Detection Of Bd Research Articles

A Universal and Efficient Detection of Chytridiomycosis Infections in Amphibians Using Novel Quantitative PCR Markers

Chytridiomycosis is an infectious disease in amphibians caused by two chytrid fungi, Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), and is the worst infectious disease known in wildlife so far. Worldwide spread of the disease has caused unprecedented loss of global amphibian diversity. Although some lineages of Bd are enzootic and are not as deadly as the pandemic lineage, nearly 40% of amphibian species are still declining globally due to chytridiomycosis. Efficient surveillance and monitoring of chytridiomycosis are the immediate safeguard against rapid declines or extinctions of amphibian populations. Previous studies showed that existing diagnostic assays were not sensitive to certain Bd haplotypes like those from Korea, China, India, Japan, and Brazil and thereby, there is a need for a universal, sensitive, specific, reproducible, and affordable diagnostic assay. We designed a one-step SYBR green-based quantitative polymerase chain reaction (nSYBR qPCR) for robust detection of Bd. It amplifies an 82 base-pair segment between the 5.8S rRNA and ITS2 of the Bd genome. The primer pair was tested in-silico on 40 isolates from four known Bd lineages. Using skin swab samples of wild amphibians and cultured zoospores from Australia and Panama, we compared the clinical specificity and sensitivity of the newly described primers to the existing TaqMan-based qPCR assay. From India, we used samples which had been previously tested with Nested PCR to validate the new primer pairs. The newly described primer pair was then tested on swab samples from Anura, Caudata and Gymnophiona from India. We report widespread chytridiomycosis with varying infection loads on them. The new assay showed comparable efficiency to the TaqMan-based qPCR assay. This diagnostic assay can facilitate widespread surveillance of chytridiomycosis where it has been previously absent, which may reveal several reservoirs of the pathogen and can improve our understanding of this important wildlife disease.

First Survey of the Pathogenic Fungus Batrachochytrium dendrobatidis in Wild Populations of the Yunnan Caecilian (Ichthyophis bannanicus) in Guangxi, China

Batrachochytrium dendrobatidis (Bd), which causes chytridiomycosis, mainly infects Anura and Caudata but is poorly known in Gymnophiona. We conducted a survey of Bd in the Yunnan caecilian (Ichthyophis bannanicus) and found that 6 of 71 samples (8.4%) tested positive for Bd. To our knowledge, this is the first detection of Bd in wild I. bannanicus.

Rapid detection of amphibian chytrid fungus Batrachochytrium dendrobatidis using in situ DNA extraction and a handheld mobile thermocycler.

The amphibian chytrid fungus (Bd) has caused declines and some extinctions of amphibian populations worldwide. Early and accurate Bd detection is essential for management of susceptible anurans. We analyzed the effectiveness of in situ DNA extraction with a handheld mobile quantitative PCR (qPCR) thermocycler to detect Bd on frog skin swabs and in water samples using environmental DNA (eDNA). We collected duplicate eDNA samples and skin swabs from 3 Bd-positive Rana sierrae populations. We processed one set of samples using a field protocol (a handheld thermocycler) and the other half using a standard lab protocol. We detected Bd DNA in all R. sierrae swabbed using both the field and lab protocols. We also detected Bd DNA in eDNA samples at all sites, although the field and lab protocols failed to detect Bd eDNA at separate singular sites; results from the field and lab eDNA protocol did not match. The probability of detecting Bd DNA in the technical replicates was lower for the field protocol than the lab protocol, suggesting the field protocol has lower sensitivity and may not detect low quantities of DNA. Our results suggest that the field extraction protocol using a handheld qPCR platform is a promising tool for rapid detection of Bd in susceptible amphibian populations, yielding accurate results in less than 60 min. However, the applied field protocol may be prone to false negatives when analyzing low-quantity DNA samples such as eDNA water samples or frog swabs with low pathogen loads.

