Tuberculosis (TB) is a chronic inflammatory and zoonotic disease caused by Mycobacterium tuberculosis complex (MTBC) members, affecting several domestic animals, wildlife species and humans. The preliminary investigation was aimed to detect antibody against MTBC among indigenous wildlife which are free-ranged wild boar, free-ranged wild macaques and captive Asian elephants in selected areas of Selangor and elephant conservation centre in Pahang, respectively. The results indicate that MTBC serodetection rate in wild boar was 16.7% (7.3-33.5 at 95% confidence interval (CI)) using an in-house ELISA bPPD IgG and 10% (3.5-25.6 at 95% CI) by DPP®VetTB assay, while the wild macaques and Asian elephant were seronegative. The univariate analysis indicates no statistically significant difference in risk factors for sex and age of wild boar but there was a significant positive correlation (P<0.05) between bovine TB in dairy cattle and wild boar seropositivity in the Sepang district.
Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by a selective predisposition to clinical disease caused by the Bacille Calmette-Guérin (BCG) vaccine and environmental mycobacteria. The known genetic etiologies of MSMD are inborn errors of IFN-γ immunity due to mutations of 15 genes controlling the production of or response to IFN-γ. Since the first MSMD-causing mutations were reported in 1996, biallelic mutations in the genes encoding IFN-γ receptor 1 (IFN-γR1) and IFN-γR2 have been reported in many patients of diverse ancestries. Surprisingly, mutations of the gene encoding the IFN-γ cytokine itself have not been reported, raising the remote possibility that there might be other agonists of the IFN-γ receptor. We describe 2 Lebanese cousins with MSMD, living in Kuwait, who are both homozygous for a small deletion within the IFNG gene (c.354_357del), causing a frameshift that generates a premature stop codon (p.T119Ifs4*). The mutant allele is loss of expression and loss of function. We also show that the patients' herpesvirus Saimiri-immortalized T lymphocytes did not produce IFN-γ, a phenotype that can be rescued by retrotransduction with WT IFNG cDNA. The blood T and NK lymphocytes from these patients also failed to produce and secrete detectable amounts of IFN-γ. Finally, we show that human IFNG has evolved under stronger negative selection than IFNGR1 or IFNGR2, suggesting that it is less tolerant to heterozygous deleterious mutations than IFNGR1 or IFNGR2. This may account for the rarity of patients with autosomal-recessive, complete IFN-γ deficiency relative to patients with complete IFN-γR1 and IFN-γR2 deficiencies.
Bacille Calmette-Guerin (BCG) vaccination for protection against tuberculosis has been in use for long. Although the vaccine is safe, its administration can result in complications such as BCG adenitis. We report here a series of children with BCG adenitis with a view to recognise and manage this condition. It is hoped that this case series would encourage the increased identification of this condition.
Study site: Paediatric Surgical Unit, Department of Surgery, Hospital Tengku Ampuan Afzan, Kuantan, Pahang, Malaysia
Live attenuated Mycobacterium bovis (M. bovis), marketed as Bacille Calmette-Guérin is the only FDA-approved vaccine against tuberculosis. The prerequisite of cold chain storage between 2 and 8 °C hinders the global vaccination effort. The study aims to investigate the effect of trehalose, sucrose and glycerol combinations in enhancing the stability of M. bovis. The bacilli were formulated in various ratios of trehalose-glycerol, sucrose-glycerol, trehalose-sucrose-glycerol systems (test samples) and sodium glutamate (control), freeze-dried and stored for 28 days at 4 °C, 25 °C and 37 °C. Bacteria viability at pre-, post-freeze-drying and after storage were quantified by its density in colony-forming unit per milliliter (CFU/mL) as obtained through the pour plate method. Formulations were characterized using differential scanning calorimetry. Structural collapsed cakes were found on all freeze-dried formulations because of the low Tg'. Comparing between binary and ternary formulations, trehalose-sucrose-glycerol was found to be a superior lyoprotectant. Upon storage, the viability of bacteria in disaccharide-polyol formulations was highest when stored at 4 °C followed by 25 °C. The lowest viability was found after storage at 37 °C. While the ternary disaccharide-polyol system may be used as a thermoprotectant up to 25 °C, sodium glutamate has a superior thermoprotective effect at temperature above 25 °C.
