Malaria PCR Detection in Cambodian Low-Transmission Settings: Dried Blood Spots versus Venous Blood Samples

In the context of malaria elimination, novel strategies for detecting very low malaria parasite densities in asymptomatic individuals are needed. One of the major limitations of the malaria parasite detection methods is the volume of blood samples being analyzed. The objective of the study was to compare the diagnostic accuracy of a malaria polymerase chain reaction assay, from dried blood spots (DBS, 5 μL) and different volumes of venous blood (50 μL, 200 μL, and 1 mL). The limit of detection of the polymerase chain reaction assay, using calibrated Plasmodium falciparum blood dilutions, showed that venous blood samples (50 μL, 200 μL, 1 mL) combined with Qiagen extraction methods gave a similar threshold of 100 parasites/mL, ∼100-fold lower than 5 μL DBS/Instagene method. On a set of 521 field samples, collected in two different transmission areas in northern Cambodia, no significant difference in the proportion of parasite carriers, regardless of the methods used was found. The 5 μL DBS method missed 27% of the samples detected by the 1 mL venous blood method, but most of the missed parasites carriers were infected by Plasmodium vivax (84%). The remaining missed P. falciparum parasite carriers (N = 3) were only detected in high-transmission areas.     

A novel approach for curve evolution in segmentation of medical images

A new joint parametric and nonparametric curve evolution algorithm is proposed for medical image segmentation. In this algorithm, both the nonlinear space of level set function (nonparametric model) and the linear subspace of level set function spanned by the principle components (parametric model) are employed in the evolution procedure. The nonparametric curve evolution can drive the curve precisely to object boundaries while the parametric model acts as a statistical constraint based on the Bayesian framework in order to match object shape more robustly. As a result, our new algorithm is as robust as the parametric curve evolution algorithms and at the same time, yields more accurate segmentation results by using the shape prior information. Comparative results on segmenting ventricle frontal horns and putamen shapes in MR brain images confirm the advantages of the proposed joint curve evolution algorithm.     

Sexually transmitted infection health-care seeking behaviour in the Netherlands: general practitioner attends to the majority of sexually transmitted infection consultations

Health-care seeking behaviour for sexually transmitted infection (STI)-related symptoms is not well known in the Netherlands. Within the framework of a large representative study, the second National Survey of General Practice (NIVEL 2001), 9687 persons aged 18 years and older were interviewed about their STI and STI-related health-care seeking behaviour. In total, 1.2% of the interviewees reported STI-related symptoms in the past year (18-24 years: 5%). A (lifetime) history of STI was reported by 2.7% (18-44 years: 4%). In all, 63% of interviewees visited their general practitioner (GP) for these complaints; 20% went to an STI-clinic and/or municipal public health services and 8% to a different care-provider. A total of 9% did not undertake any action. The majority of persons with STI-related symptoms in the Netherlands visit the GP. Reported history of STI-related symptoms was twice lower in the Netherlands compared with the UK National Sexual Health Survey. Appropriate attention for sexual health in primary care is needed.     

Cross-sectional studies of tuberculosis prevalence in Cambodia between 2002 and 2011

Objective

To measure trends in the pulmonary tuberculosis burden between 2002 and 2011 and to assess the impact of the DOTS (directly observed treatment, short-course) strategy in Cambodia.

Methods

Cambodia’s first population-based nationwide tuberculosis survey, based on multistage cluster sampling, was conducted in 2002. The second tuberculosis survey, encompassing 62 clusters, followed in 2011. Participants aged 15 years or older were screened for active pulmonary tuberculosis with chest radiography and/or for tuberculosis symptoms. For diagnostic confirmation, sputum smear and culture were conducted on those whose screening results were positive.

Findings

Of the 40 423 eligible subjects, 37 417 (92.6%) participated in the survey; 103 smear-positive cases and 211 smear-negative, culture-positive cases were identified. The weighted prevalences of smear-positive tuberculosis and bacteriologically-positive tuberculosis were 271 (95% confidence interval, CI: 212–348) and 831 (95% CI: 707–977) per 100 000 population, respectively. Tuberculosis prevalence was higher in men than women and increased with age. A 38% decline in smear-positive tuberculosis (P = 0.0085) was observed with respect to the 2002 survey, after participants were matched by demographic and geographical characteristics. The prevalence of symptomatic, smear-positive tuberculosis decreased by 56% (P = 0.001), whereas the prevalence of asymptomatic, smear-positive tuberculosis decreased by only 7% (P = 0.7249).

Conclusion

The tuberculosis burden in Cambodia has declined significantly, most probably because of the decentralization of DOTS to health centres. To further reduce the tuberculosis burden in Cambodia, tuberculosis control should be strengthened and should focus on identifying cases without symptoms and in the middle-aged and elderly population.     

Mice deficient in stem cell antigen‐1 (Sca1, Ly‐6A/E) develop normal primary and memory CD4+ and CD8+ T‐cell responses to virus infection

Stem cell antigen‐1 (Sca1, Ly‐6A/E) is a well‐established marker of murine hematopoietic stem cells, and also is expressed on memory T cells. It has been suggested that the functional maintenance of T‐cell memory requires the expression of Sca1 on a specialized population of memory T cells termed “memory stem cells”. Here, we evaluate the requirement for Sca1 in the primary T‐cell response to virus infection, and in the establishment and maintenance of T‐cell memory. We find that Sca1 expression increases on almost all CD4⁺ and CD8⁺ T cells during virus infection, and remains high on virus‐specific memory cells. However, Sca1‐deficient (Sca1KO) mice generate normal primary T‐cell responses to infection; the kinetics, the immunodominance hierarchy, and the absolute numbers of CD4⁺ and CD8⁺ T cells are essentially indistinguishable from those observed in WT mice. Furthermore, by several criteria, primary and memory T cells in Sca1‐deficient mice are phenotypically and functionally normal. These data indicate that Sca1, although perhaps a useful marker of virus‐specific memory T cells, is not required for the regulation of T‐cell quantity or quality, or for the development of a competent pool of memory cells.     

