Animal tuberculosis (TB) caused by infection with Mycobacterium bovis and closely related members of the M. tuberculosis complex (MTC), is often reported in the Eurasian wild boar (Sus scrofa). Tests detecting antibodies against MTC antigens are valuable tools for TB monitoring and control in suids. However, only limited knowledge exists on serology test performance in 2-6 month-old piglets. In this age-class, recent infections might cause lower antibody levels and lower test sensitivity. We examined 126 wild boar piglets from a TB-endemic site using 6 antibody detection tests in order to assess test performance. Bacterial culture (n=53) yielded a M. bovis infection prevalence of 33.9%, while serum antibody prevalence estimated by different tests ranged from 19% to 38%, reaching sensitivities between 15.4% and 46.2% for plate ELISAs and between 61.5% and 69.2% for rapid immunochromatographic tests based on dual path platform (DPP) technology. The Cohen kappa coefficient of agreement between DPP WTB (Wildlife TB) assay and culture results was moderate (0.45) and all other serological tests used had poor to fair agreements. This survey revealed the ability of several tests for detecting serum antibodies against the MTC antigens in 2-6 month-old naturally infected wild boar piglets. The best performance was demonstrated for DPP tests. The results confirmed our initial hypothesis of a lower sensitivity of serology for detecting M. bovis-infected piglets, as compared to older wild boar. Certain tests, notably the rapid animal-side tests, can contribute to TB control strategies by enabling the setup of test and cull schemes or improving pre-movement testing. However, sub-optimal test performance in piglets as compared to that in older wild boar should be taken into account.
Delivering novel antimycobacterial agents through the pulmonary route using nanoparticle-based systems shows promise for treating diseases like tuberculosis. However, creating dry powder inhaler (DPI) with suitable aerodynamic characteristics while preserving nanostructure integrity and maintaining bioactivity until the active ingredient travels deeply into the lungs is a difficult challenge. We developed DPI formulations containing levofloxacin-loaded solid lipid nanoparticles (SLNs) via spray-drying technique with tailored aerosolization characteristics for effective inhalation therapy. A range of biophysical techniques, including transmission electron microscopy, confocal microscopy, and scanning electron microscopy were used to measure the morphologies and sizes of the spray-dried microparticles that explored both the geometric and aerodynamic properties. Spray drying substantially reduced the particle sizes of the SLNs while preserving their nanostructural integrity and enhancing aerosol dispersion with efficient mucus penetration. Despite a slower uptake rate compared to plain SLNs, the polyethylene glycol modified formulations exhibited enhanced cellular uptake in both A549 and NR8383 cell lines. The percent viability of Mycobacterium bovis had dropped to nearly 0 % by day 5 for both types of SLNs. Interestingly, the levofloxacin-loaded SLNs demonstrated a lower minimum bactericidal concentration (0.25 µg/mL) compared with pure levofloxacin (1 µg/mL), which indicated the formulations have potential as effective treatments for tuberculosis.
Even though vaccines are effective for prevention of diseases, compliance with recommended schedules of immunisation in early childhood is not always complete. Identification of potential risk factors associated with under-immunisation may help increase vaccination uptake. The objectives of this study were to determine the prevalence and risk factors of under-immunisation in hospitalised preschool children in Universiti Sains Malaysia Hospital (HUSM). Children aged 1 month to 2 years admitted between December 1998 and December 2000 in medical paediatric wards at HUSM were studied. Parents/guardians were interviewed during admission regarding immunisation status and potential risk factors. Children who did not have home-based cards during admission were excluded. Among 401 children, 37.9% had complete immunisation, 43.4% had delayed immunisation and 34.9% had incomplete immunisation. The uptake of BCG, DPT/OPV 1, DPT/OPV2, Hepatitis B3, DPT/OPV3, and measles were 98.5%, 90.7%, 83.2%, 81.4%,79.2% and 72.9% respectively. Vaccines scheduled to be given at later ages were significantly less likely to be taken (p=0.002) compared with vaccines scheduled to be given at an earlier age. Significant independent risk factors for under-immunisation were elderly mothers (3 40 years old) (OR 3.0, 95% CI: 1.2-8.0), low birth weight £2.0 kg (OR 6.2; 95% CI: 1.8-21.4) and presence of chronic illness in the child (OR 4.5; 95% CI: 2.5-7.9). In children whose father's education was low (primary school or no education), low maternal education was significantly associated with lower rate of under-immunisation (p = 0.005, OR 0.11 95%CI 0.02-0.51).
