SCREENING WITH MASS SPECTROMETRY FOR MYCOBACTERIA PRIOR TO CARDIOTHORACIC SURGERY
20230135821 · 2023-05-04
Assignee
Inventors
Cpc classification
G01N2800/245
PHYSICS
G01N33/6851
PHYSICS
H01J49/0418
ELECTRICITY
International classification
Abstract
The invention provides a method for identifying a subject suitable for receiving a lung transplant, the method comprising: subjecting a sample to mass spectrometry analysis (in the negative ion mode or positive ion mode; preferably in the positive ion mode) and generating a mass spectrum output; wherein said sample is an isolated sample obtained from a subject that is a candidate for receiving a lung transplant; detecting one or more peak set in said mass spectrum output; wherein the presence of said one or more peak set indicates that the subject is not a suitable candidate for receiving a lung transplant; or wherein the absence of said one or more peak set indicates that the subject is a suitable candidate for receiving a lung transplant. The invention relies on the detection of peak sets specific to the membrane of pathogens, in particular mycobacteria.
Claims
1. A method for identifying a subject suitable for receiving a lung transplant, the method comprising: a. subjecting a sample to mass spectrometry analysis and generating a mass spectrum output; wherein said sample is an isolated sample obtained from a subject that is a candidate for receiving a lung transplant; and b. detecting one or more peak set in said mass spectrum output; c. wherein the presence of said one or more peak set indicates that the subject is not a suitable candidate for receiving a lung transplant; or d. wherein the absence of said one or more peak set indicates that the subject is a suitable candidate for receiving a lung transplant.
2. A method for screening operating theatre equipment for use in cardiothoracic surgery of a subject, the method comprising: a. subjecting a sample to mass spectrometry analysis and generating a mass spectrum output; wherein said sample is obtained from operating theatre equipment; and b. detecting one or more peak set in said mass spectrum output; c. wherein the presence of said one or more peak set indicates that the operating theatre equipment is not suitable to be used during cardiothoracic surgery of a subject; or d. wherein the absence of said one or more peak set indicates that the operating theatre equipment is suitable to be used during cardiothoracic surgery of a subject.
3. A method for identifying the presence or absence of a bacterial infection in a subject subsequent to receiving cardiothoracic surgery, the method comprising: a. subjecting a sample to mass spectrometry analysis and generating a mass spectrum output; wherein said sample is an isolated sample obtained from a subject that has received cardiothoracic surgery; and b. detecting one or more peak set in said mass spectrum output; c. wherein the presence of said one or more peak set is indicative of the presence of a bacterial infection in the subject subsequent to receiving cardiothoracic surgery; or d. wherein the absence of said one or more peak set is indicative of the absence of a bacterial infection in a subject subsequent to receiving cardiothoracic surgery.
4. The method according to any one of the preceding claims, wherein the sample is subjected to mass spectrometry analysis in the positive ion mode, and wherein said one or more peak set is selected from: i. a first peak set comprising at least two defined peaks selected from: a peak having a m/z of from 1256-1259 m/z, a peak having a m/z of from 1284-1287 m/z, a peak having a m/z of from 1402-1405 m/z, and a peak having a m/z of from 1430-1433 m/z; ii. a second peak set comprising at least two defined peaks selected from: a peak having a mass-to-charge ratio (m/z) of from 1220-1223 m/z, a peak having a m/z of from 1459-1462 m/z, a peak having a m/z of from 1739-1742 m/z, and a peak having a m/z of from 1741-1744 m/z; iii. a third peak set comprising at least two defined peaks selected from: a peak having a m/z of from 1242-1245 m/z, a peak having a m/z of from 1280-1283 m/z, a peak having a m/z of from 1294-1297 m/z, and a peak having a m/z of from 1923-1926 m/z; iv. a fourth peak set comprising at least two defined peaks selected from: a peak having a m/z of from 1012-1015 m/z, a peak having a m/z of from 1121-1124 m/z, a peak having a m/z of from 1122-1125 m/z, a peak having an m/z of from 1123-1126 m/z, and a peak having a m/z of from 1149-1152 m/z; v. a fifth peak set comprising at least two defined peaks selected from: a peak having a m/z of from 1311-1314 m/z, a peak having a m/z of from 1339-1342 m/z, a peak having a m/z of from 1474-1477 m/z, a peak having a m/z of from 1488-1491 m/z, and a peak having a m/z of from 1823-1826 m/z; vi. a sixth peak pet comprising at least two defined peaks selected from: a peak having a m/z of from 1242-1245 m/z, a peak having a m/z of from 1256-1259 m/z, a peak having a m/z of from 1270-1273 m/z, a peak having a m/z of from 1318-1321 m/z, and a peak having a m/z of from 1346-1349 m/z; vii. a seventh peak set comprising at least two defined peaks selected from: a peak having a m/z of from 1294-1297 m/z, a peak having a m/z of from 1296-1299 m/z, a peak having a m/z of from 1308-1311 m/z, and a peak having a m/z of from 1322-1325 m/z; viii. an eighth peak set comprising at least two defined peaks selected from: a peak having a m/z of from 1370-1373 m/z, and a peak having a m/z of from 1412-1415 m/z; ix. a ninth peak set comprising at least two defined peaks selected from: a peak having a m/z of from 2129-2132 m/z, a peak having a m/z of from 2171-2174 m/z, a peak having a m/z of from 2247-2250 m/z, and a peak having a m/z of from 2261-2264 m/z; x. a tenth peak set comprising at least two defined peaks selected from: a peak having a m/z of from 2273-2276 m/z, a peak having a m/z of from 2275-2278 m/z, a peak having a m/z of from 2551-2554 m/z, and a peak having a m/z of from 2553-2556 m/z; xi. an eleventh peak set comprising at least two defined peaks selected from: a peak having a m/z of from 1339-1342 m/z, a peak having a m/z of from 1353-1356 m/z, a peak having a m/z of from 1367-1370 m/z, and a peak having an m/z of from 1381-1384 m/z; xii. a twelfth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1242-1245 m/z, a peak having an m/z of from 1256-1259 m/z, a peak having an m/z of from 1270-1273 m/z, and a peak having an m/z of from 1298-1301 m/z; xiii. a thirteenth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1458-1461 m/z, a peak having an m/z of from 2718-2721 m/z, a peak having an m/z of from 2760-2763 m/z, and a peak having an m/z of from 2788-2791 m/z; xiv. a fourteenth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1172-1175 m/z, a peak having an m/z of from 1174-1177 m/z, a peak having an m/z of from 1883-1886 m/z, a peak having an m/z of from 