Stevens Johnson Syndrome and Toxic Epidermal Necrolysis: Maternal and Foetal Outcomes in Twenty-Two Consecutive Pregnant HIV Infected Women

Introduction

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) form a spectrum of a rare and life-threatening cutaneous drug reaction. SJS/TEN in pregnancy poses largely unknown risk factors and outcomes for both the mother and foetus compared to the general population.

Methods

We conducted a study of consecutive pregnant women admitted to single tertiary referral centre in South Africa with SJS/TEN over a 3 year period. They were all managed by the same medical team using the same protocols. We evaluated their underlying illnesses, offending drugs and the course of pregnancy and outcomes to determine factors influencing maternal and foetal outcomes.

Results

We identified twenty-two women who developed SJS/TEN while pregnant, all of them HIV-infected. Their median age was 29 years. The majority 16/22 (73%) had SJS, the milder variant of the disease affecting < 10% body surface area. Nevirapine was the offending drug in 21/22 (95%) cases. All 22 of the mothers survived with 3/22 (14%) developing postpartum sepsis. Pregnancy outcomes were known in 18/22 women and 9/18 (50%) babies were delivered by caesarean section. There were 2 foetal deaths at 21 and 31 weeks respectively and both were associated with post-partum sepsis. Postnatal complications occurred in 5 cases, 3 involving the respiratory system and the other two being low birth weight deliveries. Eight placentae and one foetus were sent for histology and none showed macroscopic or microscopic features of SJS/TEN. On follow-up, only 12/20 children were tested for HIV at 6 weeks post-delivery and none of them were HIV-infected. All had received prophylactic ARVs including nevirapine.

Conclusions

TEN, the severe form of the disease, was associated with poorer foetal outcomes. SJS/TEN-associated mortality is not increased in HIV-infected pregnant women. Maternal SJS/TEN does not seem to commonly manifest in the foetus.     

Effects of pruning on the concentration of trace elements in Colophospermum mopane leaves

Despite the nutritional value of Colophospermum mopane to browser's diets, there is still insufficient knowledge on the effect of browsers on concentrations of these trace elements. A field experiment was conducted in Musina Nature Reserve, Limpopo Province, South Africa, to determine the effect of pruning on the concentration of trace elements in mopane leaves. Samples were analysed for iron (Fe), manganese (Mn), boron (B), molybdenum (Mo), copper (Cu), zinc (Zn), cobalt (Co), fluoride (F) and selenium (Se) using the inductively coupled plasma atomic emission spectrometry technique. The effect of pruning was tested using the two‐tailed t‐test: two‐sample assuming equal variance and two‐tailed Mann–Whitney U‐test. Results showed that the concentration of trace elements in the control and pruned trees varies slightly through the year. Fe, Mn, Mo, Cu, Zn and Se are higher during leaf flush, but declined as the leaves matured and aged. This study concluded that simulated browsing had no significant effect on the concentration of trace elements in the mopane leaves. Seasonal variation in the amount of trace elements has implications on the distribution of browsers in the mopane woodland.     

Microbial pathogen-induced necrotic cell death mediated by the inflammasome components CIAS1/cryopyrin/NLRP3 and ASC

Cryopyrin (CIAS1, NLRP3) and ASC are components of the inflammasome, a multiprotein complex required for caspase-1 activation and cytokine IL-1beta production. CIAS1 mutations underlie autoinflammation characterized by excessive IL-1beta secretion. Disease-associated cryopyrin also causes a program of necrosis-like cell death in macrophages, the mechanistic details of which are unknown. We find that patient monocytes carrying disease-associated CIAS1 mutations exhibit excessive necrosis-like death by a process dependent on ASC and cathepsin B, resulting in spillage of the proinflammatory mediator HMGB1. Shigella flexneri infection also causes cryopyrin-dependent macrophage necrosis with features similar to the death caused by mutant CIAS1. This necrotic death is independent of caspase-1 and IL-1beta, and thus independent of the inflammasome. Furthermore, necrosis of primary macrophages requires the presence of Shigella virulence genes. While similar proteins mediate pathogen-induced cell death in plants, this report identifies cryopyrin as an important host regulator of programmed pathogen-induced necrosis in animals, a process we term pyronecrosis.     

Label‐free differentiation of human immunodeficiency virus‐1 infected from uninfected cells using transmission measurement

Transmission measurement has been perceived as a potential candidate for label‐free investigation of biological material. It is a real‐time, label‐free and non‐invasive optical detection technique that has found wide applications in pharmaceutical industry as well as the biological and medical fields. Combining transmission measurement with optical trapping has emerged as a powerful tool allowing stable sample trapping, while also facilitating transmittance data analysis. In this study, a near‐infrared laser beam emitting at a wavelength of 1064 nm was used for both optical trapping and transmission measurement investigation of human immunodeficiency virus 1 (HIV‐1) infected and uninfected TZM‐bl cells. The measurements of the transmittance intensity of individual cells in solution were carried out using a home built optical trapping system combined with laser transmission setup using a single beam gradient trap. Transmittance spectral intensity patterns revealed significant differences between the HIV‐1 infected and uninfected cells. This result suggests that the transmittance data analysis technique used in this study has the potential to differentiate between infected and uninfected TZM‐bl cells without the use of labels. The results obtained in this study could pave a way into developing an HIV‐1 label‐free diagnostic tool with possible applications at the point of care .     

