Evaluation of histopathology, PCR, and qPCR to detect Mikrocytos mackini in oysters Crassostrea gigas using Bayesian latent class analysis.

Latent class analysis (LCA) is a common method to evaluate the diagnostic sensitivity (DSe) and specificity (DSp) for pathogen detection assays in the absence of a perfect reference standard. Here we used LCA to evaluate the diagnostic accuracy of 3 tests for the detection of Mikrocytos mackini in Pacific oysters Crassostrea gigas: conventional polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), and histopathology. A total of 802 Pacific oysters collected over 12 sampling events from 9 locations were assessed. Preliminary investigations indicated that standard LCA assumptions of test independence and constant detection accuracy across locations were likely unrealistic. This was mitigated by restructuring the LCA in a Bayesian framework to include test-derived knowledge about pathogen prevalence and load for categorizing populations into 2 classes of infection severity (low or high) and assessing separate DSe and DSp estimates for each class. Median DSp estimates were high (>96%) for all 3 tests in both population classes. DSe estimates varied between tests and population classes but were consistently highest for qPCR (87-99%) and lowest for histopathology (21-51%). Acknowledging that detection of M. mackini may be fitted to multiple diagnostic and management purposes, qPCR had the highest DSe while maintaining similar DSp to both conventional PCR and histopathology and thus is generally well-suited to most applications.

First detection of Francisella halioticida in mussels Mytilus spp. experiencing mortalities in France.

This note describes the first detection of the bacteria Francisella halioticida in mussels Mytilus spp. from locations in Normandy and northern Brittany (France) experiencing high mussel mortalities, while it was not detected in the Bay of St Brieuc (northern Brittany), an area which was not affected by abnormal mussel mortality. The distribution of the bacteria in mussels seems to be restricted to inflammatory granulomas as observed in Yesso scallops Mizuhopecten yessoensis from Canada and Japan. F. halioticida has been identified as being involved in mass (>80%) mortality of abalones Haliotis gigantea in Japan and high (up to 40%) mortality of Yesso scallops Mizuhopecten yessoensis in Canada as well as in lesions reducing marketability of Yesso scallops in Japan. The impact of this bacterium on the health of mussels needs to be investigated in future research, especially since the cause of high mussel mortalities that have been occurring in France for the past few years is still undetermined.

Parallel studies confirm Francisella halioticida causes mortality in Yesso scallops Patinopecten yessoensis.

Francisella halioticida is a marine bacterium originally described as the causative agent of mass mortality among giant abalone Haliotis gigantea. Recent field studies in Canada and Japan have suggested that this bacterium is also the cause of adductor muscle lesions and high mortality of Yesso scallops Patinopecten yessoensis, although a causal relationship has not been established. In the present study, the pathogenicity of F. halioticida in Yesso scallops was assessed in both Canada and Japan using bacteria isolated from diseased Yesso scallops in each respective country. Independent laboratory experiments revealed that scallops challenged with F. halioticida via bath exposure resulted in high mortality and histological lesions characterized by massive haemocyte infiltration. The presence of F. halioticida was confirmed using PCR, and F. halioticida was re-isolated from a portion of dead and surviving specimens. These results fulfill Koch's classic criteria for establishing disease causation and provide conclusive evidence that F. halioticida causes adductor muscle lesions and high mortality in Yesso scallops.

Patterns of Chemotherapy Use in a U.S.-Based Cohort of Patients with Metastatic Pancreatic Cancer.