1909-1912 m/z, and a peak having an m/z of from 1911-1914 m/z; xv. a fifteenth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1202-1205 m/z, a peak having an m/z of from 1224-1227 m/z, a peak having an m/z of from 1226-1229 m/z, and a peak having an m/z of from 1240-1243 m/z; xvi. a sixteenth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1318-1321 m/z, a peak having an m/z of from 1332-1335 m/z, a peak having an m/z of from 1346-1349 m/z, a peak having an m/z of from 1360-1363 m/z, and a peak having an m/z of from 1374-1377 m/z; xvii. a seventeenth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1308-1311 m/z, a peak having an m/z of from 1310-1313 m/z, a peak having an m/z of from 1324-1327 m/z, and a peak having an m/z of from 1338-1341 m/z; xviii. an eighteenth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1012-1015 m/z, a peak having an m/z of from 1028-1031 m/z, a peak having an m/z of from 1697-1700 m/z, and a peak having an m/z of from 1698-1701 m/z; xix. a nineteenth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1226-1229 m/z, and a peak having an m/z of from 1240-1243 m/z; xx. a twentieth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1414-1417 m/z, a peak having an m/z of from 1443-1446 m/z, a peak having an m/z of from 1445-1448 m/z, a peak having an m/z of from 1515-1518 m/z, and a peak having an m/z of from 1543-1546 m/z; xxi. a twenty-first peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1036-1039 m/z, a peak having an m/z of from 1259-1262 m/z, a peak having an m/z of from 1374-1377 m/z, a peak having an m/z of from 2497-2500 m/z, and a peak having an m/z of from 2835-2838 m/z; xxii. a twenty-second peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1057-1060 m/z, a peak having an m/z of from 1498-1501 m/z, a peak having an m/z of from 1512-1515 m/z, and a peak having an m/z of from 1526-1529 m/z xxiii. a twenty-third peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1432-1435 m/z, a peak having an m/z of from 1446-1449 m/z, a peak having an m/z of from 1460-1463 m/z, and a peak having an m/z of from 1474-1477 m/z; and xxiv. a twenty-fourth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1242-1245 m/z, a peak having an m/z of from 1256-1259 m/z, a peak having an m/z of from 1270-1273 m/z, and a peak having an m/z of from 1284-1287 m/z; and xxv. a twenty-fifth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1242-1245 m/z, a peak having an m/z of from 1256-1259 m/z, a peak having an m/z of from 1270-1273 m/z, and a peak having an m/z of from 1284-1287 m/z;
5. The method according to claim 4, wherein: a. said first peak set comprises one or more further defined peak having a m/z of from 1198-1201 m/z, 1200-1203 m/z, 1210-1213 m/z, 1212-1215 m/z, 1214-1217 m/z, 1224-1227 m/z, 1226-1229 m/z, 1228-1231 m/z, 1238-1241 m/z, 1240-1243 m/z, 1242-1245 m/z, 1252-1255 m/z, 1254-1257 m/z, 1266-1269 m/z, 1268-1271 m/z, 1270-1273 m/z, 1273-1276 m/z, 1280-1283 m/z, 1282-1285 m/z, 1296-1299 m/z, 1298-1301 m/z, 1300-1303 m/z, 1312-1315 m/z, 1326-1329 m/z, 1342-1345 m/z, 1344-1347 m/z, 1346-1349 m/z, 1356-1359 m/z, 1360-1363 m/z, 1370-1373 m/z, 1372-1375 m/z, 1374-1377 m/z, 1384-1387 m/z, 1386-1389 m/z, 1388-1391 m/z, 1398-1401 m/z, 1400-1403 m/z, 1412-1415 m/z, 1414-1417 m/z, 1415-1418 m/z, 1416-1419 m/z, 1427-1430 m/z, 1428-1431 m/z, 1429-1432 m/z, 1443-1446 m/z, 1444-1447 m/z, 1446-1449 m/z, 1457-1460 m/z, 1458-1461 m/z, 1473-1476 m/z, 1474-1477 m/z, or a combination thereof; b. said second peak set comprises one or more further defined peak having a m/z of from 1025-1028 m/z, 1053-1056 m/z, 1081-1084 m/z, 1109-1112 m/z, 1178-1181 m/z, 1187-1190 m/z, 1206-1209 m/z, 1234-1237 m/z, 1236-1239 m/z, 1248-1251 m/z, 1259-1262 m/z, 1275-1278 m/z, 1287-1290 m/z, 1288-1291 m/z, 1301-1304 m/z, 1302-1305 m/z, 1303-1306 m/z, 1304-1307 m/z, 1445-1448 m/z, 1473-1476 m/z, 1475-1478 m/z, 1480-1483 m/z, 1487-1490 m/z, 1501-1504 m/z, 1517-1520 m/z, 1697-1700 m/z, 1699-1702 m/z, 1711-1714 m/z, 1713-1716 m/z, 1725-1728 m/z, 1753-1756 m/z, 1754-1757 m/z, 1755-1758 m/z, 1756-1759 m/z, 1767-1770 m/z, 2107-2110 m/z, or a combination thereof; c. said third peak set comprises one or more further defined peak having a m/z of from 1030-1033 m/z, 1210-1213 m/z, 1214-1217 m/z, 1218-1221 m/z, 1224-1227 m/z, 1225-1228 m/z, 1226-1229 m/z, 1228-1231 m/z, 1232-1235 m/z, 1234-1237 m/z, 1236-1239 m/z, 1238-1241 m/z, 1240-1243 m/z, 1245-1248 m/z, 1246-1249 m/z, 1248-1251 m/z, 1250-1253 m/z, 1252-1255 m/z, 1254-1257 m/z, 1256-1259 m/z, 1258-1261 m/z, 1260-1263 m/z, 1262-1265 m/z, 1263-1266 m/z, 1264-1267 m/z, 1266-1269 m/z, 1268-1271 m/z, 1270-1273 m/z, 1272-1275 m/z, 1273-1276 m/z, 1274-1277 m/z, 1282-1285 m/z, 1284-1287 m/z, 1285-1288 m/z, 1286-1289 m/z, 1296-1299 m/z, 1298-1301 m/z, 1308-1311 m/z, 1310-1313 m/z, 1312-1315 m/z, 1322-1325 m/z, 1324-1327 m/z, 1338-1341 m/z, 1368-1371 m/z, 1370-1373 m/z, 1384-1387 m/z, 1897-1900 m/z, 1909-1912 m/z, 1937-1940 m/z, 1939-1942 m/z, 1951-1954 m/z, 1954-1957 m/z, 1965-1968 m/z, 1967-1970 m/z, or a combination thereof; d. said fourth peak set comprises one or more further defined peak having a m/z of from 1021-1024 m/z, 1131-1134 m/z, 1134-1137 m/z, 1147-1150 m/z, 1150-1153 m/z, 1198-1201 m/z, 1878-1881 m/z, 1880-1883 m/z, 2131-2134 m/z, 2275-2278 m/z, 2553-2556 m/z, or a combination thereof; e. said fifth peak set comprises one or more further defined peak having a m/z of from 1253-1256 m/z, 1267-1270 m/z, 1269-1272 m/z, 1279-1282 m/z, 1281-1284 m/z, 1283-1286 m/z, 1295-1298 m/z, 1297-1300 m/z, 1299-1302 m/z, 1300-1303 m/z, 1307-1310 m/z, 1309-1312 m/z, 1313-1316 m/z, 1323-1326 m/z, 1325-1328 m/z, 1327-1330 m/z, 1329-1332 m/z, 1341-1344 m/z, 1355-1358 m/z, 1357-1360 m/z, 1429-1432 m/z, 1432-1435 m/z, 1442-1445 m/z, 1443-1446 m/z, 1445-1448 m/z, 1446-1449 m/z, 1456-1459 m/z, 1457-1460 m/z, 1458-1461 m/z, 1460-1463 m/z, 1469-1472 m/z, 1471-1474 m/z, 1472-1475 m/z, 1477-1480 m/z, 1484-1487 m/z, 1485-1488 m/z, 1486-1489 m/z, 1497-1500 m/z, 1499-1502 m/z, 1500-1503 m/z, 1502-1505 m/z, 1513-1516 m/z, 1514-1517 m/z, 1516-1519 m/z, 1518-1521 m/z, 1528-1531 m/z, 1529-1532 m/z, 1530-1533 m/z, 1766-1769 m/z, 1781-1784 m/z, 1795-1798 m/z, 1809-1812 m/z, 1811-1814 m/z, 1815-1818 m/z, 1821-1824 m/z, 1829-1832 m/z, 1837-1840 m/z, 1839-1842 m/z, 1849-1852 m/z, 1851-1854 m/z, or a combination thereof; f. said sixth peak set comprises one or more further defined peak having a m/z of from 1127-1130 m/z, 1140-1143 m/z, 1144-1147 m/z, 1158-1161 m/z, 1168-1171 m/z, 1169-1172 m/z, 1172-1175 m/z, 1184-1187 m/z, 1186-1189 m/z, 1198-1201 m/z, 1199-1202 m/z, 1200-1203 m/z, 1212-1215 m/z, 1214-1217 m/z, 1224-1227 m/z, 1226-1229 m/z, 1227-1230 m/z, 1228-1231 m/z, 1230-1233 m/z, 1240-1243 m/z, 1252-1255 m/z, 1254-1257 m/z, 1259-1262 m/z, 1268-1271 m/z, 1272-1275 m/z, 1282-1285 m/z, 1284-1287 m/z, 1286-1289 m/z, 1290-1293 m/z, 1296-1299 m/z, 1298-1301 m/z, 1300-1303 m/z, 