Diff-Quik Research Articles

Algorithm proposal based on the C. Diff Quik Chek Complete ICT device for detecting Clostridium difficile infection

To assess a new immunochromatography (ICT) test that detects glutamate dehydrogenase (GDH) antigen and Clostridium difficile toxin A/B simultaneously, and to propose an algorithm for the diagnosis of C. difficile infection (CDI) based on this test. We analysed 970 stool samples. Discrepant results between GDH and toxin A/B were resolved using toxigenic culture as the reference. This test enabled us to obtain a conclusive result in <30min in 93.8% of the samples. Among the discrepant results (GDH (+)/Toxin A/B (-)), 41.7% (25/60) were found to be toxigenic C. difficile by toxigenic culture. This test has a high sensitivity and specificity for the diagnosis of CDI.

Clostridium difficile Testing Algorithms Using Glutamate Dehydrogenase Antigen and C. difficile Toxin Enzyme Immunoassays with C. difficile Nucleic Acid Amplification Testing Increase Diagnostic Yield in a Tertiary Pediatric Population

We evaluated the performance of the rapid C. diff Quik Chek Complete's glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches for a tertiary pediatric population: algorithm 1 entailed initial testing with GDH/CDT followed by loop-mediated isothermal amplification (LAMP), and algorithm 2 entailed GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDT-negative results. A true positive (TP) was defined as positivity by CCNA or positivity by LAMP plus another test (GDH, CDT, or the Premier C. difficile toxin A and B enzyme immunoassay [P-EIA]). A total of 141 specimens from 141 patients yielded 27 TPs and 19% prevalence. Sensitivity, specificity, positive predictive value, and negative predictive value were 56%, 100%, 100%, and 90% for P-EIA and 81%, 100%, 100%, and 96% for both algorithm 1 and algorithm 2. In summary, GDH-based algorithms detected C. difficile infections with superior sensitivity compared to P-EIA. The algorithms allowed immediate reporting of half of all TPs, but LAMP or CCNA was required to confirm the presence or absence of toxigenic C. difficile in GDH-positive/CDT-negative specimens.

Production of large numbers of plasmacytoid dendritic cells with functional activities from CD34+ hematopoietic progenitor cells: Use of interleukin-3

Plasmacytoid dendritic cells (pDC), a subset of dendritic cells characterized by a rapid and massive type-I interferon secretion through the Toll-like receptor pathway in response to viral infection, play important roles in the pathogenesis of several diseases, such as chronic viral infections (e.g., hepatitis C virus, human immunodeficiency virus), autoimmunity (e.g., psoriasis, systemic lupus erythematosus), and cancer. As pDC represent a rare cell type in the peripheral blood, the goal of this study was to develop a new method to efficiently generate large numbers of cells from a limited number of CD34(+) cord blood progenitors to provide a tool to resolve important questions about how pDC mediate tolerance, autoimmunity, and cancer. Human CD34(+) hematopoietic progenitor cells isolated from cord blood were cultured with a combination of Flt3-ligand (Flt3L), thrombopoietin (TPO), and one of the following cytokine: interleukin (IL)-3, interferon-β(IFN-β), or prostaglandin E2(PGE(2)). Cells obtained in the different culture conditions were analyzed for their phenotype and functional characteristics. The addition of IL-3 cooperates with Flt3L and TPO in the induction of pDC from CD34(+) hematopoietic progenitor cells. Indeed, Flt3L/TPO alone or supplemented with prostaglandin E2 or interferon-β produced smaller amounts of pDC from hematopoietic progenitor cells. In addition, pDC generated in Flt3L/TPO/IL-3 cultures exhibited morphological, immunohistochemical, and functional features of peripheral blood pDC. We showed that IL-3, in association with Flt3L and TPO, provides an advantageous tool for large-scale generation of pDC. This culture condition generated, starting from 2 × 10(5) CD34(+) cells, up to 2.6 × 10(6) pDC presenting features of blood pDC.

