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Acute systemic melioidosis. Autopsy findings in four patients.
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Trimethoprim as a possible therapy for nocardiosis and melioidosis.
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[Accelerated diagnostic cultivation of pathogenic environmental microbes. II. Accelerated diagnostic cultivation of the causative agents of anthropozoonotic plague, glamders, melioidosis, brucellosis and tularemia].
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[Mobilization of a cryptic plasmid from the melioidosis pathogen in heterologous species of microorganisms].
Cryptic plasmids with different molecular weights have been detected in B. pseudomallei strains. Mobilization of B. pseudomallei plasmid DNA in heterologous B. mallei species was performed by the conjugate plasmid RP1::Tn10. Possibility of detecting phenotypical characteristics of plasmids and behavior of B. pseudomallei non-chromosomal replicons in B. mallei have been determined.
[The properties of the extracellular alkaline phosphatase of the causative agent of melioidosis].
After growing P. pseudomallei VPA on solid medium extracellular alkaline phosphatase with a molecular weight of 93,000 AMU was isolated, and practically purified from the extract of this medium by precipitation with ammonium sulfate, subsequent gel chromatography and concentration on membrane filters. The optimum conditions for enzymatic reaction were found to be pH 9.0 and a temperature of 50 degrees C. The enzyme was resistant to freezing and to heating at a temperature of up 60 degrees C for 30 minutes, as well as to the action of pH 3.0-10.5, but became completely inactivated after heating at 90 degrees C for 10 minutes and incubation at pH 2.0 for 20 hours.
[Comparative characteristics of the ultrastructure of the causative agents of glanders and melioidosis].
The authors examined the ultrastructure of the causative agents of glanders and melio idosis. It was revealed that the structure of their cell wall and of the cytoplasmic membrane was characteristic of Gram negative bacteria. The cytoplasm of both types of the causative agents showed the presence of ribosomes, membrane structure, nucleoid, and also osmiophilic and osmiophobic inclusions.
[The acid exopolysaccharide of the causative agent of melioidosis].
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In vitro activity of sparfloxacin against selected enterobacteriaceae and pseudomonaceae including Ps. pseudomallei (melioidosis).
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Burkholderia pseudomallei: the unbeatable foe?
Although melioidosis has been recognized in Thailand for many years and considerable progress in term of diagnosis and treatment was achieved, B. pseudomallei is still "the unbeatable foe", for several reasons as outlined here: under-recognition, high case-fatality rate, unacceptable relapse rate and a "time-bomb" for sero-positive patients. Melioidosis is largely restricted to certain geographical areas. In Thailand, it had long been considered a rare disease in Thailand until ten cases with culture-proven melioidosis were reported by Sompone Punyagupta and his associates at a meeting of the Infectious Disease Group of Thailand. Since then awareness of young physicians and laboratory personnel for melioidosis has been increased. The most dramatic consequence was seen at Sappasitprasong Hospital in Ubon Ratchathani where over 100 strains of B. pseudomallei are isolated each year. But the frequent isolation of B. pseudomallei is surprisingly restricted to some provinces in the northeast, namely Khon Kaen and Ubon Ratchathani provinces and only 1-10 cases or none from adjacent provinces. The discrepancy was well illustrated by mapping the number of isolations by province. Thus many cases of septicemic melioidosis are certain to receive inappropriate chemotherapy and nearly half of them probably leave this world without proper diagnosis in area where under-recognition unfortunately still prevails. Mortality in disseminated septicemic melioidosis used to occur in 82-87% of the patients who were treated with doxycycline, chloramphenicol, cotrimoxazole and kanamycin and in non-disseminated septicemic melioidosis about 20%. With ceftazidime therapy, the mortality rate was cut by half to 35-40%. About 50% of the patients deteriorated rapidly and died within the first few days of fever. Fatalities are related to the speed of positive results of blood culture. Accordingly, awareness of the disease, familiarity of clinical syndrome compatible with septicemic melioidosis, gram-staining of exudate to include or exclude melioidosis, are all crucial factors to