1304-1307 m/z, 1310-1313 m/z, 1314-1317 m/z, 1316-1319 m/z, 1328-1331 m/z, 1330-1333 m/z, 1331-1334 m/z, 1332-1335 m/z, 1342-1345 m/z, 1344-1347 m/z, 1345-1348 m/z, 1356-1359 m/z, 1358-1361 m/z, 1360-1363 m/z, 1368-1371 m/z, 1370-1373 m/z, 1372-1375 m/z, 1374-1377 m/z, 1386-1389 m/z, 1388-1391 m/z, 1402-1405 m/z, 1414-1417 m/z, 1416-1419 m/z, 1428-1431 m/z, 1430-1433 m/z, 1519-1522 m/z, 1545-1548 m/z, 1547-1550 m/z, or a combination thereof; g. said seventh peak set comprises one or more further defined peak having a m/z of from 1266-1269 m/z, 1268-1271 m/z, 1310-1313 m/z, 1324-1327 m/z, 1338-1341 m/z, or a combination thereof; h. said ninth peak set comprises one or more further defined peak having a m/z of from 2115-2118 m/z, 2145-2148 m/z, 2157-2160 m/z, 2187-2190 m/z, 2188-2191 m/z, 2205-2208 m/z, 2219-2222 m/z, 2233-2236 m/z, 2245-2248 m/z, 2259-2262 m/z, 2277-2280 m/z, or a combination thereof; i. said tenth peak set comprises one or more further defined peak having a m/z of from 1848-1851 m/z, 2215-2218 m/z, 2231-2234 m/z, 2233-2236 m/z, 2235-2238 m/z, 2243-2246 m/z, 2244-2247 m/z, 2245-2248 m/z, 2247-2250 m/z, 2257-2260 m/z, 2260-2263 m/z, 2261-2264 m/z, 2262-2265 m/z, 2271-2274 m/z, 2285-2288 m/z, 2287-2290 m/z, 2289-2292 m/z, 2291-2294 m/z, 2389-2392 m/z, 2511-2514 m/z, 2549-2552 m/z, 2569-2572 m/z, or a combination thereof; j. said eleventh peak set comprises one or more further defined peak having a m/z of from 1311-1314 m/z, 1325-1328 m/z, 1369-1372 m/z, 1397-1400 m/z, or a combination thereof; k. said twelfth peak set comprises one or more further defined peak having a m/z of from 1228-1231 m/z, 1252-1255 m/z, 1254-1257 m/z, 1258-1261 m/z, 1268-1271 m/z, 1280-1283 m/z, 1282-1285 m/z, 1284-1287 m/z, 1286-1289 m/z, 1288-1291 m/z, 1294-1297 m/z, 1296-1299 m/z, 1308-1311 m/z, 1310-1313 m/z, 1314-1317 m/z, 1322-1325 m/z, 1324-1327 m/z, 1336-1339 m/z, or a combination thereof; l. said thirteenth peak set comprises one or more further defined peak having a m/z of from 1220-1223 m/z, 1303-1306 m/z, 1317-1320 m/z, 1345-1348 m/z, 1442-1445 m/z, 1456-1459 m/z, 1474-1477 m/z, 1485-1488 m/z, 1487-1490 m/z, 1695-1698 m/z, 1697-1700 m/z, 1723-1726 m/z, 1725-1728 m/z, 1739-1742 m/z, 2107-2110 m/z, 2132-2135 m/z, 2133-2136 m/z, 2147-2150 m/z, 2161-2164 m/z, 2175-2178 m/z, 2300-2303 m/z, 2316-2319 m/z, 2454-2457 m/z, 2496-2499 m/z, 2524-2527 m/z, 2586-2589 m/z, 2628-2631 m/z, 2656-2659 m/z, 2732-2735 m/z, 2736-2739 m/z, 2746-2749 m/z, 2774-2777 m/z, 2777-2780 m/z, 2786-2789 m/z, 2802-2805 m/z, 2804-2807 m/z, 2850-2853 m/z, 2892-2895 m/z, 2920-2923 m/z, or a combination thereof; m. said fourteenth peak set comprises one or more further defined peak having a m/z of from 1146-1149 m/z, 1160-1163 m/z, 1190-1193 m/z, 1196-1199 m/z, 1561-1564 m/z, 1881-1884 m/z, 1885-1888 m/z, 1897-1900 m/z, 1923-1926 m/z, 1925-1928 m/z, 1935-1938 m/z, 1937-1940 m/z, 1939-1942 m/z, 1951-1954 m/z, or a combination thereof; n. said fifteenth peak set comprises one or more further defined peak having a m/z of from 1184-1187 m/z, 1243-1246 m/z, or a combination thereof; o. said sixteenth peak set comprises one or more further defined peak having a m/z of from 1330-1333 m/z, 1344-1347 m/z, 1358-1361 m/z, 1372-1375 m/z, 1386-1389 m/z, 1388-1391 m/z, 1400-1403 m/z, 1402-1405 m/z, 1414-1417 m/z, or a combination thereof; p. said seventeenth peak set comprises one or more further defined peak having a m/z of from 1294-1297 m/z, 1296-1299 m/z, or a combination thereof; q. said eighteenth peak set comprises one or more further defined peak having a m/z of from 1723-1726 m/z, 1814-1817 m/z, or a combination thereof; r. said nineteenth peak set comprises a further defined peak having a m/z of from 1238-1241 m/z; s. said twentieth peak set comprises one or more further defined peak having a m/z of from 1039-1042 m/z, 1074-1077 m/z, 1124-1127 m/z, 1150-1153 m/z, 1184-1187 m/z, 1198-1201 m/z, 1212-1215 m/z, 1214-1217 m/z, 1220-1223 m/z, 1226-1229 m/z, 1228-1231 m/z, 1240-1243 m/z, 1242-1245 m/z, 1254-1257 m/z, 1256-1259 m/z, 1268-1271 m/z, 1270-1273 m/z, 1282-1285 m/z, 1284-1287 m/z, 1294-1297 m/z, 1296-1299 m/z, 1299-1302 m/z, 1310-1313 m/z, 1312-1315 m/z, 1313-1316 m/z, 1324-1327 m/z, 1330-1333 m/z, 1340-1343 m/z, 1354-1357 m/z, 1356-1359 m/z, 1357-1360 m/z, 1358-1361 m/z, 1368-1371 m/z, 1370-1373 m/z, 1372-1375 m/z, 1374-1377 m/z, 1382-1385 m/z, 1384-1387 m/z, 1386-1389 m/z, 1388-1391 m/z, 1398-1401 m/z, 1400-1403 m/z, 1417-1420 m/z, 1426-1429 m/z, 1429-1432 m/z, 1430-1433 m/z, 1440-1443 m/z, 1457-1460 m/z, 1459-1462 m/z, 1471-1474 m/z, 1473-1476 m/z, 1474-1477 m/z, 1487-1490 m/z, 1501-1504 m/z, 1529-1532 m/z, 1531-1534 m/z, 1541-1544 m/z, 1559-1562 m/z, 1571-1574 m/z, or a combination thereof; t. said twenty-first peak set comprises one or more further defined peak having a m/z of from 1042-1045 m/z, 1052-1055 m/z, 1124-1127 m/z, 1150-1153 m/z, 1204-1207 m/z, 1220-1223 m/z, 1244-1247 m/z, 1273-1276 m/z, 1287-1290 m/z, 1296-1299 m/z, 1390-1393 m/z, 1392-1395 m/z, 1427-1430 m/z, 1430-1433 m/z, 1442-1445 m/z, 1444-1447 m/z, 1458-1461 m/z, 1474-1477 m/z, 1475-1478 m/z, 1488-1491 m/z, 1534-1537 m/z, 1680-1683 m/z, 1697-1700 m/z, 1698-1701 m/z, 1707-1710 m/z, 1722-1725 m/z, 1723-1726 m/z, 1779-1782 m/z, 1819-1822 m/z, 1821-1824 m/z, 1838-1841 m/z, 2091-2094 m/z, 2103-2106 m/z, 2107-2110 m/z, 2117-2120 m/z, 2118-2121 m/z, 2119-2122 m/z, 2145-2148 m/z, 2159-2162 m/z, 2175-2178 m/z, 2356-2359 m/z, 2373-2376 m/z, 2397-2400 m/z, 2439-2442 m/z, 2455-2458 m/z, 2483-2486 m/z, 2495-2498 m/z, 2512-2515 m/z, 2513-2516 m/z, 2777-2780 m/z, 2780-2783 m/z, 2793-2796 m/z, 2807-2810 m/z, 2809-2812 m/z, 2821-2824 m/z, 2822-2825 m/z, 2834-2837 m/z, 2849-2852 m/z, 2851-2854 m/z, or a combination thereof; u. said twenty-second peak set comprises one or more further defined peak having a m/z of from 1043-1046 m/z, 1071-1074 m/z, 1073-1076 m/z, 1079-1082 m/z, 1468-1471 m/z, 1474-1477 m/z, 1476-1479 m/z, 1482-1485 m/z, 1484-1487 m/z, 1490-1493 m/z, 1496-1499 m/z, 1504-1507 m/z, 1510-1513 m/z, or a combination thereof; v. said twenty-third peak set comprises one or more further defined peak having a m/z of from 1306-1309 m/z, 1404-1407 m/z, 1418-1421 m/z, 1420-1423 m/z, 1430-1433 m/z, 1444-1447 m/z, 1448-1451 m/z, 1458-1461 m/z, 1462-1465 m/z, 1472-1475 m/z, 1488-1491 m/z, 1490-1493 m/z, 1502-1505 m/z, 1511-1514 m/z, or a combination thereof; w. said twenty-fourth peak set comprises one or more further defined peak having a m/z of from 1198-1201 m/z, 1312-1315 m/z, 1388-1391 m/z, 1402-1405 m/z, 1430-1433 m/z, 1458-1461 m/z, or a combination thereof; and/or x. said twenty-fifth peak set comprises one or more further defined peak having an m/z of from 1198-1201 m/z, 1224-1227 m/z, 1228-1231 m/z, 1242-1245 m/z, 1256-1259 m/z, 1252-1255 m/z, 1268-1271 m/z, 1270-1273 m/z, 1284-1287 m/z, 1374-1377-m/z, 1388-1391 m/z, 1402-1405 m/z, 1416-1419 m/z, and 1430-1433 m/z.
