The oryx bacilli are Mycobacterium tuberculosis complex organisms for which phylogenetic position and host range are unsettled. We characterized 22 isolates by molecular methods and propose elevation to subspecies status as M. orygis. M. orygis is a causative agent of tuberculosis in animals and humans from Africa and South Asia.

The formation of oospores of Phytophthora infestans was studied in tomato and potato crops and volunteer plants under field conditions, and in laboratory tests with leaf discs of potato cultivars differing in their level of racenonspecific resistance. Oospores were readily detected in blight-affected tomato leaflets and fruits, and in leaflets of field crops and volunteer potato plants. Oospores extracted from blighted potato leaflets yielded 13 oospore-derived progeny. Oospores were also produced following inoculation of leaf discs of eight potato cultivars expressing different levels of race-nonspecific resistance with a mixture of sporangia of A1 and A2 isolates. The highest numbers of oospores were produced in cvs Bintje (susceptible) and Pimpernel (resistant), and the lowest in Nicola (intermediate resistance). The relationship between lesions per leaflet and oospore incidence, affected by varying A1 : A2 ratios, was explored using a simple mathematical model, and validated by comparing actual oospore production in leaflets with multiple lesions of the race-nonspecific-resistant potato clone Lan 22-21 with the predictions generated by the model. Survival of oospores was investigated after their incorporation in either a sandy or a light clay soil in buried clay pots exposed to the local weather conditions. Over 6 years these soils were regularly assessed for their infection potential using floating leaflets in a spore-baiting bioassay. Sandy and clay soils contaminated with oospores remained infectious for 48 and 34 months, respectively, when flooded. Infections of floating potato leaflets occurred within 84±92 h and ceased after 11 days. Soil samples remained infective if dried and re-flooded on two, but not more, occasions.

BackgroundPatients with Mycobacterium tuberculosis isolates sharing identical DNA fingerprint patterns can be epidemiologically linked. However, municipal health services in the Netherlands are able to confirm an epidemiological link in only around 23% of the patients with isolates clustered by the conventional variable number of tandem repeat (VNTR) genotyping. This research aims to investigate whether whole genome sequencing (WGS) is a more reliable predictor of epidemiological links between tuberculosis patients than VNTR genotyping.MethodsVNTR genotyping and WGS were performed in parallel on all Mycobacterium tuberculosis complex isolates received at the Netherlands National Institute for Public Health and the Environment in 2016. Isolates were clustered by VNTR when they shared identical 24-loci VNTR patterns; isolates were assigned to a WGS cluster when the pair-wise genetic distance was ≤ 12 single nucleotide polymorphisms (SNPs). Cluster investigation was performed by municipal health services on all isolates clustered by VNTR in 2016. The proportion of epidemiological links identified among patients clustered by either method was calculated.ResultsIn total, 535 isolates were genotyped, of which 25% (134/535) were clustered by VNTR and 14% (76/535) by WGS; the concordance between both typing methods was 86%. The proportion of epidemiological links among WGS clustered cases (57%) was twice as common than among VNTR clustered cases (31%).ConclusionWhen WGS was applied, the number of clustered isolates was halved, while all epidemiologically linked cases remained clustered. WGS is therefore a more reliable tool to predict epidemiological links between tuberculosis cases than VNTR genotyping and will allow more efficient transmission tracing, as epidemiological investigations based on false clustering can be avoided.

Pyrazinamide is important in the treatment of tuberculosis. Unfortunately, the diagnosis of pyrazinamide resistance is hampered by technical difficulties. We hypothesized that mutation analysis combined with the mycobacterial growth indicator tube (MGIT) phenotypic method would be a good predictor of pyrazinamide resistance. We prospectively analyzed 1,650 M. tuberculosis isolates referred to our tuberculosis reference laboratory in 2008 and 2009. In our laboratory, the MGIT 960 system was used for pyrazinamide resistance screening. If a pyrazinamide-resistant strain was detected, we performed a pncA gene mutation analysis. A second MGIT 960 susceptibility assay was performed afterwards to evaluate the accuracy of the pncA mutation analysis to detect true-or false-positive MGIT results. We observed pyrazinamide resistance in 69 samples using the first MGIT 960 analysis. In a second MGIT 960 analysis, 47 of the 69 samples proved susceptible (68% false positivity). Sensitivity of nonsynonymous pncA mutations for detecting resistant isolates was 73% (95% confidence interval [CI], 61% to 73%), and specificity was 100% (95% CI, 95% to 100%). A diagnostic algorithm incorporating phenotypic and molecular methods would have a 100% positive predictive value for detecting pyrazinamide-resistant isolates, indicating that such an algorithm, based on both methods, is a good predictor for pyrazinamide resistance in routine diagnostics.

