Identification of Pseudomonas pseudomallei in clinical practice: use of simple screening tests and API 20NE.

The API 20NE kit and a simple screening system involving Gram's stain, the oxidase reaction, colistin and gentamicin resistance, and colonial characteristics on a differential agar medium, were used to test 400 strains of Pseudomonas pseudomallei. The API kit identified 390 (97.5%) strains correctly on first testing and all but one of the remainder on second testing. Only one strain was initially misidentified (as Ps cepacia). The screening system was 100% accurate in identifying Ps pseudomallei. In non-endemic areas the API 20NE kit may be used to identify sporadic imported strains of Ps pseudomallei. Such kits may also help to delineate the geographical distribution of melioidosis. In endemic areas the screening tests described offer a cheap, simple, and accurate means of presumptively identifying Ps pseudomallei from clinical specimens.

Introduction and spread of multi-resistant Shigella dysenteriae I in Thailand.

Outbreaks of Shigella dysenteriae I occurred in northeastern Thailand in the fall of 1986 and again in the spring and fall of 1987 for the first time in over 20 years. The epidemic strain of S. dysenteriae I was resistant to tetracycline, streptomycin, chloramphenicol, and trimethoprim-sulfamethoxazole, but susceptible to ampicillin. Trimethoprim resistance was chromosomally encoded by type I dihydrofolate reductase. In Ubon Province, where 10,000 cases of dysentery were reported, there were 3-5 cases of dysentery per 1,000 residents during the peak months, with 2-5 hospitalizations per 100 cases of reported dysentery. There were 2 deaths among 101 hospitalized, culture-confirmed cases. The overall case-fatality rate among reported cases of dysentery in this province was 0.9%. In contrast to S. flexneri infections, which occurred predominantly among children less than 5 years old, S. dysenteriae I infections occurred in all age groups. The large number of susceptibles appeared to be important in allowing rapid spread of S. dysenteriae I. In 1 village, 46% of 434 villagers reported dysentery; S. dysenteriae I was isolated from 24 out of 81 (30%) individuals cultured. Based on the prevalence of IgG antibody to S. dysenteriae I lipopolysaccharide, it was estimated that 76% of the villagers had been infected.

Monoclonal antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia.

A monoclonal antibody-based latex agglutination (MAb-LA) test was employed for the rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia. These patients were admitted to 12 hospitals in the northeastern part of Thailand which is a region known to be endemic for melioidosis. Blood samples were collected and immediately added to the blood culture bottles which were incubated in either automated (five hospitals) or manual (seven hospitals) culture systems. Of a total of 1369 culture-positive specimens, 204 specimens were culture-positive for B. pseudomallei. Of those, 194 (95%) were positive by MAb-LA and the type of blood culture system did not affect positivity rates. The performance of the MAb-LA test on these specimens was highly satisfactory compared with culture detection and confirmation by biochemical test, with 95.1% sensitivity, 99.7% specificity and 98.8% and 99.2% for positive and negative predictive values, respectively. The method described is highly reproducible, simple to perform even by inexperienced laboratory personnel and does not require expensive or elaborate equipment.

Evolution of cell-surface acid phosphatase of Burkholderia pseudomallei.

Acid phosphatase active fractions were obtained from cell-free extract, outermembrane fraction and culture filtrate of Burkholderia pseudomallei by column chromatography with sepharose 6B and DEAE cellulose. The comparison of the elution patterns of protein, sugar and enzymatic activity among these three components suggested that the enzyme is a glycoprotein evolving from premature proteins through glycosylation and that the enzyme is translocated during glycosylation from the cytoplasm to the outer membrane and finally excreted into the environment. When tunicamycin, a glycosylation inhibitor, was added to the culture, the peaks of sugar and enzymatic activity were lowered concomitantly leaving the protein peak unchanged in the elution pattern of the culture filtrate. The affinity of the bacterial surface to antienzyme sera was demonstrated by immuno-fluorescence microscopy.

Separation of antigenic glycoprotein fractions from cell-free homogenate of Pseudomonas pseudomallei and characterization as tyrosine phosphatase.

Cell-free extracts were prepared from Pseudomonas pseudomallei cells by freezing-thawing, sonication, and differential ultracentrifugation. The extracts were subjected to column chromatography with DEAE-sepharose to obtain glycoprotein fractions. The fractions showed acid phosphatase activity to p-nitrophenyl phosphate, tyrosine phosphate, serine phosphate, but not to threonine phosphate. They were highly antigenic when tested by immunofluorescence assay with the sera of melioidosis patients.

