Poor continuity of care for TB diagnosis and treatment in Zambian Prisons: a situation analysis.

OBJECTIVES: Prisons act as infectious disease reservoirs. We aimed to explore the challenges of TB control and continuity of care in prisons in Zambia. METHODS: We evaluated treatment outcomes for a cohort of inmates diagnosed with TB during a TB REACH funded screening programme initiated by the Zambia Prisons Service and the Centre for Infectious Disease Research in Zambia. RESULTS: Between October 2010 and September 2011, 6282 inmates from six prisons were screened for TB, of whom 374 (6.0%) were diagnosed. TB treatment was initiated in 345 of 374 (92%) inmates. Of those, 66% were cured or completed treatment, 5% died and 29% were lost to follow-up. Among those lost to follow-up, 11% were released into the community and 13% were transferred to other prisons. CONCLUSIONS: Weak health systems within the Zambian prison service currently undermines continuity of care, despite intensive TB screening and case-finding interventions. To prevent TB transmission and the development of drug resistance, we need sufficient numbers of competent staff for health care, reliable health information systems including electronic record keeping for prison facilities, and standard operating procedures to guide surveillance, case-finding and timely treatment initiation and completion.

Bites by the Monocled Cobra, Naja kaouthia, in Chittagong Division, Bangladesh: Epidemiology, Clinical Features of Envenoming and Management of 70 Identified Cases.

AbstractWe describe 70 cases of monocled cobra (Naja kaouthia) bite admitted to Chittagong Medical College Hospital, Bangladesh. The biting snakes were identified by examining the dead snake and/or detecting N. kaouthia venom antigens in patients' serum. Bites were most common in the early morning and evening during the monsoon (May-July). Ligatures were routinely applied to the bitten limb before admission. Thirty-seven patients consulted traditional healers, most of whom made incisions around the bite site. Fifty-eight patients experienced severe neurotoxicity and most suffered swelling and pain of the bitten limb. The use of an Indian polyvalent antivenom in patients exhibiting severe neurotoxicity resulted in clinical improvement but most patients experienced moderate-to-severe adverse reactions. Antivenom did not influence local blistering and necrosis appearing in 19 patients; 12 required debridement. Edrophonium significantly improved the ability of patients to open the eyes, endurance of upward gaze, and peak expiratory flow rate suggesting that a longer-acting anticholinesterase drug (neostigmine) could be recommended for first aid. The study suggested that regionally appropriate antivenom should be raised against the venoms of the major envenoming species of Bangladesh and highlighted the need to improve the training of staff of local medical centers and to invest in the basic health infrastructure in rural communities.

High prevalence of tuberculosis in newly enrolled HIV patients in Zambia: need for enhanced screening approach.

SETTING: Tuberculosis (TB) remains a leading cause of morbidity and mortality in sub-Saharan Africa. In Zambia, smear microscopy and chest radiography (CXR) are the primary TB diagnostic tools, and most cases are not bacteriologically confirmed. OBJECTIVE: We implemented enhanced screening to determine the TB burden among new human immunodeficiency virus (HIV) clinic enrollees. DESIGN: Consecutive adult HIV clinic enrollees were screened, regardless of symptoms. All underwent microscopy (Ziehl-Neelsen/fluorescence microscopy) on three sputum specimens, physical examination, and digital CXR. Sputum, blood and urine specimens were cultured. Xpert(®) MTB/RIF testing was performed retrospectively. RESULTS: From July 2011 to April 2012, 399 patients were enrolled. The median age was 34.4 years; body mass index was 20.8 kg/m(2), CD4 count was 202 cells/µl and 86% were symptomatic. Culture-confirmed TB was diagnosed in 72/399 (18%) patients; an additional 31/399 (8%) were culture-negative but diagnosed clinically. Symptom screening for any cough, fever, weight loss or night sweats had high sensitivity (95%) but low specificity (14%) for detecting culture-confirmed cases. Among culture-confirmed cases, 35/72 (49%) were missed clinically and detected only by culture. Xpert was 64% sensitive and 98% specific. CONCLUSIONS: High TB prevalence was found in Zambians newly enrolled into HIV care. Screening with sensitive diagnostics should be considered with culture when feasible in this population.

Equine grass sickness, but not botulism, causes autonomic and enteric neurodegeneration and increases soluble N-ethylmaleimide-sensitive factor attachment receptor protein expression within neuronal perikarya.

