Conceived and designed the experiments: GB KLH BK MB FXW AD TL JN. Performed the experiments: KLH EP KH EF JN. Analyzed the data: GB KLH MB EP K-HH KA AB-K KH JN. Contributed reagents/materials/analysis tools: GB KB K-HH FXW AB-K AD TL. Wrote the paper: GB KLH MB K-HH JN. Planning and realization of training workshops for CLTs and health staff in Togo: GB KLH BK EP FXW KA AB-K AD JN. Planning and realization of outreach activities for case finding in Togo: KLH BK EP FXW KA AB-K AD JN. Critical review of manuscript: K-HH TL.
The authors have declared that no competing interests exist.
Since the early 1990s more than 1,800 patients with lesions suspicious for Buruli ulcer disease (BUD) have been reported from Togo. However, less than five percent of these were laboratory confirmed. Since 2007, the Togolese National Buruli Ulcer Control Program has been supported by the German Leprosy and Tuberculosis Relief Association (DAHW). Collaboration with the Department for Infectious Diseases and Tropical Medicine (DITM), University Hospital, Munich, Germany, allowed IS
The DAHW integrated active BUD case finding in the existing network of TB/Leprosy Controllers and organized regular training and outreach activities to identify BUD cases at community level. Clinically suspected cases were referred to health facilities for diagnosis and treatment. Microscopy was carried out locally, external quality assurance (EQA) at DITM. Diagnostic samples from 202 patients with suspected BUD were shipped to DITM, 109 BUD patients (54%) were confirmed by PCR, 43 (29.9%) by microscopy. All patients originated from Maritime Region. EQA for microscopy resulted in 62% concordant results.
This study presents a retrospective analysis of the first cohort of clinically suspected BUD cases from Togo subjected to systematic laboratory analysis over a period of three years and confirms the prevalence of BUD in Maritime Region. Intensified training in the field of case finding and sample collection increased the PCR case confirmation rate from initially less than 50% to 70%. With a PCR case confirmation rate of 54% for the entire study period the WHO standards (case confirmation rate ≥50%) have been met. EQA for microscopy suggests the need for intensified supervision and training. In January 2011 the National Hygiene Institute, Lomé, has assumed the role of a National Reference Laboratory for PCR confirmation and microscopy.
Buruli ulcer disease (BUD) is an emerging disease particularly affecting children under the age of 15 years. Due to scarring and contractures BUD may lead to severe functional disability. Introduction of antimycobacterial treatment necessitated the laboratory confirmation of BUD, and WHO recommends confirmation of at least 50% of patients with suspected BUD by polymerase chain reaction (PCR). In Togo, cases have been reported since the early 1990s. However, less than five percent were laboratory confirmed. Since 2007, the German Leprosy and Tuberculosis Relief Organization (DAHW) has supported the Togolese National Buruli Ulcer Control Program in the area of training, treatment and laboratory confirmation of BUD. In close collaboration of DAHW and the Department for Infectious Diseases and Tropical Medicine, University Hospital, Munich (DITM), diagnostic samples from Togolese patients with suspected BUD were subjected to PCR. Out of 202 suspected BUD cases 109 BUD patients (54%) were PCR confirmed over a period of three years. Whereas the PCR case confirmation rate initially was below 50%, intensified training measures for health staff in the field of clinical diagnosis and collection of diagnostic samples ultimately resulted in 69% PCR confirmed cases. Our findings confirm the prevalence of BUD in Maritime Region.
Buruli ulcer disease (BUD), caused by
BUD involves the skin and the subcutaneous adipose tissue. The disease starts as painless papule, plaque or nodule that evolves into large painless ulcerations with characteristically undermined edges and may be accompanied by edema of the surrounding skin. Large ulcers may affect the subjacent bones resulting in osteomyelitis
Since 2004, antimycobacterial treatment (if necessary followed by surgical intervention) has been considered the treatment of choice
Currently available diagnostic laboratory tests include microscopic examination, culture, IS
Since the early 1990s patients with lesions clinically suspicious for BUD have been treated in Togolese hospitals. The first two laboratory-confirmed and well documented BUD patients from Togo were described in 1996 by Meyers and colleagues
In 1999, Togo established its National Buruli Ulcer Control Program (Programme National de Lutte contre L'Ulcère de Buruli [PNLUB], since 2010: Programme National de Lutte contre L'Ulcère de Buruli – Lèpre et Pian [PNLUB-LP]). Initially limited resources hampered the progress of program activities, however, collaboration with non-governmental organizations (Handicap International [HI], France; German Leprosy and Tuberculosis Relief Association [DAHW], Germany) enhanced the efficiency of BUD control. In 2007, a five year strategic plan was developed to intensify treatment, case detection, laboratory confirmation and surveillance of BUD, initially focusing on Maritime and Central Region. The Centre Hospitalier Régionale (CHR) Tsévié, Maritime Region, was appointed National Reference Centre for BUD in Togo, and recently the Centre Hospitalier Préfectoral (CHP) Sotouboua, Central Region, was turned into an outpost of the National Reference Centre. The DAHW in particular supports training, treatment and laboratory confirmation of patients with suspected BUD [“Plan Stratégique de Lutte contre L'Ulcère de Buruli, 2008–2012”. Ministère de la Santé, République Togolaise, Lomé 2007; 18].