Inactivation of DsbA alters the behaviour of Shigella flexneri towards murine and human-derived macrophage-like cells

The mutants of Shigella flexneri, Sh4 (dsbA::kan) and Sh42 (dsbA33G), behave differently towards murine and human-derived macrophage-like cells in vitro. Sh4 was trapped in the phagocytic vacuoles of the murine J774 cells as evidenced by its colony forming units plus and minus chloroquine exposure in a gentamicin protection assay, and by light and transmission electron microscopy (TEM). Sh42, similar to the wild-type M90TS, was able to escape from the vacuoles and kill host cells presumably by inducing apoptosis. In U937 cells, unlike M90TS that was free in the cytosol, both Sh4 and Sh42 grew poorly. TEM revealed that Sh4 and Sh42 were trapped within the U937 phagocytic vacuoles. Furthermore, the two mutants induced different patterns of interleukin-1beta and tumour necrosis factor-alpha expression, which might explain why they possess different immunogenic properties in vivo.     

An AFLP-based database of Shigella flexneri and Shigella sonnei isolates and its use for the identification of untypable Shigella strains

Amplified fragment length polymorphism (AFLP) can be used to assess the genetic diversity of closely related microbial genomes. In this study, the first of its kind for identification of Shigella, the high discriminatory power of AFLP has been used to determine the genetic relatedness of 230 isolates of Shigella flexneri and Shigella sonnei strains. An AFLP database was generated to demonstrate its utility in the discrimination of closely related strains. Based on AFLP, S. flexneri strains could be grouped into separate clusters according to their serotypes. Within each serotype, strains demonstrated 80-100% similarity indicating that identical strains and closely related strains could be distinguished by this technique. S. flexneri 6 formed a distinct cluster with 55% similarity to the rest of the S. flexneri strains showing significant divergence from the rest of the S. flexneri strains. Significantly, S. sonnei isolates formed a distinct group and showed approximately the same level of genetic linkage to S. flexneri as Escherichia coli strains. Untypable isolates that showed conflicting agglutination reactions with conventional typing sera were identifiable by AFLP. Thus AFLP can be used for genetic fingerprinting of Shigella strains and aid in the identification of variant untypable isolates.     

Developing live Shigella vaccines using lambda Red recombineering

Live attenuated Shigella vaccines have shown promise in inducing protective immune responses in human clinical trials and as carriers of heterologous antigens from other mucosal pathogens. In the past, construction of Shigella vaccine strains relied on classical allelic exchange systems to genetically engineer the bacterial genome. These systems require extensive in vitro engineering of long homologous sequences to create recombinant replication-defective plasmids or phage. Alternatively, the lambda red recombination system from bacteriophage facilitates recombination with as little as 40 bp of homologous DNA. The process, referred to as recombineering, typically uses an inducible lambda red operon on a temperature-sensitive plasmid and optimal transformation conditions to integrate linear antibiotic resistance cassettes flanked by homologous sequences into a bacterial genome. Recent advances in recombineering have enabled modification of genomic DNA from bacterial pathogens including Salmonella, Yersinia, enteropathogenic Escherichia coli, or enterohemorrhagic E. coli and Shigella. These advances in recombineering have been used to systematically delete virulence-associated genes from Shigella, creating a number of isogenic strains from multiple Shigella serotypes. These strains have been characterized for attenuation using both in vivo and in vitro assays. Based on this data, prototypic Shigella vaccine strains containing multiple deletions in virulence-associated genes have been generated.     

Laser‐enhanced drug delivery of antiretroviral drugs into human immunodeficiency virus‐1 infected TZMbl cells

The introduction of highly active antiretroviral therapy (HAART) has significantly increased life expectancy and improved management of the human immunodeficiency virus‐1 (HIV‐1) disease globally. This well‐established treatment regime has shown to reduce viral capacity to undetectable limits when using traditional clinical assays. The establishment of viral reservoirs during the early stages of infection are the major contributors to failure of the current regimens to eradicate HIV‐1 infection since the reservoirs are not affected by antiretroviral drugs (ARVs). Therefore, advanced modification of the present treatment and investigation of novel antiretroviral drug delivery system are needed. The aim of this study was to use femtosecond (fs) laser pulses to deliver ARVs into HIV‐1 infected TZMbl cells. Different ARVs were translocated into TZMbl cells using fs pulsed laser (800 nm) with optimum power of 4 μW and 10 ms laser to cell exposure time. Changes in cellular processes were evaluated using cellular morphology, viability, cytotoxicity and luciferase activity assays. Cells treated with the laser in the presence of ARVs showed a significant reduction in viral infectivity, cell viability and an increase in cytotoxicity. This study demonstrated that fs laser pulses were highly effective in delivering ARVs into HIV‐1 infected TZMbl cells, causing a significant reduction in HIV‐1 infection.     

