Exudate collection using wound sponges-An easy, non-invasive and reliable method to explore protease activities in ulcers.

Proteases are important for wound healing, but in excessive amounts or left uncontrolled, they may cause healing impairment or other severe wound complications. Point-of-care testing for protease activities in wounds may be useful for monitoring the effectiveness of treatment, and for early identification of wounds that potentially fail to heal. Here we describe an easy, noninvasive method to collect wound fluid for evaluating the protease milieu of wounds. Wound fluids were collected using sterile sponges applied between wound surface and normal wound dressing. Wound fluid could be easily squeezed or centrifuged out of the sponges and was tested for gelatinase (MMP-2 and MMP-9) activities by gel zymography. In addition, we measured polymorphonuclear granulocyte elastase levels by ELISA. Both gelatinases were remarkably stable in sponge derived fluids, as no significant loss was observed even when samples were stored for 3 days at room temperature. Protease levels were highly diverse amongst patients and, in some cases, showed substantial variations in the course of the treatment. The here described wound sponge approach represents a patient-friendly and reliable method to collect wound fluid for evaluating wound healing relevant biomarkers, such as matrix metalloproteinases.

Multiple nucleotide substitutions in the Neisseria meningitidis serogroup C ctrA gene cause false-negative detection by real-time PCR.

Two strains of Neisseria meningitidis serogroup C with disparate sequences of ctrA were isolated. The nucleotide substitutions did not alter the corresponding protein sequences, but they impeded the detection of these meningococcal isolates by real-time PCR.

Evaluation of a PfHRP-2 based rapid diagnostic test versus microscopy method among HIV-positive and unknown serology patients in Ouagadougou, Burkina Faso.

We evaluated the performance of a malaria rapid diagnostic test (RDT; Malaria Quick Test(®); Cypress Diagnostic) compared with the standard thick-smear microscopy method using blood samples from human immunodeficiency virus (HIV)-infected individuals and individuals of unknown HIV status collected in Ouagadougou, Burkina Faso. Our results show that 42.1% of 114 HIV-infected patients were concordantly RDT- and thick smear-positive, and 55.3% were concordantly negative. Sensitivity and specificity of the RDT test were 100.0% and 95.4%, respectively, with 5.9% false-positive results and a total agreement of 97.4%; 127 patients with unknown HIV serology were analyzed; of them, 40.9% were RDT- and thick smear-positive, and 46.4% concordantly negative. Sensitivity and specificity were 100.0% and 78.6%, respectively, with 23.5% false-positive results and a total agreement of 87.4%. Malaria Quick Test(®) is rapid and effective for the diagnosis of malaria and has a high sensitivity, confirming its use in general and HIV patients in particular.

Immunohistochemical monitoring of wound healing in antibiotic treated Buruli ulcer patients.

BACKGROUND: While traditionally surgery has dominated the clinical management of Buruli ulcer (BU), the introduction of the combination chemotherapy with oral rifampicin and intramuscular streptomycin greatly improved treatment and reduced recurrence rates. However management of the often extensive lesions after successful specific therapy has remained a challenge, in particular in rural areas of the African countries which carry the highest burden of disease. For reasons not fully understood, wound healing is delayed in a proportion of antibiotic treated BU patients. Therefore, we have performed immunohistochemical investigations to identify markers which may be suitable to monitor wound healing progression. METHODOLOGY/PRINCIPAL FINDINGS: Tissue specimens from eight BU patients with plaque lesions collected before, during and after chemotherapy were analyzed by immunohistochemistry for the presence of a set of markers associated with connective tissue neo-formation, tissue remodeling and epidermal activation. Several target proteins turned out to be suitable to monitor wound healing. While α-smooth muscle actin positive myofibroblasts were not found in untreated lesions, they emerged during the healing process. These cells produced abundant extracellular matrix proteins, such as pro-collagen 1 and tenascin and were found in fibronectin rich areas. After antibiotic treatment many cells, including myofibroblasts, revealed an activated phenotype as they showed ribosomal protein S6 phosphorylation, a marker for translation initiation. In addition, healing wounds revealed dermal tissue remodeling by apoptosis, and showed increased cytokeratin 16 expression in the epidermis. CONCLUSION/SIGNIFICANCE: We have identified a set of markers that allow monitoring wound healing in antibiotic treated BU lesions by immunohistochemistry. Studies with this marker panel may help to better understand disturbances responsible for wound healing delays observed in some BU patients.

Late onset of the serological response against the 18 kDa small heat shock protein of Mycobacterium ulcerans in children.

A previous survey for clinical cases of Buruli ulcer (BU) in the Mapé Basin of Cameroon suggested that, compared to older age groups, very young children may be less exposed to Mycobacterium ulcerans. Here we determined serum IgG titres against the 18 kDa small heat shock protein (shsp) of M. ulcerans in 875 individuals living in the BU endemic river basins of the Mapé in Cameroon and the Densu in Ghana. While none of the sera collected from children below the age of four contained significant amounts of 18 kDa shsp specific antibodies, the majority of sera had high IgG titres against the Plasmodium falciparum merozoite surface protein 1 (MSP-1). These data suggest that exposure to M. ulcerans increases at an age which coincides with the children moving further away from their homes and having more intense environmental contact, including exposure to water bodies at the periphery of their villages.

Mycobacterium ulcerans persistence at a village water source of Buruli ulcer patients.

Buruli ulcer (BU), a neglected tropical disease of the skin and subcutaneous tissue, is caused by Mycobacterium ulcerans and is the third most common mycobacterial disease after tuberculosis and leprosy. While there is a strong association of the occurrence of the disease with stagnant or slow flowing water bodies, the exact mode of transmission of BU is not clear. M. ulcerans has emerged from the environmental fish pathogen M. marinum by acquisition of a virulence plasmid encoding the enzymes required for the production of the cytotoxic macrolide toxin mycolactone, which is a key factor in the pathogenesis of BU. Comparative genomic studies have further shown extensive pseudogene formation and downsizing of the M. ulcerans genome, indicative for an adaptation to a more stable ecological niche. This has raised the question whether this pathogen is still present in water-associated environmental reservoirs. Here we show persistence of M. ulcerans specific DNA sequences over a period of more than two years at a water contact location of BU patients in an endemic village of Cameroon. At defined positions in a shallow water hole used by the villagers for washing and bathing, detritus remained consistently positive for M. ulcerans DNA. The observed mean real-time PCR Ct difference of 1.45 between the insertion sequences IS2606 and IS2404 indicated that lineage 3 M. ulcerans, which cause human disease, persisted in this environment after successful treatment of all local patients. Underwater decaying organic matter may therefore represent a reservoir of M. ulcerans for direct infection of skin lesions or vector-associated transmission.