See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/51766027 Combining PCR with Microscopy to Reduce Costs of Laboratory Diagnosis of Buruli Ulcer Article  in  The American journal of tropical medicine and hygiene · November 2011 DOI: 10.4269/ajtmh.2011.11-0362 · Source: PubMed CITATIONS READS 19 25 5 authors, including: Dorothy kyerewah Yeboah-Manu Adwoa Wiredu Noguchi Memorial Institute for Medical Research Noguchi Memorial Institute for Medical Research 215 PUBLICATIONS   1,898 CITATIONS    40 PUBLICATIONS   260 CITATIONS    SEE PROFILE SEE PROFILE Kobina Ampah Edwin Ampadu University of Basel Ghana Health Service 24 PUBLICATIONS   144 CITATIONS    38 PUBLICATIONS   891 CITATIONS    SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Bioassay guided isolation and structure elucidation of antimycobacterial compounds View project On the Origins of Mycobacterium Ulcerans View project All content following this page was uploaded by Dorothy kyerewah Yeboah-Manu on 29 January 2016. The user has requested enhancement of the downloaded file. Am. J. Trop. Med. Hyg., 85(5), 2011, pp. 900–904 doi:10.4269/ajtmh.2011.11-0362 Copyright © 2011 by The American Society of Tropical Medicine and Hygiene C ombining PCR with Microscopy to Reduce Costs of Laboratory Diagnosis of Buruli Ulcer Dorothy Yeboah-Manu ,* Adwoa Asante-Poku , K obina Asan-Ampah , Emelia Danso Edwin A mpadu, and G erd Pluschke Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana; National Buruli Ulcer Control Program, Ghana Health Service, Accra, Ghana; Swiss Tropical and Public Health Institute, University of Basel, Basel, Switzerland A bstract. The introduction of antibiotic therapy as first-line treatment of Buruli ulcer underlines the importance of laboratory confirmation of clinical diagnosis. Because smear microscopy has very limited sensitivity, the technically demanding and more expensive I S2404 diagnostic polymerase chain reaction (PCR) has become the main method for confirmation. By optimization of the release of mycobacteria from swab specimen and concentration of bacterial suspen- sions before smearing, we were able to improve the detection rate of acid-fast bacilli by microscopy after Ziehl–Neelsen staining. Compared with I S2404 PCR, which is the gold standard diagnostic method, the sensitivity and specificity of microscopy with 100 concentrated specimens were 58.4% and 95.7%, respectively. We subsequently evaluated a stepwise laboratory confirmation algorithm with detection of AFB as first-line method and I S2404 PCR performed only with those samples that were negative in microscopic analysis. This stepwise approach reduced unit cost by more than 50% to $5.41, and the total costs were reduced from $917 to $433. I NTRODUCTION apy with rifampicin and streptomycin as first-line treatment.1 4 With the introduction of this antimycobacterial treatment, Buruli ulcer (BU) is a chronic necrotizing infection of sub- laboratory confirmation of clinically suspected cases became cutaneous tissue caused by Mycobacterium ulcerans. The even more crucial for the clinical management of BU. We, disease has been identified in more than 30 tropical and sub- therefore, evaluated the usefulness of microscopy for analyz- tropical countries in the world, with the majority of cases ing swab and fine-needle aspiration (FNA) samples to reduce being identified in West Africa. In terms of numbers, BU is the cost of laboratory confirmation of clinical diagnosis. third most important mycobacterial disease of public health importance globally; however, in endemic countries such as Ghana, it ranks second after tuberculosis. 1 BU affects people MATERIALS AND METHODS from impoverished communities, where there are significant S ource and collection of specimen. P athological samples resource constraints, especially for diagnosis and treatment. A were collected from 100 suspected BU patients from eight key element in the management of BU is the early diagnosis districts and three regions in Ghana. The majority of the before the development of large ulcers. However, most cases samples (69) were from the West and South districts in the are seen late at health facilities, resulting in increased patient 2 Greater Accra region; 29 were from the Akwapim North and suffering and prolonged hospital stays. South, Atiwa, West Akim, and Manya Kobo districts, all in the L aboratory diagnostic tests available for confirmation of Eastern region, and 2 were from Nkoranza North district in the BU are smear microscopy for detection of acid-fast bacilli Brong-Ahafo regions. Forty-seven of the cases were females; (AFB), M . ulcerans isolation by culture, histopathology, and age range was between 3 and 82 years. Arithmetic mean age polymerase chain