The first detection of the fungal pathogen batrachochytrium dendrobatidis in Norway with no evidence of population declines for great crested and smooth newts based on modeling on traditional trapping data

AbstractThe pathogenic fungus Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis in amphibians across the world, often leading to population declines and species extinctions. Here, we present the first detection of Bd in Norway, diagnosed using environmental DNA (eDNA) in water samples. In a genetic screening of 31 ponds in southeastern Norway for Bd, great crested newt (Triturus cristatus) and smooth newt (Lissotriton vulgaris), we detected Bd in five different localities. Resampling in one location revealed that time of sampling seemingly had a strong effect on the concentration of Bd eDNA in the water, which increased from May till June. In contrast, eDNA concentration of great crested newt decreased during the same period. Following a traditional sampling approach with traps, newts in the investigated ponds have been sampled for population estimates since 2012. We imposed a model on the catch data and contrasted the Bd‐positive ponds to the Bd‐negative ponds, finding no signs of population declines in the Bd‐positive ponds. This could suggest either recent infection events, with animals still having low prevalence of Bd and with little time for Bd to affect the present breeding generations of newts, and/or that the two newt species are infection‐tolerant. This study further highlights eDNA as a tool for early detection of invasive species.

Optimized Batrachochytrium dendrobatidis DNA extraction of swab samples results in imperfect detection particularly when infection intensities are low.

Accurate detection of the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd) is critical for wildlife disease research; however, false negatives in detection do occur. Here we compared different DNA extraction methods to determine the threshold for Bd detection and identify an optimal extraction method to improve detection and quantification of the pathogen. We extracted both lab-created cell suspension standards using PrepMan Ultra, Chelex resin, and 3 spin column DNA extraction kits (Qiagen DNeasy Blood and Tissue, Zymo Quick DNA miniprep, and IBI gMAX mini kit), and further compared extraction methods using field-collected samples. We found that when extracting Bd DNA from cells in lab-created culture, the spin column extraction methods and PrepMan Ultra were equivalent, while the resin method detected higher Bd DNA quantities, especially at higher loads. However, when swabs from live animals were analyzed, low Bd quantities were more than twice as likely to be detected using a spin column extraction than with the PrepMan Ultra extraction method. All tested spin column extraction methods performed similarly across both field and lab samples. Samples containing low Bd quantities yielded inconsistent detection and quantification of Bd DNA copies regardless of extraction method. To manage imperfect detection of Bd, we suggest that presence/absence analyses are more informative than attempting to quantify Bd DNA when quantities are low. Overall, we recommend that a cost-benefit analysis of target species susceptibility and epidemiology be taken into consideration when designing an experiment to determine the most appropriate DNA extraction method to be used, because sometimes detecting low Bd quantities is imperative to the study, whereas in other situations, detecting low DNA quantities is less important.

Development of a multiplex real-time PCR surveillance assay for monitoring the health status of Ecuadorian amphibians at risk of extinction

Chytrid fungi and viruses within the genus Ranavirus have been associated with mass mortality events and declines in amphibian populations worldwide. The fungus Batrachochytrium dendrobatidis (Bd) was reported in Ecuador; however, other chytrid fungi like Batrachochytrium salamandrivorans (Bsal) or ranaviruses have not been described in the country so far. To prevent the introduction of pathogens into amphibian populations under conservation programs and to implement a successful disease surveillance program, the development of a sensitive and specific diagnostic assay was required. We describe here the optimization of one TaqMan probe-based multiplex quantitative polymerase chain reaction (qPCR) assay that enables the simultaneous detection of Bsal and ranavirus, and one monoplex TaqMan qPCR assay for the detection of Bd. Standard curves, with a high linear correlation (r2 > 0.995), were generated using a synthetic genome template (gBlocks®) containing the target sequences from all three pathogens. Different samples from skin, liver, kidney, spleen, and lung from six different amphibian species were tested, and both qPCR assays showed highly reproducible and reliable results. To our knowledge, this method is the first multiplex qPCR system developed in Ecuador for identifying amphibian pathogens and represents a valuable tool for the early detection of these pathogens and for infection and co-infection monitoring in future epidemiological surveillance of amphibian species at risk of extinction.

A pH-Controlled Kit for Total and Direct Bilirubin Built on Mimetic Peroxidase CoFe2O4-DOPA-Catalyzed Fluorescence Enhancement.

