Viability and Burden of Leishmania in Extralesional Sites during Human Dermal Leishmaniasis

Understanding of the dynamics and distribution of Leishmania in the human host is fundamental to the targeting of control measures and their evaluation. Amplification of parasite gene sequences in clinical samples from cutaneous leishmaniasis patients has provided evidence of Leishmania in blood, other tissues and sites distinct from the lesion and of persistence of infection after clinical resolution of disease. However, there is uncertainty about the interpretation of the presence of Leishmania DNA as indicative of viable parasites. Because RNA is short-lived and labile, its presence provides an indicator of viability. We amplified Leishmania 7SLRNA, a molecule involved in intracellular protein translocation, to establish viability and estimate parasite load in blood monocytes, tonsil swab samples, and tissue fluid from healthy skin of patients with dermal leishmaniasis. Results showed that during active dermal leishmaniasis, viable Leishmania are present in blood monocytes, tonsils and normal skin in quantities similar to that in lesions, demonstrating widespread dissemination of infection and subclinical involvement of tissues beyond the lesion site. Leishmania 7SLRNA will be useful in deciphering the role of human infection in transmission

Phase II Evaluation of Sensitivity and Specificity of PCR and NASBA Followed by Oligochromatography for Diagnosis of Human African Trypanosomiasis in Clinical Samples from D.R. Congo and Uganda

Diagnosis plays a central role in the control of human African trypanosomiasis (HAT) whose mainstay in disease control is chemotherapy. However, accurate diagnosis is hampered by the absence of sensitive techniques for parasite detection. Without concentrating the blood, detection thresholds can be as high as 10,000 trypanosomes per milliliter of blood. The polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) are promising molecular diagnostics that generally yield high sensitivity and could improve case detection. Recently, these two tests were coupled to oligochromatography (OC) for simplified and standardized detection of amplified products, eliminating the need for electrophoresis. In this study, we evaluated the diagnostic accuracy of these two novel tests on blood specimens from HAT patients and healthy endemic controls from D.R. Congo and Uganda. Both tests exhibited good sensitivity and specificity compared to the current diagnostic tests and may be valuable tools for sensitive and specific parasite detection in clinical specimens. These standardized molecular test formats open avenues for improved case detection, particularly in epidemiological studies and in disease diagnosis at reference centres

Absence of the MGMT protein as well as methylation of the MGMT promoter predict the sensitivity for temozolomide

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) can cause resistance to the alkylating drug temozolomide (TMZ). The purpose of this study was to determine the relationship between the MGMT status, determined by means of several techniques and methods, and the cytotoxic response to TMZ in 11 glioblastoma multiforme (GBM) cell lines and 5 human tumour cell lines of other origins. Cell survival was analysed by clonogenic assay. The MGMT protein levels were assessed by western blot analysis. The MGMT promoter methylation levels were determined using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and quantitative real-time methylation-specific PCR (qMSP). On the basis of the results of these techniques, six GBM cell lines were selected and subjected to bisulphite sequencing. The MGMT protein was detected in all TMZ-resistant cell lines, whereas no MGMT protein could be detected in cell lines that were TMZ sensitive. The MS-MLPA results were able to predict TMZ sensitivity in 9 out of 16 cell lines (56%). The qMSP results matched well with TMZ sensitivity in 11 out of 12 (92%) glioma cell lines. In addition, methylation as detected by bisulphite sequencing seemed to be predictive of TMZ sensitivity in all six cell lines analysed (100%). The MGMT protein expression more than MGMT promoter methylation status predicts the response to TMZ in human tumour cell line

Mucosal Leishmaniasis Caused by Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in the Brazilian Amazon

Background: Leishmania (Viannia) braziliensis is a parasite recognized as the most important etiologic agent of mucosal leishmaniasis (ML) in the New World. In Amazonia, seven different species of Leishmania, etiologic agents of human Cutaneous Leishmaniasis, have been described. Isolated cases of ML have been described for several different species of Leishmania: L. (V.) panamensis, L. (V.) guyanensis and L. (L.) amazonensis. Methodology: Leishmania species were characterized by polymerase chain reaction (PCR) of tissues taken from mucosal biopsies of Amazonian patients who were diagnosed with ML and treated at the Tropical Medicine Foundation of Amazonas (FMTAM) in Manaus, Amazonas state, Brazil. Samples were obtained retrospectively from the pathology laboratory and prospectively from patients attending the aforementioned tertiary care unit. Results: This study reports 46 cases of ML along with their geographical origin, 30 cases caused by L. (V.) braziliensis and 16 cases by L. (V.) guyanensis. This is the first record of ML cases in 16 different municipalities in the state of Amazonas and of simultaneous detection of both species in 4 municipalities of this state. It is also the first record of ML caused by L. (V.) guyanensis in the states of Para, Acre, and Rondonia and cases of ML caused by L. (V.) braziliensis in the state of Rondonia. Conclusions/Significance: L. (V.) braziliensis is the predominant species that causes ML in the Amazon region. However, contrary to previous studies, L. (V.) guyanensis is also a significant causative agent of ML within the region. The clinical and epidemiological expression of ML in the Manaus region is similar to the rest of the country, although the majority of ML cases are found south of the Amazon River.SUFRAMA[016/2004

