Imported case of Leishmania tropica cutaneous leishmaniasis in a 10-year-old child in Malaysia

@#The present paper reported a first imported case of cutaneous leishmaniasis in a 10-yearold child who returned from Saudi Arabia to Malaysia. Six weeks after his travel to Malaysia, two erythematous dermal nodules were developed over his right cheek and chin. Occurrence of intracellular amastigote of Leishmania was observed through examination of skin biopsy with hematoxylin and eosin stain. Furthermore, molecular analysis of ribosomal internal transcribed spacer 1 (ITS1) of Leishmania spp. confirmed the child was infected with Leishmania tropica. The child was given oral fluconazole and he had a 80% recovery before he went back to Saudi Arabia.

Serological review of Bartonella henselae and Bartonella quintana infection among Malaysian patients with unknown causes of febrile illnesses

@#Limited information is available on human exposure to Bartonella infection, i.e., Bartonella henselae (causative agent of cat scratch disease) and Bartonella quintana (causative agent of trench fever) in West Malaysia. This study reports a review of serological findings obtained from patients attending to a teaching hospital in Klang Valley, Malaysia. An indirect immunofluorescence assay (IFA) was used to determine IgG and IgM antibody titers against B. henselae and B. quintana. In a pilot study conducted between 2013-2015, IgG antibodies against Bartonella spp. (either B. quintana and B. henselae) were detected in 14 (36.8%) of 38 patients who were clinically suspected of rickettsial infections, while IgM antibody was detected in 4 (10.5%) patients. This has prompted us to investigate the serologic responses of patients who were clinically suspected of other febrile causes besides rickettsial infection. Of the 59 serum samples analysed in a follow-up investigation, Bartonella IgG antibodies were detected from 7 (11.9%) patients, of which 5 (27.8%) and 2 (18.2%) patients were clinically suspected of rickettsial infection (n=18) and dengue (n=11), respectively. None of the sera obtained from the leptospirosis (n=10), legionellosis (n=10) and mycoplasma infection (n=10) groups were seropositive to Bartonella spp. The review of Bartonella serological findings in this study highlights that Bartonella infection is not uncommon and should be considered as one of the causes for febrile illness in Malaysia.

Investigation of possible rickettsial infection in patients with malaria

@#Rickettsioses are a common health problem in many geographical areas, including rural areas in Southeast Asia. Co-infection of rickettsioses and malaria has been reported in Africa, where common reservoir and vectors are available. In this study, blood samples of Malaysian patients microscopically positive (n=148) and negative (n=88) for malaria parasites (Plasmodium knowlesi, Plasmodium malariae, Plasmodium falciparum, and Plasmodium vivax) were screened for the presence of rickettsial DNA, using PCR assays targeting specific genes. A partial fragment of rickettsial ompB gene was successfully amplified and sequenced from a patient microscopically positive for Plasmodium spp. and PCR-positive for P. vivax. BLAST analysis of the ompB sequence demonstrated the highest sequence similarity (99.7% similarity, 408/409nt) with Rickettsia sp. RF2125 (Genbank accession no. JX183538) and 91.4% (374/409 nt) similarity with Rickettsia felis URRWXCal2 (Genbank accession no. CP000053). This study reports rickettsial infection in a malaria patient for the first time in the Southeast Asia region.

Genetic diversity, antifungal susceptibility and enzymatic characterisation of Malaysian clinical isolates of Candida glabrata

@#Candida glabrata has been reported as the second or third most common yeast species isolated from patients with vaginitis and invasive candidiasis. This study was aimed to determine the genetic diversity, antifungal susceptibility and enzymatic profiles of C. glabrata isolated from vaginal and blood samples in the Medical Microbiology Diagnostic Laboratory, University Malaya Medical Centre. A random amplified polymorphic DNA (RAPD) analysis method, using M13 and (GTG)5 primers, was used for strain differentiation of C. glabrata isolates. Antifungal susceptibility testing of C. glabrata isolates was determined using E-test against amphotericin B, caspofungin, fluconazole and voriconazole and microbroth dilution method against clotrimazole. The enzymic profiles of C. glabrata were determined using APIZYM semi-quantitation kit and egg-yolk agar method. A total of 14 RAPD patterns were identified amongst C. glabrata isolates investigated this study. Susceptibility to amphotericin B, caspofungin, fluconazole and voriconazole was noted. Approximately one third of the isolates demonstrated resistance to clotrimazole (MIC>1 μg/ml). A single isolate of C. glabrata was resistant to caspofungin (MIC:1.5 μg/ml). Enzymatic activities of acid and alkaline phosphatase, aminopeptidases, esterase and lipase and phospholipase were detected in the C. glabrata isolates. The genetic diversity and antifungal susceptibility profiles of C. glabrata isolates were presented in this study. Continued surveillance and monitoring of the incidence and antifungal resistance in C. glabrata isolates is necessary.

