Shrimp MultiPath™ multiplexed PCR white spot syndrome virus detection in penaeid shrimp.

White spot syndrome virus (WSSV), which causes white spot disease, is one of the notoriously feared infectious agents in the shrimp industry, inflicting estimated production losses world-wide of up to US$1 billion annually. Cost-effective accessible surveillance testing and targeted diagnosis are key to alerting shrimp industries and authorities worldwide early about WSSV carrier status in targeted shrimp populations. Here we present key validation pathway metrics for the Shrimp MultiPathTM (SMP) WSSV assay as part of the multi-pathogen detection platform. With superior throughput, fast turn-around time, and extremely low cost per test, the SMP WSSV assay achieves a high level of analytical sensitivity (~2.9 copies), perfect analytical specificity (~100%), and good intra- and inter-run repeatability (coefficient of variation <5%). The diagnostic metrics were estimated using Bayesian latent class analysis on data from 3 experimental shrimp populations from Latin America with distinct WSSV prevalence and yielded a diagnostic sensitivity of 95% and diagnostic specificity of 99% for SMP WSSV, which was higher than these parameters for the TaqMan quantitative PCR (qPCR) assays currently recommended by the World Organisation for Animal Health and the Commonwealth Scientific and Industrial Research Organisation. This paper additionally presents compelling data for the use of synthetic double-stranded DNA analyte spiked into pathogen-naïve shrimp tissue homogenate as a means to substitute clinical samples for assay validation pathways targeting rare pathogens. SMP WSSV shows analytical and diagnostic metrics comparable to qPCR-based assays and demonstrates fit-for-purpose performance for detection of WSSV in clinically diseased and apparently healthy animals.

Enterocytozoon hepatopenaei real-time and Shrimp MultiPathTM PCR assay validation for South-East Asian and Latin American strains of Penaeid shrimp.

Enterocytozoon hepatopenaei (EHP) infections are a global challenge for the Penaeid shrimp industry with a sharp rise in prevalence over the last 10 yr. EHP is known to cause sub-optimal growth, large size variation and reduced survival of shrimp. Molecular methods development has mainly focussed on 18S rRNA or spore wall protein 1 (SWP1). Due to the specificity and sensitivity issues with previously designed assays for both targets, new molecular assays are needed by the global shrimp industry and regulators to help manage the risks posed by EHP. This paper describes new real-time PCR (qPCR) methods developed for the novel EHP gene targets polar tube protein 2 (PTP2) and spore wall protein 26 (SWP26), whilst also presenting performance metrics of the new Shrimp MultiPathTM technology EHP assay. qPCR assays PTP2G and SWP26G show high amplification efficiency, a limit of detection (LOD) of between 1 and 4 copies, low assay variation and high diagnostic sensitivity (DSe) and specificity (DSp) compared to imperfect reference assays. Similar performance is seen with Shrimp MultiPathTM EHP showing an LOD of 8 copies, low assay variation and high DSe and DSp. These novel molecular targets for EHP and Shrimp MultiPathTM EHP strengthen global efforts to monitor and mitigate risks of EHP infections and outbreaks. Moreover, this study presents novel data on distribution of EHP in shrimp populations from South-East Asia and Latin America, and how sequence variations need to be considered when monitoring EHP in different geographies.

Detection and genetic diversity of porcine rotavirus A, B and C in eastern Australian piggeries.