Whereas microscopic analysis has been locally carried out at CHR Tsévié, facilities for the diagnostic IS
Laboratory confirmation and treatment of BUD patients was covered by a skeleton agreement between the DAHW and the Ministry of Health, Togo. As all activities fall under routine patient management, ethical clearance by the Committee of Bioethics in Research, Ministry of Health, Togo, was not required. In accordance with standard practice customary in Togo, from 2007 through 2008 patients with suspected BUD were verbally informed on the need for collection of diagnostic samples and treatment, and verbal consent was obtained from the patients. In 2009, the PNLUB-LP introduced informed consent forms. Written informed consent (signature or thumb print, in case of minors given by legal representatives) was obtained from the majority of patients with suspected BUD attending CHR Tsévié. Publication of pseudonymized data and results obtained during the study period was authorized by the PNLUB-LP.
To integrate active BUD case finding into the existing Togolese network of TB and Leprosy District and Regional Controllers (Contrôleur Lèpre–TB–Buruli, CLT), the DAHW conducted two initial training workshops for CLT and health staff at CHR Tsévié and CHP Sotouboua in 2007, followed by regular re-training from 2008 through 2010 (four workshops in Maritime Region, one workshop in Central and Kara Region each). Supported by CHR Tsévié hospital staff, the CLT teams conducted quarterly sensitization campaigns and outreach activities to identify cases at community level under coordination of the PNLUB-LP. Clinically suspected BUD cases were referred to peripheral health posts (Unité de Soins Périphérique, USP), CHP Sotouboua or CHR Tsévié for collection of diagnostic samples and treatment. Passive case finding included patients presenting at BUD treatment centers (USPs, CHR-Tsévié and CHP Sotouboua).
From September 2007 through August 2010, 202 suspected BUD cases from three different study sites in Togo (CHR Tsévié, Maritime Region, n = 187; CHP Sotouboua, Central Region, n = 14, USP Agbetiko, Maritime Region, n = 1) were included in the study (
Type of lesion |
Study site | Suspected cases | MIC |
PCR |
||||
Confirmed cases [N] | Suspected cases subjected to MIC [N] | Case confirmation rate (%) | Confirmed cases [N] | Suspected cases subjected to PCR [N] | Case confirmation rate (%) | |||
|
Tsévié | 49 | 9 | 23 | (39.1) | 38 | 49 | (77,6) |
Sotouboua | 2 | NA |
NA | NA | 0 | 2 | (0.0) | |
Agbetiko | 0 | NA | NA | NA | NA | NA | NA | |
Total | 51 | 9 | 23 | (39.1) | 38 | 51 | (74.5) | |
|
Tsévié | 138 | 34 | 120 | (28.3) | 71 | 138 | (51.4) |
Sotouboua | 12 | 0 | 1 | (0.0) | 0 | 12 | (0.0) | |
Agbetiko | 1 | NA | NA | NA | 0 | 1 | (0.0) | |
Total | 151 | 34 | 121 | (28.1) | 71 | 151 | (47.0) | |
|
202 | 43 | 144 | (29.9) | 109 | 202 | (54.0) |
Non-ulcerative lesions: FNA (fine needle aspiration) samples, punch biopsy samples and surgical biopsy samples were analyzed; ulcerative lesions: swab samples, FNA (fine needle aspiration) samples, punch biopsy samples and surgical biopsy samples were analyzed;
Test: MIC, microscopic examination for the detection of acid fast bacilli; swab samples and FNA samples were analyzed;
Test: PCR, polymerase chain reaction, gel-based IS
NA, not available;
Diagnostic samples were collected according to standardized procedures which have been developed in the context of previous studies on laboratory diagnosis of BUD in Ghana
To facilitate sampling, standardized specimen collection bags including swabs, biopsy punches, syringes and needles, containers with transport media (700 µl CLS® [cell lysis solution, Qiagen, Hilden, Germany] for PCR samples) and data entry forms (BU01 form
Type of Treatment | Type of lesion | Diagnostic test | Transport medium | Swab | FNA |
Punch biopsy | Surgical biopsy |
|
Non-ulcerative | MIC |
NA |
NA | yes | NA | NA |
PCR |
CLS |
NA | yes | yes | yes | ||
Ulcerative | MIC | NA | yes | yes | NA | NA | |
PCR | CLS | yes | yes | yes | yes | ||
|
Non-ulcerative | MIC | NA | NA | yes | NA | NA |
PCR | CLS | NA | yes | yes | NA | ||
Ulcerative | MIC | NA | yes | yes | NA | NA | |
PCR | CLS | yes | yes | yes | NA |
FNA, fine needle aspiration;
MIC, microscopic examination for the detection of acid fast bacilli;
NA, not applicable;
PCR, IS
CLS, cell lysis solution (Qiagen, Germany).