Pregnancy outcome after intracytoplasmic sperm injection with strontium oocyte activation in a globozoospermic patient

Pregnancy outcome after intracytoplasmic sperm injection with strontium oocyte activation in a globozoospermic patient

Detection of toxigenic Clostridium difficile in pediatric stool samples: an evaluation of Quik Check Complete Antigen assay, BD GeneOhm Cdiff PCR, and ProGastro Cd PCR assays

The performance of C. Diff Quik Chek Complete (QCC), BD GeneOhm Cdiff PCR (BD), and ProGastro Cd PCR (PG) assays was evaluated in detecting Clostridium difficile infection (CDI) in children using 200 frozen stool specimens. The results of the tests were compared to the toxigenic culture (TC) as ‘gold standard.’ The sensitivity, specificity, positive predictive value, and negative predictive value were as follows. QCC antigen (GDH + Toxin-A/B) = 70.8%, 97.4%, 89.5%, and 91.4%; BD PCR = 89.6%, 96.7%, 89.6%, and 96.7%; PG PCR = 100%, 93.4%, 82.8%, and 100%. Polymerase chain reaction (PCR) assays detected an additional 11 positives missed by TC, 7 of which were confirmed positive by an alternate tcdB gene PCR assay. However, retrospective clinical chart review indicated CDI in only 3 of the 11 patients in whom C. difficile was detected by PCR only. A 2-step algorithm utilizing QCC antigen test as a screening test followed by confirmation of GDH-positive and toxin-negative samples with either BD or PG PCR assay will provide rapid and accurate results for majority of the samples and reduce laboratory testing cost.

Comparison of the cytobrush, cottonswab, and low-volume uterine flush techniques to evaluate endometrial cytology for diagnosing endometritis in chronically infertile mares

Endometritis is the most important cause of infertility in barren mares. The quick method of endometrial cytology (EC) has a relatively high reliability in diagnosing endometrial inflammation in the mare. For reliable cytological results, a collection technique that yields many well-preserved cells representative of a large uterine surface area without causing harm to the reproductive tract is required. The aim of the study was to compare three usually employed techniques for collection of endometrial and inflammatory cells (guarded cotton swab, uterine lavage, and cytobrush) in chronically infertile mares. Twenty Standardbred mares were used. In each mare, samples for EC were collected, first by a cotton swab (DGS), then by a cytobrush (CB), and finally by low volume flush (LVF). The slides were stained using the Diff Quick stain. The following parameters were assessed for each tested technique: background content of the slides; quality of the cells harvested; total cellularity; neutrophils; ratio PMN/uterine epithelial cells; inflammatory cells; vaginal epithelium cells. Categorical variables were compared using contingency tables and Pearson Chi-square tests, whereas continuous variables were compared using one-way analysis of variance (ANOVA); P < 0.05 was considered significant. Samplings by DGS and CB resulted easy and quick to perform via a single operator in all cases. LVF was performed easily, but required the presence of 2–3 players and took more time. The background content of the slides prepared by DGS appeared proteinaceous, slides prepared by LVF appeared contaminated by red blood cells or debris, whereas slides prepared by CB appeared clear. All smears showed a good total cellularity. The CB yielded significantly more cells (P < 0.0001) than DGS and LVF. The DGS produced significant more cells than LVF (P < 0.0001). The DGS produced significantly more (P = 0.003) intact cells than CB and LVF. Distorted cells were significantly (P = 0.001) more frequent in smears by LVF. The CB harvested significantly (P = 0.009) more fragmented cells. CB and LVF produced significantly (P < 0.0001; P = 0.02) more PMNs/HPF than DGS. In smears collected by LVF the proportion of PMNs/uterine epithelial cells was significantly (P = 0.0062; P = 0.0023) higher than in smears by CB and DGS. CB collected a significantly higher (P = 0.0011) proportion of PMNs than DGS. Acute endometritis was diagnosed in 50% (10/20) of the mares by DGS cytological samples, 25% (5/20) by CB, and 75% (15/20) by LVF. Inflammatory cells other than PMN (lymphocytes, macrophages, eosinophils) were collected exclusively by CB method. Epithelial cells from the vagina were only detected in LVF slides. The agreement of the diagnosis of endometritis between the three techniques of collection and between the different criteria adopted to evaluate smears obtained with the same technique was poor (k ≤ 0.3). In conclusion, results show that cytobrush and flush specimens were superior in all parameters to cotton swab smears. Even though the cytobrush technique requires specialized equipment, sample collection by this method was easier, more consistent, and quicker than the lavage method, indicating that the brush would be the preferred collection method for use on field in the mare. More studies are needed to establish criteria for interpretation of inflammation in the mare on cytobrush samples.