REASONS FOR PERFORMING STUDY: Equine grass sickness (EGS) is of unknown aetiology. Despite some evidence suggesting that it represents a toxico-infection with Clostridium botulinum types C and/or D, the effect of EGS on the functional targets of botulinum neurotoxins, namely the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, is unknown. Further, while it is commonly stated that, unlike EGS, equine botulism is not associated with autonomic and enteric neurodegeneration, this has not been definitively assessed. OBJECTIVES: To determine: 1) whether botulism causes autonomic and enteric neurodegeneration; and 2) the effect of EGS on the expression of SNARE proteins within cranial cervical ganglion (CCG) and enteric neuronal perikarya. STUDY DESIGN: Descriptive study. METHODS: Light microscopy was used to compare the morphology of neurons in haematoxylin-eosin stained sections of CCG and ileum from 6 EGS horses, 5 botulism horses and 6 control horses. Immunohistochemistry was used to compare the expression of synaptosomal-associated protein-25, synaptobrevin (Syb) and syntaxin within CCG neurons, and of Syb in enteric neurons, from horses with EGS, horses with botulism and control horses. The concentrations of these SNARE proteins in extracts of CCG from EGS and control horses were compared using quantitative fluorescent western blotting. RESULTS: EGS, but not botulism, was associated with autonomic and enteric neurodegeneration and with increased immunoreactivity for SNARE proteins within neuronal perikarya. Quantitative fluorescent western blotting confirmed increased concentrations of synaptosomal-associated protein-25, Syb and syntaxin within CCG extracts from EGS vs. control horses, with the increases in the latter 2 proteins being statistically significant. CONCLUSIONS: The occurrence of autonomic and enteric neurodegeneration, and increased expression of SNARE proteins within neuronal perikarya, in EGS but not botulism, suggests that EGS may not be caused by botulinum neurotoxins. Further investigation of the aetiology of EGS is therefore warranted.

Mating system and early viability resistance to habitat fragmentation in a bird-pollinated eucalypt.

Habitat fragmentation has been shown to disrupt ecosystem processes such as plant-pollinator mutualisms. Consequently, mating patterns in remnant tree populations are expected to shift towards increased inbreeding and reduced pollen diversity, with fitness consequences for future generations. However, mating patterns and phenotypic assessments of open-pollinated progeny have rarely been combined in a single study. Here, we collected seeds from 37 Eucalyptus incrassata trees from contrasting stand densities following recent clearance in a single South Australian population (intact woodland=12.6 trees ha(-1); isolated pasture=1.7 trees ha(-1); population area=10 km(2)). 649 progeny from these trees were genotyped at eight microsatellite loci. We estimated genetic diversity, spatial genetic structure, indirect contemporary pollen flow and mating patterns for adults older than the clearance events and open-pollinated progeny sired post-clearance. A proxy of early stage progeny viability was assessed in a common garden experiment. Density had no impact on mating patterns, adult and progeny genetic diversity or progeny growth, but was associated with increased mean pollen dispersal. Weak spatial genetic structure among adults suggests high historical gene flow. We observed preliminary evidence for inbreeding depression related to stress caused by fungal infection, but which was not associated with density. Higher observed heterozygosities in adults compared with progeny may relate to weak selection on progeny and lifetime-accumulated mortality of inbred adults. E. incrassata appears to be resistant to the negative mating pattern and fitness changes expected within fragmented landscapes. This pattern is likely explained by strong outcrossing and regular long-distance pollen flow.

Integrating active tuberculosis case finding in antenatal services in Zambia.

SETTING: Three out-patient antenatal care (ANC) clinics in Lusaka, Zambia. OBJECTIVE: To estimate tuberculosis (TB) prevalence in human immunodeficiency virus (HIV) infected and symptomatic, non-HIV-infected pregnant women and explore the feasibility of routine TB screening in ANC settings. DESIGN: Peer educators administered TB symptom questionnaires to pregnant women attending their first ANC clinic visit. Presumptive TB patients were defined as all HIV-infected women and symptomatic non-HIV-infected women. Sputum samples were tested using smear microscopy and culture to estimate TB prevalence. RESULTS: All 5033 (100%) women invited to participate in the study agreed, and 17% reported one or more TB symptoms. Among 1152 presumed TB patients, 17 (1.5%) had previously undiagnosed culture-confirmed TB; 2 (12%) were smear-positive. Stratified by HIV status, TB prevalence was 10/664 (1.5%, 95%CI 0. 7-2.8) among HIV-infected women and 7/488 (1.4%, 95%CI 0.6-2.9) among symptomatic non-HIV-infected women. In HIV-infected women, the only symptom significantly associated with TB was productive cough; symptom screening was only 50% sensitive. CONCLUSION: There is a sizable burden of TB in pregnant women in Zambia, which may lead to adverse maternal and infant outcomes. TB screening in ANC settings in Zambia is acceptable and feasible. More sensitive diagnostics are needed.