Rotaviruses (RV) have a high prevalence in piggeries worldwide and are one of the major pathogens causing severe diarrhoea in young pigs. RV species A, B, and C have been linked to piglet diarrhoea in Australian pig herds, but their genetic diversity has not been studied in detail. Based on sequencing of the structural viral protein 7 (VP7) RVA G genotypes G3, G4 and G5, and RVC types G1, G3, G5, and G6 have been identified in Australian piggeries in previous studies. Although occurrence of RVB was reported in Australia in 1988, no further genetic analysis has been conducted. To improve health management decisions in Australian pig herds, more information on RV prevalence and genetic diversity is needed. Here, 243 enteric samples collected from 20 pig farms within Eastern Australia were analysed for the presence of RV in different age groups using a novel PCR-based multiplex assay (Pork MultiPath™ enteric panel). RVA, RVB, and RVC were detected in 10, 14, and 14 farms, respectively. Further sequencing of VP7 in selected RV-positive samples revealed G genotypes G2, G5, G9 (RVA), G6, G8, G14, G16, G20 (RVB), and G1, G3, G5, G6 (RVC) present. RVA was only detected in young (<10 weeks old) pigs whereas RVB and RVC were also detected in older animals (>11 weeks old). Interestingly, RVB and RVC G-type occurrence differed between age groups. In conclusion, this study provides new insights on the prevalence and diversity of different RV species in pig herds of Eastern Australia whilst demonstrating the ability of the Pork MultiPath™ technology to accurately differentiate between these RV species.

Obstructive sleep apnea in adults with tonsillar hypertrophy.

Tonsillar enlargement is a more common cause of obstructive sleep apnea (OSA) in the pediatric population than it is in adults. The small number of reported cases suggests that tonsillectomy for adult patients with this association may be as a successful as it is known to be for children. We compared polysomnographic findings and/or symptomatology both before and after tonsillectomy in six patients aged 22 to 52 years, who had adenotonsillar hypertrophy and OSA. Tonsillectomy provided lasting relief of OSA in four patients. We review the literature for available polygraphic data from similar patients before and after tonsillectomy. Our combined experience indicates that many adults with OSA can clearly benefit from removal of hypertrophied tonsils. Such success may, for some adults, be transient or may not occur. This should be considered during initial patient counseling and when planning postoperative follow-up.

Mycobacteria other than tuberculosis. Pulmonary involvement in patients with acquired immunodeficiency syndrome.

A normal host can be colonized by mycobacteria other than tuberculosis (MOTT), resulting in bronchoscopic isolates of no clinical significance. In the acquired immunodeficiency syndrome (AIDS), Mycobacterium avium-intracellulare, Mycobacterium kansasii, and Mycobacterium xenopi have caused widely disseminated infection. To determine the usefulness of fiberoptic bronchoscopy (FB) in evaluating MOTT infection in AIDS, we reviewed MOTT cultures from 36 FBs, correlated these to clinical course, and identified MOTT isolates from cultures of all other sources in these patients. Of ten bronchoscopic MOTT isolates, seven were not related to lung disease or to dissemination within one month of FB. Of the four Mycobacterium fortuitum and seven M avium-intracellulare that did disseminate within one month, only two were reflected in bronchoscopic cultures. In patients with AIDS, bronchopulmonary MOTT colonization does occur. We recommend that standard criteria for pulmonary mycobacterial disease be applied. Negative bronchopulmonary cultures do not seem to exclude dissemination.

A mixed-model approach for the analysis of cDNA microarray gene expression data from extreme-performing pigs after infection with Actinobacillus pleuropneumoniae.

We proposed a novel statistical approach for the analysis of cDNA experiments based on mixed-model methodology combined with mixtures of distributions. Our objective was to detect genes that may be involved in conferring heritable differences in susceptibility to common infections in intensive pig production. We employed a microarray expression profiling strategy and a mixed-model approach to the analysis of the expression data. A cDNA microarray of pig with 6,420 probes from immune tissues and cells was used to compare gene expression in peripheral blood leukocytes of two pigs showing extreme performance in their response to infection with Actinobacillus pleuropneumoniae. Principal components analyses were used to identify the two most extreme-performing pigs after infection (i.e., pigs whose measured responses to infection fell at the extremes). Blood samples and expression profiles from 0 to 24 h after infection were compared using a bivariate, mixed-model approach, in which the effect gene x immunological status interaction was treated as a random effect. Bayesian model-based clustering via mixtures of normal distributions of the resulting BLUP of the random interaction was approached and resulted in a list of 307 differentially expressed genes, of which 179 were down-regulated in the susceptible pig. The majority of the differentially expressed genes were derived from a cDNA library of leukocytes of A. pleuropneumoniae-challenged pigs that were subtracted against leukocytes before the challenge. These results provide evidence that the proposed statistical approach was useful in enhancing the knowledge of the mechanisms involved in the genetics of the immune response.

