Digital Image Analysis for the Detection of Group B Streptococcus from ChromID Strepto B Medium Using PhenoMatrix Algorithms.

Group B Streptococcus (GBS) can be found to colonize about 25% of all healthy, adult women and is the leading infectious cause of early neonatal morbidity and mortality in the United States. This study evaluated the clinical performance of PhenoMatrix (PM) chromogenic detection module (CDM) digital imaging software in detection of GBS from LIM broth plated on ChromID Strepto B chromogenic medium (ChromID) using the WASP automated processor. The performance of the PM CDM was compared to manual culture review of the digital images and molecular detection of GBS. ChromID alone had a sensitivity and specificity of 84.5% and 94.7%, respectively, after 48 h compared to nucleic acid amplification testing (NAAT). Compared to the composite reference for positivity, when PM CDM was used to detect GBS from ChromID, the sensitivity was 100%, with no true-positive GBS isolates missed by 48 h of incubation. Overall, evaluating all three methods for the detection of GBS, the sensitivities of NAAT, ChromID plus PM CDM at 48 h, and ChromID alone at 48 h were 96.8%, 95.5%, and 90.3%, respectively. The specificities of NAAT, ChromID plus PM CDM, and ChromID alone were 97.7%, 63.0%, and 95.4%, respectively. The sensitivity of ChromID in combination with the PM CDM was similar to the sensitivity of molecular detection. Further, the algorithm never called a culture negative that was determined to be positive by manual reading, and it identified an additional eight true positive specimens that were missed by manual digital image culture reading.

Diagnostic performance of two molecular assays for the detection of vaginitis in symptomatic women.

The three main causes of vaginitis are bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), and trichomoniasis (TV). Two multiplex assays are commercially available for detection of DNA from organisms associated with vaginitis: BD Affirm™ VPIII Microbial Identification Test (Affirm) and BD MAX™ Vaginal Panel (MAX VP). Here, the performance of MAX VP was compared to that of Affirm, which was considered the standard of care. Four vaginal swabs were collected from each subject with the following: BD Affirm™ VPIII Ambient Temperature Transport System (ATTS), BD MAX™ UVE Specimen Collection Kit, Hologic Aptima® Vaginal Swab Specimen Collection Kit, and BD ESwab™ collection and transport system (ESwab). Candida culture, Gram stain followed by Nugent scoring, and the Hologic Aptima® Trichomonas vaginalis assay were used for discordant analysis. Results were considered true positive if there were at least two tests positive for any vaginitis target. A total of 200 symptomatic women were evaluated in the study. The sensitivity and specificity of MAX VP for BV was 96.2% and 96.1%, respectively, compared to 96.2% and 81.6% for Affirm. The sensitivity and specificity of MAX VP for Candida spp. was 98.4% and 95.4%, respectively, compared to 69.4% and 100% for Affirm. MAX VP and Affirm showed 100% concordance for detection of TV. These results demonstrate improved accuracy of MAX VP compared to Affirm for the detection of BV and Candida spp. and no difference for detection of TV between the two tests.

Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinically Important Bacteria and Yeasts.

OBJECTIVES: A report on the multicenter evaluation of the Bruker MALDI Biotyper CA System (MBT-CA; Bruker Daltonics, Billerica, MA) for the identification of clinically important bacteria and yeasts. METHODS: In total, 4,399 isolates of medically important bacteria and yeasts were assessed in the MBT-CA. These included 2,262 aerobic gram-positive (AGP) bacteria, 792 aerobic gram-negative (AGN) bacteria 530 anaerobic (AnA) bacteria, and 815 yeasts (YSTs). Three processing methods were assesed. RESULTS: Overall, 98.4% (4,329/4,399) of all bacterial and yeast isolates were correctly identified to the genus and species/species complex level, and 95.7% of isolates were identified with a high degree of confidence. The percentage correctly identified and the percentage identified correctly with a high level of confidence, respectively, were as follows: AGP bacteria (98.6%/96.5%), AGN bacteria (98.5%/96.8%), AnA bacteria (98.5%/97.4%), and YSTs (97.8%/87.6%). The extended direct transfer method was only minimally superior to the direct transfer method for bacteria (89.9% vs 86.8%, respectively) but significantly superior for yeast isolates (74.0% vs 48.9%, respectively). CONCLUSIONS: The Bruker MALDI Biotyper CA System accurately identifies most clinically important bacteria and yeasts and has optional processing methods to improve isolate characterization.

Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinical Aerobic Gram-Negative Bacterial Isolates.

