The seroprevalence of various Orientia tsutsugamushi (OT) strains among Malaysian patients with suspected scrub typhus infections was determined using an indirect immunoperoxidase (IIP) assay. IgG against a single OT strain were detected in six sera (3 Karp, 1 Gilliam and 2 TC586), whereas IgM antibodies against a single OT strain (Gilliam) were noted in 3 sera (Gilliam). IgG reactive to all OT strains were present in 33 (47.1%) of the 70 sera and IgM reactive to all OT strains were present in 22 (78.6%) of the 28 sera. The fact that most sera were reactive to multiple OT strains suggests that group-specific antigens are involved in scrub typhus infections, whereas very few were due to strain-specific epitopes present on these strains. Peak IgG and IgM titers were noted more frequently against Gilliam, Karp, and TA763 strains: this suggests that these strains may be the commonest infecting strains among Malaysian patients. Two predominant OT polypeptides consistently reacted with patients' sera were the 70 kDa and 56 kDa proteins.
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.
A study was carried out on 49 H. pylori-positive and 11 H. pylori-negative patients to determine the reactivity of peripheral blood lymphocytes (PBL) to phytohemagglutinin (PHA) and acid glycine extract (AGE) of H. pylori, and to identify cells responsible for imunosuppression. Based on response to PHA stimulation, cell-mediated immunity of all patients were competent. In some patients, however, response to AGE of H. pylori was suppressed by plastic adherent cells. This study provided evidence of the presence of plastic adherent suppressor cells which suppressed PBL response to AGE of H. pylori but not to PHA suggesting that immunosuppression is antigen specific. There is also an indication that immunosuppression may be species-specific as PBL devoid of plastic adherent cells only responded to stimulation by AGE of H. pylori but not that to AGE of C. jejuni.
A chromatographic immunoassay cholera antigen detection kit, the Cholera Spot test, was evaluated. The test was found to be specific with a sensitivity of 10(6) cfu/ml for the direct detection of V. cholerae in simulated stool specimens and 10 cfu/ml in simulated cotton-tipped swab specimens after overnight incubation in alkaline peptone water. This enables early recognition of cholera cases and their contacts so that prevention and control measures can be promptly instituted.
Streptococcus pyogenes ST4547 is an opacity factor negative strain, which has been recently reported as a new emm type from Malaysia. Nucleotide sequencing of the mga regulon of this strain showed the existence of two emm-like genes. The emm gene located upstream of the scpA gene comprises 1305 nucleotides encoding the putative precursor M protein of 435 amino acids in length with an M(r) of 49 kDa. or a predicted mature protein of 394 amino acids with an M(r) of 44.8 kDa. Another gene mrpST4547 was located upstream of the emm gene and downstream of the mga gene. The sequence of this mrp gene comprises 1167 nucleotides encoding a predicted protein of 388 amino acids in length with an M(r) of 42.2 kDa. or a predicted mature protein of 347 amino acids with an M(r) of 37.9 kDa. The mga regulon of strain ST4547 has a mosaic structure comprising segments, which originated from different OF positive and OF negative strains. The sequences flanking the hyper-variable and C repeats of the emmST4547 gene showed high similarity to corresponding regions in the mga regulon of OF positive strains notably M15, M4, M22 and M50. In contrast, the sequence within the hyper-variable and C repeat regions of the emmST4547 gene revealed high similarity to equivalent regions in the OF negative strains. These data indicates that horizontal transfer of emm-like gene could have occurred between OF positive and OF negative strains resulting in architectural divergence in the mga regulon.
A case of respiratory infection in a child due to Chlamydia pneumoniae is reported. The diagnosis was made by the detection of chlamydial antigen in the tracheal secretion and a significant increase in C. pneumoniae antibody titre. The infection responded well to erythromycin therapy.
Data are presented for 2382 children investigated for fever in a Malaysian hospital between 1984 and 1987 when Widal tests and blood cultures were a routine part of every fever screen. There were 145 children who were culture positive (TYP-CP) for Salmonella typhi, while 166 were culture negative but were diagnosed as having typhoid (TYP-CN). Analyses of the sensitivity and specificity of combinations of initial Widal titres in predicting a positive S. typhi culture in a febrile child (culture positive vs the rest) showed the best model to be an O- and/or H-titre of > or = 1 in 40 (sensitivity 89%; specificity 89%). While the negative predictive value of the model was high (99.2%) the positive predictive value remained below 50% even for very high titres of O and H (> 1 in 640), at which point the specificity was 98.5%, supporting the clinical view that a high proportion of the TYP-CN patients really were typhoid but were missed by culture. The TYP-CN patients showed a very similar clinical and age profile to TYP-CP patients. The length of history of fever did not affect the initial Widal titre in culture positive cases. The Widal test in children remains a sensitive and specific 'fever screen' for typhoid although it will not identify all cases. In children, lower cut-off points for O- and H-titres should be used than are generally recommended.
