Comparative Diagnosis of Mycobacterium ulcerans Infections by Ziehl-Neelson Microscopy, Insertion Sequence 2404 Nested Polymerase Chain Reaction and LoopMediated Isothermal Amplification Technique

Buruli ulcer (BU) is a chronic skin disease caused by Mycobacterium ulcerans. The disease is often diagnosed clinically at peripheral health centres. World Health Organization has recommended that clinically suspected cases be confirmed in the laboratory. This study was designed to determine the sensitivity of loop-mediated isothermal amplification (LAMP) molecular assay, the Ziehl-Neelson (ZN) microscopy and ZN modifications as techniques for laboratory diagnosis of Buruli ulcer disease (BUD) and the field applicability of LAMP molecular assay. Clinical specimens for the study were collected from five (5) BU treatment centres in Ghana. In all, 141 clinical specimens were collected. The specimens were collected from 93 (66.0%) males and 48 (34.0%) females (p=0.0847) (aged 2-86 years; mean age 33.6 years and modal age 9 years); 103 (73.0%) swab specimens and 38 (27.0) aspirates were collected. Category I, II and III lesions were 64 (45.4%), 24 (17.0%) and 53 (37.6%) respectively. Dried smears from direct clinical specimens, specimen cellular suspensions and smears from chemically digested smears were examined with ZN technique. Suspected lesions were confirmed with dry-reagent based IS2404 nested PCR, first run IS2404 PCR and BULAMP molecular assay. The detection limits of the BU-LAMP, IS2404 nested PCR and the microscopic techniques were determined. The best molecular method with the highest positivity rate was nested IS2404 PCR 122 (86.5%) followed by BU-LAMP assay of 111 (78.7%). Kappa analysis indicated a good agreement between IS2404 nested PCR and BU-LAMP (κ=0.72). The sensitivity of the IS2404 nested PCR and BU-LAMP molecular assays was 30 and 3 copies of insertion sequences respectively. Direct smearing of cellular suspension had a relative higher positivity rate of 46.1%. Centrifugation and sedimentation of the cellular specimen suspension with phenol ammonium sulphate solution was the best microscopy protocol. They had positivity rate of 73 (51.7%), mean AFB yield per 100 high power field of 23.7 (95% CI, 21.1-26) and 75 (53.2%), mean AFB yield per high power field of 30.3, (95% CI, 27.0-33.8) respectively. The respective detection limits of these protocols were 16 and 4 AFBs in 1 ml of suspension. The sensitivities of the techniques were BU-LAMP molecular assay (91.5%), first run IS2404 PCR (85.2%), sedimentation with phenol ammonium sulphate (61.5%), centrifugation with phenol ammonium sulphate (59.8%), sedimentation with phosphate buffered saline (55.7%), centrifugation with phosphate buffered saline (54.1%), direct smear microscopy using specimen cellular suspension (53.3%), sedimentation with 3.5% sodium hypochlorite (51.6%), centrifugation with 3.5% sodium hypochlorite (50.0%) and direct specimen smear (41.8%). The specificity and the positive predictive values for the techniques were 100.0%. The agreement between the M. ulcerans DNA detection ability of IS2404 nested PCR and BU-LAMP from swab specimens and fine needle aspirates were perfect; κ=0.89 and κ=0.93 respectively. The best direct method for detecting AFBs in swabs and aspirates was smears from specimen cellular suspension and the best modified method for detecting AFBs in swabs and aspirates were centrifugation and sedimentation with phenol ammonium sulphate. BU-LAMP assay has a great potential in diagnosing M. ulcerans in clinical specimen but it is recommended that the assay methodology be improved to detect DNA from clinical specimens to enable the assay be used for field studies.

Source: KNUST