Increased de novo copy number variants in the offspring of older males.

The offspring of older fathers have an increased risk of neurodevelopmental disorders, such as schizophrenia and autism. In light of the evidence implicating copy number variants (CNVs) with schizophrenia and autism, we used a mouse model to explore the hypothesis that the offspring of older males have an increased risk of de novo CNVs. C57BL/6J sires that were 3- and 12-16-months old were mated with 3-month-old dams to create control offspring and offspring of old sires, respectively. Applying genome-wide microarray screening technology, 7 distinct CNVs were identified in a set of 12 offspring and their parents. Competitive quantitative PCR confirmed these CNVs in the original set and also established their frequency in an independent set of 77 offspring and their parents. On the basis of the combined samples, six de novo CNVs were detected in the offspring of older sires, whereas none were detected in the control group. Two of the CNVs were associated with behavioral and/or neuroanatomical phenotypic features. One of the de novo CNVs involved Auts2 (autism susceptibility candidate 2), and other CNVs included genes linked to schizophrenia, autism and brain development. This is the first experimental demonstration that the offspring of older males have an increased risk of de novo CNVs. Our results support the hypothesis that the offspring of older fathers have an increased risk of neurodevelopmental disorders such as schizophrenia and autism by generation of de novo CNVs in the male germline.

Comparison of a multiplexed MassARRAY system with real-time allele-specific PCR technology for genotyping of methicillin-resistant Staphylococcus aureus.

The Sequenom MassARRAY iPLEX single-nucleotide polymorphism (SNP) typing platform uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with single-base extension PCR for high-throughput multiplex SNP detection. In this study, we investigated the use of iPLEX MassARRAY technology for methicillin-resistant Staphylococcus aureus (MRSA) genotyping. A 16-plex MassARRAY iPLEX GOLD assay (MRSA-iPLEX) was developed that targets a set of informative SNPs and binary genes for MRSA characterization. The method was evaluated with 147 MRSA isolates, and the results were compared with those of an established SYBR Green-based real-time PCR system utilizing the same SNP-binary markers. A total of 2352 markers belonging to 44 SNP-binary profiles were analysed by both real-time PCR and MRSA-iPLEX. With real-time PCR as the reference standard, MRSA-iPLEX correctly assigned 2298 of the 2352 (97.7%) markers. Sequence variation in the MRSA-iPLEX primer targets accounted for the majority of MRSA-iPLEX erroneous results, highlighting the importance of primer target selection. MRSA-iPLEX provided optimal throughput for MRSA genotyping, and was, on a reagent basis, more cost-effective than the real-time PCR methods. The 16-plex MRSA-iPLEX is a suitable alternative to SYBR Green-based real-time PCR typing of major sequence types and clonal complexes of MRSA.

Health status of refugees from Vietnam, Laos, and Cambodia.

More than 0.5 million refugees from Southeast Asia have immigrated to the United States. We undertook a prospective evaluation of 709 refugees within two months of their resettlement in San Diego. The sample included 164 Vietnamese, 356 Cambodians, 139 Laotians, and 50 Hmong. The prevalence of abnormalities was high: intestinal parasites, 61%; positive tuberculin test (PPD) results 55%; anemia, 37%; hepatitis B antigenemia, 14%; and abnormal VDRL test results, 12%. Except for hepatitis, significant differences were noted among the Vietnamese, Cambodian, Laotian, and Hmong subjects on each of these health status indicators. The refugee population should not be considered a homogeneous group of Indochinese, particularly by those responsible for their health care.

Parasite screening and treatment among Indochinese refugees. Cost-benefit/utility and the General Health Policy Model.

The General Health Policy Model and the Quality of Well-being scale are used to describe a "cost-benefit/utility" evaluation of a screening and treatment program for intestinal parasites among indochinese refugees in the United States. Cost-benefit/utility analysis subsumes conventional cost-effectiveness by explicitly adding social utility factors to the dollar dimension. Using actual data on parasite prevalence and program costs from one screening project and estimated figures for other factors, this article demonstrates calculation of the cost-benefit/utility outcome measure, dollars per well-year. Dollars per well-year for parasite screening are calculated for a number of examples. Further analysis and final conclusions on the worth of parasite screening and treatment programs await more reliable data for some terms of the developed model.