6. The method according to any one of claim 2, 4 or 5, wherein the operating theatre equipment is in fluid contact with the subject during cardiothoracic surgery and/or comes into direct contact with the subject during cardiothoracic surgery.
7. The method according to any one of the preceding claims, wherein said defined peak is indicative of the presence of a bacterial membrane lipid.
8. The method according to any one of the preceding claims, wherein said defined peak is indicative of the presence of a bacterial membrane lipid and wherein said defined peak is absent from a corresponding mass spectrum output for a sample lacking said bacterial membrane lipid.
9. The method according to any one of the preceding claims, wherein the presence of said one or more peak set is indicative of the presence of a bacterial infection in the subject and the absence of said one or more peak set is indicative of the absence of a bacterial infection in the subject.
10. The method according to any one or the preceding claims, wherein said sample is admixed with a matrix solution prior to subjecting the sample to mass spectrometry analysis, wherein the matrix solution allows for the ionisation of a bacterial membrane lipid as an integral component of a bacterial membrane.
11. The method according to any one of the preceding claims, wherein said sample is admixed with a matrix solution prior to subjecting the sample to mass spectrometry analysis, wherein said matrix solution comprises a matrix suspended in an organic solvent.
12. The method according to any one of the preceding claims, wherein said sample is admixed with a matrix solution prior to subjecting the sample to mass spectrometry analysis, wherein said matrix solution comprises 2,5-dihydroxybenzoic acid (DHB) suspended in an organic solvent.
13. The method according to claim 11 or 12, wherein the organic solvent is present at a concentration of less than 50% (v/v).
14. The method according to any one of claims 11-13, wherein said organic solvent is ethanol.
15. The method according to any one of claims 10-14, wherein said matrix solution comprises DHB at a concentration of 5-10 mg/ml and ethanol at a concentration of 20%-35% (v/v); and
16. The method according to any one of claims 10-15, wherein said matrix solution comprises DHB at a concentration of 10 mg/ml and ethanol at a concentration of 25% (v/v).
17. The method according to any one of the preceding claims, wherein the sample comprise a bacterial membrane or a fragment thereof, wherein the fragment comprises a non-bacterial membrane lipid component.
18. The method according to any one of the preceding claims, wherein said sample comprises a bacterial membrane lipid present as an integral component of a bacterial membrane, or a fragment thereof; preferably a bacterial membrane lipid present as an integral component of a bacterial membrane, or a fragment thereof, of an infectious bacterium.
19. The method according to any one of the preceding claims, wherein the sample is obtained from sputum, or the sample is obtained from blood, or a combination thereof.
20. The method according to any one of the preceding claims, wherein the subject has one or more disorder selected from Cystic Fibrosis, Chronic Obstructive Pulmonary Disease (COPD), Bronchiectasis, gastro-oesophageal reflux disease (GORD), an immunodeficiency disorder (e.g. HIV), heart disease, or a combination thereof.
21. The method according to any one of the preceding claims, wherein said mass spectrometry analysis comprises (or consists of) MALDI-TOF mass spectrometry analysis.
22. The method according to any one of claims 7-21, wherein said bacterial membrane lipid is one or more selected from a sulfoglycolipid (SGL), a phosphatidyl-myo-inositol (PI), phosphatidyl-myo-inositol-dimannosides (PIM), a pentaacyltrehalose (PAT), a glycerol monomycolate (GroMM), a phenolic glycolipid (PGL), triacylglycerol (TAG), a glycopeptidolipid (GPL), and a C-mycoside.
23. The method according to any one of the preceding claims, wherein the sample comprises between at least about 10.sup.1 bacterial cells, preferably about 10.sup.1-10.sup.10 bacterial cells.
24. The method according to any one of the preceding claims, further comprising the step of recording on a suitable data carrier the data obtained in a step of subjecting the sample to mass spectrometry analysis to provide a mass spectrum output.
25. A method for detecting the presence or absence of a Mycobacterium in a sample, the method comprising: a. subjecting a sample to mass spectrometry analysis in the positive ion mode and generating a mass spectrum output; and b. detecting one or more peak set in said mass spectrum output; c. wherein said one or more peak set is selected from: i. a fourth peak set comprising at least two defined peaks selected from: a peak having a m/z of from 1012-1015 m/z, a peak having a m/z of from 1121-1124 m/z, a peak having a m/z of from 1122-1125 m/z, a peak having an m/z of from 1123-1126 m/z, and a peak having a m/z of from 1149-1152 m/z; ii. a fifth peak set comprising at least two defined peaks selected from: a peak having a m/z of from 1311-1314 m/z, a peak having a m/z of from 1339-1342 m/z, a peak having a m/z of from 1474-1477 m/z, a peak having a m/z of from 1488-1491 m/z, and a peak having a m/z of from 1823-1826 m/z; iii. a seventh peak set comprising at least two defined peaks selected from: a peak having a m/z of from 1294-1297 m/z, a peak having a m/z of from 1296-1299 m/z, a peak having a m/z of from 1308-1311 m/z, and a peak having a m/z of from 1322-1325 m/z; iv. an eighth peak set comprising at least two defined peaks selected from: a peak having a m/z of from 1370-1373 m/z, and a peak having a m/z of from 1412-1415 m/z; v. a ninth peak set comprising at least two defined peaks selected from: a peak having a m/z of from 2129-2132 m/z, a peak having a m/z of from 2171-2174 m/z, a peak having a m/z of from 2247-2250 m/z, and a peak having a m/z of from 2261-2264 m/z; vi. a tenth peak set comprising at least two defined peaks selected from: a peak having a m/z of from 2273-2276 m/z, a peak having a m/z of from 2275-2278 m/z, a peak having a m/z of from 2551-2554 m/z, and a peak having a m/z of from 2553-2556 m/z; vii. an eleventh peak set comprising at least two defined peaks selected from: a peak having a m/z of from 1339-1342 m/z, a peak having a m/z of from 1353-1356 m/z, a peak having a m/z of from 1367-1370 m/z, and a peak having an m/z of from 1381-1384 m/z; viii. a twelfth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1242-1245 m/z, a peak having an m/z of from 1256-1259 m/z, a peak having an m/z of from 1270-1273 m/z, and a peak having an m/z of from 1298-1301 m/z; ix. a fifteenth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1202-1205 m/z, a peak having an m/z of from 1224-1227 m/z, a peak having an m/z of from 1226-1229 m/z, and a peak having an m/z of from 1240-1243 m/z; x. a seventeenth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1308-1311 m/z, a peak having an m/z of from 1310-1313 m/z, a peak having an m/z of from 1324-1327 m/z, and a peak having an m/z of from 1338-1341 m/z; xi. an eighteenth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1012-1015 m/z, a peak having an m/z of from 1028-1031 m/z, a peak having an m/z of from 1697-1700 m/z, and a peak having an m/z of from 1698-1701 m/z; xii. a nineteenth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1226-1229 m/z, and a peak having an m/z of from 1240-1243 m/z; xiii. a twenty-first peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1036-1039 m/z, a peak having an m/z of from 1259-1262 m/z, a peak having an m/z of from 1374-1377 m/z, a peak having an m/z of from 2497-2500 m/z, and a peak having an m/z of from 2835-2838 m/z; xiv. a twenty-second peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1057-1060 m/z, a peak having an m/z of from 1498-1501 m/z, a peak having an m/z of from 1512-1515 m/z, and a peak having an m/z of from 1526-1529 m/z; xv. a twenty-fourth peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1242-1245 m/z, a peak having an m/z of from 1256-1259 m/z, a peak having an m/z of from 1270-1273 m/z, and a peak having an m/z of from 1284-1287 m/z; and xvi. a thirty-seventh peak set comprising at least two defined peaks selected from: a peak having an m/z of from 1242-1245 m/z, a peak having an m/z of from 1256-1259 m/z, a peak having an m/z of from 1270-1273 m/z, and a peak having an m/z of from 1284-1287 m/z; d. wherein the presence of said one or more peak set is indicative of the presence of a Mycobacterium; and e. wherein the absence of said one or more peak set is indicative of the absence of a Mycobacterium.