Zoonotic tuberculosis caused by Mycobacterium bovis (M. bovis), a member of Mycobacterium tuberculosis complex (MTBC) has increasingly gathered attention as a public health risk, particularly in developing countries with higher disease prevalence. M. bovis is capable of infecting multiple hosts encompassing a number of domestic animals, in particular cattle as well as a broad range of wildlife reservoirs. Humans are the incidental hosts of M. bovis whereby its transmission to humans is primarily through the consumption of cattle products such as unpasteurized milk or raw meat products that have been contaminated with M. bovis or the transmission could be due to close contact with infected cattle. Also, the transmission could occur through aerosol inhalation of infective droplets or infected body fluids or tissues in the presence of wound from infected animals. The zoonotic risk of M. bovis in humans exemplified by miscellaneous studies across different countries suggested the risk of occupational exposure towards M. bovis infection, especially those animal handlers that have close and unreserved contact with cattle and wildlife populations These animal handlers comprising of livestock farmers, abattoir workers, veterinarians and their assistants, hunters, wildlife workers as well as other animal handlers are at different risk of contracting M. bovis infection, depending on the nature of their jobs and how close is their interaction with infected animals. It is crucial to identify the underlying transmission risk factors and probable transmission pathways involved in the zoonotic transmission of M. bovis from animals to humans for better designation and development of specific preventive measures and guidelines that could reduce the risk of transmission and to protect these different occupational-related/populations at risk. Effective control and disease management of zoonotic tuberculosis caused by M. bovis in humans are also hindered by various challenges and factors involved at animal-human interface. A closer look into factors affecting proper disease control and management of M. bovis are therefore warranted. Hence, in this narrative review, we have gathered a number of different studies to highlight the risk of occupational exposure to M. bovis infection and addressed the limitations and challenges underlying this context. This review also shed lights on various components and approaches in tackling M. bovis infection at animal-human interface.
DNA fingerprinting with the insertion sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The insertion sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.
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.
We assessed the suitability of targeted removal as a means for tuberculosis (TB) control on an intensely managed Eurasian wild boar (Sus scrofa) hunting estate. The 60km(2) large study area included one capture (treatment) site, one control site, and one release site. Each site was fenced. In the summers of 2012, 2013 and 2014, 929 wild boar were live-captured on the treatment site. All wild boar were micro-chipped and tested using an animal side lateral flow test immediately after capture in order to detect antibodies to the Mycobacterium tuberculosis complex (MTC). The wild boar were released according to their TB status: Seropositive individuals onto the release site (hunted after summer), and seronegative individuals back onto the treatment site. The annual summer seroprevalence of antibodies to the MTC declined significantly in live-captured wild boar piglets from the treatment site, from 44% in 2012 to 27% in 2013 (a reduction of 39%). However, no significant further reduction was recorded in 2014, during the third capture season. Fall-winter MTC infection prevalence was calculated on the basis of the culture results obtained for hunter-harvested wild boar. No significant changes between hunting seasons were recorded on either the treatment site or the control site, and prevalence trends over time were similar on both sites. The fall-winter MTC infection prevalence on the release site increased significantly from 40% in 2011-2012 to 64% in 2012-2013 and 2013-2014 (60% increase). Recaptures indicated a persistently high infection pressure. This experiment, the first attempt to control TB in wild boar through targeted removal, failed to reduce TB prevalence when compared to the control site. However, it generated valuable knowledge on infection pressure and on the consequences of translocating TB-infected wild boar.