Examination of some factors affecting sensitivity and reproducibility in radioimmunoassay of thyrotropin.

Conditions for measuring human thyrotropin by radioimmunoassay have been investigated, to improve the sensitivity and reproducibility of the assay. The Chloramine-T method of iodination was used, the reaction time being 20 s. Doubling the iodination reaction volume from 55 to 95 mul did not affect the immunoreactivity. Purification of labeled hormone by use of anion-exchange resin followed by silica (Quso G-32) instead of Sephadex gel-filtration or anion exchange alone yielded a product that was the least (less than 4%) contaminated with Na125 I. Human serum albumin (2.5 g/liter) in phosphate-buffered saline, instead of bovine serum, should be used as diluent for unknowns; within-assay variance was 3% for the former, 62% for the latter. The assay worked equally well for both pregnant and nonpregnant patients, with use of 50 to 100 mul of serum per determination. A five-week-old labeled hormone yielded higher values than did two-week-old material. Use of sequential saturation techniques (tracer added on day 3) resulted in a greater than 50% drop in B/B-0 ratio in the standard curve between 0.39 and 50 microunits/ml. Somatotropin, choriomammotropin, and procine insulin did not cross react in this system. Human follitropin and lutropin did cross react, and this cross reaction could not be prevented with as much as 40 international units of human choriogonadotropin per tube. With a total reaction volume of 0.4 ml before addition of second antibody, 0.2 ml of 10-fold diluted second antibody yielded a standard curve with lower nonspecific binding and higher maximum precipitation than one constructed by using only 20 mul of second antibody. In 29 euthyroid patients the mean thyrotropin value was 5.7 microunits/ml (range 2.8-11); nine hypothyroid patients had a mean of 112 microunits/ml (range 38-267); and 13 hyperthyroid subjects showed suppressed thyrotropin with a mean of 3.1 microunits/ml (range 2.2-4.5). Following these suggestions, one can expect a more highly purified immunoreactive tracer and a more sensitive assay than is obtained with the procedure from the Pituitary Agency of the National Institutes of Health.

Diagnosis and treatment of metastatic ameloblastoma.

Although there are several theoretic mechanisms for metastasis of ameloblastoma, hematogenous and lymphangitic spread are the most likely. Metastatic ameloblastoma in the lung can be accurately diagnosed by transbronchial biopsy. Surgical excision of metastatic ameloblastoma is one choice for treatment, but there is no good evidence that this benefits the patient. Treatment of metastatic ameloblastoma with previously used chemotherapeutic regimens has shown little clinical response and has always been followed by disease progression. There may be a role for our chemotherapy regimen for local control in dangerous locations, thereby facilitating further surgical removal and minimizing postoperative sequelae.

Abnormal adolescent primiparous pregnancy: association of race, human chorionic somatomammotropin production, and smoking.

Abnormal pregnancies (33 per cent) were studied in 417 consecutive, unmarried, primiparous women, age 12 to 18 years (black, 54 per cent; white, 46 per cent). Black patients had no increase in abnormal pregnancies but had more infants weighting less than 2,500 grams (p less than 0.01). Black patients with pre-eclampsia gained less weight than white patients (24 versus 34 pounds, p less than 0.05) and had smaller infants (2,834 versus 3,316 grams). In white patients, the mean age at menarche was earlier in women who lost their infants (11.0 versus 12.7 years; p less than 0.01). Smoking was more prevalent in normal pregnancies (47 per cent) than abnormal pregnancies (38 per cent). Spontaneous abortion and fetal loss occurred more often in smokers (p less than 0.01). White women smoked more than blacks (60 versus 37 per cent). In mothers of infants weighting less than 2,500 grams, there were fewer smokers. Unexpectedly, smokers had less hypertension and pre-eclampsia. Human chorionic somatomammotropin values correlated with infant size. Levels of less than 4 mug per milliliter after 30 weeks' gestation were identical in normal and abnormal pregnancies (4.4 per cent). They were associated with small infants but no fetal deaths and did not predict outcome of pregnancy.