Derivation of a tuberculosis screening rule for sub-Saharan African prisons.

SETTING: Lusaka Central Prison, Zambia. OBJECTIVE: To derive screening rules for tuberculosis (TB) using data collected during a prison-wide TB and human immunodeficiency virus (HIV) screening program. DESIGN: We derived rules with two methodologies: logistic regression and classification and regression trees (C&RT). We evaluated the performance of the derived rules as well as existing World Health Organization (WHO) screening recommendations in our cohort of inmates, as measured by sensitivity, specificity, and positive and negative predictive values. RESULTS: The C&RT-derived rule recommended diagnostic testing of all inmates who were underweight (defined as body mass index [BMI] < 18.5 kg/m(2)] or HIV-infected; the C&RT-derived rule had 60% sensitivity and 71% specificity. The logistic regression-derived rule recommended diagnostic testing of inmates who were underweight, HIV-infected or had chest pain; the logistic regression-derived rule had 74% sensitivity and 57% specificity. Two of the WHO recommendations had sensitivities that were similar to our logistic regression rule but had poorer specificities, resulting in a greater testing burden. CONCLUSION: Low BMI and HIV infection were the most robust predictors of TB in our inmates; chest pain was additionally retained in one model. BMI and HIV should be further evaluated as the basis for TB screening rules for inmates, with modification as needed to improve the performance of the rules.

Transcutaneous immunization with a Vibrio cholerae O1 Ogawa synthetic hexasaccharide conjugate following oral whole-cell cholera vaccination boosts vibriocidal responses and induces protective immunity in mice.

A shortcoming of currently available oral cholera vaccines is their induction of relatively short-term protection against cholera compared to that afforded by wild-type disease. We were interested in whether transcutaneous or subcutaneous boosting using a neoglycoconjugate vaccine made from a synthetic terminal hexasaccharide of the O-specific polysaccharide of Vibrio cholerae O1 (Ogawa) coupled to bovine serum albumin as a carrier (CHO-BSA) could boost lipopolysaccharide (LPS)-specific and vibriocidal antibody responses and result in protective immunity following oral priming immunization with whole-cell cholera vaccine. We found that boosting with CHO-BSA with immunoadjuvantative cholera toxin (CT) or Escherichia coli heat-labile toxin (LT) following oral priming with attenuated V. cholerae O1 vaccine strain O395-NT resulted in significant increases in serum anti-V. cholerae LPS IgG, IgM, and IgA (P < 0.01) responses as well as in anti-Ogawa (P < 0.01) and anti-Inaba (P < 0.05) vibriocidal titers in mice. The LPS-specific IgA responses in stool were induced by transcutaneous (P < 0.01) but not subcutaneous immunization. Immune responses following use of CT or LT as an adjuvant were comparable. In a neonatal mouse challenge assay, immune serum from boosted mice was associated with 79% protective efficacy against death. Our results suggest that transcutaneous and subcutaneous boosting with a neoglycoconjugate following oral cholera vaccination may be an effective strategy to prolong protective immune responses against V. cholerae.

Ultrastructural evidence for the uptake of a neurotoxic snake venom phospholipase A2 into mammalian motor nerve terminals.

A mutant form of ammodytoxin A, a neurotoxic phospholipase A(2) from the venom of the long nosed viper Vipera ammodytes ammodytes, was prepared by site-directed mutagenesis, conjugated to a nanogold particle and inoculated into the antero-lateral aspect of one hind limb of female mice. Eight hours later the mice were killed, the soleus muscles of both ipsi- and contra-lateral hind limbs were removed, exposed to a silver enhancing medium and then prepared for transmission electron microscopy. Silver-enhanced particles were subsequently found concentrated in the peri-synaptic area, particularly within the synaptic gutter and the deep synaptic folds, and in many cases had been taken up into the cytoplasm of the terminal boutons of the motor axon. The results suggest that the presynaptic neurotoxicity of snake venom phospholipases A(2) involves several components of the neuromuscular apparatus, including intracellular organelles of the motor nerve terminal.

A variant in long palate, lung and nasal epithelium clone 1 is associated with cholera in a Bangladeshi population.

