Development and validation of a real-time quantitative PCR assay for the detection and quantification of Perkinsus marinus in the Eastern oyster, Crassostrea virginica

Author Posting. © National Shellfisheries Association, 2009. This article is posted here by permission of National Shellfisheries Association for personal use, not for redistribution. The definitive version was published in Journal of Shellfish Research 28 (2009): 459-464, doi:10.2983/035.028.0306.Perkinus marinus causes a devastating disease, known as Dermo, in the Eastern oyster Crassostrea virginica. Routine detection of the disease is traditionally accomplished by the use of the Ray/Makin assay, using Fluid Thioglycollate Medium (RFTM). A simple real-time quantitative PCR assay was developed as a diagnostic tool to detect and quantify P. marinus, to complement and serve as an alternate to the RFTM method. Using a dual-labeled probe approach, a sensitive assay was designed to accurately detect a range of one to several thousand P. marinus organisms present in oyster tissues. A simple extraction method was used to increase throughput of the assay. Cultured P. marinus cells were quantified prior to DNA extraction, generating a standard curve and allowing cell counts to be derived from PCR cycle threshold values. Direct comparison of the RFTM and real-time PCR methods was accomplished by using tissue samples from the same oyster for both tests. Plotting cycle threshold values against the known Mackin index value generated a standard curve with a coefficient of regression of 0.9. Our results indicate that correlations could be made between this molecular based approach and traditional methods, allowing results generated from the PCR assay to be easily translated into the understood Mackin scale

PCR and Mosquito dissection as tools to monitor filarial infection levels following mass treatment

BACKGROUND: Entomological methods may provide important tools for monitoring the progress of lymphatic filariasis elimination programs. In this study, we compared dissection of the vector, Culex quinquefasciatus, with the polymerase chain reaction (PCR) to assess filarial infection levels in mosquitoes in the context of a lymphatic filariasis elimination program in Leogane, Haiti. METHODS: Mosquitoes were collected using gravid traps located in 4 sentinel communities with Wuchereria bancrofti microfilaria prevalence that ranged from 0.8% to 15.9%. Captured mosquitoes were divided between dissection, to enumerate W. bancrofti larvae (L1, L2, L3) and desiccation for later analysis by PCR. PCR was conducted on DNA extracts from pooled mosquitoes (1–15 pooled females) utilizing a competitive PCR system with primers specific for the Ssp I repeat. PCR products were analyzed with a hybridization ELISA using probes specific for a control sequence and the Ssp I repeat. RESULTS: The prevalence of mosquito infection with W. bancrofti ranged from 0%–3.66% by dissection (L1–L3) and point estimates of infection prevalence, as assayed by PCR, ranged from 0.25% – 9.16%. Following mass treatment, W. bancrofti infection prevalence dropped significantly as determined by PCR and dissection in 2 of the 4 sentinel sites (Leogane and Barrier Jeudi, P = 0.04 and P = 0.005, respectively). Although transmission declined in the other two sites, larval recoveries were low and these changes were not statistically significant. DISCUSSION: Our results suggest that a single round of mass treatment can have an impact on transmission of lymphatic filariasis. The use of entomologic methods as a tool to monitor filariasis programs and the statistical limitations of mosquito trapping are discussed

Epidemiologic Differences Between Cyclosporiasis and Cryptosporidiosis in Peruvian Children

We compared the epidemiologic characteristics of cyclosporiasis and cryptosporidiosis in data from a cohort study of diarrhea in a periurban community near Lima, Peru. Children had an average of 0.20 episodes of cyclosporiasis/year and 0.22 episodes of cryptosporidiosis/year of follow-up. The incidence of cryptosporidiosis peaked at 0.42 for 1-year-old children and declined to 0.06 episodes/child-year for 5- to 9-year-old children. In contrast, the incidence of cyclosporiasis was fairly constant among 1- to 9-year-old children (0.21 to 0.28 episodes/child-year). Likelihood of diarrhea decreased significantly with each episode of cyclosporiasis; for cryptosporidiosis, this trend was not statistically significant. Both infections were more frequent during the warm season (December to May) than the cooler season (June to November). Cryptosporidiosis was more frequent in children from houses without a latrine or toilet. Cyclosporiasis was associated with ownership of domestic animals, especially birds, guinea pigs, and rabbits

Routine delivery of artemisinin-based combination treatment at fixed health facilities reduces malaria prevalence in Tanzania: an observational study

BACKGROUND Artemisinin-based combination therapy (ACT) has been promoted as a means to reduce malaria transmission due to their ability to kill both asexual blood stages of malaria parasites, which sustain infections over long periods and the immature derived sexual stages responsible for infecting mosquitoes and onward transmission. Early studies reported a temporal association between ACT introduction and reduced malaria transmission in a number of ecological settings. However, these reports have come from areas with low to moderate malaria transmission, been confounded by the presence of other interventions or environmental changes that may have reduced malaria transmission, and have not included a comparison group without ACT. This report presents results from the first large-scale observational study to assess the impact of case management with ACT on population-level measures of malaria endemicity in an area with intense transmission where the benefits of effective infection clearance might be compromised by frequent and repeated re-infection. METHODS A pre-post observational study with a non-randomized comparison group was conducted at two sites in Tanzania. Both sites used sulphadoxine-pyrimethamine (SP) monotherapy as a first-line anti-malarial from mid-2001 through 2002. In 2003, the ACT, artesunate (AS) co-administered with SP (AS + SP), was introduced in all fixed health facilities in the intervention site, including both public and registered non-governmental facilities. Population-level prevalence of Plasmodium falciparum asexual parasitaemia and gametocytaemia were assessed using light microscopy from samples collected during representative household surveys in 2001, 2002, 2004, 2005 and 2006. FINDINGS Among 37,309 observations included in the analysis, annual asexual parasitaemia prevalence in persons of all ages ranged from 11% to 28% and gametocytaemia prevalence ranged from <1% to 2% between the two sites and across the five survey years. A multivariable logistic regression model was fitted to adjust for age, socioeconomic status, bed net use and rainfall. In the presence of consistently high coverage and efficacy of SP monotherapy and AS + SP in the comparison and intervention areas, the introduction of ACT in the intervention site was associated with a modest reduction in the adjusted asexual parasitaemia prevalence of 5 percentage-points or 23% (p < 0.0001) relative to the comparison site. Gametocytaemia prevalence did not differ significantly (p = 0.30). INTERPRETATION The introduction of ACT at fixed health facilities only modestly reduced asexual parasitaemia prevalence. ACT is effective for treatment of uncomplicated malaria and should have substantial public health impact on morbidity and mortality, but is unlikely to reduce malaria transmission substantially in much of sub-Saharan Africa where individuals are rapidly re-infected.Financial support for IMPACT-Tz came primarily from CDC, the U.S. Agency for International Development and the Wellcome Trust