In this dataset, we report the genome assembly and data analysis of Mycobacterium tuberculosis strain SIT745/EAI1-MYS. Previously, this strain was isolated from a Malaysian patient with extra-pulmonary tuberculosis, and identification of this strain is done by spoligotype patterns with fifteen known Shared International Type (SITs). Further analysis showed that this strain has a remarkable phylogeographical specificity for Malaysia. Based on the National Center for Biotechnology Information (NCBI) nucleotide database information, the complete genome consists of 150 contigs with various sequence lengths and was not assembled. In this assembly, the aforementioned contigs along with reference sequence from Mycobacterium tuberculosis strain H37Rv and Mycobacterium bovis strain AF2122/97 was used for gap closures, were assembled into a single circular chromosome length of approximately 4.42 Mega bases (Mb) with an average GC content of 65.6%. The single circular chromosome was shown to contain 4,009 protein-coding sequences, 3 ribosomal RNAs, 45 transfer RNAs, and 12 superclasses distributed with 277 subsystems which constitute nearly 1900 genes, respectively. The genome information will provide fundamental knowledge of this organism as well as insight for understanding genomic and proteomic profiling, phylogenetic relationship.
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 tuberculosis, the causing agent of tuberculosis, comes second only after HIV on the list of infectious agents slaughtering many worldwide. Due to the limitations behind the conventional detection methods, it is therefore critical to develop new sensitive sensing systems capable of quick detection of the infectious agent. In the present study, the surface modified cadmium-telluride quantum dots and gold nanoparticles conjunct with two specific oligonucleotides against early secretory antigenic target 6 were used to develop a sandwich-form fluorescence resonance energy transfer-based biosensor to detect M. tuberculosis complex and differentiate M. tuberculosis and M. bovis Bacille Calmette-Guerin simultaneously. The sensitivity and specificity of the newly developed biosensor were 94.2% and 86.6%, respectively, while the sensitivity and specificity of polymerase chain reaction and nested polymerase chain reaction were considerably lower, 74.2%, 73.3% and 82.8%, 80%, respectively. The detection limits of the sandwich-form fluorescence resonance energy transfer-based biosensor were far lower (10 fg) than those of the polymerase chain reaction and nested polymerase chain reaction (100 fg). Although the cost of the developed nanobiosensor was slightly higher than those of the polymerase chain reaction-based techniques, its unique advantages in terms of turnaround time, higher sensitivity and specificity, as well as a 10-fold lower detection limit would clearly recommend this test as a more appropriate and cost-effective tool for large scale operations.
Diagnosis of active mycobacterial disease in orangutans (Pongo pygmaeus) has been impeded by high levels of non-specific intradermal skin test reactivity to mycobacterial antigens. This may be due in part to cross reactivity between antigens, tuberculin concentrations used or other species-specific factors. Antigen 85 (Ag85) complex proteins are major secretory products of actively growing mycobacteria, and measurement of serum Ag85 could provide a method for determining active mycobacterial infections that was not dependent on host immunity. Serum Ag85 was measured by dot-immunobinding assay using monoclonal anti-Ag85, purified Ag85 standard and enhanced chemiluminescence technology in coded serum samples from 14 captive orangutans from a zoo in Colorado, 15 semi-captive orangutans in Malaysia, and 19 free-ranging wild orangutans in Malaysia. Orangutans from Colorado (USA) were culture negative for Mycobacterium tuberculosis and M. avium, although all had laboratory suspicion or evidence of mycobacterial infection; median serum Ag85 was 10 microU/ml (range, <0.25-630 microU/ml). Of the semi-captive orangutans, six were skin test reactive and two were culture positive for M. avium on necropsy. Median serum Ag85 for this group was 1,880 microU/ml (0.75-7,000 microU/ml), significantly higher than that of Colorado zoo or free-ranging Malaysian orangutans. Median serum Ag85 in the latter group was 125 microU/ml (range, 0.75-2,500 microU/ml). These data suggest that suggest that additional studies using more specific reagents and more samples from animals of known status are appropriate.