In conclusion this study showed that a large proportion of hospitalised preschool children were under-immunised, and vaccines scheduled to be given at a later age had lower uptakes. The situation can be rectified if every opportunity is taken to immunise all children, who have lapsed in their immunisation schedule.
A 17-year-old male student of Indonesian parentage presented with two weeks history of progressive painless bilateral visual deterioration. There was no contact with tuberculosis (TB)-infected patients and parents claimed that all immunization including BCG was completed. However, BCG scar was not apparent. Visual acuity was 6/36 and 6/60 in the right and left eyes respectively. The anterior and vitreous chambers were quiet. Funduscopic examination revealed retinal vasculitis with perivascular exudates, branch vein occlusion, neovascularization and macular oedema. Fluorescein angiography confirmed large areas of capillary non-perfusion and leaking new vessels. Mantoux test was positive and full regime anti-TB therapy was instituted. HIV screening was negative. Three days later, an immunosuppressive dose of oral steroid was started. Both eyes received intensive laser photocoagulation.Interestingly, there was no development of vitritis throughout.
Mycobacterium bovis bacille Calmette-Guèrin (BCG) represents one of the most promising live vectors for the delivery of foreign antigens to the immune system. A recombinant BCG containing a synthetic gene coding for the malarial epitopes namely, the fragment 2 of region II of EBA-175 (F2R(II)EBA) and the repeat sequence of the circumsporozoite protein NANP generated in favour of mycobacterium codon usage using assembly PCR was constructed. Two T-cell epitopes of the 6-kDa M. tuberculosis early-secreted antigenic target (ESAT-6) antigen were also clone in the same construct. Expression of the synthetic gene was driven by the heat shock protein 65 (hsp65) promoter from M. tuberculosis and the signal peptide from the MPT63 antigen of M. tuberculosis. Expression of the composite epitopes was detected by Western blotting of the cell extract and culture supernatant of the recombinant clones using a specific rabbit polyclonal antibody against F2R(II)EBA. This study demonstrates the possibility of cloning and expressing immunogenic epitopes from causative agents of two important diseases: malaria and tuberculosis (TB) in a single recombinant BCG construct.
Vaccine development against the blood-stage malaria parasite is aimed at reducing the pathology of the disease. We constructed a recombinant Mycobacterium bovis bacille Calmette Guerin (rBCG) expressing the 19 kDa C-terminus of Plasmodium falciparum merozoite surface protein-1 (MSP-1(19)) to evaluate its protective ability against merozoite invasion of red blood cells in vitro. A mutated version of MSP-1(19), previously shown to induce the production of inhibitory but not blocking antibodies, was cloned into a suitable shuttle plasmid and transformed into BCG Japan (designated rBCG016). A native version of the molecule was also cloned into BCG (rBCG026). Recombinant BCG expressing the mutated version of MSP-1(19) (rBCG016) elicited enhanced specific immune response against the epitope in BALB/c mice as compared to rBCG expressing the native version of the epitope (rBCG026). Sera from rBCG016-immunized mice contained significant levels of specific IgG, especially of the IgG2a subclass, against MSP-1(19) as determined by enzyme-linked immunosorbent assay. The sera was reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA) and inhibited merozoite invasion of erythrocytes in vitro. Furthermore, lymphocytes from rBCG016-immunized mice demonstrated higher proliferative response against the MSP-1(19) antigen as compared to those of rBCG026- and BCG-immunized animals. rBCG expressing the mutated version of MSP-1(19) of P. falciparum induced enhanced humoral and cellular responses against the parasites paving the way for the rational use of rBCG as a blood-stage malaria vaccine candidate.
Cerebral demyelination and optic neuritis are often seen in children with acute disseminated encephalomyelitis following various infections and immunisations. An eight month old girl presented with a left axillary lymph node swelling and an erythematous lace-like rash over her cheeks and trunk. She then developed acute encephalopathy, bilateral nystagmus, right hemiparesis and left facial nerve palsy. Her electroencephalogram showed an encephalopathic process and visual evoked response study were grossly abnormal. Her MRI brain showed hyperintensities in the midbrain, pons and bilateral cerebellar peduncles. She was treated as postinfectious cerebral demyelination with intravenous antibiotics, methylprednisolone and immunoglobulin. Left axillary lymph node excision biopsy and GeneXpert test detected Mycobacterium tuberculosis complex that prompted initiation of antituberculous therapy. Her chest X-ray and cerebrospinal fluid examinations for tuberculosis were normal. She showed significant recovery after 2 weeks. This case illustrates a rare presentation of cerebral demyelination and bilateral optic neuritis following suppurative BCG lymphadenitis.