Burkholderia pseudomallei lectins: occurrence and expression

Lectins, also known as sugar binding proteins, play an essential role in the initiation of bacterial infections and biofilm production. To date, several lectins of Gram-negative bacteria such as Pseudomonas aeruginosa, Burkholderia cenocepacia, Ralstonia solanacearum and Chromobacterium violaceum have been identified. There are no published reports on the presence of lectins in Burkholderia pseudomallei, the causative agent of melioidosis. The aim of this study was to identify possible lectin genes of B. pseudomallei and generate recombinant proteins for assessment of hemagglutinating activity. Seven hypothetical lectins of B. pseudomallei were retrieved from the UniProt database. Four lectin domains, i.e., ricin B, C-type, H-type and Bulb-type lectins were identified. In silico analysis using a ligand binding site prediction server (3DLigandSite) predicted the presence of Nacetylglucosamine and calcium binding sites in two C-type lectins. Four recombinant proteins with the molecular weights of 11.7, 30.2, 36.2 and 46.4 kDa were expressed from the cloned genes; however none of them expressed any hemagglutinating activity. Further characterization of B. pseudomallei lectins may be able to provide insights into bacterial-host interaction that are required to initiate infections.

Molecular survey and sequence analysis of Anaplasma spp. in cattle and ticks in a Malaysian farm

This study was conducted to determine the occurrence of Anaplasma spp. in the blood samples of cattle, goats, deer and ticks in a Malaysian farm. Using polymerase chain reaction (PCR) and sequencing approach, Anaplasma spp. was detected from 81(84.4%) of 96 cattle blood samples. All blood samples from 23 goats and 22 deer tested were negative. Based on the analysis of the Anaplasma partial 16S ribosomal RNA gene, four sequence types (genotypes 1 to 4) were identified in this study. Genotypes 1-3 showed high sequence similarity to those of Anaplasma platys/ Anaplasma phagocytophilum, whilst genotype 4 was identical to those of Anaplasma marginale/ Anaplasma centrale/ Anaplasma ovis. Anaplasma DNA was detected from six (5.5%) of 109 ticks which were identified as Rhipicephalus (formely known as Boophilus) microplus ticks collected from the cattle. This study reported for the first time the detection of four Anaplasma sequence types circulating in the cattle population in a farm in Malaysia. The detection of Anaplasma DNA in R. microplus ticks in this study provides evidence that the ticks are one of the potential vectors for transmission of anaplasmosis in the cattle.

Investigation on the conA binding properties of Klebsiella pneumoniae

Klebsiella pneumoniae is a healthcare-associated bacterial pathogen which causes severe diseases in immunocompromised individuals. Concanavalin A (conA), a lectin which recognizes proteins with mannose or glucose residues, has been reported to agglutinate K. pneumoniae and hence, is postulated to have therapeutical potential for K. pneumoniaeinduced liver infection. This study investigated the conA binding properties of a large collection of clinical isolates of K. pneumoniae. ConA agglutination reaction was demonstrated by 94 (51.4%) of 183 K. pneumoniae isolates using a microtiter plate assay. The conA agglutination reactions were inhibited in the presence of 2.5 mg/ml D-mannose and 2.5 mg/ml glucose, and following pretreatment of the bacterial suspension with protease and heating at 80ºC. Majority of the positive isolates originated from respiratory specimens. Isolation of conA-binding proteins from K. pneumoniae ATCC 700603 strain was performed using conA affinity column and the conA binding property of the eluted proteins was confirmed by western blotting analysis using conA-HRP conjugates. Proteins with molecular weights ranging from 35 to 60 kDa were eluted from the conA affinity column, of which four were identified as outer membrane protein precursor A (37 kDa), outer membrane protein precursor C (40 kDa), enolase (45 kDa) and chaperonin (60 kDa) using mass spectrometry analysis. Several conA binding proteins (including 45 and 60 kDa) were found to be immunogenic when reacted with rabbit anti-Klebsiella antibody. The function and interplay of the conA binding proteins in bacterium-host cell relationship merits further investigation.