Evaluation of the bacterial microflora of the conjunctival sac of healthy dogs and dogs with atopic dermatitis

The aim of this case-control study was to evaluate and compare the bacterial microflora from the conjunctival sac of dogs with atopic dermatitis and healthy dogs. Twenty-one atopic dogs without clinical and/or cytopathological signs of bacterial blepharoconjunctivitis and 21 breed-matched healthy dogs were enrolled. Under topical anaesthesia, the inferior conjunctival sac of one eye was scraped twice. Material was collected with a Kimura spatula, spread over a slide and stained with a Diff Quick(®) -type stain (Medion Diagnostics GmbH, Düdingen, Switzerland) for cytological examination. An area of 0.5 cm(2) was examined at ×1000 magnification, and the types and numbers of cells and bacteria were recorded. A bacterial swab was collected and inoculated into culture media for the growth of aerobic bacteria. Before sampling, each atopic dog was evaluated for severity of cutaneous lesions, pruritus and conjunctival inflammation. Significant differences were observed between atopic and healthy dogs for the presence of bacteria on cytology (P = 0.015), keratinized (P = 0.001) and nonkeratinized epithelial cells (P = 0.013), eosinophils (P = 0.019) and lymphocytes (P = 0.008). Bacteria were recovered from 12 atopic dogs and three healthy dogs (P = 0.004). Staphylococcus pseudintermedius was the most commonly isolated species in atopic dogs (seven of 12). In atopic dogs, no significant relation was found between conjunctival bacterial colonization (on cytology and culture) and the severity of any of the clinical parameters. This study suggests differences in conjunctival bacterial colonization and cytological features between atopic and healthy dogs.

Comparison of Five Assays for Detection of Clostridium difficile Toxin

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended.

Chronic organizing retroperitoneal abscess caused by Klebsiella oxytoca masquerading as sarcoma: Recognition by Diff–Quik stain on FNA material

Chronic organizing retroperitoneal abscess caused by <i>Klebsiella oxytoca</i> masquerading as sarcoma: Recognition by Diff–Quik stain on FNA material

KRAS testing on colo‐rectal carcinoma cytological imprints

Anti-EGFR monoclonal antibodies, cetuximab, and panitumumab, are administrated under the condition that advanced colo-rectal cancer (CRC) carries a wild-type KRAS gene. Thus, clinicians request pathologists to genotype KRAS before treatment. In the near future routine mutation testing at the same time of the surgery may be implemented. The reliability of a rapid KRAS testing on ex vivo cytological samples obtained by direct scraping of the colon tumour tissue is here evaluated. A consecutive series of 20 surgically resected, primary CRC specimens was analysed. Fresh tissue from CRC was scraped with a scalpel blade, smeared on uncoated glass slides, air-dried and Diff-Quik stained to ensure malignant cell presence. The same tissue area was also histologically processed. Exon 2 KRAS gene mutations were evaluated on both cytological and histological specimens by dideoxy sequencing and by the DxS KRAS Mutation Test Kit (DxS, Manchester, England). Data obtained on on imprint cytology and matched histological samples showed full concordance; however, the mutation frequency was slightly higher (35%) by the DxS KRAS Mutation Test Kit than by the dideoxy sequencing (30%). Thus, colon cancer imprint cytology sample is a reliable biospecimen for both dideoxy-sequencing and DxS KRAS Mutation Test Kit analysis and it may be useful to abbreviate the KRAS assay turnaround time.

Ultrasound gel artifact on diff–quik stained thyroid fine‐needle aspirate smears

Ultrasound gel artifact on diff–quik stained thyroid fine‐needle aspirate smears

The staining pattern of human sperm with Diff Quik: relationship with sperm head morphology and a sperm chromatin structure assay (SCSA)

The dark staining of human sperm heads by Diff Quik is significantly correlated with abnormal sperm head morphology (r=0.51, p<0.0001), but is not associated with changes in sperm chromatin detected by a sperm chromatin structure assay (SCSA; r=0.18, p>0.09). Whilst valuable in the assessment of head morphology, it is concluded that Diff Quik staining is not a useful substitute for the SCSA to assess human sperm chromatin.