26. The method according to claim 25, wherein: a. said fourth peak set comprises one or more further defined peak having a m/z of from 1021-1024 m/z, 1131-1134 m/z, 1134-1137 m/z, 1147-1150 m/z, 1150-1153 m/z, 1198-1201 m/z, 1878-1881 m/z, 1880-1883 m/z, 2131-2134 m/z, 2275-2278 m/z, 2553-2556 m/z, or a combination thereof; b. said fifth peak set comprises one or more further defined peak having a m/z of from 1253-1256 m/z, 1267-1270 m/z, 1269-1272 m/z, 1279-1282 m/z, 1281-1284 m/z, 1283-1286 m/z, 1295-1298 m/z, 1297-1300 m/z, 1299-1302 m/z, 1300-1303 m/z, 1307-1310 m/z, 1309-1312 m/z, 1313-1316 m/z, 1323-1326 m/z, 1325-1328 m/z, 1327-1330 m/z, 1329-1332 m/z, 1341-1344 m/z, 1355-1358 m/z, 1357-1360 m/z, 1429-1432 m/z, 1432-1435 m/z, 1442-1445 m/z, 1443-1446 m/z, 1445-1448 m/z, 1446-1449 m/z, 1456-1459 m/z, 1457-1460 m/z, 1458-1461 m/z, 1460-1463 m/z, 1469-1472 m/z, 1471-1474 m/z, 1472-1475 m/z, 1477-1480 m/z, 1484-1487 m/z, 1485-1488 m/z, 1486-1489 m/z, 1497-1500 m/z, 1499-1502 m/z, 1500-1503 m/z, 1502-1505 m/z, 1513-1516 m/z, 1514-1517 m/z, 1516-1519 m/z, 1518-1521 m/z, 1528-1531 m/z, 1529-1532 m/z, 1530-1533 m/z, 1766-1769 m/z, 1781-1784 m/z, 1795-1798 m/z, 1809-1812 m/z, 1811-1814 m/z, 1815-1818 m/z, 1821-1824 m/z, 1829-1832 m/z, 1837-1840 m/z, 1839-1842 m/z, 1849-1852 m/z, 1851-1854 m/z, or a combination thereof; c. said seventh peak set comprises one or more further defined peak having a m/z of from 1266-1269 m/z, 1268-1271 m/z, 1310-1313 m/z, 1324-1327 m/z, 1338-1341 m/z, or a combination thereof; d. said ninth peak set comprises one or more further defined peak having a m/z of from 2115-2118 m/z, 2145-2148 m/z, 2157-2160 m/z, 2187-2190 m/z, 2188-2191 m/z, 2205-2208 m/z, 2219-2222 m/z, 2233-2236 m/z, 2245-2248 m/z, 2259-2262 m/z, 2277-2280 m/z, or a combination thereof; e. said tenth peak set comprises one or more further defined peak having a m/z of from 1848-1851 m/z, 2215-2218 m/z, 2231-2234 m/z, 2233-2236 m/z, 2235-2238 m/z, 2243-2246 m/z, 2244-2247 m/z, 2245-2248 m/z, 2247-2250 m/z, 2257-2260 m/z, 2260-2263 m/z, 2261-2264 m/z, 2262-2265 m/z, 2271-2274 m/z, 2285-2288 m/z, 2287-2290 m/z, 2289-2292 m/z, 2291-2294 m/z, 2389-2392 m/z, 2511-2514 m/z, 2549-2552 m/z, 2569-2572 m/z, or a combination thereof; f. said eleventh peak set comprises one or more further defined peak having a m/z of from 1311-1314 m/z, 1325-1328 m/z, 1369-1372 m/z, 1397-1400 m/z, or a combination thereof; g. said twelfth peak set comprises one or more further defined peak having a m/z of from 1228-1231 m/z, 1252-1255 m/z, 1254-1257 m/z, 1258-1261 m/z, 1268-1271 m/z, 1280-1283 m/z, 1282-1285 m/z, 1284-1287 m/z, 1286-1289 m/z, 1288-1291 m/z, 1294-1297 m/z, 1296-1299 m/z, 1308-1311 m/z, 1310-1313 m/z, 1314-1317 m/z, 1322-1325 m/z, 1324-1327 m/z, 1336-1339 m/z, or a combination thereof; h. said fifteenth peak set comprises one or more further defined peak having a m/z of from 1184-1187 m/z, 1243-1246 m/z, or a combination thereof; i. said seventeenth peak set comprises one or more further defined peak having a m/z of from 1294-1297 m/z, 1296-1299 m/z, or a combination thereof; j. said eighteenth peak set comprises one or more further defined peak having a m/z of from 1723-1726 m/z, 1814-1817 m/z, or a combination thereof; k. said nineteenth peak set comprises a further defined peak having a m/z of from 1238-1241 m/z; l. said twenty-first peak set comprises one or more further defined peak having a m/z of from 1042-1045 m/z, 1052-1055 m/z, 1124-1127 m/z, 1150-1153 m/z, 1204-1207 m/z, 1220-1223 m/z, 1244-1247 m/z, 1273-1276 m/z, 1287-1290 m/z, 1296-1299 m/z, 1390-1393 m/z, 1392-1395 m/z, 1427-1430 m/z, 1430-1433 m/z, 1442-1445 m/z, 1444-1447 m/z, 1458-1461 m/z, 1474-1477 m/z, 1475-1478 m/z, 1488-1491 m/z, 1534-1537 m/z, 1680-1683 m/z, 1697-1700 m/z, 1698-1701 m/z, 1707-1710 m/z, 1722-1725 m/z, 1723-1726 m/z, 1779-1782 m/z, 1819-1822 m/z, 1821-1824 m/z, 1838-1841 m/z, 2091-2094 m/z, 2103-2106 m/z, 2107-2110 m/z, 2117-2120 m/z, 2118-2121 m/z, 2119-2122 m/z, 2145-2148 m/z, 2159-2162 m/z, 2175-2178 m/z, 2356-2359 m/z, 2373-2376 m/z, 2397-2400 m/z, 2439-2442 m/z, 2455-2458 m/z, 2483-2486 m/z, 2495-2498 m/z, 2512-2515 m/z, 2513-2516 m/z, 2777-2780 m/z, 2780-2783 m/z, 2793-2796 m/z, 2807-2810 m/z, 2809-2812 m/z, 2821-2824 m/z, 2822-2825 m/z, 2834-2837 m/z, 2849-2852 m/z, 2851-2854 m/z, or a combination thereof; m. said twenty-second peak set comprises one or more further defined peak having a m/z of from 1043-1046 m/z, 1071-1074 m/z, 1073-1076 m/z, 1079-1082 m/z, 1468-1471 m/z, 1474-1477 m/z, 1476-1479 m/z, 1482-1485 m/z, 1484-1487 m/z, 1490-1493 m/z, 1496-1499 m/z, 1504-1507 m/z, 1510-1513 m/z, or a combination thereof; n. said twenty-fourth peak set further comprises one or more peak having a m/z of from 1198-1201 m/z, 1312-1315 m/z, 1388-1391 m/z, 1402-1405 m/z, 1430-1433 m/z, 1458-1461 m/z, or a combination thereof; and/or o. said twenty-fifth peak set comprises one or more further defined peak having an m/z of from 1198-1201 m/z, 1224-1227 m/z, 1228-1231 m/z, 1242-1245 m/z, 1256-1259 m/z, 1252-1255 m/z, 1268-1271 m/z, 1270-1273 m/z, 1284-1287 m/z, 1374-1377-m/z, 1388-1391 m/z, 1402-1405 m/z, 1416-1419 m/z, 1430-1433 m/z, or a combination thereof.