reaction (PCR) for detection of the inser- was 25.1 years, median was 14 years, and 10 years was the mode. tion sequence I S2404. Although both histopathology and cul- The remaining 53 cases were males between 4 months and tivation are technically very demanding and time-consuming 82 years; the arithmetic mean was 26.1 years, the median was diagnostic approaches, the fast and less demanding smear 15.5 years, and the mode was 10 years. Among the 100 cases, microscopy has limited sensitivity. Therefore, I S2404 PCR has 99 were new cases, and 1 was recorded as a recurrent case. The become the gold standard diagnostic tool,4 although its routine majority of the suspects had ulcers (86): 74 had only ulcers, 9 had use in resource-poor settings is limited by the high costs and edema with ulcers, 1 had ulcers with oesteomylitis, and 2 had need for sophisticated laboratory infrastructure. Therefore, nodules and ulcers. Only 14 cases were pre-ulcerative: 12 nodules, PCR tests are usually not performed at the treating facili- 1 plaque, and 1 edema. The lesion category was recorded ties but in bulk at national reference laboratories or outside for 80 cases, and of these cases, 31 (38.75%), 22 (27.5%), and the endemic country at international reference laboratories.5 29 (33.75%) were categories I, II, and III, respectively. These shortcomings have reduced microbiological confirma- Specimens were collected using standard procedures as tion to a quality control tool for clinical diagnosis. Although prescribed by the WHO. From cases with ulcerative lesions, the general perception is that diagnosis based on clinical judg- 6 two swab samples were collected by swabbing the surface ment alone can be adequate, incidents of misdiagnosis have 7–9 beneath the entire undermined edges per each lesion to make been reported. BU cases have been missed initially, but pre- 10–13 sure that the areas with the highest load of bacteria are sam-sumed BU lesions have proven to be other conditions. pled. In the case of pre-ulcerative lesions, one FNA speci- In the past, surgery was used as the only means of BU men was collected at the center of the lesion.1 5 We used a case management; however, the World Health Organization 21/23-gauge needle attached to a 5-mL syringe. Swabs were (WHO) currently recommends an 8-week combination ther- either transported dry or inserted into 15-mL falcon tubes containing transport medium to cover the entire tip of the swab, whereas FNA specimens were immediately drained * Address correspondence to Dorothy Yeboah-Manu, Noguchi Memorial Institute for Medical Research, University of Ghana, PO into an Epperndorf tube containing 500 μL phosphate buff- Box LG581, Accra, Ghana. E-mail: d yeboah-manu@noguchi.mim ered saline (PBS). A specimen collection form was used to com.org obtain detailed information about the specimen, and each 900 MICROSCOPY AND BURULI ULCER DIAGNOSIS 901 pair of specimens and matching specimen collections were were concentrated by centrifuging at 3,000 × g for 15 minutes. packed in a zip-locked bag and then transported to the Noguchi About 1.5 mL supernatant were carefully taken out, and 100 Memorial Institute for Medical Research (NMIMR) for pro- and 400 μL sediment were used to prepare slides for Ziehl– cessing and analysis. The procedures used for sample collection Neelson (ZN) microscopy and I S2404 PCR, respectively. DNA and analysis conform to the WHO standards, and they form was extracted using the Qiagen DNA mini prep kit (Qiagen, part of the national Buruli ulcer control program system of Hilden, Germany), and the manufacturer’s instruction was laboratory confirmation of BU cases. Ethical approval of the followed. study was obtained from the Institutional Review Committee Smear microscopy. Slides were allowed to dry and were fixed of the NMIMR. In addition, consent was obtained from all by heat. The smears were then stained as previously described adult participants and legal guardians of all minors. using the ZN procedure and graded by the International Union Laboratory analysis. Optimization of bacteria release against Tuberculosis and Lung Diseases grading system. A from swab specimen. S wab samples that are received in our slide is graded as negative only after reading at least 100 high laboratory usually come as dry swabs and have spent between fields without the detection of AFB under oil immersion and 2 days and 1 month in transit. To optimize bacteria release confirmation by a second reader. from the swab sample, two procedures were evaluated using Quality control. O ne-tenth of all the slides were blindly 20 paired samples. The first process, swab specimen(s) from the evaluated by an independent technician. A discordant reading same lesion were