The interaction of asbestos fibres with human mesothelial cells: a combined investigation exploiting microscopic and nanoscopic techniques

Introduction . The exposure to asbestos fibres is associated with the development of severe diseases such as lung cancer and pleural mesothelioma. The interaction mechanism of these fibres with the mesothelial cells is still debated.(1) This work aims at obtaining information about the interaction of crocidolite fibres with mesothelial cells, for a better understanding of the processes that trigger cell transformation. For this reason we combine optical microscopy and SEM, with nanoscopic techniques as near-field optical (SNOM) and atomic force microscopy (AFM). These two latter techniques, thanks to their high sensitivity and non-invasiveness, are suitable for investigating phenomena occurring at the cell membrane with nanometric resolution.(2) In addition, SNOM provides simultaneous topography and optical image with a resolution beyond the light diffraction limit. This allows a direct coupling of the morphological features with the optical properties of the sample. Materials and Methods . Mesothelial cell line (MET5A from ATCC) are grown in RPMI with FCS 10%, 2 mM glutamine. Cells are exposed to 5µg/cm2 crocidolite for 3, 6 or 12 h. For optical microscopy cells are stained with Diff-Quick. The samples after fixation with PFA 4% are prepared for SEM, SNOM and AFM observations that are carried out by using a Leica Stereoscan 430i, a A-100 AFM and TriA-SNOM microscope (A.P.E.Research, Trieste, Italy). Results and Discussion . By analysing the optical data we estimate that fibres are associated with 75% of mesothelial cells. SEM images confirm these results and allow distinguishing that some fibres are on cell surface, while others appears to be clearly inside the cells, in some cases even deforming the cell morphology. A deeper investigation is achieved by SNOM and AFM. By comparing the SNOM topography with the simultaneous transmission and reflection images, we can define the position of the fibres respect to the cell membrane, owing to difference in optical properties between the crocidolite and the cell material. In addition, high-resolution AFM images highlight the entrance site of the nanometre-size fibres at cell membrane. In conclusion the combination of our findings provides an accurate description about the interaction of mesothelial cells with crocidolite fibres having different size. Importantly, SNOM optical images can disclose details about such interaction not observed up to now. 1. Arch. Biochem. Biophys. 2010, 502: 1. 2. J. Cell Sci. 2001, 114: 4153.

Evaluation of a simultaneous detection kit for the glutamate dehydrogenase antigen and toxin A/B in feces for diagnosis of Clostridium difficile infection

Rapid detection kits for toxin A/B in feces are widely used as a diagnostic tool for Clostridium difficile infection (CDI). Their low sensitivity, however, has been considered a problem. In this study, we evaluated a new rapid diagnostic kit for simultaneous detection of the glutamate dehydrogenase (GDH) antigen and toxin A/B, C. DIFF QUIK CHEK COMPLETE. A total of 60 stool specimens from 60 patients with antibiotic-associated diarrhea were examined. Using C. difficile culture as the reference method, the GDH portion of this kit indicated a sensitivity, specificity, and negative predictive value of 100, 93.3, and 100%, respectively. The toxin A/B portion showed a sensitivity and specificity of 78.6 and 96.9%, respectively, compared to the culture results of toxin B-positive C. difficile (toxigenic culture). Of the 23 specimens that showed "dual positives" for GDH and toxin A/B, 22 were toxigenic culture positive, whereas C. difficile culture was negative in all the 28 specimens that showed "dual negatives" for GDH and toxin A/B. Of the nine "GDH-positive and toxin A/B-negative" specimens, six exhibited positive results by toxigenic culture. Results showing "dual positives" and "dual negatives" for GDH and toxin A/B can be reported as "true positive" and "true negative," respectively, whereas additional testing for confirmation, such as toxigenic culture, is required for specimens with discrepant results. Diagnostic algorithms, utilizing the simultaneous detection kit for GDH and toxin A/B as an initial screening test, may be useful for accurate and efficient diagnosis of CDI as well as the control of healthcare-associated infections.