PURPOSE: Few population studies have examined patterns of systemic therapy administration in metastatic pancreatic cancer (MPC) or the predictors associated with specific treatment choices. PATIENTS AND METHODS: We assessed 4,011 consecutive MPC patients who received chemotherapy between January 2005 and December 2015 at academic, private, and community-based oncology practices subscribing to a U.S.-wide chemotherapy order entry system capturing disease, patient, provider, and treatment data. Multivariate analyses of these prospectively recorded characteristics identified significant predictors of specific therapeutic choices. RESULTS: Overall, 100 different regimens were used in first-line treatment of MPC. First-line gemcitabine monotherapy usage fell steadily from 72% in 2006 to 16% in 2015. This steep decline mirrored increases in first-line usage of both 5 fluorouracil, leucovorin, irinotecan and oxaliplatin (FOLFIRINOX) and gemcitabine + nab-paclitaxel. Younger male patients were more likely to receive FOLFIRINOX as first-line treatment, whereas patients treated at community practices and by oncologists with lower MPC patient volume were more likely to receive gemcitabine plus nab-paclitaxel (all p ≤ .05). Among all patients receiving first-line chemotherapy for MPC, 49% went on to receive second-line therapy and 19% received third-line therapy; administration of second- and third-line therapies increased steadily over the time course of follow-up. Younger patients and those treated by oncologists with higher MPC patient volume were more likely to receive second- and third-line therapies. CONCLUSION: This population-based study provides insight into treatment patterns of MPC in the U.S. Usage patterns varied greatly according to patient and provider characteristics. IMPLICATIONS FOR PRACTICE: This study examined real world metastatic pancreatic cancer treatment patterns in the United States with the goals of understanding changes in chemotherapy treatment frequencies over time and determining the individual predictors that underlie the chemotherapy choices oncologists make for their patients. Our data set is unique in that it captured not only patient-level data, but also oncologist-level data. It also captured data from private and community practices as well as academic centers. To our knowledge, this is the only data set that can give this degree of insight into oncologist decision making practices.

Rare occurrence of heart lesions in Pacific oysters Crassostrea gigas caused by an unknown bacterial infection.

On rare occasions, small cream-coloured cysts have been observed in the heart and pericardial cavity of Pacific oysters Crassostrea gigas from British Columbia, Canada. Histopathology revealed the presence of large colonies of bacteria (up to 800 µm in diameter) causing significant host response and hypertrophy of the heart epithelium. The causative bacteria were characterized as follows: Gram-negative, coccoid to small rod-shaped, typically <1.5 µm in size, cell walls highly endowed with surface fimbriae and division via binary fission. Although these bacteria shared some morphological characteristics with the order Rickettsiales, they did not require an intracellular existence for multiplication. Unfortunately, a cultured isolate was not available, and a retrospective attempt to further characterize the bacteria using DNA sequence analysis of a fragment from the 16S rDNA region proved to be uninformative.

Disease and mortality among Yesso scallops Patinopecten yessoensis putatively caused by infection with Francisella halioticida.

During the fall of 2015, up to 40% mortality occurred in juvenile Yesso scallops Patinopecten yessoensis at an aquaculture site in Baynes Sound, British Columbia, Canada. Macroscopic lesions were present in 11% of the scallops, and histopathology consisting of multifocal and diffuse haemocyte infiltration was observed in 44% of the specimens examined. Histologically, small Gram-negative intracellular bacteria-like particles were observed within necrotic haemocytes of the lesions, suggesting a bacterial aetiology. DNA was extracted from adductor muscle lesions of diseased scallops, and the 16s rDNA gene as well as the DNA-directed RNA polymerase beta subunit (rpoB) were amplified by PCR. Sequence analyses of the resulting 413 and 925 bp fragments were a 100% match to the reference sequence for Francisella halioticida, originally described as the cause of mortality in abalone from Japan. Isolation and culture of the bacteria was not possible at the time, as no further diseased specimens were available. Results from in situ hybridization assays as well as examination by transmission electron microscopy provide further evidence supporting the hypothesis that F. halioticida was the most probable causative agent of the lesions and mortality.

Seawater detection and biological assessments regarding transmission of the oyster parasite Mikrocytos mackini using qPCR.

Mikrocytos mackini is an intracellular parasite of oysters and causative agent of Denman Island disease in Pacific oysters Crassostrea gigas. Although M. mackini has been investigated for decades, its natural mode of transmission, mechanism for host entry, and environmental stability are largely unknown. We explored these biological characteristics of M. mackini using a recently described quantitative PCR (qPCR) assay. We detected M. mackini in the flow-through tank water of experimentally infected oysters and during disease remission in host tissues following 6 wk of elevated water temperature. Waterborne exposure of oysters to M. mackini further confirmed the potential for extracellular seawater transmission of this parasite and also identified host gill to have the highest early and continued prevalence for M. mackini DNA compared to stomach, mantle, labial palps, or adductor muscle samples. However, infections following waterborne challenge were slow to develop despite a substantial exposure (>106 M. mackini l-1 for 24 h), and further investigation demonstrated that M. mackini occurrence and infectivity severely declined following extracellular seawater incubation of more than 24 h. This study demonstrates a potential for using qPCR to monitor M. mackini in wild or farmed oyster populations during periods of disease remission or from environmental seawater samples. This work also suggests that gill tissues may provide a primary site for waterborne entry and possibly shedding of M. mackini in oysters. Further, although extracellular seawater transmission of M. mackini was possible, poor environmental stability and infection efficiency likely restricts the geographic transmission of M. mackini between oysters in natural environs and may help to explain localized areas of infection.

