Evaluation of the fluorescent-thin layer chromatography (f-TLC) for the diagnosis of Buruli ulcer disease in Ghana

BACKGROUND: Buruli ulcer is a tissue necrosis infection caused by an environmental mycobacterium called Mycobacterium ulcerans (MU). The disease is most prevalent in rural areas with the highest rates in West and Central African countries. The bacterium produces a toxin called mycolactone which can lead to the destruction of the skin, resulting in incapacitating deformities with an enormous economic and social burden on patients and their caregivers. Even though there is an effective antibiotic treatment for BU, the control and management rely on early case detection and rapid diagnosis to avert morbidities. The diagnosis of Mycobacterium ulcerans relies on smear microscopy, culture histopathology, and PCR. Unfortunately, all the current laboratory diagnostics have various limitations and are not available in endemic communities. Consequently, there is a need for a rapid diagnostic tool for use at the community health centre level to enable diagnosis and confirmation of suspected cases for early treatment. The present study corroborated the diagnostic performance and utility of fluorescent-thin layer chromatography (f-TLC) for the diagnosis of Buruli ulcer. METHODOLOGY/PRINCIPAL FINDINGS: The f-TLC method was evaluated for the diagnosis of Buruli ulcer in larger clinical samples than previously reported in an earlier preliminary study Wadagni et al. (2015). A total of 449 patients suspected of BU were included in the final data analysis out of which 122 (27.2%) were positive by f-TLC and 128 (28.5%) by PCR. Using a composite reference method generated from the two diagnostic methods, 85 (18.9%) patients were found to be truly infected with M. ulcerans, 284 (63.3%) were uninfected, while 80 (17.8%) were misidentified as infected or noninfected by the two methods. The data obtained was used to determine the discriminatory accuracy of the f-TLC against the gold standard IS2404 PCR through the analysis of its sensitivity, specificity, positive (+LR), and negative (–LR) likelihood ratio. The positive (PPV) and negative (NPV) predictive values, area under the receiver operating characteristic curve Azevedo et al. (2014), and diagnostic odds ratio were used to assess the predictive accuracy of the f-TLC method. The sensitivity of f-TLC was 66.4% (85/128), specificity was 88.5% (284/321), while the diagnostic accuracy was 82.2% (369/449). The AUC stood at 0.774 while the PPV, NPV, +LR, and–LR were 69.7% (85/122), 86.9% (284/327), 5.76, and 0.38, respectively. The use of the rule-of-thumb interpretation of diagnostic tests suggests that the method is good for use as a diagnostic tool. CONCLUSIONS/SIGNIFICANCE: Larger clinical samples than previously reported had been used to evaluate the f-TLC method for the diagnosis of Buruli ulcer. A sensitivity of 66.4%, a specificity of 88.5%, and diagnostic accuracy of 82.2% were obtained. The method is good for diagnosis and will help in making early clinical decisions about the patients as well as patient management and facilitating treatment decisions. However, it requires a slight modification to address the challenge of background interference and lack of automatic readout to become an excellent diagnostic tool

Virulence potential of Staphylococcus aureus isolates from Buruli ulcer patients

Buruli ulcer (BU) is a necrotizing infection of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. BU wounds may also be colonized with other microorganisms including Staphylococcus aureus. This study aimed to characterize the virulence factors of S. aureus isolated from BU patients. Previously sequenced genomes of 21 S. aureus isolates from BU patients were screened for the presence of virulence genes. The results show that all S. aureus isolates harbored on their core genomes genes for known virulence factors like alpha-hemolysin, and the a and beta-phenol soluble modulins. Besides the core genome virulence genes, mobile genetic elements (MGEs), i.e. prophages, genomic islands, pathogenicity islands and a Staphylococcal cassette chromosome (ECG) were found to carry different combinations of virulence factors, among them genes that are known to encode factors that promote immune evasion, superantigens and Panton-Valentine Leucocidin. The present observations imply That the S. aureus isolates from BU patients harbor a diverse repertoire of virulence genes that may enhance bacterial survival and persistence in the wound environment and potentially contribute to delayed wound healing

Molecular Characterization of Staphylococcus aureus Isolates Transmitted between Patients with Buruli Ulcer

BACKGROUND:Buruli ulcer (BU) is a skin infection caused by Mycobacterium ulcerans. The wounds of most BU patients are colonized with different microorganisms, including Staphylococcus aureus. METHODOLOGY:This study investigated possible patient-to-patient transmission events of S. aureus during wound care in a health care center. S. aureus isolates from different BU patients with overlapping visits to the clinic were whole-genome sequenced and analyzed by a gene-by-gene approach using SeqSphere(+) software. In addition, sequence data were screened for the presence of genes that conferred antibiotic resistance. PRINCIPAL FINDINGS:SeqSphere(+) analysis of whole-genome sequence data confirmed transmission of methicillin resistant S. aureus (MRSA) and methicillin susceptible S. aureus among patients that took place during wound care. Interestingly, our sequence data show that the investigated MRSA isolates carry a novel allele of the fexB gene conferring chloramphenicol resistance, which had thus far not been observed in S. aureus

Detection of Mycobacterium ulcerans by the Loop Mediated Isothermal Amplification Method

