In this study, recombinant proteins that encompassed the AD I-AD III regions of 56 kDa immunodominant gene of 2 Orientia tsutsugamushi (OT) serotypes; Gilliam and TA763 were expressed in Escherichia coli. Both recombinant proteins exhibited serologic cross-reactivity with the rabbit antisera against various OT serotypes, as evaluated by enzyme-linked immunosorbent assay (ELISA), but not against other rickettsial species, including Rickettsia typhi, R. prowazekii and TT118 SFG rickettsiae. The feasibility of using the recombinant proteins as a diagnostic reagent was further evaluated by ELISA using sera from blood donors and scrub typhus patients. The results suggested a higher affinity of the antihuman IgM than IgG with both recombinant proteins. The IgM ELISA findings were agreeable with the results of indirect immunoperoxidase (IIP) assay especially with sera of high antibody (1:1600). However, more than one antigen are probably needed for development of an effective assay for serodiagnosis of scrub typhus in endemic areas.
During a 1-year period from October 1995 to September 1996, 273 isolations of Streptococcus pneumoniae were made from various types of clinical specimens. The majority of the isolates (39.2%) were from sputum whilst 27.5% were from blood, CSF and other body fluids. The organism was isolated from patients of all age groups, 31.1% from children aged 10 years and below, 64.7% of which come from children aged 2 years or below. The majority of the isolates belong to serotypes 1, 6B, 19B, 19F and 23F. Serotypes 1 and 19B were the most common serotypes associated with invasive infection. About 71.9% of the invasive infections were due to serotypes included in the available 23 valent polysaccharide vaccine. The rates of resistance to penicillin and erythromycin were 7.0 and 1.1% respectively. Our findings show that the serotypes of S. pneumoniae causing most invasive infections in Malaysia are similar to those in other parts of the world and the available vaccine may have a useful role in this population.
This study was conducted to determine the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Malaysian hospitals. A total of 264 MRSA isolates from eight hospitals were subjected to typing by pulsed-field gel electrophoresis (PFGE) of SmaI restricted DNA. Antibiotic disk susceptibility testing was also carried out to determine their resistance patterns. Thirty-one PFGE pattern types were identified. Three major pattern types A, ZC and K were found with type A the predominant profile in c. 80% of strains and present in all hospitals. Unlike type A, other DNA pattern types were unique to the hospitals in which they were isolated. PFGE type A also consisted of strains that were multiply antibiotic resistant. The presence of a single predominant PFGE type in Malaysian hospitals is an important finding which suggests that inter-hospital spread of MRSA had occurred frequently and regularly.
Isolates of 390 Staphylococcus aureus were tested against 13 different antibiotics by a disc diffusion method as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). Strains were isolated from blood (5.7%), cerebrospinal fluid (0.5%), respiratory tract (11.8%), pus and wound (73.3%), urine (1.8%), genital specimens (1.0%) and other specimens (4.3%). Only 4.6% of the isolates were fully susceptible to all the drugs tested. Resistance to penicillin was 94.1%, methicillin, 39.7%, chloramphenicol, 8.5%, ciprofloxacin, 29.2%, clindamycin, 2.1%, erythromycin, 45.9% gentamicin, 40.5%; rifampicin, 3.3% tetracycline, 47.2%, co-trimoxazole, 38.5%, mupirocin, 2.8%, fusidic acid, 3.6%. None of the isolates was resistant to vancomycin. The susceptibility of methicillin-resistant strains to erythromycin, gentamicin, tetracycline and ciprofloxacin was low, while clindamycin, fusidic acid, mupirocin, and rifampicin remained active.