lead to proper empiric chemotherapy. Since the addition of anti-cytokine and platelet activating factor receptor antagonist to current antimicrobials failed to lower the mortality rate, we need to find a new antimicrobial such as protegrin-1 which exhibits rapid microbicidal activity, especially against stationary-phase cell. We need to optimize the bactericidal action of currently used antimicrobials by examining their pharmacokinetics. With prolonged maintenance treatment with cotrimoxazole plus doxycycline or co-amoxiclav, relapse occurs in 4 to 23%. Various explanations for the relapse are the ability of the organism to produce glycocalyx, form microcolonies in damaged tissues and survive within phagocytic cells. Again, the bactericidal antimicrobial which is concentration-dependent, may be used to shorten the duration of treatment and reduce relapse. Studies so far can not relate relapse to any defect of host defense mechanism. In endemic areas, seroepidemiological surveys showed that infection, mostly latent, occurred fairly commonly since childhood as 80% of children had antibodies by the age of four years. However, clinical melioidosis is more common in the elderly which in some cases are due to reactivation of primary latent infection. Since the incubation period of the reactivation can vary from weeks to many years, a vaccine or short-course secondary chemoprophylaxis may be possible interventions for the high risk group to get rid of the "time-bomb" reactivation. The vaccine may also be used to reduce the relapse rate. We need to discover the cellular determinants which is critical to awake the host defense to the sleeping bacteria and provoke local inflammatory response to newly born bacteria before dissemination takes place again. Basic research into the pathogenic mechanisms are key to understanding how to make an effective vaccine.
Detection of immunoglobulins M and G using culture filtrate antigen of Burkholderia pseudomallei.
IgM and IgG based ELISA systems were developed using the culture filtrate antigen (CFA) of Burkholderia pseudomallei. The assays were evaluated using 95 sera from 66 septicemic cases and 47 sera from 20 cases with localized melioidosis. In addition 65 sera from culture negative cases that were also serologically negative for other endemic infections clinically suspected of melioidosis were included. These were compared with sera from 260 non-melioidosis cases, 169 sera from individuals with high risk of acquiring the infection and 48 sera from healthy controls. The IgG-ELISA was 96% sensitive and 94% specific. All sera from cases with septicemic and localized infections and 61 of 63 sera from clinically suspected melioidosis cases were positive for IgG antibody. The geometric mean titre index (GMTI) values of IgG antibody in melioidosis cases were significantly higher (p < 0.0005) compared to that of healthy subjects, high risk group and subjects with non-melioidosis infections. The sensitivity and specificity of IgM ELISA was 74 and 99% respectively. The GMTI value of IgM antibody in the sera of melioidosis cases was significantly higher as compared to that of non-melioidosis disease controls (p < or = 0.001). These results demonstrate that the detection of IgG is a better indicator of the disease in the diagnosis of melioidosis.
Polymorphism in the promoter region of tumor necrosis factor-alpha gene is associated with severe meliodosis.
Melioidosis is an important infectious disease endemic in Southeast Asia and the Northern territories of Australia. Septicemic melioidosis, is the leading cause of fatality from community acquired septicemia in northeastern part of Thailand where death often occurs within a few days after hospitalization. The present study was carried out to investigate the polymorphisms of the position -308 promoter region of the TNF-alpha gene, as well as of the intron 1 of the TNF-beta gene in patients with melioidosis compared with normal uninfected controls in the same endemic area. The gene frequency of TNF2 allele was significantly higher in melioidosis patients compared with control subjects (p = 0.0097, relative risk 2.32). The increase in TNF2 allele in melioidosis patients was found in both heterozygous and homozygous forms. In addition, the increase in TNF2 allele was most apparent in patients who had fatal outcome from septicemic melioidosis (p = 0.017), but was also observed with lesser degree in other groups of melioidosis patients. However, no difference in the frequency of TNF-beta polymorphism the melioidosis patients was observed.
Whole genome study and construction of SHERLOCK detection method for endemic strains of Burkholderia pseudomallei in Hainan based on third-generation sequencing.