Enzymatic profiling of clinical and environmental isolates of Burkholderia pseudomallei

Abstract. Melioidosis has been recognized as an important cause of sepsis in the tropics. The disease caused by an environmental saprophyte Burkholderia pseudomallei, affects mostly adults with underlying immunocompromised conditions. In this study, the enzymatic profiles of 91 clinical and 9 environmental isolates of B. pseudomallei were evaluated using the APIZYM system, in addition to assessment of protease, phospholipase C and sialidase activities using agar plate methods and other assays. The activity of 10 enzymes - alkaline phosphatase, esterase, esterase lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β-glucosaminidase were detected in >75% of the clinical isolates. The majority of B. pseudomallei isolates in this study exhibited protease and phospholipase activities. No sialidase activity was detected. Five Burkholderia thailandensis isolates had similar APIZYM profiles as B. pseudomallei clinical isolates except for the lower detection rate for N-acetyl-β-glucosaminidase. The subtle differences in the number of enzymes secreted and the levels of enzymatic activities of phenotypically identical clinical and environmental strains of B. pseudomallei give weight to the fact that the causative agent of melioidodis originates from the environment.

Diversity and killer activity of yeasts in Malaysian fermented food samples

The biodiversity and the killer activity of yeasts isolated from various types of fermented food in Malaysia were investigated in this study. Of 252 yeasts isolated from 48 fermented food samples in this study, 19 yeast species were identified based on sequence analysis of the ITS1-5.8S-ITS2 partial fragments of the yeasts. A total of 29 (11.5%) of the yeast isolates demonstrated killer activity to at least one Candida species tested in this study; including 22 isolates of Trichosporon asahii, 4 isolates of Pichia anomala, and one isolate each of Pichia norvegensis, Pichia fermentans and Issatchenkia orientalis, respectively. The presence of killer yeasts reflects antagonism that occurs during microbial interaction in the fermented food, whereby certain yeasts produce killer toxins and possibly other toxic substances in competition for limited nutrients and space. The anti-Candida activity demonstrated by killer yeasts in this study should be further explored for development of alternative therapy against candidiasis.

Reduced susceptibility of Malaysian clinical isolates of Burkholderia pseudomallei to ciprofloxacin

Ciprofloxacin, a quinolone with good intracellular penetration may possibly be used for treatment of melioidosis caused by Burkholderia pseudomallei, but problems with resistance may be encountered. Amino acid substitutions in gyrA/gyrB have given rise to fluoroquinolone resistance in various microorganisms. Using published primers for gyrA and gyrB, PCR was performed on 11 isolates of B. pseudomallei with varying degrees of sensitivity to ciprofloxacin, followed by DNA sequencing to detect possible mutations. Results showed an absence of any point mutation in either gene. Local isolates have yet to develop full resistance to ciprofloxacin and probably other mechanisms of resistance may have been involved in the decreased sensitivity to ciprofloxacin.

The integron prevalence of extended-spectrum beta-lactamase Malaysian teaching hospital

The increased frequency of antibiotic resistance is known to be associated with the dissemination of integrons in the Enterobacteriaceae. This study determined the prevalence and type of integrons amongst 160 extended-spectrum beta-lactamase producing enterobacterial isolates kept in our culture collection. Integrons were detected in 98(61.3%) isolates, including 28(62.2%) Escherichia coli, 34(64.2%) Klebsiella spp., 27(61.4%), Enterobacter spp. and 9(50.0%) Citrobacter spp. investigated in this study. Restriction analysis of the integron gene fragments revealed that class I integron was the principal integron detected in 92(57.5%) of our isolates. Class II integron was detected in 6(3.8%) of our isolates, while no class III integron was detected in this study. The high rates of integron prevalence particularly of the class I integron in the E. coli and Klebsiella spp. concur with previous studies in other geographical regions. The higher (>50%) integron prevalence of Citrobacter and Enterobacter isolates comparing to previous studies suggests the potential of these isolates as sources for dissemination of resistance determinants. The finding in this study serves as a basis for further study on the antibiotic resistance mechanisms of enterobacterial species in this teaching hospital.

In vitro demonstration of the hemolytic, cytotoxic activities and induction of apoptosis in Orientia tsutsugamushi infected L929 mouse fibroblast cells.