pooled together and soaked in sterile PBS was resolved by a second assessor. for 30 minutes, which was followed by vortexing for 2 minutes. TaqMan real-time PCR. The primers and TaqMan MGB In the second process, swab specimen(s) from the same lesion probes (Applied Biosystems, California) for detecting the were pooled together and soaked for 30 minutes in a tube M . ulcerans -specific sequence, I S2404 , were as optimized in the containing sterile PBS and 10 3-mm-diameter undrilled glass work by Fyfe and others1 6 with some modifications in reac- beads (Merck, Darmstadt, Germany ). The specimen was then tion conditions. I S2404 real-time PCR mixtures contained vortexed at full speed for 2 minutes (F igure 1 ). 1× Qiagen master mix (containing HotstarTaq plus DNA The effectiveness of the two procedures was evaluated semi- polymerase, deoxyribonucleotide triphosphate (dNTP) mix, quantitatively by microscopy and real-time PCR. Thereafter, and PCR buffer), 1 μL extracted template DNA, 0.5 μM the better of the two procedures was used for subsequent sam- concentrations of each primer, and 0.2 μM probe, 1× TaqMan ple processing for analysis. exogenous internal positive control (IPC), and probe reagents P rocessing of swab and FNA specimens. T he bacteria sus- (Applied Biosystems) in a total volume of 20 μL. Ampli- pensions obtained as already described from swab specimens fication and detection were performed with the Rotor-Gene (Qiagen, Hilden, Germany) using the following program: 1 cycle of 95°C for 5 minutes and 40 cycles of 95°C for 15 seconds and 60°C for 15 seconds. Each PCR run contains two non-template controls and an I S2404 positive control. Conventional PCR. The I S2404 sequence was amplified in a 20-μL reaction volume using the Qiagen Fast cycling PCR kit. The primer sets used for the amplification were as described previously.1 7 The reaction mixture contained a 0.5-μM concentration of each primer, a 200-μM concentration of each deoxynucleoside triphosphate, an optimized concentration of MgCl2, 1× PCR buffer, 1 U HotstarTaq plus DNA polymerase, and approximately 50 ng DNA. Thermocycling parameters were 95°C for 5 minutes and 35 cycles of 5 seconds at 96°C, 5 seconds at 60°C, 15 seconds at 60°C, and 1 minute at 72°C; 10 μL amplified DNA were subjected to electrophoresis in a 2% agarose gel and detected by ethidium bromide staining and ultraviolet (UV) transillumination. To ensure the quality of diagnostic procedure, all batches of extraction included controls, and amplification procedures included negative and positive controls. The amplicons were sized by comparing with a 1-kb ladder. Stepwise confirmation of BU. In this case, a case was confirmed by first analyzing with ZN microscopy, which is cheaper and technologically less expensive, and PCR was done only when microscopy was negative (approach B), which contrasts the previous approach of analyzing all samples by PCR (approach A). We assumed that, in already described endemic areas, the specificity of microscopy will be very high, which is in accordance with the diagnosis of other mycobacterial disease in Ghana. Data handling and analysis. The positivity rates of each of the two methods were determined by dividing the number of Figure 1. Work flow. positive tests by the total number of tests. The sensitivity and 902 YEBOAH-MANU AND OTHERS specificity of the microscopy test were calculated using I S2404 S ensitivity and specificity of the optimized procedure for PCR as the gold standard. microscopic detection of AFB in swab and FNA samples. In Cost analysis. The definition used to calculate the cost was the next step, we analyzed 105 swab samples from 86 suspected defined as the value of resources used to produce something BU cases with ulcerative lesions; 68.6% of the samples (72/105) (hence, the costs incurred by smear microscopy and PCR), and were positive by conventional I S2404 PCR. From 19 lesions, the cost was calculated based on the direct cost of reagents and two samples were analyzed; in six cases, both samples were materials used to arrive at the result. We compared the cost positive, in five cases, both were negative, and in eight cases, of the routine PCR analysis of all samples (approach A) with PCR results were diverging. Testing more than one sample, the cost of our proposing strategy of first microscopy followed thus, increased lesion positivity from 58% to 66% for PCR. by PCR only on smear negative samples (approach B) using Microscopy testing of more than one sample increased the another batch of received specimen. P value for statistical positivity from 32% to 39%. significance of difference in cost of the two approaches I n addition, FNA samples collected from 14 suspected BU was determined