Impact of diabetes mellitus on clinical parameters and treatment outcomes of newly diagnosed pulmonary tuberculosis patients in Thailand.

BACKGROUND: To assess the clinical and laboratory parameters, response to therapy and development of antituberculosis (TB) drug resistance in pulmonary TB (PTB) patients with diabetes mellitus (DM) and without DM. METHODS: Using a prospective design, 227 of 310 new cases of culture-positive PTB diagnosed at the Queen Savang Vadhana Memorial Hospital and the Chonburi Hospital between April 2010 and July 2012 that met the study criteria were selected. Data regarding clinical and laboratory parameters, drug susceptibility and treatment outcomes were compared between PTB patients with DM and those without DM. To control for age, the patients were stratified into two age groups (< 50 and ≥ 50 years) and their data were analysed. RESULTS: Of the 227 patients, 37 (16.3%) had DM, of which 26 (70.3%) had been diagnosed with DM prior to PTB diagnosis and 11 (29.7%) had developed DM at PTB diagnosis. After controlling for age, no significant differences were found between the two groups regarding mycobacterium burden, sputum-culture conversion rate, evidence of multidrug-resistant tuberculosis, frequency of adverse drug events from anti-TB medications, treatment outcomes and relapse rate. The presenting symptoms of anorexia (p = 0.050) and haemoptysis (p = 0.036) were observed significantly more frequently in PTB patients with DM, while the presenting symptom of cough was observed significantly more frequently in PTB patients without DM (p = 0.047). CONCLUSIONS: Plasma glucose levels should be monitored in all newly diagnosed PTB patients and a similar treatment regimen should be prescribed to PTB patients with DM and those without DM in high TB-burden countries.

A human volunteer challenge model using frozen bacteria of the new epidemic serotype, V. cholerae O139 in Thai volunteers.

A total of 35 volunteers were recruited for an IRB-approved inpatient dose-escalation challenge. The goal was to identify a dose that produced an observed cholera attack rate > or =80% and an illness of sufficient severity during the defined study period such that the model would be useful for determining vaccine protection. Volunteers were challenged in groups of 5 with V. cholerae O139 that had been reconstituted immediately before use. Only 2 out of 5 volunteers who received the lowest dose (4.3 x 10(4) cfu) had diarrhea. As the inoculum size increased, the attack rate of diarrhea increased to 3-4 of 5 volunteers. At the highest dose tested, approximately 5 x 10(7) cfu, the attack rate was 73%. We recommend the use of frozen V. Cholera O139 in a human experimental challenge model to assess cholera vaccine efficacy (VE) in a cholera endemic area but with 4 days observation period before initiation of tetracycline to allow assessment of severity.

Cutaneous manifestations in HIV positive patients.

Cutaneous manifestations are common clinical findings among HIV positive patients. The causes may be bacteria, viruses, fungi and other non-infectious agents. This study was conducted at the Pramongkutklao Hospital skin clinic to determine the frequency distribution of cutaneous manifestations in HIV positive patients. A total of 147 patients with HIV seropositivity were recruited and divided into a retrospective group and a prospective study group. For the retrospective study, hospital records of 129 patients who attended from January 1995 to November 1998 were recruited. The prospective study was carried out from November 1998 to January 1999 and 18 patients were recruited. Cutaneous finding among patients in the two studies were evaluated. There were ten common cutaneous manifestations observed in the retrospective and prospective study including pruritic papular eruptions (PPE) (51.2%, 50%), oral candidiasis (16.7%, 21.7%), herpes zoster (10.9%, 5.6%), oral hairy leukoplakia (10%, 5.6%), unclassified eczema (9%, 11.1%), urticaria (5.6%, 3.1%), seborrheic dermatitis (4.7%, 16.7%), folliculitis (4.7%, 5.6%), prurigo simplex (4.7%, 5.6%), and Steven-Johnson syndrome (3.9%, 0%). However, the distribution of cutaneous manifestations in the two studies were not significantly different. These findings may be useful as baseline data for common cutaneous manifestations in HIV positive patients.

Diagnosis of Penicillium marneffei infection by quantitation of urinary antigen by using an enzyme immunoassay.