Denman Island disease in Washington State, USA: distribution and prevalence in Pacific and Olympia oysters.

We sampled over 2400 wild, feral, and cultured Pacific oysters Crassostrea gigas and Olympia oysters Ostrea lurida in Washington State, USA, from 2002 to 2006 to estimate the prevalence of infection with Mikrocytos mackini, the causative agent of Denman Island disease. Both histology and qualitative PCR methods were used. Estimates of true prevalence of M. mackini infection in C. gigas, after accounting for imperfect test sensitivity, ranged from mean values of 0 to 10.0% by histology and 0 to 8.4% based on pooled PCR samples. M. mackini was not detected in any of the O. lurida samples. Results suggest a lower prevalence of the pathogen and severity of this oyster disease in Washington than that indicated in previous reports from British Columbia, Canada, potentially attributable to higher seawater temperatures in the Washington sample locations.

Review of Mikrocytos microcell parasites at the dawn of a new age of scientific discovery.

The genus Mikrocytos is traditionally known for Mikrocytos mackini, the microcell parasite that typically infects Pacific oysters along the west coast of North America. Multiple factors have conspired to create difficulty for scientific research on Mikrocytos parasites. These include their tiny cell size, infections that are often of light intensity, lack of suitable cell lines and techniques for in vitro culture, and the seasonal nature of infections. The extreme rate of molecular evolution in Mikrocytos stymied new species discovery and confounded attempts to resolve its phylogenetic position for many years. Fortunately, 2 recent landmark studies have paved the way forward for future research by drastically changing our understanding of the evolution and diversity of these parasites. No longer an orphan eukaryotic lineage, the phylogenetic placement of Mikrocytos has been confidently resolved within Rhizaria and as sister taxon to Haplosporidia. The genus has also found a taxonomic home within the newly-discovered order, Mikrocytida - a globally distributed lineage of parasites infecting a wide range of invertebrate hosts. Here we review available scientific information on Mikrocytos parasites including their evolution and diversity, host and geographic ranges, epizootiology, and detection of the regulated pathogen, M. mackini. We also make recommendations towards a consistent taxonomic framework for this genus by minimally suggesting the use of 18S rDNA sequence, host species information, and histopathological presentation in new species descriptions. This is timely given that we are likely embarking on a new era of scientific advancements, including species discovery, in this genus and its relatives.

Ameson metacarcini sp. nov. (Microsporidia) infecting the muscles of Dungeness crabs Metacarcinus magister from British Columbia, Canada.

The Dungeness crab Metacarcinus magister supports a large and valuable fishery along the west coast of North America. Since 1998, Dungeness crabs exhibiting pink- to orange-colored joints and opaque white musculature have been sporadically observed in low prevalence from the Fraser River delta of British Columbia, Canada. We provide histological, ultrastructural, and molecular evidence that this condition is caused by a new microsporidian parasite. Crabs displaying gross symptoms were confirmed to have heavy infections of ovoid-shaped microsporidian spores (~1.8 × 1.4 µm in size) within muscle bundles of the skeletal musculature. The parasite apparently infected the outer periphery of each muscle bundle, and then proliferated into the muscle fibres near the centre of each infected bundle. Light infections were observed in heart tissues, and occasionally spores were observed within the fixed phagocytes lining the blood vessels of the hepatopancreas. Transmission electron microscopy (TEM) revealed multiple life stages of a monokaryotic microsporidian parasite within the sarcoplasm of muscle fibres. Molecular analysis of partial small subunit rRNA sequence data from the new species revealed an affinity to Ameson, a genus of Microsporidia infecting marine crustaceans. Based on morphological and molecular data, the new species is distinct from Nadelspora canceri, a related microsporidian that also infects the muscles of this host. At present, little is known about the distribution, seasonality, and transmission of A. metacarcini in M. magister.