Myelin oligodendrocyte glycoprotein peptide-induced experimental allergic encephalomyelitis and T cell responses are unaffected by immunoproteasome deficiency

The inoculation of MOG peptides into C57BL/6 mice induces CD4(+) and CD8(+) T cells, and recent work has shown that adoptive transfer of the latter population, after extensive in vitro stimulation, can cause EAE in na?ve recipient mice. Herein, we have evaluated the incidence and severity of EAE, and the induction of CD4(+) and CD8(+) T cells, following MOG peptide inoculation of wt mice and of LMP-2KO mice that lack an intact immunoproteasome, a cytoplasmic organelle that is induced by chronic inflammation and that may be important for the presentation of MHC class I epitopes to CD8(+) T cells. We report that EAE, evaluated by both clinical and histological criteria, is similar in LMP-2KO mice and wildtype C57B/6 mice (wt) in response to immunization with MOG peptides MOG(35-55) and MOG(40-54), suggesting that the immunoproteasome does not play a key role in the development of demyelinating disease. Furthermore, and consistent with previous reports, peptide-specific CD8(+) T cells were barely detectable in the CNS of peptide-immunized mice, although peptide-specific CD4(+) T cells were abundant. Therefore, we used a new technique to look for autoreactive CD8(+) T cells in MOG peptide-immunized mice, and we report the identification of CD4(+) and CD8(+) T cells that, as late as 19 days after peptide injection, are actively producing IFNgamma in vivo, in response to in vivo antigen contact.     

Rollout of Xpert? MTB/RIF in Northwest Cambodia for the diagnosis of tuberculosis among PLHA

Objective: To describe the implementation and utilization of the Xpert^® MTB/RIF (Xpert) assay to diagnose tuberculosis (TB) among people living with the human immunodeficiency virus/acquired immune-deficiency syndrome (HIV/AIDS, PLHA) in Cambodia.Design: Following the rollout of Xpert, an evaluation was conducted in four provinces of Cambodia from March to December 2012 to determine the utilization, performance, and turnaround time (TAT) of Xpert among PLHA. Data were collected from paper-based patient registers.Results: Of 497 PLHA with a positive TB symptom screen, 357 (72%) were tested with smear microscopy, and 250 (50%) with Xpert; 25 (10%) PLHA tested with Xpert were positive for TB and none were rifampicin-resistant. The utilization of Xpert increased from 23% to 75%, with a median TAT of 1 day. Across districts, utilization ranged from zero to 85%, while the TAT ranged from zero to 22 days.Conclusion: While early data show increasing utilization of Xpert for PLHA with a positive symptom screen, most patients underwent smear microscopy as an initial diagnostic test. Training delays and challenges associated with specimen referral may have contributed to variability in Xpert uptake and TAT, particularly for sites without onsite Xpert testing. Enhanced programmatic support, particularly for specimen referral and results reporting, may facilitate appropriate utilization.     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> dihydroartemisinin-piperaquine failures in Cambodia are associated with mutant K13 parasites presenting high survival rates in novel piperaquine in vitro assays: retrospective and prospective investigations

Background

The declining efficacy of dihydroartemisinin-piperaquine against Plasmodium falciparum in Cambodia, along with increasing numbers of recrudescent cases, suggests resistance to both artemisinin and piperaquine. Available in vitro piperaquine susceptibility assays do not correlate with treatment outcome. A novel assay using a pharmacologically relevant piperaquine dose/time exposure was designed and its relevance explored in retrospective and prospective studies.

Methods

The piperaquine survival assay (PSA) exposed parasites to 200 nM piperaquine for 48 hours and monitored survival 24 hours later. The retrospective study tested 32 culture-adapted, C580Y-K13 mutant parasites collected at enrolment from patients treated with a 3-day course of dihydroartemisinin-piperaquine and having presented or not with a recrudescence at day 42 (registered ACTRN12615000793516). The prospective study assessed ex vivo PSA survival rate alongside K13 polymorphism of isolates collected from patients enrolled in an open-label study with dihydroartemisinin-piperaquine for uncomplicated P. falciparum malaria in Cambodia (registered ACTRN12615000696594).

Results

All parasites from recrudescent cases had in vitro or ex vivo PSA survival rates ≥10 %, a relevant cut-off value for piperaquine-resistance. Ex vivo PSA survival rates were higher for recrudescent than non-recrudescent cases (39.2 % vs. 0.17 %, P <1?×?10^?7). Artemisinin-resistant K13 mutants with ex vivo PSA survival rates ≥10 % were associated with 32-fold higher risk of recrudescence (95 % CI, 4.5–224; P?=?0.0005).

Conclusion

PSA adequately captures the piperaquine resistance/recrudescence phenotype, a mainstay to identify molecular marker(s) and evaluate efficacy of alternative drugs. Combined ex vivo PSA and K13 genotyping provides a convenient monitor for both artemisinin and piperaquine resistance where dihydroartemisinin-piperaquine is used.