Use of endotoxin antigens in enzyme-linked immunosorbent assay for the diagnosis of P. pseudomallei infections (melioidosis).

An enzyme-linked immunosorbent assay (ELISA) with endotoxin preparations of P. pseudomallei as antigen was developed for detection of IgG antibodies specific to melioidosis. Forty-seven sera of bacteriologically confirmed melioidosis patients, 55 non-melioidosis sera and 50 sera of healthy blood donors from non-endemic areas were subjected to this assay in comparison with indirect hemagglutination assay (IHA). The data were treated by receiver operating characteristics analysis. The sensitivity, specificity and accuracy in this ELISA were 95.7%, 94.2%, and 94.7%, respectively, with cut-off value of OD = 0.312 at 490 nm. Meanwhile, those in IHA were 81.0%, 91.4%, and 88.1%, respectively, with a cut-off value of > or = 1:160. From these results, the ELISA was judged to be more reliable than IHA as the seroassay for diagnosis of melioidosis.

Application of indirect immunofluorescence microscopy to colony identification of Pseudomonas pseudomallei.

Indirect immunofluorescence microscopy was used as a colony identification method of Pseudomonas pseudomallei isolates. The antisera against lipopolysaccharide and protein fractions of P. pseudomallei were prepared in guinea pigs and rabbits. With these antisera and fluorescence-labelled anti-guinea pig IgG and anti-rabbit IgG prepared in sheep (goat), indirect immunofluorescence microscopy was conducted on the colonies of P. pseudomallei and other species of bacteria. The overall results indicated that this method is efficient, rapid and specific for identification of P. pseudomallei colonies from clinical specimens.

Chlamydophila (Chlamydia) pneumoniae as a cause of community-acquired pneumonia in Thailand.

Chlamydophila (Chlamydia) pneumoniae infection is increasingly reported worldwide nowadays. We studied twelve Thai adults presenting with the clinical symptoms and signs of community-acquired pneumonia (CAP) due to C. pneumoniae (TWAR) at Pramongkutklao Hospital in Bangkok, Thailand. Their mean age was 38 (range 21-73) years. Six patients lived in Bangkok. Seven patients had comorbid diseases (four cases with allergic asthma, one each with diabetes mellitus, chronic obstructive pulmonary disease and coronary artery disease). C. pneumoniae pneumonia presented as subacute pneumonia in 6 patients. The clinical manifestations were mild (IDSA risk class I-III) except in 4 patients who had preexisting allergic asthma, COPD and coronary heart disease. The diagnosis of C. pneumoniae pneumonia was based on microimmunofluorescence (MIF) antibody technique (IgM titer > or = 1:16, IgG > or = 1:512, IgA > or = 1:256 with or without fourfold rises). The clinical conditions were consistent with the primary infection (IgM titer of 1:16 or higher) in 6 patients and reinfection (IgG titer of 1:512, IgA titer of 1:256 or higher without rises of IgM titer) in the other 6 patients. Minimal bilateral pleural effusion was detected in only one patient. Coinfection was demonstrated in 2 patients (one each with S. pneumoniae and K. pneumoniae). All patients markedly improved after a 2-week course of macrolide, doxycycline or newest fluoroquinolone therapy. All patients had done well at one year of follow-up. C. pneumoniae infection has been recently recognized and a high seroprevalence (37%) in Thai school children and 100 per cent in young male Thai military conscripts has been reported. This report suggests that this infection, C. pneumoniae, may be a common pathogen of CAP in Thailand.

Gold blot for detection of immunoglobulin M (IgM)- and IgG-specific antibodies for rapid serodiagnosis of melioidosis.

Gold blot tests for rapid serodiagnosis of melioidosis were developed and evaluated with sera from 40 melioidosis patients and 159 normal controls. The sensitivity and specificity were 87.5 and 88%, respectively, for the immunoglobulin M (IgM) test and 100 and 91%, respectively, for the protein A test for IgG. Combination of the IgM gold blot and protein A gold blot yielded 97.5% sensitivity and 94.3% specificity. The tests were rapid and simple.

Rapid antigen detection assay for identification of Burkholderia (Pseudomonas) pseudomallei infection.

A simple antigen detection test was developed for rapid diagnosis of melioidosis caused by Burkholderia (Pseudomonas) pseudomallei infection. The method was based on the use of a specific monoclonal antibody in a sandwich enzyme-linked immunosorbent assay to capture antigen in clinical specimens culture positive for B. pseudomallei. The results showed the sensitivity and specificity of the test to be 75 and 98%, respectively.