The emergence and spread of multiresistant methicillin-resistant Staphylococcus aureus (MRSA) strains, especially those resistant to fusidic acid and rifampicin, in Malaysian hospitals is of concern. In this study DNA fingerprinting by PFGE was performed on fusidic acid- and rifampicin-resistant isolates from Malaysian hospitals to determine the genetic relatedness of these isolates and their relationship with the endemic MRSA strains. In all, 32 of 640 MRSA isolates from 9 Malaysian hospitals were resistant to fusidic acid and rifampicin. Seven PFGE types (A, ZC, ZI, ZJ, ZK, ZL and ZM) were observed. The commonest type was type ZC, seen in 72% of isolates followed by type A, seen in 13%. Each of the other types (ZI, ZJ, ZK, ZL and ZM) was observed in a single isolate. Each type, even the commonest, was found in only one hospital. This suggests that the resistant strains had arisen from individual MRSA strains in each hospital and not as a result of the transmission of a common clone.
The molecular types and genetic heterogeneity of Cryptococcus neoformans and C. gattii clinical isolates in Malaysia were determined in this study. Of 44 C. neoformans collected between 1980 and 2003, 42 (95.5%) were molecular type VNI, 2 (4.5%) were molecular type VNII. Of 17 C.gattii isolates, 13 (76.5%) were molecular type VGI, and 4 (23.5%) were molecular type VGII. A difference was noted when comparing the molecular types of cryptococcal isolates in the earlier and recent cases of cryptococcosis. While both molecular types VNI and VGI were equally predominant in the earlier cases of cryptococcosis, VNI was the most predominant molecular type isolated from the recent cases. VNII was a new molecular type, isolated from 5.1% of the recent cases. All the bird dropping isolates were molecular type VNI. The genetic heterogeneity of the two predominant molecular types, i.e., VNI, VGI clinical isolates and bird dropping isolates of C. neoformans were further determined by polymerase chain reaction (PCR) fingerprinting method, using (GTG)5 as single primer. Two clusters of cryptococcal isolates were distinguished at 68.5% of similarity, with cluster I consisting of VNI isolates and cluster II consisting of VGI isolates. Each cluster was further subdivided into three subtypes at >/=80% of similarity. Fourteen bird dropping isolates were grouped into a subtype within VN1, sharing 82.7% of similarity with the clinical isolates. A higher degree of similarities, ranging from 93.4-97.6% was noted between 3 bird dropping isolates with the clinical isolates in another subtype. This study demonstrated the existence of various molecular types of C. neoformans isolates in Malaysia and the genetic heterogeneity within the predominant molecular types. The study also provides evidence for genetic relatedness of clinical isolates with bird dropping isolates in the environment.
The occurrence of Cryptococcus neoformans in bird excreta in Klang valley, Malaysia was determined in this study. Of 544 samples of bird excreta collected from a local zoo, pet shops and public areas, 20 strains of C. neoformans were isolated. All C. neoformans strains were serotype A and thus identified as C. neoformans variety grubii. All did not produce color changes on canavanine-glycine-bromothymol blue agar. All were of alpha-mating types, as determined by a pheromone-specific PCR assay. The antifungal susceptibility testing using agar diffusion method Neo-sensitabs showed that all were susceptible to amphotericin B, fluconazole and itraconazole.
The in vitro susceptibilities of Malaysian clinical isolates of Cryptococcus neoformans var. grubii and C . gattii to five antifungal drugs (amphotericin B, flucytosine, fluconazole, itraconazole and ketoconazole) were determined using the Etest method. None of the Malaysian isolates was resistant to amphotericin B and ketoconazole. Isolates resistant to flucytosine, fluconazole and itraconazole were observed in this study. Minimum inhibition concentrations (MICs) of > or = 32 microg ml(-1) against flucytosine, > or = 64 microg ml(-1) against fluconazole and > or = 1 microg ml(-1) against itraconazole were noted in four (8.3%), two (4.2%) and one (2.1%) isolates respectively. There was no significant difference in the MICs for both Cryptococcus species (P > 0.05), indicating that C. gattii was as susceptible as var. grubii to all the antifungal drugs tested. No significant difference in the MICs for both Cryptococcus species collected from 1980 to 1990 and 2002 to 2004 were observed (P > 0.05).