As shown in
Number of positive samples/total number of samples tested [N(%)] | |||||||||
Type of lesion | Study site | Swab | FNA |
Punch biopsy | Surgical biopsy | ||||
MIC [N(%)] |
PCR [N(%)] |
MIC [N(%)] | PCR [N(%)] | MIC [N(%)] | PCR [N(%)] | MIC [N(%)] | PCR [N(%)] | ||
|
Tsévié | ND |
ND | 9/23 (39.1) | 27/49 (55.1) | NA |
32/50 (64.0) | NA | 2/3 (66.7) |
Sotouboua | ND | ND | NA | 0/1 (0) | NA | NA | NA | 0/2 (0) | |
Agbetiko | ND | ND | NA | NA | NA | NA | NA | NA | |
Total | ND | ND | 9/23 (39.1) | 27/50 (54.0) | NA | 32/50 (64.0) | NA | 2/5 (40.0) | |
|
Tsévié | 27/115 (23.5) | 63/142 (44.4) | 31/92 (33.7) | 45/111 (40.5) | NA | 49/121 (40.5) | NA | 12/44 (27.3) |
Sotouboua | 0/1 (0.0) | 0/9 (0.0) | NA | 0/5 (0.0) | NA | 0/6 (0.0) | NA | 0/2 (0.0) | |
Agbetiko | NA | 0/1 (0.0) | NA | 0/1 (0.0) | NA | 0/2 (0.0) | NA | NA | |
Total | 27/116 (23.3) | 63/152 (41.5) | 31/92 (33.7) | 45/117 (38.5) | NA | 49/129 (38.0) | NA | 12/46 (26.1) | |
|
27/116 (23.3) | 63/152 (41.5) | 40/115 (34.8) | 72/167 (43.1) | NA | 81/179 (45.3) | NA | 14/51 (27.5) |
FNA, fine needle aspiration;
MIC, microscopic examination for the detection of acid fast bacilli;
PCR, IS
ND, not done;
NA, not available;
The turnaround time between shipment of samples and availability of results averaged approximately two weeks. Results were communicated by email to DAHW and distributed to the hospitals.
Clinical and epidemiological information derived from laboratory data entry and BU01 forms as well as diagnostic results obtained at DITM and CHR Tsévié were stored in a database (Access 2003, Microsoft Cooperation, Redmond, WA). For analysis, the study period was divided into three observation periods (September 2007 through August 2008, September 2008 through August 2009, September 2009 through August 2010), for clarification selected data are also indicated per calendar year. Beside epidemiological data, the analysis included case confirmation rates (number of laboratory confirmed BUD patients divided by the total number of suspected BUD cases included in the study) per diagnostic test, and positivity rates (number of positive samples divided by the total number of samples tested) per sample type and diagnostic test. Approximative tests (χ2-tests) and t-tests as parametric tests were conducted using Stata software, version 9.0 (Stata Corporation, College Station, TX) and EpiInfo, version 3.3.2 (Centers for Disease Control and Prevention, CDC, Atlanta, GA).
206 sets of specimens from 202 suspected BUD cases were collected for laboratory confirmation. Fifty-one suspected cases (25.2%) had non-ulcerative lesions, 151 (74.8%) had ulcerative lesions. Four suspected cases (2.0%) had two lesions. From 13 of the 202 study participants 13 sets of follow-up specimens were available.
The patients with suspected BUD originated from ten districts in three regions (Maritime, Central and Plateaux). Most of the suspected cases (82.2%) were detected in districts Zio (n = 89 [44.1%]) and Yoto (n = 77 [38.1%]) of Maritime Region. The age range of the suspected cases was 1–72 years (mean = 24.8 years, median = 17 years) and 39.6% of the suspected cases were in age group 5–14 years, 114 of the suspected cases (56.4%) were male.
Out of the 202 patients with suspected BUD 109 were laboratory confirmed as BUD patients. Out of them 43 (39.5%) were confirmed by two and 66 (60.6%) by at least one positive laboratory test. Out of the 13 study participants followed over time twelve were laboratory confirmed at their first presentation at hospital (also the second sample collection rendered positive results). For one of the 13 study participants followed over time neither the first nor the second sample collection rendered positive results.
