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1.  Primary cultivation: factors affecting contamination and Mycobacterium ulcerans growth after long turnover time of clinical specimens 
BMC Infectious Diseases  2014;14(1):636.
Background
While cultivation of pathogens represents a foundational diagnostic approach in the study of infectious diseases, its value for the confirmation of clinical diagnosis of Buruli ulcer is limited by the fact that colonies of Mycobacterium ulcerans appear only after about eight weeks of incubation at 30°C. However, for molecular epidemiological and drug sensitivity studies, primary isolation of M. ulcerans remains an essential tool. Since for most of the remote Buruli ulcer endemic regions of Africa cultivation laboratories are not easily accessible, samples from lesions often have to be stored for extended periods of time prior to processing. The objective of the current study therefore was to determine which transport medium, decontamination method or other factors decrease the contamination rate and increase the chance of primary isolation of M. ulcerans bacilli after long turnover time.
Methods
Swab and fine needle aspirate (FNA) samples for the primary cultivation were collected from clinically confirmed Buruli ulcer patients in the Mapé Basin of Cameroon. The samples were either stored in the semi-solid transport media 7H9 or Amies or dry for extended period of time prior to processing. In the laboratory, four decontamination methods and two inoculation media were evaluated and statistical methods applied to identify factors that decrease culture contamination and factors that increase the probability of M. ulcerans recovery.
Results
The analysis showed: i) that the use of moist transport media significantly increased the recovery rate of M. ulcerans compared to samples kept dry; ii) that the choice of the decontamination method had no significant effect on the chance of M. ulcerans isolation; and iii) that Löwenstein-Jensen supplemented with antibiotics as inoculation medium yielded the best results. We further found that, ten extra days between sampling and inoculation lead to a relative decrease in the isolation rate of M. ulcerans by nearly 20%. Finally, collection and processing of multiple samples per patient was found to significantly increase the M. ulcerans isolation rate.
Conclusions
Based on our analysis we suggest a procedure suitable for the primary isolation of M. ulcerans strains from patients following long delay between sample collection and processing to establish a M. ulcerans strain collection for research purposes.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-014-0636-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s12879-014-0636-7
PMCID: PMC4264541  PMID: 25433390
Buruli ulcer; Mycobacterium ulcerans; Primary cultivation; Long turnover time
2.  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.
Author Summary
Although M. ulcerans, the causative agent of Buruli ulcer (BU), was identified in 1948, its transmission pathways and environmental reservoirs remain poorly understood. The occurrence of M. ulcerans infections in endemic countries in West and Central Africa is highly focal and associated with stagnant and slow flowing water bodies. BU is often described as a disease mainly affecting children <15 years of age. However, taking the population age distribution into account, our recent longitudinal survey for BU in the Mapé Dam Region of Cameroon revealed that clinical cases of BU among children <5 years are relatively rare. In accordance with these findings, data of the present sero-epidemiological study indicate that children <4 years old are less exposed to M. ulcerans than older children. Sero-conversion is associated with age, which may be due to age-related changes in behavioural factors, such as a wider movement radius of older children, including more frequent contact with water bodies at the periphery of their villages.
doi:10.1371/journal.pntd.0002904
PMCID: PMC4031220  PMID: 24853088
3.  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.
Author Summary
Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans which affects mainly children in West Africa. Although it is commonly believed that the infection originates from an environmental source, both the reservoir of M. ulcerans and the mode of transmission to human patients remain to be elucidated. Previous investigations indicated that transmission likely takes place away from the homes of patients. We therefore screened the farms as well as village and farm water locations of 46 laboratory confirmed BU patients of the Mapé Basin of Cameroon for the presence of M. ulcerans DNA by real-time PCR. In this analysis three positive village water locations were identified. By studying one of these locations in great detail we found that M. ulcerans DNA persists in underwater detritus in one section of the village water location even after all local cases had been treated. The detritus may represent a reservoir of M. ulcerans from where infection could take place through either direct contamination of skin lesions or through contamination or colonization of insect vectors.
doi:10.1371/journal.pntd.0002756
PMCID: PMC3967953  PMID: 24675964
4.  Geographic Distribution, Age Pattern and Sites of Lesions in a Cohort of Buruli Ulcer Patients from the Mapé Basin of Cameroon 
Buruli ulcer (BU), a neglected tropical disease of the skin, caused by Mycobacterium ulcerans, occurs most frequently in children in West Africa. Risk factors for BU include proximity to slow flowing water, poor wound care and not wearing protective clothing. Man-made alterations of the environment have been suggested to lead to increased BU incidence. M. ulcerans DNA has been detected in the environment, water bugs and recently also in mosquitoes. Despite these findings, the mode of transmission of BU remains poorly understood and both transmission by insects or direct inoculation from contaminated environment have been suggested. Here, we investigated the BU epidemiology in the Mapé basin of Cameroon where the damming of the Mapé River since 1988 is believed to have increased the incidence of BU. Through a house-by-house survey in spring 2010, which also examined the local population for leprosy and yaws, and continued surveillance thereafter, we identified, till June 2012, altogether 88 RT-PCR positive cases of BU. We found that the age adjusted cumulative incidence of BU was highest in young teenagers and in individuals above the age of 50 and that very young children (<5) were underrepresented among cases. BU lesions clustered around the ankles and at the back of the elbows. This pattern neither matches any of the published mosquito biting site patterns, nor the published distribution of small skin injuries in children, where lesions on the knees are much more frequent. The option of multiple modes of transmission should thus be considered. Analyzing the geographic distribution of cases in the Mapé Dam area revealed a closer association with the Mbam River than with the artificial lake.