UNLABELLED: Burkholderia pseudomallei (Bp) is a gram-negative bacterium found in soil and surface water. It is also the pathogen that causes melioidosis disease in humans and animals. This study aimed to obtain the whole genome sequence of the endemic strain of Bp in Hainan, using third-generation sequencing (TGS) technology, and elucidate the genome structure, function, and genetic evolution. Additionally, the study aimed to achieve rapid and specific identification of these endemic strains using specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) detection technology, providing a new strategy for the early diagnosis of melioidosis. Utilizing the PacBio platform for TGS technology, we completed whole genome sequencing of 16 Bp strains from Hainan. High-precision and complete genome sequences were obtained through quality control and genome assembly of the sequencing data. Additionally, we established a nucleic acid detection technology platform based on SHERLOCK, which could be completed from nucleic acid extraction to result reading within 1-2 hours, demonstrating good sensitivity and specificity (both are 100%). The lateral chromatography strip method does not require special equipment and holds promise as an immediate screening method for the early diagnosis of melioidosis. IMPORTANCE: Melioidosis is a highly pathogenic infectious disease caused by a gram-negative bacterium of Burkholderia pseudomallei (Bp). The traditional gold standard for diagnosing melioidosis is still isolation and culture from clinical samples. Although this method has high specificity, it has low sensitivity and is time-consuming, which often leads to misdiagnosis or missed diagnosis of melioidosis, affecting subsequent treatment. In this study, recombinase polymerase amplification technology and clustered regularly interspaced short palindromic repeats/Cas13a technology were combined to establish the Specific High-sensitivity Enzymatic Reporter Unlocking detection technology, which can achieve rapid and accurate identification of Bp, providing a new method for the early diagnosis of melioidosis.
[Infection with Pseudomonas pseudomallei].
Melioidosis, a severe, often fatal disease caused by infection with Pseudomonas pseudomallei, has been thought to be a rare endemic disease relatively limited to the areas 20 degrees on either side of the equator. However, an increasing number of people travelling to these areas are reportedly suffering from this disease. It is timely to review this disease for doctors who are unfamiliar with this disease. P. pseudomallei, first discovered by Whitmore and Krishnaswami in 1912, is a gram-negative aerobic rod, motile due to polar flagella, isolated from soil and natural waters in endemic areas, and presumably transmitted to human beings through skin abrasion, ingestion and inhalation. Associated underlying conditions must be searched for, such as diabetes mellitus and chronic renal failure. Clinical classification ranges from disseminated septicemic melioidosis, the most serious form, to subclinical melioidosis, the least serious form. Disseminated septicemic type is associated with high fever, multiple organ lesions with septic shock and high fatality rate within a few days after symptoms develop. This type of infection requires prompt institution of antimicrobial therapy as well as surgical intervention such as drainage. Antimicrobial agents should be carefully selected according to the susceptibility results of the isolates. During the suspected stage, ceftazidime is a drug of choice. Subclinical melioidosis associated with positive serologic test alone should be closely followed up against the potential reactivation of dormant infection with P. pseudomallei. We must certainly be aware of melioidosis and diagnose melioidosis as early as possible by completing the initial routine diagnostic procedures to febrile patients.
Genotype analysis of Burkholderia pseudomallei using randomly amplified polymorphic DNA (RAPD): indicative of genetic differences amongst environmental and clinical isolates.
Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease common in the tropics. Melioidosis is most prevalent in the northeastern part of Thailand. The diseases has diverse clinical manifestations ranging from mild localized to fatal septicemic forms. The bacterial genetic factors contributing to the severity of melioidosis have not been completely identified. We have developed a genotyping method based upon randomly amplified polymorphic DNA (RAPD) analysis. Eighteen deca-oligo nucleotide primers with 70% GC content, eight previously published 60%GC RAPD primers, and four random deca oligomers were tested on nine strains of B. pseudomallei isolated from five patients with localized and four with septicemic melioidosis. The RAPD patterns were analyzed by polyacrylamide gel electrophoresis using a laser based automated fragment analyzer, GS2000. Based upon the pattern complexity, seven pairs consisting of eight primers were chosen for further analysis. Six hundred and thirty-two samples, including duplicates/triplicates, of B. pseudomallei isolated from melioidosis patients and the environment were analyzed. Two controls were included in each run of the test samples. All the samples were tested and patterns analyzed by blinded technical staff. Apparently, the method is reproducible. This is indicated by the RAPD patterns of the two controls of between run assay. Interestingly, some RAPD patterns were more prevalent in the clinical isolates than the environmental specimens and vice versa. For example, Q162KKU4-0 and Q162KKU1-0 were found 3. 5 and 3.3 times more often in the clinical specimens (P<0.025). Likewise, Q162KKU1-1 and Q162KKU4-1 were found 18 and 37 times more often in the environment (P<0.0000001). In addition, there was a bias in the distribution of arabinose positive strains and particular RAPD patterns; RAPD patterns of B. pseudomallei that were found frequently in septicemic patients were less likely to be arabinose positive. The data suggest the existence of bacterial genetic differences between the clinical and environmental isolates of B. pseudomallei. Further analysis of the RAPD patterns searching for common polymorphic DNA fragments and systemic comparative genomic analysis of B. pseudomallei in accordance with the clinical data should reveal genetic factors involved in severity and bacterial pathogenesis of B. pseudomallei in melioidosis.