Using cultured mouse fibroblast L929 cells, this study demonstrated the hemolytic and cytotoxic activities and induction of apoptosis in cells infected with Orientia tsutsugamushi. Low levels of hemolytic activity were detected using heavily infected cells. No hemolysin or cytotoxin were detected in the infected culture fluid regardless of the pathogenicity of the O. tsutsugamushi strains in mice. Using propidium iodide uptake assay, acridine orange/ethidium bromide staining and terminal deoxynucleotide transferase-mediated dUTP-digoxigenin nick-end labeling assay, apoptosis was observed in L929 cells infected with Karp and Gilliam strains.

Antibody prevalence of Orientia tsutsugamushi, Rickettsia typhi and TT118 spotted fever group rickettsiae among Malaysian blood donors and febrile patients in the urban areas.

The seroprevalence of Orientia tsutsugamushi (OT), Rickettsia typhi (RT) and TT118 spotted fever group rickettsiae (SFGR) among blood donors and febrile Malaysian patients in the urban areas was determined. Of the 240 blood donors, 5.4%, 9.2% and 1.7% had either present or previous exposure to OT, RT and SFG rickettsiae, respectively. Patients admitted to an urban hospital had high seroprevalences of OT (43.5%) and RT (22.9%), as compared to SFGR (11.6%). Antibody levels suggestive of recent infections of scrub typhus, murine typhus and tick typhus were detected in 16.8%, 12.7% and 8.2% of patients respectively. No significant difference was noted in the distribution of rickettsial antibodies among urban patients from 2 geographical locations. However, the serologic patterns of rickettsial infection in the urban areas were different form those of rural areas.

Borrelia burgdorferi (strain B. afzelii) antibodies among Malaysian blood donors and patients.

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.

Isolation and PCR detection of rickettsiae from clinical and rodent samples in Malaysia.

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.

Antigenic types of Orientia tsutsugamushi in Malaysia.

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.

Virulence of Malaysian isolates of Orientia tsutsugamushi in mice.

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.

The use of the indirect immunoperoxidase test for the serodiagnosis of rickettsial diseases in Malaysia.

The indirect immunoperoxidase (HP) test has been used extensively in most government hospitals in Malaysia for the serodiagnosis of scrub typhus, murine typhus and tick typhus during the 1990s. The test was used to determine the IgG and IgM antibody titers in patients' sera for three rickettsial species, ie Orientia tsutsugamushi OT; the causative agent of scrub typhus), Rickettsia typhi (RT; the causative agent of murine typhus), and TT118 spotted fever group rickettsiae (TT; the causative agent of tick typhus). The serological findings obtained from Malaysian hospitals using the IIP test (1994-1999) were analyzed. During the six-year period, a total of 61,501 patients' sera were tested, of which 9.6%, 10.5%, and 12.9% had antibody (IgG and/or IgM of > or = 1:50) for OT, RT and TT respectively. A total of 8.6%, 9.8%, and 9.7% of sera had IgG antibody of > or = 1:50 for OT, RT, and TT respectively, indicating past infection. A total of 3.4%, 3.8%, and 6.4 % of sera had IgM antibody of > or = 1:50 for OT, RT, and TT respectively, indicating recent infection. A total of 2,986 (4.9%), 1,882 (3.1%), and 1,574 (2.6%) of sera had IgG and/or IgM antibody titers of > or = 1:400 for OT, RT, and TT respectively, suggesting active rickettsial infection. The seropositivity rates of OT, RT and TT varied according to geographical locations. While the seropositivity of OT remained constant during the six-year period, a reduction in the seropositivity of both RT and TT was noted during recent years. The serological findings reflect the endemicity of rickettsial diseases, including tick typhus, and endemic typhus in various parts of Malaysia. Awareness of these diseases by health and medical staff and by the general public is important if the mortality and morbidity associated with scrub typhus, tick typhus, and murine typhus in Malaysia, are to be reduced.

Detection of Mycoplasma pneumoniae from cerebrospinal fluid samples of pediatric patients.

Central nervous system manifestations are probably the most frequent extrapulmonary complications of infections due to Mycoplasma pneumoniae, occur mostly in children. In this study, we attempted to isolate M. pneumoniae and to detect the organism by polymerase chain reaction (PCR) from cerebrospinal fluid samples (CSF) of pediatric patients. Of the 244 CSF samples, no M. pneumoniae was isolated. Six (2.5%) of the CSF samples were positive by PCR amplification. More effort are necessary to isolate the organism from CSF samples in order to ascertain the role of M. pneumoniae in causing neurological complications.