 3w; N = 30), DOX 200mg/d for 3 weeks plus albendazole (ALB) 800mg/d for 3 days (DOX 3w + ALB 3d, N = 32), DOX 200mg/d for 3 weeks (DOX 3w, N = 31) and ALB 800mg for 3 days (ALB 3d, N = 30). Out of 158 randomized participants, 116 (74.4%) were present for the follow-up at 6 months of whom 99 participants (63.5%) followed the treatment per protocol and underwent surgery. Histological analysis of the adult worms in the extirpated nodules revealed absence of Wolbachia in 98.8% (DOX 4w), 81.4% (DOX 3w + ALB 3d), 72.7% (MIN 3w), 64.1% (DOX 3w) and 35.2% (ALB 3d) of the female worms. All 4 treatment regimens showed superiority to ALB 3d (p < 0.001, p < 0.001, p = 0.002, p = 0.008, respectively), which was confirmed by real-time PCR. Additionally, DOX 4w showed superiority to all other treatment arms. Furthermore DOX 4w and DOX 3w + ALB 3d showed a higher amount of female worms with degenerated embryogenesis compared to ALB 3d (p = 0.028, p = 0.042, respectively). These results confirm earlier studies that DOX 4w is sufficient for Wolbachia depletion and the desired parasitological effects. The data further suggest that there is an additive effect of ALB (3 days) on top of that of DOX alone, and that MIN shows a trend for stronger potency than DOX. These latter two results are preliminary and need confirmation in a fully randomized controlled phase 2 trial. TRIAL REGISTRATION: ClinicalTrials.gov #06010453.

Induction of chronic colitis in IL-10 deficient mice requires IL-4.

Th 1 cells activated by IL-12 and secreting IFN-gamma have been described as the main mediators for onset and maintenance of chronic colitis in IL-10 deficient mice. It was therefore surprising that mice deficient for IL-4 in addition to IL-10 showed intestinal pathology very rarely, whereas IL-10 KO mice developed rectal prolapse in most cases. To investigate the underlying mechanisms, we studied changes of ongoing inflammatory processes in mice deficient for IL-4, IL-10 or both cytokines. Levels of IFN-gamma, IL-12p40 and MHCII mRNA were elevated to a much higher degree in colonic tissue of IL-10 KO compared to IL-4/10 KO at the onset of colitis. Furthermore, the influx of eosinophils, a marker for Th2 responses, was investigated. Only IL-10 deficient mice displayed a significant increase of eosinophils in the lamina propria of the colon and rectum. In contrast, IL-4/10 deficient mice had eosinophil levels comparable to wildtype controls and IL-4 KO. Together these results indicate an important role of IL-4 for the onset of colitis in IL-10 KO mice by promoting a Th1 response and induction of a deleterious Th2 effector response.

Multivariable Regression Analysis in Schistosoma mansoni-Infected Individuals in the Sudan Reveals Unique Immunoepidemiological Profiles in Uninfected, egg+ and Non-egg+ Infected Individuals.

BACKGROUND: In the Sudan, Schistosoma mansoni infections are a major cause of morbidity in school-aged children and infection rates are associated with available clean water sources. During infection, immune responses pass through a Th1 followed by Th2 and Treg phases and patterns can relate to different stages of infection or immunity. METHODOLOGY: This retrospective study evaluated immunoepidemiological aspects in 234 individuals (range 4-85 years old) from Kassala and Khartoum states in 2011. Systemic immune profiles (cytokines and immunoglobulins) and epidemiological parameters were surveyed in n = 110 persons presenting patent S. mansoni infections (egg+), n = 63 individuals positive for S. mansoni via PCR in sera but egg negative (SmPCR+) and n = 61 people who were infection-free (Sm uninf). Immunoepidemiological findings were further investigated using two binary multivariable regression analysis. PRINCIPAL FINDINGS: Nearly all egg+ individuals had no access to latrines and over 90% obtained water via the canal stemming from the Atbara River. With regards to age, infection and an egg+ status was linked to young and adolescent groups. In terms of immunology, S. mansoni infection per se was strongly associated with increased SEA-specific IgG4 but not IgE levels. IL-6, IL-13 and IL-10 were significantly elevated in patently-infected individuals and positively correlated with egg load. In contrast, IL-2 and IL-1ß were significantly lower in SmPCR+ individuals when compared to Sm uninf and egg+ groups which was further confirmed during multivariate regression analysis. CONCLUSIONS/SIGNIFICANCE: Schistosomiasis remains an important public health problem in the Sudan with a high number of patent individuals. In addition, SmPCR diagnostics revealed another cohort of infected individuals with a unique immunological profile and provides an avenue for future studies on non-patent infection states. Future studies should investigate the downstream signalling pathways/mechanisms of IL-2 and IL-1ß as potential diagnostic markers in order to distinguish patent from non-patent individuals.

