Real-time PCR in HIV/Trypanosoma cruzi coinfection with and without Chagas disease reactivation: association with HIV viral load and CD4 level

Background

Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed.

Methodology

Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not.

Principal Findings

qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman''s correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4⁺ cells or the CD4⁺/CD8⁺ ratio.

Conclusions

qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4⁺ cells/mm³ and the CD4⁺/CD8⁺ ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.     

The anxiety-like phenotype of 5-HT receptor null mice is associated with genetic background-specific perturbations in the prefrontal cortex GABA-glutamate system

A deficit in the serotonin 5-HT(1A) receptor has been found in panic and post-traumatic stress disorders, and genetic inactivation of the receptor results in an anxiety-like phenotype in mice on both the C57Bl6 and Swiss-Webster genetic backgrounds. Anxiety is associated with increased neuronal activity in the prefrontal cortex and here we describe changes in glutamate and GABA uptake of C57Bl6 receptor null mice. Although these alterations were not present in Swiss-Webster null mice, we have previously reported reductions in GABA(A) receptor expression in these but not in C57Bl6 null mice. This demonstrates that inactivation of the 5-HT(1A) receptor elicits different and genetic background-dependent perturbations in the prefrontal cortex GABA/glutamate system. These perturbations can result in a change in the balance between excitation and inhibition, and indeed both C57Bl6 and Swiss-Webster null mice show signs of increased neuronal excitability. Because neuronal activity in the prefrontal cortex controls the extent of response to anxiogenic stimuli, the genetic background-specific perturbations in glutamate and GABA neurotransmission in C57Bl6 and Swiss-Webster 5-HT(1A) receptor null mice may contribute to their shared anxiety phenotype. Our study shows that multiple strains of genetically altered mice could help us to understand the common and individual features of anxiety.     

The reproductive biology of Parazoanthus parasiticus (Hexacorallia: Zoanthidea) in Bermuda

The zooxanthellate macrocnemic zoanthid Parazoanthus parasiticus lives at densities of 3–10 cm⁻² in the chimney sponge Callyspongia vaginalis in Bermuda. It is gonochoric and oviparous. Small oocytes appear in the mesenteries in February–March, grow slowly at first, then increase volume rapidly as seawater temperature passes 27 °C in July. In 1993, oocytes were found to have been shed over 2–3 days around the full moon on 1 September, with inferred small spawnings over the preceding and following full moons. Reproduction had finished by November. In 1999, spawning of azooxanthellate eggs ∼ 250 μm diameter took place on 28 August, two nights after full moon. Spawnings therefore precede those known for four oviparous scleractinians and one gorgonian in Bermudian waters by about a week. The eggs are not shed in bundles and lack zooxanthellae. The unknown embryonic development and larval type in macrocnemic zoanthids is discussed and remains to be resolved by further study with P. parasiticus. Contribution number 1662 from the Bermuda Biological Station for Research.     

The nature ofd,l-fenfluramine-induced 5-HT reuptake transporter loss in rats

The administration of the anorexigenic drugd,l-fenfluramine (Ponderax^®) to laboratory animals results in a dose-dependent reduction in presynaptically located serotonergic reuptake transporter protein. This long-term effect may represent an altered mechanism of synthesis of the transporter (downregulation). Alternatively, fenfluramine may destroy the serotonergic terminals on which 5-HT transporters are located. To distinguish between these two alternatives, we applied an assay of neurotransmitter-specific nerve endings (α) to brain tissue from two animal models of reduced 5-HT transporter density. In Model 1, serotonergic nerve terminals were destroyed (rats received 5,7-dihydroxytryptamine [5,7-DHT] intracisternally); in Model 2, there was a loss of 5-HT transportersper se on otherwise intact serotonergic nerve terminals. The manner in which α declined as transporter density was decreased (reducingV _max values) in animal Models 1 and 2 was found to be significantly different. In rats treated with fenfluramine, the association between 5-HT transporter density and α was the same as in the neurotoxic model.     

Larvae of related Diptera species from thermally contrasting habitats exhibit continuous up-regulation of heat shock proteins and high thermotolerance

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name ‘soldier flies’, occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 °C, have constitutively high concentrations of the normally inducible heat-shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi-aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat-shock treatment of S. japonica larvae activates heat-shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.     

Pancreatic secretory trypsin inhibitor I reduces the severity of chronic pancreatitis in mice overexpressing interleukin-1β in the pancreas

IL-1β is believed to play a pathogenic role in the development of pancreatitis. Expression of human IL-1β in pancreatic acinar cells produces chronic pancreatitis, characterized by extensive intrapancreatic inflammation, atrophy, and fibrosis. To determine if activation of trypsinogen is important in the pathogenesis of chronic pancreatitis in this model, we crossed IL-1β transgenic [Tg(IL1β)] mice with mice expressing a trypsin inhibitor that is normally produced in rat pancreatic acinar cells [pancreatic secretory trypsin inhibitor (PTSI) I]. We previously demonstrated that transgenic expression of PSTI-I [Tg(Psti1)] increased pancreatic trypsin inhibitor activity by 190%. Tg(IL1β) mice were found to have marked pancreatic inflammation, characterized by histological changes, including acinar cell loss, inflammatory cell infiltration, and fibrosis, as well as elevated myeloperoxidase activity and elevated pancreatic trypsin activity, as early as 6 wk of age. In contrast to Tg(IL1β) mice, pancreatitis was significantly less severe in dual-transgenic [Tg(IL1β)-Tg(Psti1)] mice expressing IL-1β and PSTI-I in pancreatic acinar cells. These findings indicate that overexpression of PSTI-I reduces the severity of pancreatitis and that pancreatic trypsin activity contributes to the pathogenesis of an inflammatory model of chronic pancreatitis.