27. The method according to any one of the preceding claims, wherein the sample is subjected to mass spectrometry in the positive ion mode, and the method further comprises confirming the presence or absence of one or more of the following defined peak(s) in the mass spectrum output: a peak having a m/z of from 1210-1213 m/z (preferably about 1211.8 m/z), 1326-1329 m/z (preferably about 1327.8 m/z), 1356-1359 m/z (preferably about 1357.9 m/z), 1370-1373 m/z (preferably about 1371.9 m/z), 1384-1387 m/z (preferably about 1385.9 m/z), 1398-1401 m/z (preferably about 1399.9 m/z), 1400-1403 m/z (preferably about 1401.9 m/z), 1412-1415 m/z (preferably about 1413.9 m/z), 1414-1417 m/z (preferably about 1415.9 m/z), 1426-1429 m/z (preferably about 1427.9 m/z), 1428-1431 m/z (preferably about 1429.9 m/z), 1442-1445 m/z (preferably about 1443.9 m/z), 1444-1447 m/z (preferably about 1445.9 m/z), 1456-1459 m/z (preferably about 1457.9 m/z), 1458-1461 m/z (preferably about 1459.9 m/z), and/or 1472-1475 m/z (preferably about 1474 m/z); a. wherein the presence of said one or more peak confirms the presence of M. abscessus subsp. abscessus; or wherein the presence of said one or more peak preferably is indicative of the absence of M. abscessus subsp. abscessus; and/or b. wherein the presence of said one or more peak indicates that the subject is not a suitable candidate for receiving a lung transplant; and/or c. wherein the presence of said one or more peak indicates that the operating theatre equipment is not suitable to be used during cardiothoracic surgery of a subject; and/or d. wherein the presence of said one or more peak is indicative of the presence of a bacterial infection in the subject subsequent to receiving cardiothoracic surgery.
28. A method according to any one of the preceding claims, wherein the sample is subjected to mass spectrometry in the negative ion mode, and the method further comprises confirming the presence or absence of one or more of the following defined peak(s) in the mass spectrum output: a peak having a m/z of from 681-684 m/z (preferably about 682 m/z) and/or 696-699 m/z (preferably about 697 m/z); a. wherein the presence of said one or more peak confirms the presence of M. abscessus subsp. abscessus; or wherein the absence of said one or more peak is indicative of the presence of M. abscessus subsp. abscessus; and/or b. wherein the presence of said one or more peak indicates that the subject is not a suitable candidate for receiving a lung transplant; and/or c. wherein the presence of said one or more peak indicates that the operating theatre equipment is not suitable to be used during cardiothoracic surgery of a subject; and/or d. wherein the presence of said one or more peak is indicative of the presence of a bacterial infection in the subject subsequent to receiving cardiothoracic surgery.
29. A method according to any one of the preceding claims, wherein the sample is subjected to mass spectrometry in the negative ion mode, and the method further comprises confirming the presence or absence of one or more of the following defined peak(s) in the mass spectrum output: a peak having a m/z of from 520-523 m/z (preferably about 521 m/z), 534-537 m/z (preferably about 535.9 m/z), 535-538 m/z (preferably about 536.9 m/z), 1403-1406 m/z (preferably about 1404 m/z), 1565-1568 m/z (preferably about 1566.9 m/z), 1568-1571 m/z (preferably about 1569 m/z), 1578-1581 m/z (preferably about 1579 m/z), 1581-1584 m/z (preferably about 1582 m/z), 1621-1624 m/z (preferably about 1622.1 m/z), 1623-1626 m/z (preferably about 1624.1 m/z), 1637-1640 m/z (preferably about 1638.1 m/z), 1638-1641 m/z (preferably about 1639.1 m/z), 1647-1650 m/z (preferably about 1648.1 m/z), 1648-1651 m/z (preferably about 1649.1 m/z), 1649-1652 m/z (preferably about 1650.1 m/z), 1663-1666 m/z (preferably about 1664.1 m/z), 1664-1667 m/z (preferably about 1665.1 m/z), 2296-2299 m/z (preferably about 2297.3 m/z), and/or 2297-2300 m/z (preferably about 2298.3 m/z); a. wherein the presence of said one or more peak confirms the presence of M. abscessus subsp. bolletii; or wherein the absence of said one or more peak is indicative of the absence of M. abscessus subsp. bolletii; and/or b. wherein the presence of said one or more peak indicates that the subject is not a suitable candidate for receiving a lung transplant; and/or c. wherein the presence of said one or more peak indicates that the operating theatre equipment is not suitable to be used during cardiothoracic surgery of a subject; and/or d. wherein the presence of said one or more peak is indicative of the presence of a bacterial infection in the subject subsequent to receiving cardiothoracic surgery.
30. A method according to any one of the preceding claims, wherein the sample is subjected to mass spectrometry in the positive ion mode, and a method of the invention may further comprise confirming the absence of one or more (preferably two or more; more preferably three or more) of the following (e.g. further) defined peak(s) in the mass spectrum output: a peak having a m/z of from 1072-1075 m/z (preferably about 1073.6 m/z), 1248-1251 m/z (preferably about 1249.9 m/z), 1260-1263 m/z (preferably about 1261.9 m/z), 1262-1265 m/z (preferably about 1263.9 m/z), 1274-1277 m/z (preferably about 1275.9 m/z), 1276-1279 m/z (preferably about 1277.9 m/z), 1288-1291 m/z (preferably about 1289.9 m/z), 1290-1293 m/z (preferably about 1291.9 m/z), 1294-1297 m/z (preferably about 1295.9 m/z), 1304-1307 m/z (preferably about 1305.9 m/z), 1308-1311 m/z (preferably about 1309.9 m/z), 1310-1313 m/z (preferably about 1311.9 m/z), 1314-1317 m/z (preferably about 1315.9 m/z), 1318-1321 m/z (preferably about 1319.9 m/z), 1320-1323 m/z (preferably about 1321.9 m/z), 1322-1325 m/z (preferably about 1323.9 m/z), 1324-1327 m/z (preferably about 1325.9 m/z), 1332-1335 m/z (preferably about 1333.9 m/z), 1336-1339 m/z (preferably about 1337.9 m/z), 1338-1341 m/z (preferably about 1339.9 m/z), 1719-1722 m/z (preferably about 1720.1 m/z), 1735-1738 m/z (preferably about 1736.1 m/z), 1747-1750 m/z (preferably about 1748.1 m/z), 1761-1764 m/z (preferably about 1762.1 m/z), 1764-1767 m/z (preferably about 1765.1 m/z), and/or 1789-1792 m/z (preferably about 1790.1 m/z); a. wherein the absence of said one or more peak confirms the absence of M. abscessus subsp. massiliense; and/or b. wherein the absence of said one or more peak indicates that the subject is not a suitable candidate for receiving a lung transplant; and/or c. wherein the absence of said one or more peak indicates that the operating theatre equipment is not suitable to be used during cardiothoracic surgery of a subject; and/or d. wherein the absence of said one or more peak is indicative of the absence of a M. abscessus subsp. massiliense infection in the subject subsequent to receiving cardiothoracic surgery.
31. A method according to any one of the preceding claims, wherein the sample is subjected to mass spectrometry in the negative ion mode (for example, additionally or alternatively to subjecting the sample to mass spectrometry in the positive ion mode), and method of the invention may further comprise confirming the absence of one or more (preferably two or more; more preferably three or more) of the following (e.g. further) defined peak(s) in the mass spectrum output: a peak having a m/z of from 408-411 m/z (preferably about 409 m/z), 490-493 m/z (preferably about 491 m/z), and/or 512-515 m/z (preferably about 513 m/z); a. wherein the absence of said one or more peak preferably confirms the absence of M. abscessus subsp. massiliense; and/or b. wherein the absence of said one or more peak indicates that the subject is not a suitable candidate for receiving a lung transplant (e.g. if treated for an M. abscessus subsp. massiliense infection); and/or c. wherein the absence of said one or more peak indicates that the operating theatre equipment is not suitable to be used during cardiothoracic surgery (of a subject) (e.g. if treated to remove M. abscessus subsp. massiliense); and/or d. wherein the absence of said one or more peak is indicative of the absence of a M. abscessus subsp. massiliense infection in the subject subsequent to receiving cardiothoracic surgery.
32. A kit for use in a method for identifying a subject suitable for receiving a lung transplant; and/or for use in a method for screening operating theatre equipment for use in cardiothoracic surgery of a subject; and/or for use in method for identifying the presence or absence of a bacterial infection in a subject subsequent to receiving open heart surgery and/or a heart transplant; and/or for use in a method for detecting the presence or absence of a Mycobacterium; the kit comprising: a. 2,5-dihydroxybenzoic acid and/or super 2,5-dihydroxybenzoic acid; b. an organic solvent, preferably ethanol; and c. instructions to perform a method according to any one of the preceding claims.