The overall case confirmation rate by PCR was 54.0% (109/202), and 29.9% (43/144) by microscopy (
Among the 151 suspected cases with ulcerative lesions, 71/151 (47.0%) were PCR confirmed (
The positivity rates for microscopy and PCR per type of specimen are shown in
EQA for microscopy resulted in 23/37 (62.2%) concordant results, 14 slides (37.8%) were false negative.
Out of the 109 laboratory confirmed BUD patients, 38 (34.9%) had non-ulcerative, 71 (65.1%) had ulcerative lesions, 57 (52.3%) were male, and 65 (59.6%) of them were in age group 5–14 years (age range 2–60 years, mean 17.3 years, median 12 years) (
For all patients the age was known and 65 (59.6%) of them were in age group 5–14 years. The age range was 2–60 years with a mean of 17.3 years and the median was 12 years.
The confirmed BUD patients originated from five districts of Maritime Region (Zio, n = 51; Yoto, n = 49; Vo, n = 5, Golfe, n = 1; Avé, n = 1).
In 90.8% (99/109) the lesions were located on limbs or shoulders. None of the sides was significantly more affected (right side, n = 51 and left side, n = 48).
For all 109 confirmed BUD patients the lesion sizes were known and the lesions were distributed according to WHO categories as follows
Among the BUD patients originating from Maritime Region, five pairs of siblings (two individuals each) were identified, three pairs of siblings developed BUD at the same time. Three pairs of siblings originated from the district of Yoto, two from the district of Zio, all affected families lived close to flowing water bodies (Haho River, Lili River).
The PCR case confirmation rate increased with a definite trend from 42.9% (36/84) to 69.2% (36/52) (coefficient of determination, R2 = 1) from the first through the third observation period (
The PCR case confirmation rate was 36/84 (42.86%) in the first observation period (September 2007–August 2008), 37/66 (56.06%) in the second observation period (September 2008–August 2009) and 36/52 (69.23%) in the third observation period (September 2009–August 2010). The case confirmation rate increased during the three observation periods with a definite trend (coefficient of determination, R2 = 1).
This study describes the results of a collaborative approach to implement systematic laboratory confirmation of BUD in Togo. Whereas previous data reported from Togo were largely based on clinical observations, this study proves the prevalence of laboratory confirmed BUD cases in Maritime Region. From 2007 through 2010 out of 202 suspected BUD cases 109 BUD patients were PCR confirmed, which equals an overall PCR case confirmation rate of 54%. During the decade after the description of the first two laboratory confirmed BUD patients in 1996
The strategies applied for collection of diagnostic samples and data management were originally developed in the context of an EC funded research project (project no. INCO-CT-2005-015476-BURULICO) conducted in Ghana
Initially the PCR case confirmation rate was below 50%. However, for the second (56.1%) and third (69.2%) observation period as well as for the entire study period (54.0%) the WHO criteria for PCR case confirmation rates have been met
As far as punch biopsies are concerned, meanwhile broad consensus has been reached that FNA are equal to punch biopsies for most diagnostic applications, and – in the interest of the patients - the use of punch biopsies should be restricted to a minimum
A number of limitations of this study need to be mentioned. During the study period most training workshops were held in Maritime Region – which is reflected in a continuous improvement of the quality of samples and data obtained from the catchment area of CHR Tsévié. In contrast, all diagnostic samples sent from Central Region were negative, therefore this study did not succeed to confirm the prevalence of BUD outside of Maritime Region. Further attempts to verify if the disease occurs in other regions of the country require intensified training in the field of clinical diagnosis and collection of diagnostic samples in the respective areas.
Furthermore, this study did not use specific questionnaires; patient related information was obtained from standardized BU01 forms instead. The current versions of BU01 forms however, do not contain information required for analysis of risk factors to contract the disease (e.g. information on living conditions and contact with water bodies); therefore only baseline data were available for analysis.
As PCR assessment was conducted in an external reference laboratory in Germany, a maximum number of samples were collected per patient to increase the probability for laboratory confirmation and to avoid repeated shipping of samples. To comply with recent WHO recommendations
Concerning microscopy, beside a low concordance rate and a high percentage of false negative results, for approximately 30% of the patients with suspected BUD microscopy had either not been performed, local results could not be retrieved retrospectively, or a considerable number of slides had been discarded, thus were not available for re-checking at DITM. Improvement of the performance of microscopy requires a more stringent system for external quality assurance including regular supervision of local microscopy laboratories.
Although in general - with a turnaround time of approximately two weeks between shipment of samples and availability of laboratory results - PCR assessment at an external reference laboratory in Germany worked satisfactorily, local PCR capacities are desirable. Therefore, in January 2011 the National Hygiene Institute (INH) in Lomé has assumed the role of a National Reference Laboratory for PCR confirmation and microscopy.
We thank the Togolese CLT network for their efforts in organization of sensitization and case finding activities. The article contains parts of the doctoral dissertation of Kristina Lydia Huber.