Safety and Colonization of Two Novel virG(icsA)-based Live Shigella sonnei Vaccine Strains in Rhesus Macaques (Macaca mulatta)

Shigella are gram-negative bacterium that cause bacillary dysentery (shigellosis). Symptoms include diarrhea and discharge of bloody mucoid stools, accompanied by severe abdominal pain, nausea, vomiting, malaise, and fever. Persons traveling to regions with poor sanitation and crowded conditions become particularly susceptible to shigellosis. Currently a vaccine for Shigella has not been licensed in the United States, and the organism quickly becomes resistant to medications. During the past 10 y, several live attenuated oral Shigella vaccines, including the strain WRSS1, have been tested in humans with considerable success. These Phase I vaccines lack the gene for the protein VirG also known as IcsA, which enables the organism to disseminate in the host target tissue. However, 5% to 20% of the vaccinated volunteers developed mild fever and brief diarrhea, and the removal of additional virulence-associated genes from the vaccine strain may reduce or eliminate these side effects. We administered 2 Shigella sonnei vaccines, WRSs2 and WRSs3, along with WRSS1 to compare their rates of colonization and clinical safety in groups of 5 rhesus macaques. The primate model provides the most physiologically relevant animal system to test the validity and efficacy of vaccine candidates. In this pilot study using a gastrointestinal model of infection, the vaccine candidates WRSs2 and WRSs3, which have additional deletions in the enterotoxin and LPS modification genes, provided better safety and comparable immunogenicity to those of WRSS1.Abbreviations: IcsA, intercellular spread protein A (VirG protein); Ipa, invasion plasmid antigen; LPS, lipopolysaccharideVarious species of Shigella cause bacillary dysentery, also known as shigellosis, in humans and other primates.^{1,18,31,37,49} Symptoms include diarrhea, with various degrees of mucus and hematochezia, accompanied by severe abdominal pain, nausea, vomiting, malaise, and fever. Shigella is a gram-negative bacterium whose genomic sequence is very similar to that of Escherichia coli and is phylogenetically considered a pathotype of E. coli.²³ Shigella infections are spread by the fecal–oral route through the consumption of contaminated food and water or by mechanical vectors such as insects. Persons traveling in areas with poor sanitation and crowded conditions become particularly susceptible to such diseases.^2,39,48,56 In colonies of research macaques, Shigella infections are spread by the fecal–oral route, originating from addition of animals that are asymptomatic carriers, either from the wild or from other colonies.^1,55 Currently no Shigella vaccine has been licensed in the United States, although several are under development.^{25,32,35,38,54} Antibiotics are used as treatment therapy, but many Shigella isolates are multidrug-resistant, increasing the need for a preventive vaccine for travelers, military personnel, and children in endemic areas.^{16,34,41,44,57} Rhesus macaques represent an excellent model for studying Shigella because primates are the only known animal model that simulate natural human infection, including dysentery after oral challenge.^{11-13,17,28,40,47,46} Other animal models, such as the mouse pulmonary model and the guineapig ocular and intranasal models, mimic specific, isolated steps in Shigella pathogenesis.^14,19,26There are 4 major serogroups of Shigella and one or more serotypes within each serogroup. This classification is based on antigenic differences in the O-antigen polysaccharide component of the outer membrane-associated lipopolysaccharide (LPS). Estimates from the Centers for Disease Control (Atlanta, GA) indicate that approximately 400,000 cases of shigellosis occur in the US annually, with an estimated 165 million cases worldwide each year.²² These occur predominantly in developing countries, where the population most affected is children younger than 5 y.²²Various in vitro cell culture models of infection, as well as studies in animal models including gastrointestinal infection in nonhuman primates, have contributed to the current understanding of Shigella pathogenesis.^4,42 Shigella organisms target the distal region of the colon and rectum, where the bacteria are captured by specialized M-cells located within the follicle-associated epithelium. The M-cells deliver bacterial antigens, which include bacterial LPS and invasion plasmid antigen (Ipa) proteins, to the underlying antigen-presenting macrophages and dendritic cells.³⁶ Virulent Shigella strains escape macrophages by a cytotoxic effect, causing release of the bacteria and inflammatory cytokines such as IL1β, TNFα, and IL18. The bacteria then invade adjacent intestinal epithelial cells at the basolateral side, through interaction between bacterial proteins and multiple signaling molecules within the host cell.