by normal approximation to binomial test patients with non-ulcerative lesions were analyzed. Of these calculator. patients, 11 (78.6%) were positive by PCR, and 6 (43%) of 11 were positive by microscopy. Taken together, of 100 suspected BU cases, 77 cases were RESULTS confirmed by I S2404 PCR; 45 of 77 PCR positives (58%) were O ptimization of the release of bacteria from swab samples. also confirmed by microscopy, and 1 of 23 PCR-negative sam- Efficacy of two procedures for the release of mycobacteria ples was microscopy-positive. Combining the two methods, from dry swab samples was compared. Swabs soaked in PBS 78 of 100 suspected BU cases were confirmed. Taking I S2404 were vortexed with or without glass beads. Suspensions were PCR as the gold standard diagnostic method, the sensitivity subsequently evaluated by microscopy after ZN staining and and specificity of microscopy were 57.1% (44/77) and 95.7% semiquantitative IS2404 real-time (RT) PCR. Included in (22/23), respectively. the comparative study were 19 BU lesions testing positive by Reduction of costs for laboratory diagnosis of BU by using RT-PCR (cycle threshold [Ct] ≤ 39) after sample processing the optimized microscopic procedures as first-line test. I n the with glass beads (T able 1 ). Although 12 of 13 (92%) suspensions next step, we evaluated whether microscopic analysis with with a Ct < 31 in RT-PCR were also positive by microscopy, the optimized procedures as first-line diagnostic test can only 3 of 25 (12%) suspensions with a Ct > 31 were microscopy substantially reduce costs for BU diagnosis. Monitoring the positive, showing the overall superior sensitivity of PCR. materials needed to perform the two tests, the unit cost of ZN T he addition of glass beads increased the microscopic detec- smear microscopy was $0.22, and the unit cost of I S2404 PCR tion of AFBs from 21% (4/19) to 58% (11/19); 6 of 19 (32%) was $11.24. We analyzed 80 samples from 80 patients using lesions rated negative by RT-PCR when swabs were vortexed an approach in which PCR was done only when microscopy without glass beads turned positive when glass beads were was negative. By microscopy, 53.8% (43/80) of samples were used. To increase sensitivity of microscopy, we subsequently positive, and costs of materials for the microscopic examination concentrated supernatants by centrifugation. For all subse- of all 80 samples were $17.60. PCR was performed only on the quent analyses, glass beads were added, and suspensions were 37 microscopy-negative samples, bringing material costs for concentrated. PCR of $415.88. Of these samples, 17 of 37 (43.6%) samples were PCR-positive. The total material costs of analyses were $433.48, reflecting a unit cost of $5.41. Costs for PCR analysis of all samples would have been more than two times as high Table 1 ($899.20), with a unit cost of $11.24. R elease of M. ulcerans cells from swab samples Ct RT-PCR Ct RT-PCR Microscopy Microscopy with beads without beads with beads without beads DISCUSSION 23.9 Neg + − 23.9 25.9 + + A t the onset of global BU initiative, recommendations by 27.2 29.4 + − the WHO required that a case of BU must be confirmed by at 27.4 25.2 + + least two laboratory tests.3 However, despite that recommen- 29.8 Neg + − dation, most cases, especially in Africa, were treated by surgi- 29.9 30.2 + + 30.4 35.5 + − cal excision based on clinical judgment. Although there was 30.7 31.7 + − little error by experienced clinicians in endemic areas of ulcers, 30.9 31.2 + − there were a number of reports of misdiagnosis, which could 33.4 Neg − − be as high as 30%.1 0– 14 The WHO now puts a lot of emphasis 33.7 38.5 − − on laboratory confirmation, because the introduction of anti- 33.8 34.7 + + 33.9 36.1 − − biotic therapy results in the formation of a network of labora- 34.5 35.3 + − tories that confirms BU cases. Nevertheless, more emphasis on 36.5 Neg − − laboratory support for case management is focusing on I S2404 36.9 35.5 − − PCR 15– 18 and sidelining smear microscopy, which is the corner- 37.1 37.1 − − stone of TB control.1937.6 Neg − − I S2404 PCR is another mycobacterial 39.0 Neg − − disease that is known to be cheaper, faster, and very specific. S wabs were soaked in PBS and vortexed with or without glass beads. After concentration, The overreliance on PCR, a technique that requires elabo- aliquots were used for microscopic analysis after Ziehl–Neelsen staining and I S2404 real- rate infrastructure, expertise, and costly reagents that are not time PCR after DNA extraction. Results for individual patients are shown and ordered by RT-PCR Ct values after vortexing of samples with glass beads. available at the peripheral health facilities treating BU, brings MICROSCOPY AND BURULI ULCER