Endotoxin of Pseudomonas pseudomallei detected by the body weight-decreasing reaction in mice and comparison of it with those of P. cepacia and P. aeruginosa.

The heat-treated whole cells, culture supernatants, and extracted endotoxin preparations of Pseudomonas pseudomallei were examined for endotoxin by the mouse body weight-decreasing (BWD) test. The experiments were conducted also with those of P. cepacia and P aeruginosa. Endotoxin was detected in all the samples of P. pseudomallei. Endotoxin of P. cepacia was detected in whole cells, but not in culture supernatant. The BWD activity of P. aeruginosa was 30 times as high as that of P. pseudomallei. This result was confirmed by the experiments with endotoxin preparations. In the limulus amebocyte lysate gelation (LAL) test, however, the endotoxin preparations of the two species showed the same level of activity.

Serosurveillance for double infection with Pseudomonas pseudomallei in tuberculous patients.

The serosensitivity to Pseudomonas pseudomallei antigen in pulmonary tuberculous patients was surveyed in Ubon Ratchathani Province, a melioidosis-endemic and tuberculosis-prevalent area in Thailand. Indirect hemagglutination assay (IHA) and indirect immunofluorescent assay (IFA) for IgM and IgG were employed for the measurement of antibody levels, with cut-off points set at 1:160, 1:8, and 1:32, respectively. Retrospective protocol survey of clinical data was also conducted. From these studies, however, no evidence was obtained to show that tuberculous patients have a disposition to acquire double-infected with P. pseudomallei and to develop melioidosis. The serosensitivity was never higher than that of the general population of the province as represented by healthy blood donors, nor related with the clinical severeness. Tuberculosis and melioidosis appear to be mutually independent diseases without showing interrelated prevalence in the endemic area.

Serosurveillance for Pseudomonas pseudomallei infection in Thailand.

A nation-wide survey was conducted to see the prevalence of serosensitivity to Pseudomonas pseudomallei antigens by indirect hemagglutination (IHA) and indirect immunofluorescent assay (IFA) for IgG and IgM. Serum samples were collected from blood donors in eight selected areas and bacteriologically confirmed melioidosis patients in Ubon Ratchathani province. The distribution patterns of antibody titers were compared among the survey areas with cut-off points set at 1:160 for IHA, 1:4 for IFA-IgM and 1:32 for IFA-IgG. These cut-off points were decided by ROC (Receiver Operating Characteristics) analysis. The specificity (% true negative reactions) of each serological test in the general population differed significantly among survey areas, possibly reflecting the extent of inapparent infection in each community. IFA was more successful than IHA in differentiating between negative from positive reactions. The survey classified the areas into endemic (Khon Kaen, Ubon Ratchathani), transported (Bangkok), and non-endemic (other provinces) types.

A preliminary survey for human immunodeficient virus (HIV) infections in tuberculosis and melioidosis patients in Ubon Ratchathani, Thailand.

A preliminary survey was conducted for the prevalence of HIV infections in pulmonary tuberculosis and melioidosis patients in Ubon Ratchathani province, in Thailand, the second largest province in population which supplies labors to Bangkok metropolis. In this province, tuberculosis is prevalent in a higher rate than in most other provinces and melioidosis is endemic. Four HIV-seropositives were found in a total of 551 suspected and culture-positive cases of pulmonary tuberculosis, while no HIV-seropositive was found in 121 melioidosis patients. In view of the rapidly expanding HIV-infections in Thailand, a strict watch will be needed on the future epidemiological status of HIV-infection in tuberculous patient.

PIP: HIV infection develops not only to AIDS, but it is also a leading risk factor for the development of many other infectious diseases due to the depletion of T lymphocytes such as the interrelated prevalence of tuberculosis (TB) and AIDS. Surveillance conducted in the 1988-1989 in the US and other recent studies found a serious epidemiological relation. Thailand has an endemic disease, melioidosis, caused by P. pseudomallei living in environmental soil and water. The disease takes various clinical types; localized, systemic, acute, subacute, chronic, and inapparent; presenting symptoms undistinguishable from many other infectious diseases. Pulmonary melioidosis shows a clinical feature similar to lung tuberculosis which occurs more easily in the individuals of impaired immunity, such as diabetes patients. According to available literatures, one case of recurrent melioidosis has been reported in Thailand as a complication of AIDS. The patient was a German homosexual male who had been living in the country for more than 10 years and showed a fatal course with interstitial pneumonitis. Ubon Ratchathani province, Thailand, is an area endemic for both TB and melioidosis, as well as a major supplier of laborers to Bangkok. A preliminary survey was conducted for the prevalence of HIV infections in pulmonary TB and melioidosis patients in Ubon Ratchathani province. TB was found to be prevalent in the province to a greater extent than in most other provinces and melioidosis is endemic. Four individuals were found to be HIV-seropositive amid a total 551 suspected and culture-positive cases of pulmonary TB, while no HIV-seropositive case was found among 121 melioidosis patients. In view of the rapidly expanding HIV-infections in Thailand, careful attention will have to be given to the future epidemiological status of HIV infection in TB patients.