^36,42Shigella are capable of orchestrating this uptake into nonphagocytic epithelial cells, a process termed invasion, through the secretion of proteins that are highly conserved among all virulent strains. These proteins are encoded by a large, 213-kb plasmid, referred to as the invasion plasmid or the virulence plasmid.^50-52 Each bacterium ultimately is engulfed within an endocytic vacuole. Subsequent lysis of the vacuole sets the bacterium free within the epithelial cell cytoplasm, where it replicates and moves to adjacent epithelial cells, with the help of the intercellular spread (Ics) A protein, also known as VirG.^3,24,45 The VirG(IcsA)-assisted intra- and intercellular spread of the bacteria within the epithelial tissue contributes significantly to the loss of epithelial cell integrity and accompanying tissue injury. Shigella strains with loss of the virG(icsA) gene are significantly attenuated in all animal models of virulence.⁵ Several Shigella vaccine candidates that have undergone Phase 1 clinical trials are principally attenuated due to lack of the VirG(IcsA) protein, these include S. flexneri 2a strain SC602, S. sonnei strain WRSS1, and S. dysenteriae 1 vaccine strain WRSd1.^{5,10,15,21,53}The S. sonnei first-generation vaccine candidate WRSS1 has been tested in several Phase I inpatient and outpatient trials.^15,21,33 WRSS1, like the previous S. flexneri 2a vaccine candidate, SC602, was shown to be safe in Phase I trials when orally administered at a dose of 10³ to 10⁴ CFU.^15,21,33 Both SC602 and WRSS1 colonized well as evidenced by fecal excretion of the vaccine strain for 5 to 7 d. The vigorous immune response seen with both these vaccine candidates was correlated with the robust colonization. However, 5% to 20% of the SC602- and WRSS1-immunized volunteers showed mild and transient symptoms of diarrhea and fever. In light of new knowledge about Shigella pathogenesis as well as data from other clinical trials, 2 new and presumably safer S. sonnei derivatives have been constructed (WRSs2 and WRSs3). In addition to loss of virG(icsA), WRSs2 has deletions in 2 genes, senA and senB, that are present on the virulence plasmid.^29,52 senA (also known as shet2-1 and ospD3) encodes the enterotoxin ShET2-1, which has been shown to cause fluid accumulation in rabbit ileal loops.^7,8 senB (also known as shet2-2 and ospD2) encodes a similarly sized protein as SenA and shows 40% identity at the amino acid level. The enterotoxic activity of ShET2-2 remains to be demonstrated.^20,29 Like WRSs2, WRSs3 lacks virG(icsA) and the 2 enterotoxin genes with the additional deletion of the virulence plasmid-based msbB2 gene. Lack of the msbB2 gene product has previously been shown to produce a less-toxic LPS molecule, which might help to reduce the symptoms of fever seen in WRSS1-immunized volunteers.^20,29 How these additional mutations, especially that of msbB2, will affect colonization of these strains is unclear, given that effective colonization is critical in the elicitation of immune response and protection. The primary goal of this pilot study was to compare the safety and colonization of 2 novel S. sonnei vaccine candidate strains (WRSs2 and WRSs3), with those of the clinically tested vaccine strain WRSS1, by using a gastrointestinal model of infection. The hypothesis tested was that the 2 novel Shigella vaccine strains would colonize the vaccinated animal and induce an immune response similar to that of WRSS1 but with less frequent and less severe clinical side effects.     

Effects of pruning on the concentration of macronutrients in Colophospermum mopane leaves

Despite the nutritional value of Colophospermum mopane leaves to browser's diets, there is limited knowledge on the effect of browsers on the concentration of macronutrients in mopane leaves. A field experiment was conducted in Musina Nature Reserve, Limpopo Province, South Africa to determine the effect of pruning, a proxy for browsing, on the concentration of macronutrients in mopane leaves. Samples were analysed for calcium (Ca), potassium (K), nitrogen (N), phosphorus (P), sulphur (S), chlorine (Cl), magnesium (Mg), nitrate (NO₃), sodium (Na), protein and fibre, using inductively coupled plasma atomic emission spectrometry techniques. The effect of pruning on the monthly concentration of macronutrients was tested using a two‐tailed t test: two‐sample assuming equal variance. The seasonal and annual effect of pruning on the concentration of macronutrients was tested using a one‐way ANOVA. Results showed that the amount of Ca, K, N, P, S, Cl, Na, protein and fibre increased during leaf flush in October and then declined as the leaves matured and aged. However, the concentration of Mg and NO₃ increased when the leaves reached maturity in June, particularly during senescence stage, and declined thereafter. The concentration of macronutrients between the control and pruned trees was statistically insignificant for most samples.