Description
FIGURES
[0536] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures.
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EXAMPLES
Materials and Methods
Bacterial Strains
[0572] Reference bacterial strains were obtained from the Belgian Co-ordinated Collections of Microorganisms (BOOM, Antwerp, Belgium) and from the European Centres for Disease Control (ECDC) (Table 1).
TABLE-US-00001 TABLE 1 Mycobacterium reference strains Strains (n = 29) Complex Source RGM/SGM M. abscessus M. abscessus complex BCCM C04975 RGM M. avium M. avium complex BCCM C01788 RGM M. bohemicum ECDC SGM M. celatum ECDC SGM M. chelonae Unknown 17T9705 RGM M. chimaera M. avium complex BCCM 130444 RGM M. chimaera M. avium complex BCCM 2015-01546 RGM M. fortuitum M. fortuitum complex BCCM 52847 RGM M. fortuitum M. fortuitum complex ECDC RGM M. gordonae BCCM C02565 SGM M. interjectum ECDC SGM M. intermedium ECDC RGM M. intracellulare M. avium complex ECDC SGM M. intracellulare M. avium complex BCCM 120584 SGM M. kansasii BCCM 960032 SGM M. marinum ECDC SGM M. nonchromogenicum ECDC SGM M. peregrinum M. fortuitum complex BCCM 970462 RGM M. peregrinum M. fortuitum complex ECDC RGM M. scrofulaceum BCCM C01934 SGM M. scrofulaceum ECDC SGM M. shimoidei ECDC SGM M. simiae BCCM C02428 SGM M. simiae ECDC SGM M. smegmatis BCCM M010336 SGM M. szulgai BCCM M003303 SGM M. ulcerans BCCM C05150 SGM M. xenopi ECDC SGM M. xenopi BCCM C05984 SGM RGM = rapidly growing mycobacteria; SGM = slowly growing mycobacteria.
[0573] The lyophilized BCCM strains were revived by re-suspending in a small amount (˜10 μL) of liquid medium, Middlebrook 7H9 (Sigma Aldrich). All isolates were incubated in 5 mL of 7H9 with OADC (oleic acid-albumin-dextrose-catalase) for 2-4 weeks and then 1 ml of bacterial biomass of each strain was frozen at −20° C. Then, 100 μL was cultured again on selected media and incubated at 37° C. from 5 days up to 5 weeks. Collected bacteria may be heat killed at 95° C. for 30 minutes (e.g. before leaving the BSL3 containment area) prior to mass spectrometry.
Clinical Isolates
[0574] Thirty-two anonymized M. abscessus complex clinical isolates were (originally) collected from the lung of UK cystic fibrosis (CF) patients (recovered from sputum samples), and were grown from frozen stocks in 7H9 liquid media with OADC in 37° C. for 2 weeks before testing on MALDI-ToF MS. Previously those isolates were rpoB sequenced and the data indicated 3 clusters corresponded to three M. abscessus subspecies: abscessus sensu stricto, bolletii, and massiliense.
Sample Preparation
[0575] Three variables were selected for initial evaluation of NTM strains via MALDI-ToF MS. [0576] (i) Phase of culture growth [0577] (ii) Growth media. Three media were used for mycobacterial growth [0578] Middlebrook 7H9 (Sigma Aldrich) with OADC—liquid medium [0579] Middlebrook 7H11 (Sigma Aldrich) with OADC—solid medium [0580] Kirchner (Sigma Aldrich)—liquid medium [0581] (iii) Matrix solution (composition): 10 mg/ml of 2,5-dihydroxybenzoic acid (DHB) diluted in four types of solvents to check the best performance: [0582] A—in chloroform:methanol (95:5) (C/M) [0583] B—in 50% Ethanol (EtOH) [0584] C—in 25% Ethanol (EtOH) [0585] D—in 50% Methanol (MeOH)
[0586] Additional concentrations of EtOH were also analysed, as per
[0587] About 0.5 ml-1 mL of liquid mycobacterial biomass from the bottom of the tube was pipetted to the 1 mL Eppendorf and heated in water bath at 95° C. for 30 min in containment level 3 laboratory. The samples then were transported into a containment level 2 laboratory where all further work was performed. The liquid mycobacterial biomass was centrifuged at 15,000×g for 5 min and the supernatant was discarded. The bacterial pellet was washed 4 times with 200 μL of ultra-purified water. After each washing the pellet was centrifuged and the supernatant was removed. After the last washing around 50 μL of mycobacterial biomass was left, to be used for mass spectrometry analysis (and the remaining culture was frozen at −20° C. till the further testing).
[0588] Next, 0.4 μL of sample (resuspended bacteria) was pipetted onto the MALDI-ToF target slide and mixed with 0.6 μL of matrix solution (DHB in organic solvent, as described above).
[0589] The mixtures were dried at room temperature. Samples were analyzed on 4800 MALDI TOF/TOF™ Analyzer.
[0590] An exemplary way to carry out the invention is as follows:
Step 1: mycobacteria were isolated and grown on Middlebrook 7H9 liquid medium (without glycerol) and supplemented with 10% OADC (without any detergent). The culture was incubated (with shaking as appropriate) for 2-6 weeks at 37° C. One hundred microliters (e.g. having 10.sup.4 to 10.sup.7 bacteria) of bacteria are collected and transferred into an Eppendorf tube (0.5 to 2 mL) containing 100 microliter of water (distilled or double-distilled);
Step 2: Bacteria are then heat inactivated (facultative step) using a dry bath or water bath for 30 min at 95° C.;
Step 3: bacteria were pelleted and washed at least once with 100 μl to 1,000 μl double-distilled water to remove salts excess resulting from the culture medium (this optimises removal of background peaks in the mass spectrum output);
Step 4: the washed bacteria are suspended in 50 μl of double-distilled water and 0.4 μl is loaded on the MALDI-TOF target (SmallAnchor MALDI target plate, Bruker).
Step 5: Overlay the 0.4 μl of bacterial suspension with 0.6 μl of the designed matrix solution (DHB in organic solvent, as described above). Here, matrix 2,5-dihydroxybenzoic acid (DHB) suspended in organic solvents (usually Ethanol 25%), with mass spectrometry analysis conduced in the positive ion mode (or negative ion mode).
Step 5: MALDI-TOF MS acquisition (positive or negative ion mode). Classical MALDI-TOF MS machines that are present in clinical microbiology labs are suitable (Bioyper from Bruker Daltonics and VITEK-MS from bioMerieux). The former (Bioyper) was employed here. Samples were analyzed in the positive and negative ion mode operating at 20 kV and were set to acquire mass spectral peaks with mass/charge ratio (m/z) from 400 to 4,000 mass units. Mass spectrometry data were analyzed using Data Explorer® Software version 4.9 from Applied Biosystems and assignment of MTB/NTM was performed blinded, after visual interpretation of spectra following criteria previously published
Step 6: From the obtained mass spectrum output having peaks corresponding to species-specific lipids, the inventors were able to discriminate Mtb complex, Non-Tuberculous mycobacteria.
Reproducibility
[0591] To check the technical reproducibility and the robustness of the instrument three samples were prepared from a single culture of ten ECDC strains. Additionally the thirty-two M. abscessus clinical isolates were tested blindly in duplicates/triplicates to confirm the results.
Organic Solvents Suitable for Use in a Matrix Solution
[0592] Examples of suitable solvents for matrix include acetic acid, acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 2-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, diethylene glycol, diethyl ether, diglyme (diethylene glycol dimethyl ether), 1,2-dimethoxy-ethane (glyme, DME), dimethyl-formamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, glycerine, heptane, Hexamethylphosphoramide (HMPA), Hexamethylphosphorous triamide (HMPT), hexane, methanol, methyl t-butyl ether (MTBE), methylene chloride, N-methyl pyrrolidinone (NMP), pentane, Petroleum ether (ligroine), 1-propanol, 2-propanol, pyridine, tetrahydrofuran (THF), toluene, triethyl amine, water, water heavy (D.sub.2O), o-xylene, m-xylene, and p-xylene.
Example 1
Detection of Mycobacterial Lipid on Intact Bacteria
[0593] Samples of reference strains of mycobacteria were prepared as detailed above and subjected to mass spectrometry analysis (MALDI-TOF). Peaks corresponding to a number of mycobacterial membrane lipids (e.g. C-mycosides and phosphatidyl-myo-inositols) were readily detectable in the expected range of m/z.
[0594] These results demonstrate the applicability of the methods of the invention in detecting an intact mycobacteria in a sample (e.g. a crude sample).