This study describes the isolation of Cryptococcus neoformans and Cryptococcus gattii from patients with chronic meningitis who were admitted to 16 Malaysian hospitals, from 2003 to 2004. Of the 96 cryptococcal cases reported over the 2-year period, 74 (77.1%) patients were male and 45 (46.9%) patients were between 30 and 39 years old. Cryptococcosis was uncommon in children. A total of 57 (59.4%) and 23 (24.0%) patients were Malay and Chinese respectively. Human immunodeficiency virus infection was the major underlying disease reported in 36 (37.5%) patients. C. neoformans var. grubii (serotype A and molecular type VNI) was the predominant Cryptococcus species isolated from 88.5% of cryptococcal cases in this country. Cryptococcal cases due to C. neoformans var. grubii were reported from all the five regions in Malaysia, with the most number of cases reported from the central and northern regions. Cryptococcus gattii (all were serotype B and molecular types VGI/II) was isolated from all regions except the southern region. Compared with a study conducted prior to the AIDS era, our findings show substantial changes in the demographical characteristics of patients.
Invasive Neisseria meningitidis infection is rare but carries a high mortality rate. The carriage rate in the normal population is around 10% and can be higher in confined populations. A study on the prevalence of carriage of N. meningitidis was conducted among 3195 army recruits after 2 months of intensive training in an army camp. N. meningitidis was isolated from 37.0% of these recruits. Two hundred and ten of N. meningitidis isolates were subjected to serogrouping and 100 to antibiotic sensitivity testing by the disc diffusion method and E-test for penicillin. Ten (4.8%) of 210 Neisseria meningitidis serogrouped belonged to serogroup W135, 3.33% serogroup A and 81.4% belonged to either serogroup X, Y or Z. With the agar disc diffusion method, all the N. meningitidis showed susceptiblity to chloramphenicol, rifampicin, cefotaxime and levofloxacin; 85% of the strains were resistant to cotrimoxazole and 12.5% resistant to penicillin. However, based on minimum inhibitory concentration, none of the Neisseria meningitidis tested was resistant to penicillin.
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.
The diagnostic value of adenosine deaminase (ADA) activity was studied to evaluate its use in the differential diagnosis of tuberculous meningitis in the local setting. Cerebrospinal fluid (CSF) from 119 patients with meningitis and other conditions with central nervous system symptoms were collected and ADA activity determined by the colorimetric method of Guisti read at 628 nm. The CSF was also subjected to other laboratory examinations so as to provide the aetiological diagnosis. All 14 tuberculous meningitis patients had ADA activity greater than the cut off value of 9.0 IU/L. High ADA activity was also seen in 13 of 105 non-tuberculous cases giving a specificity of 87.6%. Even though the ADA activity determination is sensitive for tuberculosis, it was not specific enough to be used as a rapid diagnostic test. However when interpreted together with clinical signs and symptoms and other laboratory tests, it is a useful adjunctive rapid marker for tuberculosis.
The in-vitro susceptibility of quinupristin/dalfopristin, levofloxacin and moxifloxacin against methicillin-resistant Staphylococcus aureus (MRSA) strains, which are also resistant to fusidic acid and rifampicin were carried out to determine whether these antibiotics can be used as an alternative treatment for multiply resistant MRSA strains. The minimum inhibitory concentrations (MIC) of these antibiotics were determined by E-test. Quinupristin/dalfopristin had good activity (MIC90 = 1 mg/L) against these strains while most of the strains showed intermediate resistance to moxifloxacin with MIC90 = 2 mg/L). However, more than 90% of these strains were resistant to levofloxacin with the MICs that ranged from 8 mg/L to 16 mg/L with the majority inhibited at 8 mg/L.
Neisseria meningitidis is one of the important cause of meningitis and has been extremely susceptible to penicillin. Nevertheless, moderately penicillin resistant strains have been reported in some parts of the world. To our knowledge, there is no such report in Malaysia. We report two clinical isolates that were found to have MICs of decreased susceptibility to penicillin by the agar dilution method.