Vibrio cholerae causes a dehydrating diarrheal illness that can be rapidly fatal in the absence of specific treatment. The organism is an historic scourge and, like similar infectious diseases, may have influenced the evolution of the human genome. We report here the results of the first candidate gene association study of cholera. In a family-based study of 76 pedigrees from Dhaka, Bangladesh, we evaluated the association between cholera and five candidate genes-the cystic fibrosis transmembrane receptor; lactoferrin; long palate, lung and nasal epithelium clone 1 (LPLUNC1); estrogen-related receptor alpha and calcium-activated chloride channel 1. We found a significant association with a marker in the promoter region of LPLUNC1 (rs11906665), a member of a family of evolutionarily conserved innate immunity proteins. An earlier microarray-based study of duodenal biopsies showed significantly increased expression of LPLUNC1 in cholera patients compared with healthy control subjects. Our results suggest that variation in host innate immune responses may influence the outcome of exposure to V. cholerae in an endemic setting.

Early lessons from the integration of tuberculosis and HIV services in primary care centers in Lusaka, Zambia.

BACKGROUND: Zambia faces overlapping tuberculosis (TB) and human immunodeficiency virus (HIV) epidemics; however, care for co-infected patients often occurs through separate, vertical programs. OBJECTIVE: To establish a program to integrate TB and HIV services in Lusaka primary care centers. METHODS: In collaboration with the Zambian Ministry of Health, TB-HIV integration activities began in December 2005 and were expanded to seven health centers by March 2007. Principal activities included developing staff capacity to manage co-infected patients, implementing HIV testing within TB departments and establishing referral systems between departments. RESULTS: Using a provider-initiated approach, 2053 TB patients were offered HIV testing. Seventy-seven per cent agreed to be tested; 69% of those tested were HIV-infected. Of these, 59% were enrolled in HIV care. The proportion of antiretroviral treatment (ART) program enrollees who were TB-HIV co-infected increased by 38% after program implementation. The median CD4 count among co-infected patients was 161 cells/microl, with 88% eligible for ART. CONCLUSION: Integration of HIV testing and referral services into urban primary care centers identified many co-infected patients and significantly increased the proportion of TB patients among people accessing HIV care. Ongoing challenges include maximizing the number of patients accepting HIV testing and overcoming barriers to enrollment into HIV care.

Envenoming bites by kraits: the biological basis of treatment-resistant neuromuscular paralysis.

Beta-bungarotoxin, a neurotoxic phospholipase A2 is a major fraction of the venom of kraits. The toxin was inoculated into one hind limb of young adult rats. The inoculated hind limb was paralysed within 3 h, and remained paralysed for 2 days. The paralysis was associated with the loss of synaptic vesicles from motor nerve terminal boutons, a decline in immunoreactivity of synaptophysin, SNAP-25 and syntaxin, a loss of muscle mass and the upregulation of NaV(1.5) mRNA and protein. Between 3 and 6 h after the inoculation of toxin, some nerve terminal boutons exhibited clear signs of degeneration. Others appeared to be in the process of withdrawing from the synaptic cleft and some boutons were fully enwrapped in terminal Schwann cell processes. By 12 h all muscle fibres were denervated. Re-innervation began at 3 days with the appearance of regenerating nerve terminals, a return of neuromuscular function in some muscles and a progressive increase in the immunoreactivity of synaptophysin, SNAP-25 and syntaxin. Full recovery occurred at 7 days. The data were compared with recently published clinical data on envenoming bites by kraits and by extrapolation we suggest that the acute, reversible denervation caused by beta-bungarotoxin is a credible explanation for the clinically important, profound treatment-resistant neuromuscular paralysis seen in human subjects bitten by these animals.

Beta-bungarotoxin-induced depletion of synaptic vesicles at the mammalian neuromuscular junction.

The neurotoxic phospholipase A(2), beta-bungarotoxin, caused the failure of the mechanical response of the indirectly stimulated rat diaphragm. Exposure to beta-bungarotoxin had no effect on the response of the muscle to direct stimulation. Resting membrane potentials of muscle fibres exposed to the toxin were similar to control values, and the binding of FITC-labelled alpha-bungarotoxin to nAChR at the neuromuscular junction was unchanged. Motor nerve terminal boutons at a third of cell junctions were destroyed by exposure to beta-bungarotoxin leaving only a synaptic gutter filled with Schwann cell processes and debris. At other junctions, some or all boutons survived exposure to the toxin. Synaptic vesicle density in surviving terminal boutons was reduced by 80% and synaptophysin immunoreactivity by >60% in preparations exposed to beta-bungarotoxin, but syntaxin and SNAP-25 immunoreactivity was largely unchanged. Terminal bouton area was also unchanged. The depletion of synaptic vesicles was completely prevented by prior exposure to botulinum toxin C and significantly reduced by prior exposure to conotoxin omega-MVIIC. The data suggest that synaptic vesicle depletion is caused primarily by a toxin-induced entry of Ca(2+) into motor nerve terminals via voltage gated Ca(2+) channels and an enhanced exocytosis via the formation of t- and v-SNARE complexes.