Fatty acid profile and acid phosphatase activity of fresh isolates of Pseudomonas pseudomallei.

Eighty-one fresh isolates of Pseudomonas pseudomallei from melioidosis patients were subjected to the analysis for the fatty acid composition by gas-liquid chromatography (GLC) and pH-dependent pattern of nonspecific phosphatase activity. All the test strains were identical in the GLC profile showing the three peaks of characteristic hydroxy acids (3-OH 14:0, 2-OH 16:0, 3-OH 16:0) and the two prominent peaks of cyclopropane acids (17:0 delta, 19:0 delta). They had also basically the same pH-dependent curves of the enzymatic activity with paranitrophenyl phosphate as substrate, showing two to three peaks or shoulders only in the acidic side of the curve. These two biochemical characteristics could differentiate P. pseudomallei distinctly from P. aeruginosa, but not from P. cepacia.

Demonstration of acid phosphatase activity in antigenic glycoprotein fractions obtained from the culture filtrate of Pseudomonas pseudomallei.

Pseudomonas pseudomallei, the causative microorganism of melioidosis, was grown in Mueller-Hinton liquid medium, and glycoprotein fractions were separated from the culture filtrate by ammonium sulfate precipitation, gel-filtration with Sephadex G-75, and column chromatography with DEAE-cellulose. The fractions revealed acid phosphatase activity, and reacted to the sera from melioidosis patient in gel-diffusion precipitation assay.

Microcapsule agglutination test for the diagnosis of leptospirosis in Thailand.

Sera from 82 cases of leptospirosis (confirmed by micro-agglutination tests or IFAT) and 108 patients with other diseases were investigated using the microcapsule agglutination test (MCAT) for leptospirosis. The overall sensitivity (90.2%), specificity (96.3%), positive predictive value (94.9%), negative predictive value (92.9%), and accuracy of the MCAT (93.7%) were encouraging. MCAT is simple, can be performed by unskilled personnel with minimum laboratory facilities, and produces results in 3 h. MCAT would be a reliable serodiagnostic test for rapidly screening individuals for leptospirosis, in various geographical areas of Thailand.

In vitro susceptibilities of Pseudomonas pseudomallei to 27 antimicrobial agents.

Clinical isolates of Pseudomonas pseudomallei isolated in Thailand from 1981 to 1989 were tested for their in vitro susceptibilities to 27 antimicrobial agents, including older and newer quinolones, broad-spectrum cephems, carbapenems, monobactams, penicillins, tetracyclines, chloramphenicol, rifamycin, sulfamethoxazole, trimethoprim, and fosfomycin. Tosufloxacin, meropenem, CS-533, and minocycline were active against P. pseudomallei at levels comparable to or even greater than those of antimicrobial agents tested in previous studies, such as ciprofloxacin, ceftazidime, imipenem, carumonam, and piperacillin. Drug-resistant P. pseudomallei was found in only 1% of the isolates. The drug-resistant P. pseudomallei isolates displayed a unique pattern of susceptibility to the above-listed drugs.

Serology and carriage of Pseudomonas pseudomallei: a prospective study in 1000 hospitalized children in northeast Thailand.

Throat swab cultures and indirect hemagglutination assay (IHA) for Pseudomonas pseudomallei were done in 1000 randomly selected children at a large hospital in northeast Thailand. During 18 months, 17 children with melioidosis were admitted (0.46% of pediatric admissions excluding neonates born in the hospital). Throat swab was positive for P. pseudomallei in 8 of these but in none of 1000 control children. IHA seroprevalence rose at a conversion rate of 24% per year, from 12% in those 1-6 months old to a plateau at approximately 80% after age 4 years. No control child < 4 had an IHA titer > 1:160. The median titer in children with melioidosis was 1:80 (range, negative [in3]-1:5120). Specificity of IHA declined with age, but high titers (> or = 1:640) remained diagnostically useful. Thus, throat carriage of P. pseudomallei indicates active melioidosis. There is no evidence for an asymptomatic carrier state in children. Environmental exposure to P. pseudomallei in endemic areas begins when the child becomes mobile.