Facile and reliable detection of total bilirubin (Bt, summation of indirect and direct bilirubin) and direct bilirubin (Bd) in human serum is of crucial importance to clinical diagnosis. However, it is still a challenge to explore an ideal recognition system for discriminating Bd and indirect bilirubin (Bi). In this work, a dual-functional sensor for Bt and Bd was first built on pH-controlled and mimetic peroxidase-catalyzed fluorescence enhancement. The fluorescence of nitrogen-doped graphene quantum dots (NGQDs) can be effectively quenched by bilirubin through the IFE process. With the catalysis of dopamine-derived magnetic ferrite nanoparticles (CoFe2O4-DOPA), both Bd and Bi were oxidized by H2O2 to colorless and fluorescent oxidates at pH 8.0. Interestingly, only Bd was oxidized at pH 3.5. The discriminating principle of Bd and Bi relied on their pH-controlled oxidation potentials. A sensitive sensor for Bt and Bd was developed on the enhanced fluorescence of the NGQDs/CoFe2O4-DOPA/H2O2 sensing system after bilirubin oxidation, which was originated from a combination of the fluorescence recovery of NGQDs and newly spawned fluorescence of bilirubin oxidates. The designed probe well quantifies Bt and Bd with the detection limits of 10 and 50 nM, respectively. Moreover, a portable diagnostic kit was fabricated and successfully used for the detection of Bt and Bd in 60 unrelated human serum samples, and the obtained results were almost consistent with those measured by biochemistry analyzer. The present kit exhibits the superiorities of high sensitivity and stability, interference-resistant, and green reagents, making it a promising candidate for bilirubin detection in the clinical diagnosis of jaundice.

The Chytrid Fungus, Batrachochytrium dendrobatidis, is Widespread Among Cuban Amphibians.

The fungus Batrachochytrium dendrobatidis (Bd) is a generalist amphibian pathogen responsible for chytridiomycosis. It was documented for the first time in Cuba in 2007, the apparent cause of the decline in one species of toad. In a recent survey, Bd was reported only for the highlands of Central Cuba. In the present study, we reexamined the geographic distribution and level of impact of Bd in Cuba by conducting an island-wide sampling in 10 localities and collecting skin swabs from 18 species and 28 environmental samples. We report detection of Bd in 60% of sampled sites and in 58% of sampled taxa. We show that Bd is associated with riparian, arboreal and terrestrial species, and it was estimated to occur in approximately 30% of the aquatic habitats we sampled. In addition, we confirmed that a dying individual of the species Eleutherodactylus casparii was severely infected with Bd. We also rise concern about the endanger toad Peltophryne longinasus and about three species of endemic riparian frogs that were not detected during our surveys. This study demonstrates that this pathogen is widespread throughout Cuba and provides relevant evidence to advance our understanding of its detection in amphibians and the aquatic environment in Cuba and about the occurrence of Bd in species with different ecologies. We provide valuable baseline information for Bd risk assessment and decision-making processes to mitigate its negative impact on Cuban amphibians.

Optimizing, validating, and field testing a multiplex qPCR for the detection of amphibian pathogens.

Amphibian populations worldwide are facing numerous threats, including the emergence and spread of infectious diseases. In the past 2 decades, Batrachochytrium dendrobatidis (Bd), a parasitic fungus, and a group of viruses comprising the genus Ranavirus have become widespread and resulted in mass mortality events and extirpations worldwide. In 2013, another novel fungus, B. salamandrivorans (Bsal), was attributed to dramatic declines in populations of fire salamander Salamandra salamandra in the Netherlands. Experimental infections demonstrated that Bsal is highly pathogenic to numerous salamander genera. In an effort to prevent the introduction of Bsal to North America, the US Fish and Wildlife Service (USFWS) listed 201 salamander species as injurious wildlife under the Lacey Act. To determine infection status and accurately assess amphibian health, the development of a sensitive and specific diagnostic assay was needed. We describe the optimization and validation of a multiplex quantitative polymerase chain reaction (qPCR) protocol for the simultaneous detection of Bd, Bsal, and frog virus 3-like ranaviruses. A synthetic genome template (gBlock®) containing the target genes from all 3 pathogens served as the positive control and allowed accurate quantification of pathogen genes. The assay was validated in the field using an established non-lethal swabbing technique to survey local amphibian populations throughout a range of habitats. This multiplex qPCR demonstrates high reproducibility, sensitivity, and was capable of detecting both Bd and ranavirus in numerous locations, species, and life stages. Bsal was not detected at any point during these sampling efforts.