Ureaplasma urealyticum was isolated from the endotracheal aspirates of 39 (21.4%) of 182 neonates with respiratory distress requiring ventilatory support. Mycoplasma hominis was isolated from one (0.5%) neonate. Bacterial cultures were negative in 123 (67.6%) neonates. Antibiotic susceptibility test carried out on ten isolates of U. urealyticum showed that all the organisms were sensitive to erythromycin but resistant to lincomycin and sulfamethoxazole trimethoprim. All, except one, U. urealyticum were sensitive to tetracycline and minocycline. Two isolates were resistant to ciprofloxacin. This study showed that U.urealyticum was a common organism isolated from the endotracheal aspirates of neonates with respiratory distress.
Melioidosis is endemic in Malaysia. Emerging resistance with new and current antimicrobial agents has underscored the need to look further for new antimicrobial agents for the treatment of melioidosis. Hence, we evaluated the in-vitro susceptibility of fifty locally isolated strains of Burkholderia pseudomallei, the causative agent of melioidosis to cefoperazone-sulbactam combination using the method of NCCLS. All the fifty strains tested were susceptible in-vitro to cefoperazone-sulbactam. The MIC90 of the organism for cefoperazone-sulbactam was 4 mg/L. The results of our findings suggested that cefoperazone-sulbactam may be useful in the treatment of melioidosis.
Isolation of rickettsiae from patients' blood samples and organ samples of wild rodents from areas with high seroprevalence of rickettsial infections was attempted using cell culture assay and animal passages. L929 mouse fibroblast cells grown in 24 well tissue culture plate were inoculated with buffy coat of febrile patients and examined for the growth of rickettsiae by Giemsa, Gimenez staining and direct immunofluorescence assay. No rickettsiae were isolated from 48 patients' blood samples. No symptomatic infections were noted in mice or guinea pigs infected with 50 organ samples of wild rodents. There was no rickettsial DNA amplified from these samples using various PCR detection systems for Orientia tsutsugamushi, typhus and spotted fever group rickettsiae.
In this study, the presence of IgG and IgM antibodies against Borrelia burgdorferi (strain B. afzelii) among Malaysian blood donors and patients admitted to hospital with various infectious diseases was determined. Sera were screened using enzyme-linked immunosorbent assay (ELISA); positive sera were then subjected to Western blot testing. All but one of the blood donors were negative for borrelial antibodies. Of 121 patients' sera, IgM antibodies were detected in 24 (19.8%) and IgG antibodies were detected in 5 (4.1%) sera. Only one of two patients with skin manifestations suggestive of Lyme disease had IgM antibody against B. afzelii. Of 30 patients with exposure to tick typhus, 4 (13.3%) were IgM positive and 1 (3.3%) was IgG positive. Based on the detection of antigenic bands by Western blot, 6 patients' sera showed positive reactions. Antigenic bands of p39, p41 and p59/62 kDa were the commonest findings of Western blotting. This study provides serological evidence of B. afzelii infections in Malaysia; further investigation is needed to correlate serological and clinical findings.
The pathogenicity of Malaysian isolates of Orientia tsutsugamushi was investigated by a mouse virulence assay. The isolates could be differentiated as low (4 isolates), moderately (3 isolates) and highly virulent (2 isolates) based on the different responses in infected mice. No direct correlation between severity of human scrub typhus infections and virulence of the O. tsutsugamushi in mice was observed. Mice infected with virulent strains of O. tsutsugamushi showed splenomegaly, ascitis accumulation and enlargement of kidneys and livers whereas avirulent O. tsutsugamushi strains were asymptomatic and exhibited ruffled fur for a short period after infection. There was low antibody response in mice infected with isolates of low pathogenicity as compared with those of highly virulent isolates. Upon dissection of the infected mice, enlargement of mouse organs such as spleen, kidney and liver was noted. Presence of rickettsemia in mice was confirmed by the growth of O. tsutsugamushi in the L929 cells when inoculated with blood from infected mice. O. tsutsugamushi was also cultured from the peritoneal exudates of the infected mice. However, DNA of O. tsutsugamushi was only detected in the peritoneal exudates (by PCR) and blood (by cell culture) and not from other tissue samples.