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.
During recent years, extensive development has been made to improving vaccines for tuberculosis. This is due to the presence of genome sequences of diverse mycobacterial species and Mycobacteroum tuberculosis (M. tuberculosis) isolates which has led to advances in the characterization of genes and antigens of M. tb and better realization of protective immune responses to the disease in both animals and humans. This review summarizes vaccine types, reasons for variable efficacy of BCG, latest advances in tuberculosis vaccine development and major vaccine design strategies.
To verify the actual immunisation coverage in Kuala Lumpur, City Hall Health Department and the Malaysian Paediatric Association (NGO ) carried out a survey. The survey revealed that the immunisation coverage determined at the child's first birthday for BCG was 95%, DPT 3 94%, OPV 3 94%, and measles = 27% (59% at 2 years). These figures correspond closer to City Hall's estimated coverage rather than the rates projected by the Ministry of Health. The main reasons for immunisation failure were, child ill 31.8% (not brought = 20.1%, brought but not given vaccine 11.7%), lack of information 28.6%, lack of motivation 9.1%, mother too busy 9.1%. Measles immunisation coverage at 1 year was low because of wrong information on schedules. Tetanus toxoid immunisation coverage of pregnant women was low. Only 27% of children were protected against neonatal tetanus although 97% of pregnant women received antenatal care and 50% had attended other health facilities as well during pregnancy. Private medical practitioners were responsible for more than 40% of all immunisations but were not submitting returns to the Health Department. Recommendations to improve immunisation coverage include education and motivation of the public and also doctors and health personnel on prevention of missed opportunities, contraindictions to immunisation and correct schedules. (Copied from article).
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.
Macrophage phagocytosis is the first line of defense of the innate immune system against malaria parasite infection. This study evaluated the immunomodulatory effects of BCG and recombinant BCG (rBCG) strains expressing the C-terminus of the merozoite surface protein-1 (MSP-1C) of Plasmodium falciparum on mouse macrophage cell line J774A.1 in the presence or absence of lipopolysaccharide (LPS) or LPS + IFN-γ. The rBCG strain significantly enhanced phagocytic activity, production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, nitric oxide (NO), and inducible nitric oxide synthase (iNOS) as compared with parental BCG strain, and these activities increased in the presence of LPS and LPS+IFN-γ. Furthermore, the rBCG strain also significantly reduced the macrophage viability as well as the rBCG growth suggesting the involvement of macrophage apoptosis. Taken together, these data indicate that the rBCG strain has an immunomodulatory effect on macrophages, thus strengthen the rational use of rBCG to control malaria infection.
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.
Proteins on the surface of Plasmodium falciparum merozoites are good targets for vaccine development against malaria because they are accessible to antibodies in the plasma. The 19 kDa C-terminus of merozoite surface protein-1 (MSP-1(19)) has been shown to induce both inhibitory as well as blocking antibodies, the latter blocking the protective effects of the former. Inhibitory antibodies bind to MSP-1(19) and inhibit merozoite invasion of red blood cells (RBC) but the binding of blocking antibodies can prevent binding of inhibitory antibodies thereby allowing the parasite to invade RBC. We constructed a synthetic version of the MSP-1(19) of the P. falciparum using mycobacterium codon usage by assembly PCR. The synthetic MSP-1(19) was mutated at various sites to promote the production of inhibitory but not blocking antibodies as previously reported. The native and mutated MSP-1(19) were cloned and expressed in Mycobacterium bovis bacille Calmette-Guerin (BCG) and the expressions of the recombinant proteins were detected by specific monoclonal antibodies (mAbs) namely, 12.10 and 1E1 against MSP-1(19) using Western blotting. The mutated MSP-1(19) protein reacted with the inhibitory mAb, 12.10, but not the blocking mAb, 1E1, paving the way for the construction of a potential recombinant BCG (rBCG) blood stage vaccine against malaria.