Author Summary
Buruli ulcer (BU) is an infectious disease caused by Mycobacterium ulcerans that is affecting mostly children in endemic areas of West Africa. Proximity to slow flowing water is a risk factor, but the exact mode of transmission of BU remains unclear. Man-made environmental changes, such as sand mining, damming of rivers and irrigation have been implicated with increases in disease incidence. Here, we report findings from a survey for BU and continued case detection thereafter in the Bankim Health District of Cameroon. In this area, the local population believed that the damming of the Mapé River has led to the emergence of BU. In 28 months we identified 88 laboratory confirmed cases of BU. Studying these cases, we found that the age adjusted cumulative incidence of BU in the elderly is similar to that in children and that the distribution pattern of BU lesions neither matches mosquito biting patterns nor the distribution of small skin injuries. Multiple modes of transmission should therefore be considered. Our data further showed that the patients appear to have closer contact to the local Mbam River than to the artificial Mapé dam reservoir.
doi:10.1371/journal.pntd.0002252
PMCID: PMC3681622  PMID: 23785529
6.  Mycolic acids as diagnostic markers for tuberculosis case detection in humans and drug efficacy in mice 
EMBO Molecular Medicine  2012;4(1):27-37.
Mycolic acids are attractive diagnostic markers for tuberculosis (TB) infection because they are bacteria-derived, contain information about bacterial species, modulate host–pathogen interactions and are chemically inert. Here, we present a novel approach based on mass spectrometry. Quantification of specific precursor → fragment transitions of approximately 2000 individual mycolic acids (MAs) resulted in high analytical sensitivity and specificity. We next used this tool in a retrospective case–control study of patients with pulmonary TB with varying disease burdens from South Korea, Vietnam, Uganda and South Africa. MAs were extracted from small volume sputum (200 µl) and analysed without the requirement for derivatization. Infected patients (70, 19 of whom were HIV+) could be separated from controls (40, 20 of whom were HIV+) with a sensitivity and specificity of 94 and 93%, respectively. Furthermore, we quantified MA species in lung tissue of TB-infected mice and demonstrated effective clearance of MA levels following curative rifampicin treatment. Thus, our results demonstrate for the first time the feasibility and clinical relevance of direct detection of mycobacterial lipids as biomarkers of TB infection.
doi:10.1002/emmm.201100185
PMCID: PMC3376831  PMID: 22147526
diagnostic marker; lipidomics; mass spectrometry; Mycobacterium tuberculosis; mycolic acids
7.  Glycerol-3-Phosphate Acyltransferases Gat1p and Gat2p Are Microsomal Phosphoproteins with Differential Contributions to Polarized Cell Growth▿ †  
Eukaryotic Cell  2009;8(8):1184-1196.
Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial step in the synthesis of all glycerolipids. It is the committed and rate-limiting step and is redundant in Saccharomyces cerevisiae, mammals, and plants. GPAT controls the formation of lipid intermediates that serve not only as precursors of more-complex lipids but also as intracellular signaling molecules. Saccharomyces cerevisiae possesses two GPATs, encoded by the GAT1 and GAT2 genes. Metabolic analysis of yeast lacking either GAT1 or GAT2 indicated partitioning of the two main branches of phospholipid synthesis at the initial and rate-limiting GPAT step. We are particularly interested in identifying molecular determinants mediating lipid metabolic pathway partitioning; therefore, as a starting point, we have performed a detailed study of Gat1p and Gat2p cellular localization. We have compared Gat1p and Gat2p localization by fluorescence microscopy and subcellular fractionation using equilibrium density gradients. Our results indicate Gat1p and Gat2p overlap mostly in their localization and are in fact microsomal GPATs, localized to both perinuclear and cortical endoplasmic reticula in actively proliferating cells. A more detailed analysis suggests a differential enrichment of Gat1p and Gat2p in distinct ER fractions. Furthermore, overexpression of these enzymes in the absence of endogenous GPATs induces proliferation of distinct ER arrays, differentially affecting cortical ER morphology and polarized cell growth. In addition, our studies also uncovered a dynamic posttranslational regulation of Gat1p and Gat2p and a compensation mechanism through phosphorylation that responds to a cellular GPAT imbalance.
doi:10.1128/EC.00085-09
PMCID: PMC2725570  PMID: 19525420

Results 1-7 (7)