T cell receptor (TCR)-like antibodies, obtained with the use of phage display technology, sandwich the best of the both arms of the adaptive immune system. In this study, in vitro selections against the latency associated Mycobacterium tuberculosis (Mtb) heat shock protein 16 kDa antigen (16 kDa) presented by HLA-A*011 and HLA-A*24 were carried out with the use of a human domain phage antibody library. TCR-like domain antibodies (A11Ab and A24Ab) were successfully generated recognizing 16 kDa epitopes presented by HLA-A*011 and HLA-A*24 molecules respectively. Both antibodies were found to be functional in soluble form and exhibited strong binding capacity against its targets. The results obtained support the future evaluation of these ligands for the development of diagnostic and therapeutic tools for tuberculosis infection.
Ehrlichia sennetsu, the causative agent of human sennetsu rickettsiosis, was successfully propagated in primary canine blood monocyte cultures. The growth cycle of this organism appears to be similar to that of Ehrlichia canis. The antigen derived from our E. sennetsu cultures was used to develop an indirect fluorescent antibody test for detection and titration of serum antibodies to the organism. Using this test system, we found that five human serum samples obtained from patients clinically diagnosed as having sennetsu rickettsiosis were positive for anti-E. sennetsu antibodies. In addition, 29% of the serum samples obtained from 200 patients having a fever of unknown origin and residing in various regions of Malaysia were also serologically positive. All sera from apparently healthy individuals were negative in the test. Dogs inoculated with cell culture-adapted E. sennetsu developed a significant specific antibody titer to E. sennetsu, and the organism was subsequently isolated from their blood. These animals showed no clinical evidence of disease. The possibility of a higher prevalence of human sennetsu rickettsiosis in Southeast Asia and the potential usefulness of the canine model for studies of human sennetsu rickettsiosis are discussed.
Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.
There is a need for identification of new infection markers against common Leptospira isolates in Malaysia. To achieve this goal, seven-day-old cultures of Leptospira interrogans serogroup Icterohemorrhagiae (L44) and Leptospira interrogans serogroup Javanica (L55) were used for antigen preparation by sequential extraction method using 40 mM Tris, 8M Urea and 2M thiourea. Immunoblot analysis of the antigens were performed using serum samples from 46 local patients with confirmed acute leptospirosis, 28 patients with other infections and 14 healthy controls. The patients serum samples used in this study contained heterologous antibody against a number of different leptospira serovars. A strong IgM reactivity to a broad diffuse band of 10-15 kDa was observed. Combining results using L44 and L55 antigens showed sensitivity of 80.4% and specificity of 95.2% for detection of leptospirosis. Proteinase K and periodate treatment indicated that the band is likely to be lipopolysaccharide (LPS) in nature. This study showed that the 10-15 kDa antigen could potentially be useful for serodiagnosis of acute leptospirosis in Malaysia.
The aim of this study was to investigate the clinico-pathology and haemato-biochemistry alterations in buffaloes inoculated with Pasteurella multocida type B:2 immunogen outer membrane protein via subcutaneous and oral routes. Nine buffalo heifers were divided equally into 3 treatment groups. Group 1 was inoculated orally with 10 mL of phosphate buffer saline (PBS); Group 2 and 3 were inoculated with 10 mL of outer membrane protein broth subcutaneously and orally respectively. Group 2 buffaloes showed typical haemorrhagic septicaemia clinical signs and were only able to survive for 72 h of the experiment. However, Group 3 buffaloes were able to survive throughout the stipulated time of 21 days of experiment. There were significant differences (p 0.05) in edema between groups except for the lung. This study was a proof that oral route infection of Pasteurella multocida type B:2 immunogen outer membrane protein can be used to stimulate host cell.
Leptospirosis is a febrile zoonotic disease. Routine diagnosis of leptospirosis is based on the detection of specific antibodies with serological tests. The aim of our study was to determine the usefulness of immunochromatographic assay (ICA), ImmuneMed Leptospira IgM Duo Rapid test kit from Korea, in rapid screening of acute leptospirosis in emergency cases with limited expertise. A total of 197 serum samples (93 positive, 104 negative) were selected randomly. The test has good diagnostic sensitivity 73% and specificity 90%. With positive predictive value of 87% and negative predictive value of 79%, this reassures patients have higher chance of correct diagnosis. This ICA is acceptable for screening of leptospirosis but confirmation with microscopic agglutination test should follow.