Recently Acquired Toxoplasma gondii Infection, Brazil

Soil exposure, eating undercooked meat, and having children are risk factors for acute infection and high rate of eye disease

Community-based care vs. centralised hospitalisation for MDR-TB patients, KwaZulu-Natal, South Africa.

CAPRISA, 2015.Abstract available in pdf

Outbreak of Cyclosporiasis Associated with Imported Raspberries, Philadelphia, Pennsylvania, 2000

An outbreak of cyclosporiasis occurred in attendees of a wedding reception held in Philadelphia, Pennsylvania, on June 10, 2000. In a retrospective cohort study, 54 (68.4%) of the 79 interviewed guests and members of the wedding party met the case definition. The wedding cake, which had a cream filling that included raspberries, was the food item most strongly associated with illness (multivariate relative risk, 5.9; 95% confidence interval, 3.6 to 10.5). Leftover cake was positive for Cyclospora DNA by polymerase chain reaction analyses. Sequencing of the amplified fragments confirmed that the organism was Cyclospora cayetanensis. The year 2000 was the fifth year since 1995 that outbreaks of cyclosporiasis definitely or probably associated with Guatemalan raspberries have occurred in the spring in North America. Additionally, this is the second documented U.S. outbreak, and the first associated with raspberries, for which Cyclospora has been detected in the epidemiologically implicated food item

National Outbreak of Acanthamoeba Keratitis Associated with Use of a Contact Lens Solution, United States

Premarket standardized testing for Acanthamoeba spp. is warranted

Feasibility and Effectiveness of Basic Lymphedema Management in Leogane, Haiti, an Area Endemic for Bancroftian Filariasis

Lymphatic filariasis is a parasitic disease that is spread by mosquitoes. In tropical countries where lymphatic filariasis occurs, approximately 14 million people suffer from chronic swelling of the leg, known as lymphedema. Repeated episodes of bacterial skin infection (acute attacks) cause lymphedema to progress to its disfiguring form, elephantiasis. To help achieve the goal of eliminating lymphatic filariasis globally, the World Health Organization recommends basic lymphedema management, which emphasizes hygiene, skin care, exercise, and leg elevation. Its effectiveness in reducing acute attack frequency, as well as the role of compressive bandaging, have not been adequately evaluated in filariasis-endemic areas. Between 1995 and 1998, we studied 175 people with lymphedema of the leg in Leogane, Haiti. During Phase I of the study, when compression bandaging was used to reduce leg volume, the average acute attack rate was 1.56 episodes per year; it was greater in people who were illiterate and those who used compression bandages. After March 1997, when hygiene and skin care were emphasized and bandaging discouraged, acute attack frequency significantly decreased to 0.48 episodes per year. This study highlights the effectiveness of hygiene and skin care, as well as limitations of compressive bandaging, in managing lymphedema in filariasis-endemic areas

Association between health systems performance and treatment outcomes in patients co-infected with MDR-TB and HIV in KwaZulu-Natal, South Africa: implications for TB programmes.

CAPRISA, 2014.Objective: To improve the treatment of MDR-TB and HIV co-infected patients, we investigated the relationship between health system performance and patient treatment outcomes at 4 decentralised MDR-TB sites. Methods: In this mixed methods case study which included prospective comparative data, we measured health system performance using a framework of domains comprising key health service components. Using Pearson Product Moment Correlation coefficients we quantified the direction and magnitude of the association between health system performance and MDR-TB treatment outcomes. Qualitative data from participant observation and interviews analysed using systematic text condensation (STC) complemented our quantitative findings. Findings: We found significant differences in treatment outcomes across the sites with successful outcomes varying from 72% at Site 1 to 52% at Site 4 (p<0.01). Health systems performance scores also varied considerably across the sites. Our findings suggest there is a correlation between treatment outcomes and overall health system performance which is significant (r = 0.99, p<0.01), with Site 1 having the highest number of successful treatment outcomes and the highest health system performance. Although the 'integration' domain, which measured integration of MDR-TB services into existing services appeared to have the strongest association with successful treatment outcomes (r = 0.99, p<0.01), qualitative data indicated that the 'context' domain influenced the other domains. Conclusion: We suggest that there is an association between treatment outcomes and health system performance. The chance of treatment success is greater if decentralised MDR-TB services are integrated into existing services. To optimise successful treatment outcomes, regular monitoring and support are needed at a district, facility and individual level to ensure the local context is supportive of new programmes and implementation is according to guidelines