Differential interactions in tuber blight attack between potato cultivars and Phytophthora infestans isolates were studied using whole tuber and tuber slice assays. Tuber blight incidence and severity were studied in a whole tuber assay, whilst necrosis and mycelium coverage were evaluated in a tuber slice assay. The overall defence reaction of the potato cultivars tested varied considerably. Cultivars like Kartel and Producent showed resistant reactions, whilst Bintje and, to a lesser extent, Astarte reacted more susceptibly after inoculation with aggressive strains of P. infestans. A highly significant cultivar by year interaction was observed when tuber blight incidence was evaluated in two successive years. Differential responses were revealed by changing ranked order of cultivars after exposure to aggressive isolates of P. infestans. The results show that cultivar by isolate interactions existed for all components of tuber blight resistance studied. The quantitative nature of the observed resistance responses suggests the presence of quantitative trait loci governing resistance to tuber blight. The consequences of differential interactions in relation to the stability of tuber resistance are discussed.

Summary assessment, fungalVariation in aggressiveness to tubers among isolates of Phytophthora hlfestans sampled from three potato growing regions in the Netherlands was compared. Variation in the ability to infect tubers of cv. Bintje was found between isolates of each of the three regional populations. The most aggressive isolate of the old population matched the average level of the new population in its ability to infect tubers. As a consequence, the commonly used rel~erence isolate VK 6C can no longer be considered to be representative of the present population of P.infestans. Therefore it is recommended that testing tuber resistance for the official list of potato cultivars with this isolate should be discontinued.Tuber infection and subsequent spread of the fungus in the tuber tissues were not found to be correlated. The components of tuber pathogenicity studied were not correlated to pathogenicity factors in the foliage, as measured under growth chamber conditions.

mIn order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a detailed evaluation on discriminatory power and agreement with findings in a cluster investigation was performed on 3,975 tuberculosis cases during the period of 2004 to 2008. The level of discrimination of the two typing methods did not differ substantially: RFLP typing yielded 2,733 distinct patterns compared to 2,607 in VNTR typing. The global concordance, defined as isolates labeled unique or identically distributed in clusters by both methods, amounted to 78.5% (n ‫؍‬ 3,123). Of the remaining 855 cases, 12% (n ‫؍‬ 479) of the cases were clustered only by VNTR, 7.7% (n ‫؍‬ 305) only by RFLP typing, and 1.8% (n ‫؍‬ 71) revealed different cluster compositions in the two approaches. A cluster investigation was performed for 87% (n ‫؍‬ 1,462) of the cases clustered by RFLP. For the 740 cases with confirmed or presumed epidemiological links, 92% were concordant with VNTR typing. In contrast, only 64% of the 722 cases without an epidemiological link but clustered by RFLP typing were also clustered by VNTR typing. We conclude that VNTR typing has a discriminatory power equal to IS6110 RFLP typing but is in better agreement with findings in a cluster investigation performed on an RFLPclustering-based cluster investigation. Both aspects make VNTR typing a suitable method for tuberculosis surveillance systems. DNA fingerprinting of Mycobacterium tuberculosis isolates has been applied for the investigation of epidemiological links between tuberculosis (TB) cases in several countries since DNA typing techniques were developed in the early 1990s (1-4). In The Netherlands, IS6110 restriction fragment length polymorphism (RFLP) typing was used nationwide from 1993 until the end of 2008. Although RFLP typing revolutionized studies on the transmission of M. tuberculosis, the method remained technically demanding and time consuming. The analysis of the complex IS6110 RFLP banding pattern requires a sophisticated computer application and a high degree of expertise (5). Moreover, in the Netherlands, it took on average 44 days to the deliver the RFLP typing result to the Municipal Health Services, after the isolate had reached the laboratory (D. van Soolingen, personal communication). This major drawback significantly limited the usefulness of DNA fingerprinting in routine examination of transmission, as it in fact only offered retrospective confirmation of suspected epidemiological links. In 2006, 24-locus variable-number tandemrepeat (VNTR) typing was favorably evaluated and proposed as the new gold standard for typing of M. tuberculosis (6). In order to switch from RFLP to VNTR typing in 2008 in the Netherlands, it was considered necessary to establish a retrospective VNTR pattern database of possible sources of infection for a pr...

No abstract