Splenic abscess: clinical features, microbiologic finding, treatment and outcome.
Splenic abscess is a rare clinical entity but may be underreported. A retrospective study at Srinagarind Hospital revealed 60 cases of splenic abscess between 1992 and 2001. The causative organisms were identified in 41 cases (68.3%). Gram negative bacilli were commonly isolated and Burkholderia pseudomallei was the most predominant. Diabetes mellitus and leukemia were common underlying diseases found in 46.3 per cent and 9.7 per cent of culture confirmed cases, respectively. The patients usually presented with fever, left upper quadrant pain, tenderness and splenomegaly. Multiple abscesses were more commonly found in the melioidosis than in the non-melioidosis group (p = 0.032), but a single abscess was more commonly found in the non-melioidosis than in the melioidosis group (p = 0.032). Concurrent liver abscesses, often multiple, were not different in both groups. Antimicrobials alone were given in 66.7 per cent of cases with melioidosis and 64.7 per cent of non-melioidosis group. Splenectomy and percutaneous aspiration were performed only in 29.3 per cent and 4.9 per cent of cases with splenic abscess. The overall mortality rate of splenic abscess was only 4.9 per cent in the present series. In conclusion, splenic abscess is not uncommon. Burkholderia pseudomalleli is the most common causative agent found in the present series. Therefore, it should be targeted in the initial empirical antibiotic therapy before the culture results are available especially when multiple lesions in the spleen and concurrent multiple liver abscesses are seen. Prolonged treatment with appropriate antimicrobials alone is usually effective. Splenectomy and/or aspiration may be useful in selected patients.
Seroprevalence of Burkholderia pseudomallei in East Timorese refugees: implications for healthcare in East Timor.
Melioidosis is a disease with protean clinical manifestations caused by the bacterium Burkholderia pseudomallei. It is endemic in countries surrounding the newly independent East Timor, but has yet to be isolated or demonstrated serologically in that country. One illness that can be clinically indistinguishable from melioidosis is pulmonary tuberculosis, a condition with a very high prevalence in East Timor. We used an indirect hemagglutination test (IHA) to measure antibodies to B. pseudomallei in 407 East Timorese evacuated to Darwin, Australia, in September 1999. Assuming a positive IHA titer as > or = 1:40, the overall seroprevalence rate was 17.0%, in keeping with other seroprevalence studies from the region. The IHA titres ranged up to 1:320. After adjusting for age, females were 2.5 times more likely to be seropositive than males (p = 0.0001). There was an inverse relationship between seropositivity and age. This study shows that exposure to B. pseudomallei occurs in East Timor melioidosis is also likely to occur. Due to the lack of laboratory facilities at present, it may be some time before a laboratory-confirmed case proves that melioidosis occurs. In the meantime, clinicians in East Timor should include melioidosis in the differential diagnosis of the many conditions that it may mimic.
Burkholderia pseudomallei infection in chronic granulomatous disease.
Melioidosis is a severe illness caused by Burkholderia pseudomallei, a sepsis-causing bacterial pathogen that is common in East Asia. We reexamined the underlying diagnosis in a 12-year-old boy who was diagnosed in the French West Indies with melioidosis when he was 4 years old. Our investigations led to the determination of chronic granulomatous disease (CGD), an inherited condition characterized by phagocytic cell dysfunction. This is the third reported case of melioidosis associated with CGD to be reported, lending support to an association between the two. The molecular determinants of the susceptibility of CGD patients to B. pseudomallei are still unknown. Our report suggests that CGD should be suspected in children with melioidosis, both in the Americas and probably in other regions world-wide.