Using environmental DNA for early detection of amphibian chytrid fungus Batrachochytrium dendrobatidis prior to a ranid die-off.

Amphibian chytridiomycosis caused by the fungus Batrachochytrium dendrobatidis (Bd) is an emerging infectious disease that has been associated with mass mortality and extinctions of amphibians worldwide. Environmental DNA (eDNA) techniques have been used to detect the presence of Bd in the environment, but not to detect Bd prior to an amphibian die-off. We collected eDNA using filtered water samples from 13 lakes across Sequoia Kings Canyon National Park. Seven of those sites had populations of mountain yellow-legged frogs, an amphibian highly susceptible to chytridiomycosis, and 3 of those populations experienced a Bd related die-off 1 mo post-eDNA sampling. We detected Bd in eDNA samples that were collected 1 mo prior to the observed Bd-caused die-off at all 3 sites affected by Bd, and we did not detect Bd at the other sites where no die-off was observed. Our study indicates the potential to use eDNA techniques for early detection of Bd in the environment.

Lowland extirpation of anuran populations on a tropical mountain.

BackgroundClimate change and infectious diseases threaten animal and plant species, even in natural and protected areas. To cope with these changes, species may acclimate, adapt, move or decline. Here, we test for shifts in anuran distributions in the Luquillo Mountains (LM), a tropical montane forest in Puerto Rico by comparing species distributions from historical (1931–1989)and current data (2015/2016).MethodsHistorical data, which included different methodologies, were gathered through the Global Biodiversity Information Facility (GBIF) and published literature, and the current data were collected using acoustic recorders along three elevational transects.ResultsIn the recordings, we detected the 12 native frog species known to occur in LM. Over a span of ∼25 years, two species have become extinct and four species suffered extirpation in lowland areas. As a consequence, low elevation areas in the LM (<300 m) have lost at least six anuran species.DiscussionWe hypothesize that these extirpations are due to the effects of climate change and infectious diseases, which are restricting many species to higher elevations and a much smaller area. Land use change is not responsible for these changes because LM has been a protected reserve for the past 80 years. However, previous studies indicate that (1) climate change has increased temperatures in Puerto Rico, and (2) Batrachochytrium dendrobatidis (Bd) was found in 10 native species and early detection of Bd coincides with anurans declines in the LM. Our study confirms the general impressions of amphibian population extirpations at low elevations, and corroborates the levels of threat assigned by IUCN.

Assessing the ability of swab data to determine the true burden of infection for the amphibian pathogen Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd) is a pathogenic fungus which causes the disease chytridiomycosis in amphibians by infecting the animals’ epidermis. The most commonly applied method for the detection of Bd is the use of a sterile swab, rubbed over the keratinized areas of an amphibian and then processed to yield DNA for detection by qPCR. This method has been used to infer a threshold of lethal infection in some species; however, how reliable and reproducible the swabbing method is at detecting the true burden of infection suffered by individuals is not known. European midwife toads, Alytes obstetricans, are susceptible to chytridiomycosis and are highly parasitised by Bd across Europe. By quantifying Bd-load throughout the entire skin and comparing this to swab results taken from the same individual, we determined whether epidermal swabs provide a quantifiable and accurate indication of the true fungal burden suffered. Further, we examined whether we could infer a threshold for lethal infection based on comparison of swab data taken from infected A. obstetricans exhibiting different clinical states. From swab data, we detected significantly higher fungal burdens from moribund metamorphs compared to visually healthy individuals; however, the ability of these swab data to provide an accurate indication of the true fungal burden was not reliable. These data suggest that fungal load dynamics play an important role in disease-induced mortality in A. obstetricans at these sites, but that using swab data to infer an exact threshold for Bd-associated mortality might be inappropriate and misleading.

Amphibian Chytrid Fungus in Madagascar neither Shows Widespread Presence nor Signs of Certain Establishment.