Human chorionic somatomammotropin in normal adolescent primiparous pregnancy. I. Effect of smoking.

PIP: Human chorionic somatomammotropin (HCS) levels were studied in normal smoking and nonsmoking primiparous adolescent pregnancies. 136 teenagers, aged 12-18 years, were divided into groups: nonsmokers, deep, and shallow inhalers, long, and short puffers, high, and low tar, and high, and low nicotin. Shallow inhaling and low nicotine exposure patients were found to have a later age of menarche than did nonsmokers (13.2 vs. 12.3 years, p=.03). The mean body weight of the mothers who smoked was slightly less (61 gm) than that of nonsmoking mothers. Except for long puffers, overall, smokers had significantly lower HCS values throughout pregnancy than noosmokers (p = .48 high tar-p = .002 low tar). However, in the third trimester those with the lowest smoking exposures had the lowest HCS values and the heavier smokers had slightly higher mean values than nonsmokers. These data suggest that HCS production may be more sensitive to low tar and nicotine exposure with possible tolerance or even stimulation occurring in larger doses.^ieng

Experimental cretinism in lambs: an intrauterine model with thyroid evaluation in surviving lambs.

The experiments in this article were designed to establish an animal model analogous to human athyrotic cretinism without altering maternal thyroid status and subsequently to observe the natural course of the disorder. The effect of ovine intrauterine fetal thyroidectomy during the second trimester was assessed by following maternal thyroid function and amniotic fluid T4 by column [T4(C)], T3 by radioimmunoassay (T3RIA), and ovine TSH (oTSH). Maternal serum T4(C) rose during pregnancy but unexpectedly both T3RIA and oTSH decreased. In amniotic fluid, T4(C) and oTSH values were lower than maternal serum values and T3RIA was not detectable. Amniotic fluid T4(C) at Cesarean section was identical in cretin lambs and twin controls and was thus not a useful diagnostic tool. Athyrotic lambs showed gross physical, behavioral, and pathologic changes of cretinism and expired during the first day of life. At birth, cretin serum T4(C) and T3RIA were markedly decreased and oTSH was elevated. Normal lambs had a gradual decline in T4(C) levels until 10 weeks of age, but T3RIA values changed erratically. In addition, one cryptocretin with a thyroid remnant had a high normal T4(C) and elevated T3RIA and oTSH at birth. Her course was normal until 7 months of age when hypothyroidism suddenly developed. These observations could prove relevant in the interpretation of thyroid function of normal versus partially or completely athyrotic human infants.

Asthma in a Vietnamese refugee population.

Asians and Pacific Islanders comprise a large and growing minority group in the United States, yet data on health status specific to these populations are scant. We conducted an epidemiologic study of asthma in a Vietnamese refugee population to estimate prevalence, evaluate risk factors, and better understand treatments of asthma among Vietnamese individuals. One hundred twenty-four asthma cases were identified from a population of 2,536 new Vietnamese refugees in San Diego (prevalence = 49 per 1,000; 4.9%). Two nonasthmatic control groups of Vietnamese refugees, matched for age and gender with the asthma cases, were recruited for a case-control study, using a questionnaire administered in Vietnamese. Vietnamese asthmatic individuals used both Western and non-Western therapies. Most subjects used traditional health practices, such as coining, cupping, and oil inhalation. As compared with current-refugee controls, the asthmatic subjects used significantly more bleeding (OR: 3.40; 95% CI: 1.06 to 10.80) and herbal ingestion (OR: 1.87; 95% CI: 1.08 to 3.19). As compared with former-refugee controls, the asthmatic subjects used significantly more oil inhalation (OR: 2.58; 95% CI: 1.45 to 4.85), bleeding (OR: 8.64, 95% CI: 1.02 to 73.70), and herbal ingestion (OR: 1.93; 95% CI: 1.02 to 3.67). The presentation and recognition of asthma among the Vietnamese subjects were similar to those in other populations. This information may be helpful in designing culture-specific health-education programs.