Hyperreactive onchocerciasis is characterized by a combination of Th17-Th2 immune responses and reduced regulatory T cells.

Clinical manifestations in onchocerciasis range from generalized onchocerciasis (GEO) to the rare but severe hyperreactive (HO)/sowda form. Since disease pathogenesis is associated with host inflammatory reactions, we investigated whether Th17 responses could be related to aggravated pathology in HO. Using flow cytometry, filarial-specific cytokine responses and PCR arrays, we compared the immune cell profiles, including Th subsets, in individuals presenting the two polar forms of infection and endemic normals (EN). In addition to elevated frequencies of memory CD4+ T cells, individuals with HO showed accentuated Th17 and Th2 profiles but decreased CD4+CD25hiFoxp3+ regulatory T cells. These profiles included increased IL-17A+, IL-4+, RORC2+ and GATA3+CD4+ T cell populations. Flow cytometry data was further confirmed using a PCR array since Th17-related genes (IL-17 family members, IL-6, IL-1ß and IL-22) and Th2-related (IL-4, IL-13, STAT6) genes were all significantly up-regulated in HO individuals. In addition, stronger Onchocerca volvulus-specific Th2 responses, especially IL-13, were observed in vitro in hyperreactive individuals when compared to GEO or EN groups. This study provides initial evidence that elevated frequencies of Th17 and Th2 cells form part of the immune network instigating the development of severe onchocerciasis.

Mice deficient in interleukin-4 (IL-4) or IL-4 receptor alpha have higher resistance to sporozoite infection with Plasmodium berghei (ANKA) than do naive wild-type mice.

BALB/c interleukin-4 (IL-4(-/-)) or IL-4 receptor-alpha (IL-4ralpha(-/-)) knockout (KO) mice were used to assess the roles of the IL-4 and IL-13 pathways during infections with the blood or liver stages of plasmodium in murine malaria. Intraperitoneal infection with the blood-stage erythrocytes of Plasmodium berghei (ANKA) resulted in 100% mortality within 24 days in BALB/c mice, as well as in the mutant mouse strains. However, when infected intravenously with the sporozoite liver stage, 60 to 80% of IL-4(-/-) and IL-4ralpha(-/-) mice survived, whereas all BALB/c mice succumbed with high parasitemia. Compared to infected BALB/c controls, the surviving KO mice showed increased NK cell numbers and expression of inducible nitric oxide synthase (iNOS) in the liver and were able to eliminate parasites early during infection. In vivo blockade of NO resulted in 100% mortality of sporozoite-infected KO mice. In vivo depletion of NK cells also resulted in 80 to 100% mortality, with a significant reduction in gamma interferon (IFN-gamma) production in the liver. These results suggest that IFN-gamma-producing NK cells are critical in host resistance against the sporozoite liver stage by inducing NO production, an effective killing effector molecule against Plasmodium. The absence of IL-4-mediated functions increases the protective innate immune mechanism identified above, which results in immunity against P. berghei infection in these mice, with no major role for IL-13.