Detecção de estro em jaguatirica (Leopardus pardalis) utilizando citologia vaginal

Utilizou-se a citologia vaginal por meio de diferentes métodos de coloração para detecção de cio em jaguatirica, Leopardus pardalis, pela estimulação hormonal exógena e pela avaliação das estruturas ovarianas por videolaparoscopia. Cinco fêmeas foram tratadas com eCG/hCG e FSH/LH a cada quatro meses pelo período de dois anos. Videolaparoscopia foi realizada após cada tratamento utilizando-se cetamina-xilazina e isoflurano. Esfregaços vaginais foram obtidos 15 dias antes e após a videolaparoscopia. As lâminas foram analisadas ao microscópio de luz quanto aos tipos celulares predominantes. Todos os animais apresentaram folículos maduros (&gt;2mm) e corpos lúteos recentes em todas as intervenções. Não houve diferença significativa entre os resultados obtidos na mesma coloração de acordo com os tratamentos utilizados. Todas as técnicas mostraram-se eficientes na detecção de células superficiais queratinizadas anucleadas e nucleadas, intermediárias, parabasais e basais. Foi possível determinar a fase de estro em Leopardus pardalis por meio da citologia vaginal

A Prospective, Single Blinded, Randomized Controlled Trial of Endoscopic Ultrasound (EUS) - Guided Fine-Needle Aspiration (FNA) With and Without a Stylet

Purpose: Background: The use of stylet during EUS-FNA is assumed to improve quality and diagnostic yield of specimens, despite lack of evidence. The use of stylet during FNA is cumbersome, time consuming and increases cost of FNA needle. Aim: To compare specimens obtained by EUS-FNA with stylet (S+) and without stylet (S-) for: 1. Cellularity, contamination, and amount of blood, and 2. Diagnostic yield of malignancy. Methods: Pts referred for EUS-FNA of solid lesions were prospectively enrolled at 2 tertiary referral centers (2 endosonographers). 22-gauge FNA needle with suction using 10 ml syringe was used. Each lesion was sampled for a minimum of 4 passes (2 S+ and 2 S-). Sequence of S+ and S- passes was determined by a preprinted randomization scheme. A cytopathologist was present in the room during the procedure. 2 slides were prepared for each needle pass (stained with Diff Quick and Pap) and numbered accordingly. Cytopathologists blinded to technique of FNA evaluated slides for: 1. Cellularity - no representative cells of target organ, representative cells in <25%, 25-50% or >50% of the slide, 2. Number of cells - <100(fair), 100-1000(good) and >1000 (excellent) per slide, 3. Contamination with cells from GI wall v none, contamination in < 25%, 25-50% or >50% of the slide, 4. Amount of blood - minimal, moderate or significant, and 5. Final diagnosis. Results: Results from the 2 groups of slides (S+ and S-) were compared using Fisher's exact test. In this pilot study, 100 pts was estimated as an adequate sample size. Results: 101 pts with 118 lesions were prospectively enrolled. Mean age 63 yrs, 69% males, 85% Caucasians. The sites of FNA were: pancreas-61, lymph node-30, liver-6, adrenal-5, others-16. Overall diagnosis was malignant in 55 lesions, benign 27, atypical/suspicious 23, inadequate specimen in 13. 236 passes each were made S+ and S-. There were no differences between S+ and S- specimens with regards to cellularity (p=0.98), no. of cells (p=0.99), contamination (p=0.92), and significant amount of blood (p=0.61). Specimen was deemed inadequate in 102/236 (43%) S+ passes vs. 89/236 (38%) S- passes (p=0.26). Diagnosis of malignancy was made in 55/236 (23%) S+ passes vs. 66/236 (28%) S- passes (p=0.74). Of the 55 malignant lesions, S+ passes were positive in 35 (64%) vs. 43 (78%) for S- passes (p=0.14). Conclusion: In this RCT comparing specimens from FNA passes made with and without stylet, no differences were seen in the cellularity, contamination, significant amount of blood and diagnostic yield of malignancy. Similar number of S+ specimens were deemed inadequate compared to S- specimens. Results of this study suggest that use of a stylet during EUS-FNA does not confer any advantage.