Molecular taxonomy of Mikrocytos boweri sp. nov. from Olympia oysters Ostrea lurida in British Columbia, Canada.

Mikrocytos mackini is a microcell parasite that usually infects Crassostrea gigas distributed along the Pacific Northwest coast of North America. For many years, M. mackini was the only known species in the genus, but there have been multiple recent findings of genetically divergent forms of Mikrocytos in different hosts and in distantly located geographic locations. This note describes M. boweri sp. nov. found in Olympia oysters Ostrea lurida collected from and native to British Columbia, Canada, primarily using a molecular taxonomic approach.

Detection of a parasitic amoeba (Order Dactylopodida) in the female gonads of oysters in Brazil.

The impacts of oocyte parasites on the reproductive success of molluscs are largely unknown. In this study, we evaluated the presence of gonad parasites in 6 species of marine bivalve molluscs native to southern Brazil. Cultured bivalves included the mangrove oyster Crassostrea gasar (sometimes called C. brasiliana), the brown mussel Perna perna, the lion's paw scallop Nodipecten nodosus and the wing pearl oyster Pteria hirundo. Another species of mangrove oyster, C. rhizophorae, and the carib pointed venus clam Anomalocardia brasiliana (syn. A. flexuosa) were collected from the wild. Molluscs were collected in winter 2009 and summer 2010 for histopathological and molecular evaluation. An unknown ovarian parasite (UOP) was observed in histopathological sections of female gonads of C. gasar and C. rhizophorae. The UOP possessed features suggestive of amoebae, including an irregular outer membrane, frothy cytoplasm, a nucleus with a prominent central nucleolus and a closely associated basophilic parasome. PCR analysis was negative for Marteilioides chungmuensis, Perkinsus spp. and Paramoeba perurans. However, real-time PCR successfully amplified DNA from oyster gonads when using universal Paramoeba spp. primers. Also, conventional PCR amplified DNA using primers specific for Perkinsela amoebae-like organisms (syn. Perkinsiella), which are considered as endosymbionts of Parameoba spp., previously thought to be the parasome. Our results suggest that this UOP is a species of amoeba belonging to 1 of the 2 families of the order Dactylopodida, possibly related to Paramoeba spp. This study represents the first report of this type of organism in oysters. We found that C. gasar and C. rhizophorae were the most susceptible molluscs to these UOPs.

A fast and high-order accurate surface perturbation method for nanoplasmonic simulations: basic concepts, analytic continuation and applications.

In this paper we demonstrate that rigorous high-order perturbation of surfaces (HOPS) methods coupled with analytic continuation mechanisms are particularly well-suited for the assessment and design of nanoscale devices (e.g., biosensors) that operate based on surface plasmon resonances generated through the interaction of light with a periodic (metallic) grating. In this connection we explain that the characteristics of the latter are perfectly aligned with the optimal domain of applicability of HOPS schemes, as these procedures can be shown to be the methods of choice for low to moderate wavelengths of radiation and grating roughness that is representable by a few (e.g., tens of) Fourier coefficients. We argue that, in this context, the method can, for instance, produce full and precise reflectivity maps in computational times that are orders of magnitude faster than those of alternative numerical schemes (e.g., the popular "C-method," finite differences, integral equations or finite elements). In this initial study we concentrate on the description of the basic principles that underlie the solution scheme, including those that relate to analytic continuation procedures. Within this framework, we explain how, in spite of conventional wisdom to the contrary, the resulting perturbative techniques can provide a most valuable tool for practical investigations in plasmonics. We demonstrate this with some examples that have been previously discussed in the literature (including treatments of the reflectivity and band gap structure of some simple geometries) and extend this to demonstrate the wider applicability of the proposed approach.

Rediscovery of the Yesso scallop pathogen Perkinsus qugwadi in Canada, and development of PCR tests.