The prompt and accurate identification of bacterial pathogens is fundamental to patient health and outcome. Recent advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have revolutionized bacterial identification in the clinical laboratory, but uniform incorporation of this technology in the U.S. market has been delayed by a lack of FDA-cleared systems. In this study, we conducted a multicenter evaluation of the MALDI Biotyper CA (MBT-CA) System (Bruker Daltonics Inc, Billerica, MA) for the identification of aerobic gram-negative bacteria as part of a 510(k) submission to the FDA. A total of 2,263 aerobic gram negative bacterial isolates were tested representing 23 genera and 61 species. Isolates were collected from various clinical sources and results obtained from the MBT-CA System were compared to DNA sequencing and/or biochemical testing. Isolates that failed to report as a "high confidence species ID" [log(score) ≥2.00] were re-tested using an extraction method. The MBT-CA System identified 96.8% and 3.1% of isolates with either a "high confidence" or a "low confidence" [log(score) value between 1.70 and <2.00] species ID, respectively. Two isolates did not produce acceptable confidence scores after extraction. The MBT-CA System correctly identified 99.8% (2,258/2,263) to genus and 98.2% (2,222/2,263) to species level. These data demonstrate that the MBT-CA System provides accurate results for the identification of aerobic gram-negative bacteria.

Determination of testicular blood flow in camelids using vascular casting and color pulsed-wave Doppler ultrasonography.

We describe the vasculature of the camelid testis using plastic casting. We also use color pulsed-wave Doppler ultrasonography to measure testicular blood flow and compare the differences between testicular blood flow in fertile and infertile camelids. The testicular artery originates from the ventral surface of the aorta, gives rise to an epididymal branch, and becomes very tortuous as it approaches the testis. Within the supratesticular arteries, peak systolic velocity (PSV) was higher in fertile males compared to infertile males (P = 0.0004). In addition, end diastolic velocity (EDV) within the supratesticular arteries was higher for fertile males when compared to infertile males (P = 0.0325). Within the marginal arteries, PSV was also higher in fertile males compared to infertile males (P = 0.0104). However, EDV within the marginal arteries was not significantly different between fertile and infertile males (P = 0.121). In addition, the resistance index was not significantly different between fertile and infertile males within the supratesticular (P = 0.486) and marginal arteries (P = 0.144). The significance of this research is that in addition to information obtained from a complete reproductive evaluation, a male camelid's fertility can be determined using testicular blood flow measured by Doppler ultrasonography.

Esophageal anatomy of the llama (Lama glama) / Anatomía del esófago de la llama (Lama glama)

This study describes gross, microscopic and muscle fiber anatomy of the esophagus of the llama, Lama glama. The esophagus was studied grossly in twenty-five adult llamas and a subset of ten with normal esophageal physiology was used for the microanatomic studies. Esophageal length was 122 +/- 7 cm with two-thirds of the length in the neck and the remainder in the thorax, consistent with the long neck of the llama. Esophageal diameter increased steadily from 2.5 +/- 0.3 cm in the cranial cervical region to 3.9 +/- 0.8 cm in the caudal thoracic region. The mucosal epithelium was keratinized stratified squamous and there were abundant submucosal glands throughout the esophagus. The entire muscularis of the esophagus was striated muscle in two general layers but also with a somewhat random orientation of fibers. The tunica muscularis steadily increased in thickness from 3.43 +/- 0.30 mm in the cranial cervical region to 4.39 +/- 0.39 mm in the middle thoracic region. In the llama Type 2 muscle fibers predominated in the esophageal musculature, with the percentage of Type 1 fibers increasing from 1 percent cranially to 33 percent in the caudal thoracic region of the esophagus. This study of the normal llama esophagus enhances our knowledge of this species and provides the basis for future study of pathological conditions of the esophagus.

Este estudio describe la anatomía morfológica, microscópica, y tipo de fibra muscular del esófago de la llama, Lama glama. Estudiamos la anatomía morfológica del esófago, con fisiología normal, en 25 llamas adultas y, adicionalmente, en 10 de ellas la anatomía microscópica. La longitud del esófago fue 122 +/- 7 cm con dos tercios en el cuello y un tercio en el tórax. El diámetro del esófago aumentó de 2,5 +/- 0,3 cm en la región craneal del cuello y a 3,9 +/- 0,8 cm en la región caudal del tórax. El epitelio de la mucosa eera escamoso estratificado queratinizado y la submucosa contenía abundantes glándulas a lo largo de todo el esófago. La muscular entera del esófago se compuso de músculo esquelético en más o menos dos capas, pero con algunas fibras orientadas al azar. La muscular aumentó de 3,43 +/- 0,30 mm en la región craneal del cuello a 4,39 +/- 0,39 mm en la región media del tórax. Fibras musculares Tipo 2 predominaron en la muscular. El porcentaje de fibras Tipo 1 aumentó de 1 por ciento al inicio del esófago a 33 por ciento en la región caudal torácica. Este estudio del esófago normal de la llama ofrece más información sobre la anatomía de la llama y proporciona una base para futuros estudios de patologías esofágicas.

Expression of estrogen receptors alpha and beta in the corpus luteum and uterus from non-pregnant and pregnant llamas.