DIAGNOSIS 903 about reliance on research laboratories (in some countries, Received June 8, 2011. Accepted for publication June 27, 2011. even at international level) and therefore, affects availability Acknowledgments: T his study was funded by the Stop Buruli of research projects. Consortium and Volkswagen Foundation. We are grateful to Kwaku W e have previously reported a sensitivity of more than Bio, William Opare, Dr. Benjamin Anku, and Isaac Lamptey of Ghana 70% for detection of AFB in stained smears of biopsy speci- Health Service for providing us with clinical specimens for the analy- sis. We also appreciate the contribution of Inna Ibrahim, Grace Kpeli, men collected during surgical excision. An important step was David Mensah, and Zuliehatu Nakobu. the concentrating of specimen before smearing. Our findings using swab and FNA samples indicate that smear microscopy Authors’ address: Dorothy Yeboah-Manu, Adwoa Asante-Poku, Kobina Asan-Ampah, Emelia Danso Edwin Ampadu, and Gerd can be used to confirm more than 50% of cases that would Pluschke, Noguchi Memorial Institute for Medical Research, Uni- have been confirmed by PCR alone. We believe that this find- versity of Ghana, Accra, Ghana, E-mails: dyeboah-manu@noguchi ing was achieved through (1) a good sample collection pro- .mimcom.org, aasante-poku@noguchi.mimcom.org, kampah@nogu cedure using recommended guidelines, (2) at least two swabs chi.mimcom.org, edanso@noguchi.mimcom.org, ghanbu@4u.com.gh, and gerd.pluschke@unibas.ch . per lesion analyzed, (3) concentration of specimen before smearing, and (4) good microscopy practice of taking care to read at least 100 high-power fields before declaring a slide REFERENCES negative. 1. A siedu K , S herpbier R, Raviglione M C , 2 000 . Buruli Ulcer By combining direct smear microscopy with PCR step- Mycobacterium ulcerans I nfection. WHO Global Buruli Ulcer wise, the cost of analysis was reduced significantly to less than Initiative. Report 2000. G eneva, Switzerland: World Health 50% of the cost if the samples were analyzed by PCR alone. Organization . 2. W ansbrough-Jones M, Phillips R, 2006 . Buruli ulcer: emerging However, it must be mentioned here that the cost analysis from obscurity . Lancet 3 67: 1 849 –1 858 . done involved only the cost of materials needed to perform 3 . Portaels F , Johnson P , M eyers WM , 2 001. D iagnosis of the experiment, without taking into consideration the techni- Mycobacterium ulcerans Disease . Geneva, Switzerland : W orld cian’s time. We are of the opinion that, because all districts/ Health Organization. subdistrict health facilities have the capacities to do ZN smear 4. 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An outreach education and treatment project in Ghana for the reduction in cost of diagnosis, giving the opportunity for the early stage of M ycobacterium ulcerans disease . T rans R Soc different sectors of healthcare to be involved in BU control Trop Med Hyg 9 7: 1 59 –1 60 . and reduce the overreliance on externally funded projects. The 9 . Coloma JN, Navarrete-Franco G, I ribe P , Lopez-Cepeda L D, 2 005 . WHO guidelines require a case to be confirmed by at least two Ulcerative cutaneous mycobacteriosis due to Mycobacterium laboratory assays; however, with the focal distribution of BU, ulcerans : report of two Mexican cases . Int J Lepr Other Mycobact Dis 7 3: 5 –1 2. we believe that a suspected case can only be confirmed by ZN 1 0. Revill WD , M orrow R H, Pike MC, Ateng J , 1 973 . A controlled trial microscopy. This assertion is supported by the high specificity of the treatment of M ycobacterium ulcerans infection with clo- of ZN in this report using I S2404 by PCR as the gold standard fazimine. L ancet 2: 873– 877 . detection method. The specificity of our study was 95.7, and 1 1. Guarner J , B artlett J , Whitney E A, Raghunathan PL , Stienstra Y , Asamoa K , Etuaful S , K lutse E , Quarshie E , van der Werf TS, this finding compares very well with a similar study conducted van der Graaf WTA , K ing CH , A shford D A , 2 003 . H istopatho- by Bretzel and others4 in 2006 that had a specificity of 96.6%.4 logic features of M ycobacterium ulcerans infection . Emerg PCR will be required as a second confirmation test only if Infect Dis 9 : 651 – 656 . the case involves a new focus. Nevertheless, the fact that one 1 2. 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M ensah-Quainoo E, Y eboah-Manu D , Asebi C , Patafuor F , O fori- versus concentrated sputum smear microscopy in HIV-infected Adjei D , Junghanss T , Pluschke G , 2008 . Diagnosis of patients suspected of having pulmonary tuberculosis . B MC Mycobacterium ulcerans infection (Buruli ulcer) at a treatment Infect Dis 9: 53 . View publication stats