Clostridium Difficile Infection: Comparison of Available Laboratory Diagnostic Methods and Clinical Correlations

Purpose: (i) To compare 3 real-time polymerase chain reaction (PCR) methods, 2 enzyme immunoassay (EIA) methods for glutamate dehydrogenase (GDH) and Toxin A/B, and cell culture neutralization assay (CCNA) to toxigenic culture (TC), and (ii) to find correlation between C. difficile infection and patient covariates including nursing home residency, proton pump inhibitor (PPI) use, immune deficiency (ID), antibiotic use, consistency and number of stool, presence of diabetes mellitus (DM), inflammatory bowel disease (IBD), sepsis and connective tissue disease (CTD). Methods: 401 patients were included in the study. The BD GeneOhm C. diff Assay (BD-PCR), Cepheid Xpert C. difficile Assay (GE-PCR), Prodesse Pro-Gastro CD Assay (PG-PCR), Meridian ImmunoCard Toxins A & B (ICTAB), TechLab C. diff Chek - 60 (GDH), TechLab C. diff Quick Chek Complete (CDQC) and CCNA were performed. TC for C. difficile was tested for toxin by CCNA. Patient groups were simplified for comparison in C. difficile positive group, which comprised all patients with positive cultures, and C. difficile negative group, which included all other patients. Clinical data was statistically analyzed by chi-square test. Statistical significance was defined as p<0.05. Results:C. difficile was isolated from 82 specimens with 72 being toxin positive. The sensitivity/specificity of the BD-PCR, GE-PCR, PG-PCR, GDH, ICTAB, CDQC and CCNA compared to toxigenic culture were 87.5%/99.7%, 94.4%/97.9%, 83.3%/99.1%, 91.7%/96.0%, 55.6%/99.1%, 58.3%/100%, and 75%/100%, respectively. The GE-PCR detected the most toxigenic C. difficile followed by the BD-PCR and then the PG-PCR. The difference in sensitivity between the methods was not statistically significant (P=0.109). All 3 PCR methods were more sensitive than the CCNA (P<0.05). The only statistically significant correlations with C. difficile infection were 3 or more stools, loose consistency of stools and antibiotic use (p<0.01). The criterion of 3 or more stools had 100% sensitivity and included all the patients with positive cultures. There was no correlation between C. difficile and nursing home residency (p<0.175), PPI use (p<0.397), DM (p<0.535), CTD (p<0.138), IBD (p<0.561), ID (p<0.320) and sepsis (p<0.141). Conclusion: All 3 PCR methods were more sensitive than the CCNA (P< 0.05) with the GE-PCR being the most sensitive, but with no statistical significance. There is no value in testing stool specimens from patients who lack the clinical criterion of 3 or more loose stools per day. The use of antibiotics and loose consistency of stool were the only other statistically significant patient covariates. There was no correlation with PPI use, nursing home residency or presence of sepsis, IBD, DM, ID or CTD.

Utilization of peripherin and S-100 immunohistochemistry in the diagnosis of Hirschsprung disease

Evaluation of rectal biopsies for ganglion cells is performed for patients suspected of having Hirschsprung disease. At times, identification of ganglion cells can be difficult, especially in newborns. To assist in diagnosis, frozen tissue can be collected for acetylcholinesterase histochemical staining. At our institution, we developed a protocol using peripherin and S-100 immunostaining as an adjunct to hematoxylin and eosin (H&E) for the identification of ganglion cells. Further, at the time of frozen section, we performed Diff Quik staining to highlight ganglion cells. One hundred and thirty eight rectal biopsies submitted for evaluation of Hirschsprung disease were compiled from the archives of the Medical College of Georgia from 2002 to 2009. Initial evaluation consisted of eight levels of H&E-stained slides and two unstained slides each for immunostaining with peripherin and S-100. If on initial evaluation, ganglion cells were not identified, additional H&E and peripherin immunostains were performed. Peripherin immunostaining was unequivocally identified in the cytoplasm of ganglion cells of patients at all ages. Of the 136 patients with diagnostic biopsies, 80% had ganglion cells. Of these, 93% of cases were diagnosed on the original eight levels. Twenty-seven cases were devoid of ganglion cells, and of these, 81% showed submucosal neural hypertrophy on S-100 staining. Twenty-six patients had confirmed aganglionic segments at the time of colonic resection. One patient had colostomy only. A total of 54 frozen sections were performed on 25 patients over this same period of time. Diff Quick staining was found to be very useful. In this study, our protocol proved to be very sensitive, specific, and efficient for the diagnosis of Hirschsprung disease.