Quantification of the response to miltefosine treatment for visceral leishmaniasis by QT-NASBA

A male patient with psoriatic arthritis and visceral Leishmania infantum infection was treated with oral miltefosine 50 mg three times a day for 4 weeks at the Academic Medical Center, Amsterdam, The Netherlands. Miltefosine plasma concentrations were measured with liquid chromatography/mass spectrometry. The parasite load was followed by quantitative nucleic acid sequence-based amplification (QT-NASBA) assay in blood. Miltefosine elicited a prompt therapeutic effect. After an initial worsening of symptoms and an increase of QT-NASBA values during the first week, recovery was rapidly achieved. QT-NASBA values declined exponentially and were negative after 6 weeks. Miltefosine plasma concentrations continued to accumulate during the 4 weeks of treatment. The terminal elimination half-life was 14.8 day

Treatment assessment by monitoring parasite load in skin biopsies from patients with cutaneous leishmaniasis, using quantitative nucleic acid sequence-based amplification

BACKGROUND: Current diagnostic methods for cutaneous leishmaniasis (CL) have low sensitivity or are not useful for treatment follow-up. We previously described the quantitative nucleic acid sequence-based amplification (QT-NASBA) method as a sensitive and specific assay for detection and quantification of Leishmania parasites in skin biopsies. This assay could be a valuable instrument for monitoring response to treatment of CL and identifying treatment failures at an early stage. AIM: QT-NASBA results of skin biopsies at the end and 6 weeks after treatment from patients with proven CL on various treatment regimens were compared with clinical outcome. METHODS: The QT-NASBA assay measured the parasite load in skin biopsies before, at the end and 6 weeks after treatment. The results were compared with treatment outcome (clinical cure, delayed healing response or treatment failure) up to 6 months after treatment. RESULTS: In total, 137 skin biopsies were obtained from 53 patients. A positive QT-NASBA result 6 weeks after treatment was significantly associated with treatment failure/delayed healing up to 6 months (P < 0.001). The positive predictive value (PPV) was 100% and the negative predictive value (NPV) was 92% (95% CI 82-100%). QT-NASBA results at the end of treatment and clinical outcome showed a less significant association (P < 0.05), with a PPV of 46% (95% CI 16-75% and an NPV of 89% (95% CI 79-99%). CONCLUSIONS: The QT-NASBA assay is a useful instrument to monitor parasite load in skin biopsies of patients with CL 6 weeks after treatment and can help to predict clinical outcom

Culicoides obsoletus extract relevant for diagnostics of insect bite hypersensitivity in horses

Insect bite hypersensitivity (IBH) is an allergic dermatitis in horses caused by the bites of Culicoides species. The aim of the present study was to evaluate the applicability of whole body extracts of C. obsoletus (the main species found feeding on horses in the Netherlands), C. nubeculosus (rarely found in The Netherlands) and C. sonorensis (typical for North America) for diagnosis of IBH in horses in The Netherlands. Blood and serum samples of 10 clinically confirmed IBH affected and 10 healthy control horses were used to evaluate the IgE titers (ELISA) against the Culicoides whole body extracts of the three Culicoides species. Basophil degranulation was assessed by histamine release test (HRT) after stimulation with these extracts at 5, 0.5 and 0.05 µg/ml. IBH affected horses had significantly higher IgE titers against C. obsoletus than against C. nubeculosus and C. sonorensis. Furthermore, C. obsoletus induced significantly higher histamine release in whole blood of IBH affected horses compared to the other extracts at 0.5 µg/ml. Western blot data revealed IgE binding to many proteins in C. obsoletus extract. This interaction was absent or weak in C. nubeculosus and C. sonorensis extracts for IBH affected horses. Results on individual level indicate that the HRT is more sensitive than ELISA in diagnosing IBH. However, ELISA is more practical as a routine test, therefore the ELISA was further evaluated using C. obsoletus extract on 103 IBH affected and 100 healthy horses, which resulted in a test sensitivity and specificity of 93.2% and 90.0%, respectively. The IgE ELISA readings enabled the analysis of the predicted probability of being IBH affected. From an optical density 450 nm value of 0.33 onwards, the probability of IBH affected was more than 0.9. The results presented in this paper show that the use of native Culicoides spp. that feed on horse, is important for improved diagnosis and that the described ELISA based on C. obsoletus can be used routinely to diagnose IBH in countries where this species is the main Culicoides feeding on horse

Miltefosine Treatment of Leishmania major Infection: An Observational Study Involving Dutch Military Personnel Returning from Northern Afghanistan

In a retrospective, observational study involving 34 patients with Leishmania major infection, 31 of whom had experienced unsuccessful treatment with intralesional antimony (ilSb(v)), miltefosine proved effective. Thirty patients experienced cure after receipt of miltefosine, 3 after receipt of additional ilSbv, and 1 after 28 daily intravenous injections of antimony. Temporary diminution of ejaculate volume was reported by 21 patient