Perkinsus qugwadi, a pathogenic protozoan parasite of Yesso scallops Patinopecten yessoensis, is found only in cultured populations in British Columbia, Canada. This pathogen was first identified in 1988 and caused significant mortalities at some locations during the early 1990s. Prevalence of infection decreased dramatically following 1995, and the disease was last reported in 1997, leading to speculation that the Yesso scallop stocks in Canada had developed resistance to the disease, or that P. qugwadi had disappeared. However, the present study revealed that infection with P. qugwadi and associated mortality is still occurring in scallops from at least one location in British Columbia. One of the PCR tests developed for P. qugwadi detected the parasite in a 105-fold dilution of DNA extracted from a heavily infected sample and detected 52% more positive scallops than histology; however, the assay also cross-reacted with P. honshuensis and P. olseni. The other PCR test was less sensitive and detected 34% more positives, but did not react to any of the other Perkinsus species tested, suggesting that these PCR tests are powerful tools for screening for the presence of P. qugwadi. Phylogenetic analysis of 1796 bp of SSU rRNA gene sequence clearly indicated that P. qugwadi is positioned basally to other Perkinsus species.

Sequence homogeneity of internal transcribed spacer rDNA in Mikrocytos mackini and detection of Mikrocytos sp. in a new location.

Mikrocytos mackini is a microcell parasite of Pacific oysters only known to occur on the Pacific coast of North America. It is the only described species in the genus, although a genetically divergent Mikrocytos sp. organism has been reported once in both the Atlantic Ocean and China. We developed methods for sequencing the internal transcribed spacer (ITS) of rDNA for the purpose of characterizing extant diversity within M. mackini throughout its known geographic range, and surveying for other evidence of Mikrocytos sp. organisms. Our specific aims were to examine relatedness of M. mackini among sites to make inferences about its recent evolutionary history, and to provide baseline data for future development of a species-specific molecular detection method. We found a total lack of genetic variation within M. mackini across the complete ITS1-5.8S-ITS2 array in over 70 samples collected throughout its range. We hypothesize that this could be a result of a founder effect if the parasite had been introduced into its known range alongside its host, which was imported from Asia beginning around 1914 to about 1961. We detected a single divergent sequence at a short stretch of 18S that was identical to the Mikrocytos sp. detected elsewhere, which adds to the recent and growing body of evidence that Mikrocytos is much more broadly distributed than the limited range of M. mackini suggests. A 1903 bp section of rDNA from Mikrocytos sp. was generated that contained regions of high divergence from M. mackini (in ITS1 and ITS2) that could be exploited for molecular diagnostics.

Resistance of the Manila clam (Venerupis philippinarum) to infection with Mikrocytos mackini.

Manila clams (Venerupis philippinarum) challenged in laboratory trials via bath exposure proved to be resistant to infections with Mikrocytos mackini (protistan parasite of unknown taxonomic affiliation), while Pacific oysters (Crassostrea gigas) challenged simultaneously using identical conditions developed infections. Although M. mackini was detected by a nucleic acid pathogen specific (PCR) assay in 10-30% of the challenged V. philippinarum that were sampled soon after exposure (0-48 h, n=40), all of the subsequent V. philippinarum (n=62) sampled 9-17 weeks post-exposure tested negative for M. mackini by PCR assay. Prevalence of infection for the exposed C. gigas (n=100) during this same period ranged from 50% to 100% by PCR assay. Infection was confirmed in the oysters (58%, n=60) by a digoxigenin-labelled DNA probe designed to detect M. mackini by in situ hybridization, but M. mackini was not found in any of the exposed Manila clams (n=63) using this technique.

Perceptually guided polygon reduction.

The properties of the human visual system are taken into account, along with the geometric aspects of an object, in a new surface remeshing algorithm and a new mesh simplification algorithm. Both algorithms have a preprocessing step and are followed by the remeshing or mesh simplification steps. The preprocessing step computes an importance map that indicates the visual masking potential of the visual patterns on the surface. The importance map is then used to guide the remeshing or mesh simplification algorithms. Two different methods are proposed for computing an importance map that indicates the masking potential of the visual patterns on the surface. The first one is based on the Sarnoff visual discrimination metric, and the second one is inspired by the visual masking tool available in the current JPEG2000 standard. Given an importance map, the surface remeshing algorithm automatically distributes few samples to surface regions with strong visual masking properties due to surface texturing, lighting variations, bump mapping, surface reflectance and inter-reflections. Similarly, the mesh simplification algorithm simplifies more aggressively where the light field of an object can hide more geometric artifacts.

A clinically feasible multiplex proteomic immunoassay as a novel functional diagnostic for pancreatic ductal adenocarcinoma.