Because estrogen may be involved in maternal recognition of pregnancy and embryonic migration in llamas, expression of estrogen receptor subtypes alpha (ERalpha) and beta (ERbeta) was evaluated in corpus luteum (CL), endometrium, and uterus using relative RT-PCR. Tissues were recovered from sterile-mated (SM) and pregnant (PG) females during Days 7-11 and 7-13 (Day 0 = day of mating), respectively, and follicular phase and juvenile females. Luteal expression of ERalpha and beta was similar (P > 0.10) in SM and PG females and within Days 7-11, however, expression of ERalpha in ovarian tissue from follicular phase females was greater (P < 0.05) than Days 7 and 9 CL. Uterus expressed less ERalpha and beta compared to endometrium (P = 0.07 and P < 0.01, respectively). Expression of ERalpha was greater (P < 0.05) in Day 7 and follicular phase uteri than Days 9 and 11, Day 13 PG and juvenile uteri. Uterine ERbeta expression was greater (P = 0.09) in PG versus SM females and in mated compared to follicular phase females (P < 0.05). Endometrial expression of ERalpha and beta did not differ (P > 0.10) between SM and PG females or by day. The presence of luteal ER during this period may mean a role for estradiol in maternal recognition of pregnancy. Observed increases in uterine ER expression with no changes in endometrium suggest expression increased in myometrium and/or perimetrium. Upregulation of myometrial ERbeta in PG females may be involved in supporting uterine migration of the embryo.

Estradiol production by preimplantation blastocysts and increased serum progesterone following estradiol treatment in llamas.

Estradiol is a potential candidate for the blastocyst signal responsible for maternal recognition of pregnancy in the llama (Lama glama). Two experiments were conducted to determine if the llama blastocyst produces estradiol during the presumed period of maternal recognition of pregnancy and if exogenous estradiol can extend the luteal phase. In Experiment 1, llamas were superovulated with eCG and mated 7 days later (Day 0=day of mating). Blastocysts were collected nonsurgically on Days 7, 9, or 11 or at necropsy on Days 13 and 15 post-mating and cultured for 48h. Conditioned medium was recovered, replaced with fresh medium at 24-h intervals, and assayed for estradiol-17beta. Estradiol production (pg/blastocyst) over the 48-h culture increased (P<0.05) by day of gestation where more estradiol (P<0.05) was produced by Day 11 compared to Day 7 blastocysts, Day 13 compared to Days 7-11 blastocysts, and Day 15 compared to Days 7-13 blastocysts. A dramatic increase was observed between Days 11 and 13 when estradiol production by Day 13 blastocysts increased (P<0.05) more than 50-fold. In Experiment 2, 30 females were induced to ovulate with hCG (Day 0=day of hCG injection). Starting on Day 7 and continuing through Day 15, animals received daily injections i.m. of 0 (n=11), 5 (n=7), or 10mg (n=12) estradiol benzoate (EB) dissolved in isopropylmyristate. Sera were collected immediately prior to each injection and on Days 16, 17, 18, 20, and 22 and analyzed for progesterone. Progesterone concentrations were greater (P<0.05) on Days 14, 15, 16, and 17 in llamas treated with 10mg EB compared to llamas treated with 0mg EB. These results demonstrate that llama blastocysts produce estradiol and exogenous estradiol can enhance and transiently extend luteal progesterone production. Estradiol produced by the preimplantation llama blastocyst may play a role in maternal recognition of pregnancy and early luteal support.

Seasonal interaction of serum vitamin D concentration and bone density in alpacas.

OBJECTIVE: To evaluate temporal changes in bone mineral density associated with seasonal variation in serum vitamin D, calcium, and phosphorus concentrations in alpacas. ANIMALS: 5 healthy mature neutered male alpacas. PROCEDURE: Metacarpal bone mineral density was measured at 4 times during a year. Each time alpacas were weighed, blood was collected for determination of serum calcium, phosphorus, and vitamin D concentrations, and samples of feed were analyzed for nutrient content. Vitamin D status was determined by use of an assay that measured serum 25-hydroxycalciferol concentration. Effects of changes in serum vitamin D, calcium, and phosphorus concentration and body weight with season on bone mineral density were determined. RESULTS: Bone mineral density, body weight, and serum vitamin D and phosphorus concentrations varied with season. Bone mineral density, serum vitamin D concentration, and body weight also varied among individual alpacas. Serum vitamin D concentration was lower in January than the previous October and increased from May to the following September. The decrease in bone mineral density lagged behind the decrease in serum vitamin D concentration and was lower in May, compared with the previous October. Body weight was lower in May than the previous October or following September. Solar radiation was highest in July and lowest in December. CONCLUSIONS AND CLINICAL RELEVANCE: Seasonal changes in bone mineral density are associated with changes in serum vitamin D concentrations in alpacas. Changes in bone mineral density associated with a decline in serum vitamin D concentration may predispose some alpacas to developing fractures minimal trauma.