Papanicolaou stain may not be necessary in majority of head and neck fine‐needle aspirations: Evidence from a correlation study between diff–quik‐based onsite diagnosis and final diagnosis in 287 head and neck fine‐needle aspirations

Fine-needle aspiration (FNA) is a useful tool for immediate assessment of palpable lesions, especially in the head and neck region. The objective of this study is to evaluate the degree of correlation between Diff-Quik-based onsite diagnosis (OD) and final diagnosis (FD) and further improve the efficiency of FNA practice. Two hundred and eighty-seven cytopathologist-performed FNAs from the head and neck region were evaluated. Number of passes, number and type of slides and correlation (agreement, modified final diagnosis and disagreement) between OD and FD were evaluated. Among 287 FNAs, the average number of passes per FNA case was 2 (range, 1-5&.rpar;). The mean number of slides reviewed per case was 5 including 2 Diff-Quik (D-Q)-stained slides, 2 Papanicolaou (Pap)-stained slides, and 1 cell block (CB)/1 cytospin (Cy). 247 of 287 (86%) cases showed agreement between OD and FD. FD on 36 out of 287 cases (12.5%) was slightly modified or refined after reviewing additional slides. A major diagnostic discrepancy was noted in four cases (1.5%), three of which were classified as squamous cell carcinoma on final diagnosis, and confirmed on surgical follow-up. Accurate diagnosis can be achieved in the majority (86%) of head and neck FNAs based on immediate examination of D-Q stained slides alone. In a small number of cases (12.5%), reviewing additional slides may refine the final diagnosis. In rare cases, especially cystic squamous lesions, Pap-stained slides appeared to be helpful. It is plausible to use D-Q-stained slides alone with most head and neck FNAs in order to provide more cost effective and efficient triaging and patient management.

Evaluation of tcdB Real-Time PCR in a Three-Step Diagnostic Algorithm for Detection of Toxigenic Clostridium difficile

Clostridium difficile is the most common infectious cause of diarrhea in hospitalized patients. The optimal approach for the detection of toxigenic C. difficile remains controversial because no single test is sensitive, specific, and affordable. We have developed a real-time PCR method (direct stool PCR [DPCR]) to detect the tcdB gene encoding toxin B directly from stool specimens and have combined it with enzyme immunoassays (EIAs) in a three-step protocol. DPCR was performed on 699 specimens that were positive for C. difficile glutamate dehydrogenase (GDH) by Wampole C Diff Quik Chek EIA (GDH-Q) and negative for toxins A and B by Wampole Tox A/B Quik Chek EIA (AB-Q), performed sequentially. The performance of this three-step algorithm was compared with a modified "gold standard" that combined tissue culture cytotoxicity (CYT) and DPCR. A separate investigation was performed to evaluate the sensitivity of the GDH-Q as a screening test, and toxigenic C. difficile was found in 1.9% of 211 GDH-Q-negative specimens. The overall sensitivity, specificity, and positive and negative predictive values, respectively, were as follows for an algorithm combining GDH-Q, AB-Q, and DPCR: 83.8%, 99.7%, 97.1%, and 97.9%. Those for CYT alone were 58.8%, 100%, 100%, and 94.9%, respectively. In comparison, the sensitivity and specificity of DPCR were estimated to be 97.5% and 99.7%, respectively, using the same modified gold standard. Neither CYT nor toxin EIA was sufficiently sensitive to exclude toxigenic C. difficile, and combining EIAs with CYT in a three-step algorithm failed to substantially improve sensitivity. DPCR is a sensitive and specific method for the detection of toxigenic C. difficile that can provide same-day results at a cost-per-positive test comparable to those of other methods. A three-step algorithm in which DPCR is used to analyze GDH EIA-positive, toxin EIA-negative specimens provides a convenient and specific alternative with rapid results for 87.7% of specimens, although this approach is less sensitive than performing DPCR on all specimens.