In order to develop a simple and rapid test that can be used to diagnose Buruli ulcer under field conditions, we modified the conventional LAMP assay by using a disposable pocket warmer as a heating device for generating a constant temperature for the test reaction and employed the use of crude sample preparations consisting of boiled and unboiled extracts of the clinical specimen instead of using purified DNA as the diagnostic specimen. Thirty clinical specimens from suspected Buruli ulcer patients were investigated by the modified LAMP (or pocket warmer LAMP) and the conventional LAMP, as well as IS2404 PCR, a reference method for the detection of Mycobacterium ulcerans. There was no significant difference in the detection rate (63–70%) in all of the methods when purified samples were used for the tests. On the other hand the use of crude specimen preparation resulted in a drop in detection rate (30–40%). This study demonstrates that the LAMP test can be used for rapid detection of M. ulcerans when purified DNA preparations are used. With further improvements in the sample reaction, as well as in specimen purification, the pocket warmer LAMP may provide a simple and rapid diagnostic test for Buruli ulcer

Methicillin Resistant Staphylococcus aureus Transmission in a Ghanaian Burn Unit:The Importance of Active Surveillance in Resource-Limited Settings

Objectives:Staphylococcus aureus infections in burn patients can lead to serious complications and death. The frequency of S. aureus infection is high in low- and middle-income countries presumably due to limited resources, misuse of antibiotics and poor infection control. The objective of the present study was to apply population genomics to precisely define, for the first time, the transmission of antibiotic resistant S. aureus in a resource-limited setting in sub-Saharan Africa.Methods:Staphylococcus aureus surveillance was performed amongst burn patients and healthcare workers during a 7-months survey within the burn unit of the Korle Bu Teaching Hospital in Ghana.Results: Sixty-six S. aureus isolates (59 colonizing and 7 clinical) were obtained from 31 patients and 10 healthcare workers. Twenty-one of these isolates were ST250-IV methicillin-resistant S. aureus (MRSA). Notably, 25 (81%) of the 31 patients carried or were infected with S. aureus within 24 h of admission. Genome comparisons revealed six distinct S. aureus clones circulating in the burn unit, and demonstrated multiple transmission events between patients and healthcare workers. Further, the collected S. aureus isolates exhibited a wide range of genotypic resistances to antibiotics, including trimethoprim (21%), aminoglycosides (33%), oxacillin (33%), chloramphenicol (50%), tetracycline (59%) and fluoroquinolones (100%).Conclusion: Population genomics uncovered multiple transmission events of S. aureus, especially MRSA, within the investigated burn unit. Our findings highlight lapses in infection control and prevention, and underscore the great importance of active surveillance to protect burn victims against multi-drug resistant pathogens in resource-limited settings

Heterogeneity among Mycobacterium ulcerans Isolates from Africa

Mycobacterium ulcerans causes Buruli ulcer, an ulcerative skin disease in tropical and subtropical areas. Despite restricted genetic diversity, mycobacterial interspersed repetitive unit–variable-number tandem repeat analysis on M. ulcerans revealed 3 genotypes from different African countries. It is the first time this typing method succeeded directly on patient samples

Draft genome sequences and antimicrobial profiles of three staphylococcus epidermidis strains from neonatal blood samples

Data on molecular characterization of coagulase-negative staphylococci causing neonatal sepsis in low-income countries are highly limited. This report highlights the isolation of three Staphylococcus epidermidis non-genome assembly strains (NGASs) from blood samples from neonates with unknown transmission sources. Pathogenic factors and sources of transmission of these strains warrant further investigation

Epidemiology of Staphylococcus aureus in a burn unit of a tertiary care center in Ghana

Background: In developing countries, hospitalized burn victims are at high risk of nosocomial infections caused by Staphylococcus aureus. Risk factors include poor infection control practices, prolonged hospitalisation and limited capacity for laboratory microbiological analyses. These problems are compounded by widespread use of antibiotics that drives the spread of multidrug resistant bacteria. Methods: During the study period (November 2014-June 2015), nasal and invasive S. aureus isolates were collected consecutively from patients and healthcare workers (HCWs) within the burn unit of the Reconstructive Plastic Surgery and Burn Center of Korle Bu Teaching Hospital in Ghana. Antibiotic prescription, antibiotic susceptibility and bacterial typing were used to assess antibiotic pressure, antibiotic resistance, and possible transmission events among patients and HCWs. Results: Eighty S. aureus isolates were obtained from 37 of the 62 included burn patients and 13 of the 29 HCWs. At admission, 50% of patients carried or were infected with S. aureus including methicillin resistant S. aureus (MRSA). Antibiotic use per 100 days of hospitalization was high (91.2 days), indicating high selective pressure for resistant pathogens. MRSA isolates obtained from 11 patients and one HCW belonged to the same spa-type t928 and multi-locus sequence type 250, implying possible transmission events. A mortality rate of 24% was recorded over the time of admission in the burn unit. Conclusion: This study revealed a high potential for MRSA outbreaks and emergence of resistant pathogens amongst burn patients due to lack of patient screening and extended empirical use of antibiotics. Our observations underscore the need to implement a system of antibiotic stewardship and infection prevention where microbiological diagnostics results are made available to physicians for timely and appropriate patient treatment

<i>spa</i>-types of 91 <i>S. aureus</i> isolates from the anterior nares and wounds of 19 BU patients.

<p><i>spa</i>-types of 91 <i>S. aureus</i> isolates from the anterior nares and wounds of 19 BU patients.</p