The humoral immune response against 43 staphylococcal antigens was compared among hospitalized patients where none of them had any staphylococcal infection on the day of admission with or without nasal Staphylococcus aureus carriage. Fifty-nine carriers and 59 matched non-carriers were studied. The carriers harbored S. aureus of 35 different spa types, including three t037/ST239 methicillin-resistant S. aureus (MRSA) (5.1%). Among the 118 patients, 31 acquired S. aureus during hospitalization. In colonized and non-colonized patients, unique patterns of S. aureus-specific immune responses were observed. The mean fluorescence indices (MFIs) of antibodies against 36/43 (83.7%) antigens were seen to be elevated among carriers. The MFI among carriers with acquisition was significantly higher for staphylococcal superantigen-like protein 5 (SSL5, p = 0.028) when compared to carriers without acquisition. High antibody levels against staphylococcal enterotoxin A (SEA) among carriers illustrate its role as a superantigen in both infection and colonization. We also report a dynamic immune response in S. aureus-carrying patients against the recently reported formyl peptide receptor-like inhibitory (FLIPr)-like protein. In the current study, the dynamics of antibodies against staphylococcal antigens among carrier patients seem quite similar to non-carrier patients. To better understand the dynamic immunogenicity during S. aureus infection and colonization, artificial colonization studies and investigation of the changes in the levels of antibodies against other staphylococcal antigens are recommended.
Tuberculosis (TB) is an airborne infection caused by Mycobacterium tuberculosis (Mtb). About one-third of the world's population is latently infected with TB and 5-15% of them will develop active TB in their lifetime. It is estimated that each case of active TB may cause 10-20 new infections. Host immune response to Mtb is influenced by interferon- (IFN-) signaling pathways, particularly by type I and type II interferons (IFNs). The latter that consists of IFN-γ has been associated with the promotion of Th1 immune response which is associated with protection against TB. Although this aspect remains controversial at present due to the lack of established correlates of protection, currently, there are different prophylactic, diagnostic, and immunotherapeutic approaches in which IFNs play an important role. This review summarizes the main aspects related with the biology of IFNs, mainly associated with TB, as well as presents the main applications of these cytokines related to prophylaxis, diagnosis, and immunotherapy of TB.
A study to determine the sensitivity and specificity of the Directogen Group A Streptococcal Test was carried out in a primary care clinic in University Hospital, Kuala Lumpur. The study also looked at the prevalence of streptococcal pharyngitis in the clinic. We found that the rapid test has a sensitivity of 90.9% and a specificity of 90.7%. Viral pharyngitis is the commonest form (63%) of pharyngitis followed by Group A Beta haemolytic streptococcal pharyngitis (14.2%). The presence of exudates, tonsillar enlargement and the presence of tender cervical lymph nodes have a significantly higher association with streptococcal pharyngitis.
Study site: Primary care clinic, University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia
Leptospirosis is a widespread zoonotic disease and a public health problem, particularly in tropical and subtropical countries. Varied clinical manifestations of the disease frequently lead to misdiagnosis resulting in life-threatening multi-organ complications. Therefore, early laboratory investigation using an appropriate diagnostic approach is crucial. In the present study, a potential protein marker was identified and evaluated for its usefulness in the serodiagnosis of acute leptospirosis.
The H5 gene of avian influenza virus (AIV) strain A/chicken/Malaysia/5744/2004(H5N1) was cloned into pcDNA3.1 vector, and Esat-6 gene of Mycobacterium tuberculosis was fused into downstream of the H5 gene as a genetic adjuvant for DNA vaccine candidates. The antibody level against AIV was measured using enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition (HI) test. Sera obtained from specific-pathogen-free chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5/Esat-6 demonstrated antibody responses as early as 2 weeks after the first immunization. Furthermore, the overall HI antibody titer in chickens immunized with pcDNA3.1/H5/Esat-6 was higher compared to the chickens immunized with pcDNA3.1/H5 (p<0.05). The results suggested that Esat-6 gene of M. tuberculosis is a potential genetic adjuvant for the development of effective H5 DNA vaccine in chickens.
Mucosal presentation of Actinomyces viscosus results in antigen-specific systemic immune suppression, known as oral tolerance. The aim of the present study was to determine the mechanism by which this oral tolerance is induced. DBA/2 mice were gastrically immunized with A. viscosus. Serum, Peyer's patch (PP) and spleen cells were transferred to syngeneic recipients which were then systemically challenged with the sameiA. viscosus strain. To determine antigen-specificity of cells from gastrically immunized mice, recipients which received immune spleen cells were also challenged with Porphyromonas gingivalis. One week after the last systemic challenge, the delayed type hypersensitivity (DTH) response was determined by footpad swelling and the level of serum IgG, IgA and IgM antibodies to A. viscosus or P. gingivalis measured by an ELISA. No suppression of DTH response or of specific serum antibodies was found in recipients which received serum from gastrically immunized mice. Systemic immune suppression to A. viscosus was observed in recipients which had been transferred with PP cells obtained 2 days but not 4 and 6 days after gastric immunization with A. viscosus. Conversely, suppressed immune response could be seen in recipients transferred with spleen cells obtained 6 days after gastric immunization. The immune response to P. gingivalis remained unaltered in mice transferred with A. viscosus-gastrically immunized cells. The results of the present study suggest that oral tolerance induced by A. viscosus may be mediated by antigen-specific suppressor cells which originate in the PP and then migrate to the spleen.