The global spread of amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd) is associated with amphibian mass mortality, population decline, and extinction. Over the past decade, concern has been expressed for the potential introduction of Bd to Madagascar, a global hotspot of amphibian biodiversity. Following years without detection, widespread Bd presence in Madagascar has now been reported (Bletz et al. 2015a), raising international conservation concern. Before reacting to this finding with a significant management response, the accuracy and context of the data warrant cautious review. Re-examination of a 10-year dataset together with results from more recent surveillance (Kolby et al. 2015) does not yet demonstrate widespread Bd presence. Detection of Bd at “positive” locations in Madagascar has been inconsistent for unknown reasons. Whether Bd is established in Madagascar (i.e. populations are self-sustaining) or instead requires continued introduction to persist also remains uncertain. The deployment of emergency conservation rescue initiatives is expected to target areas where the distribution of Bd and the risk of chytridiomycosis endangering amphibians is believed to overlap. Thus, erroneous description of Bd presence would misdirect limited conservation resources. Standardized surveillance and confirmatory surveys are now imperative to reliably characterize the distribution, potential spread, virulence and overall risk of Bd to amphibians in Madagascar.

Widespread presence of the pathogenic fungus Batrachochytrium dendrobatidis in wild amphibian communities in Madagascar.

Amphibian chytridiomycosis, an emerging infectious disease caused by the fungus Batrachochytrium dendrobatidis (Bd), has been a significant driver of amphibian declines. While globally widespread, Bd had not yet been reported from within Madagascar. We document surveys conducted across the country between 2005 and 2014, showing Bd's first record in 2010. Subsequently, Bd was detected in multiple areas, with prevalence reaching up to 100%. Detection of Bd appears to be associated with mid to high elevation sites and to have a seasonal pattern, with greater detectability during the dry season. Lineage-based PCR was performed on a subset of samples. While some did not amplify with any lineage probe, when a positive signal was observed, samples were most similar to the Global Panzootic Lineage (BdGPL). These results may suggest that Bd arrived recently, but do not exclude the existence of a previously undetected endemic Bd genotype. Representatives of all native anuran families have tested Bd-positive, and exposure trials confirm infection by Bd is possible. Bd's presence could pose significant threats to Madagascar's unique “megadiverse” amphibians.

Differential efficiency among DNA extraction methods influences detection of the amphibian pathogen Batrachochytrium dendrobatidis

Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is responsible for massive declines and extinctions of amphibians worldwide. The most common method for detecting Bd is quantitative polymerase chain reaction (qPCR). qPCR is a highly sensitive detection technique, but its ability to determine the presence and accurately quantify the amount of Bd is also contingent on the efficiency of the DNA extraction method used prior to PCR. Using qPCR, we compared the extraction efficiency of 3 different extraction methods commonly used for Bd detection across a range of zoospore quantities: PrepMan Ultra Reagent, Qiagen DNeasy Blood and Tissue Kit, and Mobio PowerSoil DNA Isolation Kit. We show that not all extraction methods led to successful detection of Bd for the low zoospore quantities and that there was variation in the estimated zoospore equivalents among the methods, which demonstrates that these methods have different extraction efficiencies. These results highlight the importance of considering the extraction method when comparing across studies. The Qiagen DNeasy kit had the highest efficiency. We also show that replicated estimates of less than 1 zoospore can result from known zoospore concentrations; therefore, such results should be considered when obtained from field data. Additionally, we discuss the implications of our findings for interpreting previous studies and for conducting future Bd surveys. It is imperative to use the most efficient DNA extraction method in tandem with the highly sensitive qPCR technique in order to accurately diagnose the presence of Bd as well as other pathogens.

Three new X-ray emitting O-type subdwarf stars discovered withChandra

X-ray observations of sdO stars are a useful tool to investigate their properties, but so far only two sdO stars were detected at X-rays. We observed a complete flux-limited sample of 19 sdO stars with the Chandra HRC-I camera to measure the count rate of the detected sources or to set a tight upper limit on it for the undetected sources. We obtained a robust detection of BD+37 1977 and Feige 34 and a marginal detection of BD+28 4211. The estimated luminosity of BD+37 1977 is above 10^31 erg/s, which is high enough to suggest the possible presence of an accreting compact companion. This possibility is unlikely for all the other targets (both detected and undetected), since in their case L_X < 10^30 erg/s. On the other hand, for all 19 targets the estimated value of L_X (or its upper limit) implies an X-ray/bolometric flux ratio that agrees with log(L_X/L_bol) = -6.7 +/- 0.5, which is the range of values typical of main-sequence and giant O stars. Therefore, for Feige 34 and BD+28 4211 the observed X-ray flux is most probably due to intrinsic emission. The same is possibile for the 16 undetected stars.