To date, targeted therapy for pancreatic ductal adenocarcinoma (PDAC) remains largely unsuccessful in the clinic. Current genomics-based technologies are unable to reflect the quantitative, dynamic signaling changes in the tumor, and require larger tumor samples that are difficult to obtain in PDAC patients. Therefore, a highly sensitive functional tool that can reliably and comprehensively inform intra-tumoral signaling events is direly needed to guide treatment decision. We tested the utility of a highly sensitive proteomics-based functional diagnostic platform, Collaborative Enzyme Enhanced Reactive-immunoassay (CEERTM), on fine-needle aspiration (FNA) samples obtained from 102 patients with radiographically-evident pancreatic tumors. Two FNA passes were collected from each patient, hybridized to customized chips coated with an array of capture antibodies, and detected using two enzyme-conjugated antibodies which emit quantifiable signals. We demonstrate that this technique is highly sensitive in detecting total and phosphorylated forms of multiple signaling molecules in FNA specimens, with reasonable correlation of marker intensities between two different FNA passes. Notably, signals of several markers were significantly higher in PDAC compared to non-cancerous samples. In PDAC samples, we found high total c-Met signal to be associated with poor survival, and confirmed this finding using an independent PDAC tissue microarray.

An evaluation of the Freedom From Smoking Online cessation program among Wisconsin residents.

OBJECTIVE: To study the effectiveness of the American Lung Association's Freedom From Smoking Online cessation program in assisting Wisconsin residents to quit smoking. METHODS: Five hundred fifty-three Wisconsin residents who signed up for the American Lung Association's Freedom From Smoking Online cessation program over a 10-month period were solicited for participation. Of these, 80 individuals completed the initial survey (response rate 14.41%). Follow-up surveys were conducted 3, 6, 9, and 12 months after participants completed the program. Fifty-two participants completed the 3-month follow-up, 43 completed the 6-month follow-up, 38 completed the 9-month follow-up, and 36 completed the 12-month follow-up. RESULTS: Initial point prevalence rates or whether participants reported that they had smoked in the previous 24-hour period revealed a quit rate of 55%. Sustained abstinence or whether they reported that they had smoked in the previous 3-month period ranged between 28.8% (3 months after program completion) and 16.3% (1 year after program completion). CONCLUSION: Quit rates compare favorably to current clinic-based smoking cessation programs. Given the low cost nature of an on-line cessation program and the ability to reach a wide audience, the evaluation undertaken of the American Lung Association's Freedom From Smoking Online cessation program revealed promising results.

Molecular detection of Mikrocytos mackini in Pacific oysters using quantitative PCR.

Mikrocytos mackini is an internationally regulated pathogen and causative agent of Denman Island disease in Pacific oysters Crassostrea gigas. Recent phylogenetic breakthroughs have placed this parasite within a highly divergent and globally distributed eukaryotic lineage that has been designated a new taxonomic order, Mikrocytida. The discovery of this new radiation of parasites is accompanied by a heightened awareness of the many knowledge gaps that exist with respect to the general biology, epizootiology, and potential impact of mikrocytid parasites on hosts, ecosystems, and commercial fisheries. It has also highlighted current shortcomings regarding our ability to detect these organisms. In this study, we developed a species-specific, sensitive, and quantitative method for detecting M. mackini DNA from host tissues using probe-based real-time qPCR technology. A limit of sensitivity between 2 and 5 genome copy equivalents was achieved in a reaction matrix containing ≥ 40 ng/µL host gDNA without inhibition. This detection proved superior to existing methods based on conventional PCR, histology or gross pathology and is the first species-specific diagnostic test for M. mackini. Quantitative assessment of parasite DNA using this assay remained accurate to between 10 and 50 copies identifying that during infection, M. mackini DNA was significantly more prevalent in hemolymph, labial palp, and mid-body cross-sections compared to mantle or adductor muscle. DNA extracted from a mid-body cross-section also provided the highest likelihood for detection during diagnostic screening of infected oysters. Taken together, these findings provide strong analytical evidence for the adoption of qPCR as the new reference standard for detecting M. mackini and give preliminary insight into the distribution of the parasite within host tissues. Standardised operating methodologies for sample collection and qPCR testing are provided to aid in the international regulatory diagnosis of M. mackini and serve as a useful platform for the future development of multiplexed or alternate mikrocytid species detection.