[0595] Detection of mycobacteria by methods of the invention was possible for both bacteria in the stationary phase of growth and the logarithmic phase of growth (although a better mass spectral signal was achieved for stationary bacteria). Our samples were kept in incubator till the relevant growth was reached.
[0596] Furthermore, any differences in the mass spectrum output based on the tested media, Middlebrook 7H11, Middlebrook 7H9, and Kirchner, were very minor. However, among media used the best quality of biomass was generated by Middlebrook 7H11 agar. For practical purposes, it is useful to note that Middlebrook 7H9 medium works well, because this is the same medium employed in commercial MGIT tubes (Mycobacterium growth indicator tube) (Becton-Dickinson Microbiology Systems, Cockeysville, Md.) used in diagnostics laboratories for drug susceptibility testing of Mycobacteria species.
Example 2
Detection of Specific Species of Mycobacteria Via Species-Specific Membrane Lipid Fingerprints
[0597] The inventors selected a panel of 29 strains of NTMs comprising 22 commonly isolated species (Table 1). They were grown on different media (see above) and tested on DHB matrix diluted with four different solvents (A, B, C, and D). This collection of isolates included 7 RGM and 15 SGM species (Table 1).
[0598] First, the 10 ECDC reference strains was grown on Middlebrook 7H9 media with OADC and three samples were prepared from a single culture to check the lipid signature and reproducibility. The isolates were tested on MALDI-TOF MS with matrixes (DHB) solubilized in solvent A, or B. The reproducibility was high with both solvents. We also noticed that the same quality or better of mass spectra was generated with matrix diluted in the solvent B (50% EtOH) (better than A (C/M 95:5)).
[0599] We further compared another two solvents, C (25% EtOH) and D (50% MeOH). All results were comparable but the best quality was obtained from samples admixed with DHB matrix diluted in solvent B and C and analyzed on MALDI-ToF in positive ion mode.
[0600] Surprisingly, not only is it possible to distinguish between M. tuberculosis and NTMs, it is also possible to detect and discriminate between individual species based on the lipid fingerprints detectable on intact bacteria. Indeed, every strain tested was associated with a readily detectable, unique lipid fingerprint. Thus, the invention makes it possible to detect specific species, such as those associated with disease or with antibiotic resistance, from a crude sample of intact bacteria without any need for lengthy and expensive lipid extraction/purification. Given the unique compatibility of the methods with clinical laboratories, the invention will have a large impact on healthcare and the assessment of clinical infections.
Example 3
Detection of the Presence of a Mycobacterium Infection in Lung Transplant Candidate
[0601] The 32 clinical isolates (of M. abscessus complex) obtained from the lung of cystic fibrosis (e.g. via recovery from sputum) were pipetted on the MALDI-ToF target slide (M. abscessus, 24; M. bolletii, 3; M. massiliense, 5). The analysis generated a strong signal with DHB matrix diluted in solvent A (Chloroform/Ethanol 95:5), B (50% Ethanol), and C (25% EtOH) (the latter is exemplified in the Figures)).
[0602] We repeated the test on the same number of frozen pellets again to test the technical reproducibility. There was 100% match to the previous results.
[0603] All M. abscessus complex isolates in this study were correctly identified to the species level.
[0604] The positive ion mass spectrum with DHB matrix diluted in solvent A, B and C is dominated by two sets of peaks. The first set of peaks from 1199.7 m/z to 1313.7 m/z with the most intense peaks at 1257.7, 1271.7, and 1285.7 m/z. And another set of peaks from 1389.7 m/z to 1459.8 m/z with the most intense peaks vary at 1389.7, 1403.8, 1431.8 m/z.
Example 4
[0605] Detection of the Presence of Mycobacterium Associated with Complications During Heart Surgery
[0606] Detection of a number of Mycobacterium infections following cardiothoracic surgery has recently become a high priority (e.g. for Public Health England). Indeed, extra measures are generally put in place prior to open heart surgery to prevent infection with mycobacteria, in particular Mycobacterium chimaera (part of the M. avium complex).
[0607] M. avium complex (MAC) comprises mainly three very closely related species, M. avium, M. intracellulare, and M. chimaera. Existing diagnostic methods attempt to distinguish between M. avium and M. intracellulare, however M. chimaera is very difficult to differentiate from M. intracellulare. Visual analysis of the MALDI-TOF mass spectra of the MAC reference strains showed characteristic peaks for all of the species. In addition, a set of peaks present in M. intracellulare from 1443 m/z to 1569 m/z with the most intense peaks at 1471 m/z, 1499 m/z and 1513 m/z were absent in both strains of M. chimaera isolates—thus, the present invention advantageously allows for specific detection (e.g. distinction) of MAC species.
[0608] Both M. chimaera strains shared a lipid fingerprint based on a range of peaks between about 1200 m/z and 1400 m/z. Surprisingly, it was further possible to discriminate between strains of M. chimaera. The M. chimaera 20215-01546 has additional set of peaks at 2040-2068-2096 m/z and the M. chimaera 130444 at 1546-1560-1588-1602-1616 m/z. These peaks advantageously allow further differentiation from M. intracellulare.
Example 5
Detection of Mycobacterium Infection in Candidate for a Lung Transplant
[0609] The ability to detect mycobacteria infection in lung transplant candidates is of vital importance, as any existing infection can spread to and colonise the transplanted lung, significantly increasing the chance of organ rejection. In particular, an infection with M. abscessus is compromises selection for a lung transplant (i.e. such infection is considered to be a contraindication for a lung transplant).
[0610] A candidate for a lung transplant, suffering from Cystic Fibrosis, was investigated for the presence of a lung infection with Mycobacterium (as per Example 3). The present invention allowed for rapid detection of a NTM in the patient, and indeed the lipid fingerprint detected was indicative of the presence of an M. abscessus. The subject was therefore marked as an unsuitable candidate for a lung transplant, and the lung could be allocated to be given to a different candidate (shown to lack a mycobacterial infection by the method of the invention) who would be at a much lower risk of rejecting the transplant.
[0611] Furthermore, the inventors have demonstrated that the following positive ion mode peaks are specific to M. abscessus subsp. massiliense 1072-1075 m/z (e.g. about 1073.6 m/z), 1248-1251 m/z (preferably about 1249.9 m/z), 1260-1263 m/z (e.g. about 1261.9 m/z), 1262-1265 m/z (e.g. about 1263.9 m/z), 1274-1277 m/z (e.g. about 1275.9 m/z), 1276-1279 m/z (e.g. about 1277.9 m/z), 1288-1291 m/z (e.g. about 1289.9 m/z), 1290-1293 m/z (e.g. about 1291.9 m/z), 1294-1297 m/z (e.g. about 1295.9 m/z), 1304-1307 m/z (e.g. about 1305.9 m/z), 1308-1311 m/z (e.g. about 1309.9 m/z), 1310-1313 m/z (e.g. about 1311.9 m/z), 1314-1317 m/z (e.g. about 1315.9 m/z), 1318-1321 m/z (e.g. about 1319.9 m/z), 1320-1323 m/z (e.g. about 1321.9 m/z), 1322-1325 m/z (e.g. about 1323.9 m/z), 1324-1327 m/z (e.g. about 1325.9 m/z), 1332-1335 m/z (e.g. about 1333.9 m/z), 1336-1339 m/z (e.g. about 1337.9 m/z), 1338-1341 m/z (e.g. about 1339.9 m/z), 1719-1722 m/z (e.g. about 1720.1 m/z), 1735-1738 m/z (e.g. about 1736.1 m/z), 1747-1750 m/z (e.g. about 1748.1 m/z), 1761-1764 m/z (e.g. about 1762.1 m/z), 1764-1767 m/z (e.g. about 1765.1 m/z), and 1789-1792 m/z (e.g. about 1790.1 m/z). Thus, detecting the absence one or more of said peaks indicates that the patient is infected with a subspecies (e.g. M. abscessus subsp. abscessus or M. abscessus subsp. bolletii) that is less susceptible (or resistant) to key antibiotics such as clarithromycin.
[0612] The first candidate was subsequently given a dose (e.g. prolonged course) of antibiotics, and removal of the infection was monitored by the methods of the invention.
Example 6
Demonstrating Utility of Matrix Solution
[0613] Colonies of M. abscessus were picked from solid agar medium (e.g. McFarland 20, 30 or 40) and subjected to mass spectrometry analysis according to a method of the invention (
[0614] All publications mentioned in the above specification are herein incorporated by reference.
[0615] Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.