Retrospective Survey of Museum Specimens Reveals Historically Widespread Presence of Batrachochytrium dendrobatidis in China

Chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has been implicated in amphibian population declines worldwide. However, no amphibian declines or extinctions associated with Bd have been reported in Asia. To investigate the history of this pathogen in China, we examined 1,007 museum-preserved amphibian specimens of 80 species collected between 1933 and 2009. Bd was detected in 60 individuals (6.0%), with the earliest case of Bd infection occurring in one specimen of Bufo gargarizans and two Fejervarya limnocharis, all collected in 1933 from Chongqing, southwest China. Although mainly detected in non-threatened native amphibians, Bd was also found in four endangered species. We report the first evidence of Bd for Taiwan and the first detection of Bd in the critically endangered Chinese giant salamander (Andrias davidianus). Bd appears to have been present at a low rate of infection since at least the 1930s in China, and no significant differences in prevalence were detected between decades or provinces, suggesting that a historical steady endemic relationship between Bd and Chinese amphibians has occurred. Our results add new insights on the global emergence of Bd and suggest that this pathogen has been more widely distributed in the last century than previously believed.

Accumulation of the pathogenic fungus Batrachochytrium dendrobatidis on the regressing tail of midwife toads Alytes obstetricans undergoing metamorphosis

Batrachochytrium dendrobatidis (Bd) is a fungus infecting the skin of amphibians. On metamorphosing animals the infection is difficult to detect because of the limited information concerning the location of Bd on the animals during this stage. Histological investigation revealed that Bd accumulated on the reabsorbing tail of metamorphosing animals. This observation may facilitate the detection of Bd in metamorphosing amphibians.

Techniques for minimizing the effects of PCR inhibitors in the chytridiomycosis assay

Chytridiomycosis is an amphibian disease of global conservation concern that is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Since the discovery of Bd in 1998, several methods have been used for detection of Bd; among these polymerase chain reaction (PCR) from skin swabs is accepted as the best method due to its noninvasiveness, high sensitivity and ease of use. However, PCR is not without problems - to be successful, this technique is dependent upon the presence of nondegraded DNA template and reaction contents that are free from inhibitors. Here, we report on an investigation of several techniques aimed at improving the reliability of the Bd PCR assay by minimizing the effects of humic acid (HA), a potent PCR inhibitor. We compared the effectiveness of four DNA extraction kits (DNeasy, QIAamp DNA Stool, PowerLyzer Power Soil and PrepMan Ultra) and four PCR methods (Amplitaq Gold, bovine serum albumin, PowerClean DNA Clean-up and inhibitor resistant Taq Polymerase). The results of this and previous studies indicate that chytridiomycosis studies that use PCR methods for disease detection may be significantly underestimating the occurrence of Bd. Our results suggest that to minimize the inhibitory effects of HA, DNeasy should be used for sample DNA extraction and Amplitaq Gold with bovine serum albumin should be used for the Bd PCR assay. We also outline protocols tested, show the results of our methods comparisons and discuss the pros and cons of each method.

A strong analogy between the dielectric breakdown of high-K gate stacks and the progressive breakdown of ultrathin oxides

The breakdown of high-K (HK) stack gate dielectrics is shown to be analogous to the failure of ultrathin SiO2-based oxides showing progressive breakdown. The breakdown of a HK stack is shown to occur in two phases. First, one or several percolation paths are formed in the SiO2-based interfacial layer. Then, these partial breakdowns propagate into the HK through defect generation in this layer. This propagation phase is equivalent to the progressive breakdown local degradation phenomenon. As a consequence of this analogy, the reliability assessment tools developed for ultrathin single-layer oxides can also be used without significant changes to assess the reliability of HK stacks. This analogy is so strong that the experimental distinction between HK propagation and conventional progressive breakdown appears to be rather difficult. However, the residual time distribution from first breakdown to final oxide failure is shown to be significantly different for both processes at low failure percentiles. This has allowed us to obtain experimental evidence of HK propagation (as opposed to conventional progressive breakdown effects) in relatively thick Hf-based gate stacks where the detection of first BD appears to be feasible.