Pharmacological strategies to block rod photoreceptor apoptosis caused by calcium overload: a mechanistic target-site approach to neuroprotection.

PURPOSE: Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited, and disease-, injury- and chemical-induced retinal degeneration. Our aims were three-fold: 1) to determine the kinetics of rod apoptosis and Ca2+ overload in Pde6b9rd1) mice and developmentally lead-exposed rats, 2) to establish a pathophysiologically-relevant model of Ca2+ overload/rod-selective apoptosis in isolated rat retina and 3) to examine different mechanistic based neuroprotective strategies that would abrogate or mollify rod Ca2+ overload/apoptosis. METHODS: Retinal morphometry and elemental calcium content ([Ca]) determined the kinetics of rod apoptosis and Ca2+ overload. A multiparametric analysis of apoptosis including rod [Ca], a live/dead assay, rod oxygen consumption, cytochrome c immunoblots and caspase assays was combined with pharmacological studies of an isolated rat retinal model of rod-selective Ca2+ overload/apoptosis. RESULTS: Ca2+ overload preceded rod apoptosis in mice and rats, although the extent and kinetics in each differed significantly. The isolated rat model of rod Ca2+ overload/apoptosis showed that blockade of Ca2+ entry through rod cGMP-activated channels with L-cis diltiazem was partially neuroprotective, whereas blockade of Ca2+ entry into rods through L-type Ca2+ channels with D-cis diltiazem or verapamil provided no protection. Inhibition of the mitochondrial Na+/Ca2+ exchanger with D-cis diltiazem provided no protection. CsA and NIM811, mitochondrial permeability transition pore (mPTP) inhibitors, blocked all Ca(2+)-induced apoptosis, whereas the caspase-3 inhibitor DEVD-fmk only blocked the downstream cytochrome c-induced apoptosis. CONCLUSIONS: The successful pharmacological neuroprotective strategies for rod Ca2+ overload/apoptosis targeted the rod cGMP-activated channels or mPTP, but not the rod L-type Ca2+ channels.

Muscle fibre breakdown in venom-induced muscle degeneration.

We studied the early stages of the degeneration of skeletal muscles using the venom of Notechis scutatus as the myotoxic agent. The venom was used at a dose equivalent to the LD50 in the mouse. There was no mortality amongst the rats. Electron microscopy was used to show the progressive hypercontraction of sarcomeres and the loss of alignment of myofibrils in individual muscle fibres. Between areas of hypercontraction sarcomeres were torn, shedding loosened myofilaments into the cytosol. Western blotting and Coomassie staining were used to compare the respective rates of loss of desmin, titin, actin, myosin and dystrophin. We showed that desmin and titin were the first proteins to be degraded with a time to 50% loss of approximately 1 h and 3 h, respectively. The loss of major contractile proteins, myosin and actin, was rather slower. The loss of dystrophin was also slower than the loss of desmin and titin. Early damage to the plasma membrane of the muscle fibre caused the cells to depolarize, probably promoting the hypercontraction of the sarcomeres, but actual loss of membrane was incomplete even at 24 h. We suggest that the early degradation of desmin and titin was responsible for the disaggregation of the sarcomeres; the liberated contractile proteins myosin and actin were shed into the cytosol, where they were degraded. Phagocytic cells that had invaded the degenerating muscle fibres were primarily involved in the clearance of damaged mitochondria.

Myotoxic phospholipases A2 and the regeneration of skeletal muscles.

The review explains why the myotoxic phospholipases A2 and cardiotoxins are such important tools in the study of the regeneration and maturation of mammalian skeletal muscle. The role of satellite cells as precursors of cell-based regeneration is discussed and recent controversies on the origin of myogenic cells involved in the regeneration of mature skeletal muscle are addressed. This is followed by discussions of sarcomere reconstruction, myosin and sarcoplasmic reticulum ATPase expression, the electrophysiological properties of regenerating muscle, and the reconstruction of the neuromuscular junction. The emphasis throughout is on the plastic changes of major structural and functional proteins that occur during regeneration, and on other influences that determine the final outcome of regenerative activity such as innervation, thyroid status, mechanical work and the functional integrity of the microcirculation. The review closes with a discussion of some of the factors--such as active regeneration--that influence the success of gene-based therapies applied to inherited muscle disease.