Penicillium marneffei is a major cause of opportunistic infection in patients with AIDS in north and northeastern Thailand. A method for the quantitation of P. marneffei antigen in urine was developed by using fluorescein isothiocyanate-labelled purified rabbit hyperimmune immunoglobulin G in an enzyme-linked immunosorbent assay. This method was evaluated with 33 patients with culture-proven penicilliosis and 300 controls (52 healthy subjects, 248 hospitalized patients without penicilliosis) from the same area in which penicilliosis is endemic. Urinary antigen was found in all 33 (100%) patients with penicilliosis, with a median titer of 1:20,480. With undiluted samples, 67 (27%) of 248 hospital patients and 3 (6%) of 52 healthy controls were reactive. At a cutoff titer of 1:40, the urine antigen detection assay had a diagnostic sensitivity of 97% and specificity of 98% (positive predictive value, 84%; negative predictive value, 99.7%). This test offers a valuable and rapid method for the diagnosis of penicilliosis in patients with AIDS and could be a useful addition to conventional diagnostic methods in areas in which penicilliosis is endemic.

Prevalence of cytomegalovirus antibodies among various age groups of Thai population.

The prevalence of CMV antibody in various groups of Thai population was studied. Pregnant women and young children had been infected with CMV more than the other studied groups. Children of both sex had equal chance of getting CMV infection while the risk of CMV infection in adult between male and female was significantly difference (p < 0.001). Pregnant women had higher chance to get CMV infection than normal women. Prevalence of CMV antibody at present was similar to previous studies.

Semi-quantitative measurement of Plasmodium falciparum antigen PfHRP2 in blood and plasma.

Plasmodium falciparum histidine rich protein 2 (PfHRP2) antigen was measured semi-quantitatively in whole blood, plasma, and supernatants and red blood cells of cultures in vitro using the dipstick ParaSight-F test and also by a quantitative antigen-capture enzyme-linked immunosorbent assay (ELISA). In vitro, PfHRP2 was secreted mainly during the second half of the asexual cycle with a marked rise during schizont development and rupture. The total PfHRP2 secreted before schizogony corresponded to approximately 4% of that contained in the red blood cells. In samples from 55 patients with acute falciparum malaria, the level of detection by ELISA corresponded to parasitaemias of 100/microL for whole blood and 1600/microL for separated plasma. Whole blood PfHRP2 levels were correlated significantly with admission parasitaemia (r = 0.76, P < 0.0001) and the stage of parasite development (r = 0.43, P < 0.01). Although whole blood PfHRP2 concentrations were higher in severe malaria, plasma concentrations of PfHRP2 were considerably higher in severe malaria (median titre 1:320, range zero to 1:1280) than in uncomplicated malaria (median titre 1:5, range zero to 1:80; P < 0.0001). The ratio of whole blood to plasma PfHRP2 was lower in severe than in uncomplicated malaria (median 4, range 0.25 to 256, versus 64, range 4 to 1280; P < 0.0001). With plasma samples the intensity of colour change on the dipstick correlated well with more precise measurement of optical density in the ELISA (r = 0.88, P < 0.0001). These results suggest that measurement of PfHRP2 in plasma could provide an alternative approach to the assessment of the parasite biomass, and thus prognosis, in severe malaria, and that this could be done simply by using the currently available dipsticks.

Simplified, rapid diagnosis of respiratory syncytial virus from clinical specimens.

DOT ELISA was compared with RT-PCR and tissue culture to detect RSV from nasopharyngeal aspirates. DOT ELISA had diagnostic sensitivity and specificity of 65.62% and 93.92%, respectively. The results indicate that DOT ELISA can be used for screening detection of RSV from clinical specimens and is suitable for small laboratories in the provincial areas of developing countries.

Monoclonal antibody-based dot-blot ELISA for the detection of Salmonella in foods.

Monoclonal antibody (MAb) produced to polysaccharides in the LPS molecule of salmonellae was used in a dot-blot ELISA for detecting Salmonella in 873 food samples, ie 100 fresh chicken, 261 frozen chicken, 78 pork, 84 beef, 100 hen eggs, 100 duck eggs, 50 sea-mussels, 50 shrimps and 50 squids in comparison with the conventional culture method. Salmonella culture from foods involved the following steps: pre-enrichment, enrichment in selective medium, isolation on selective and indicator media, followed by biochemical and serological identification of appropriate colonies, respectively. The whole culture procedure took 5 days. Food samples from the selective enrichment medium were also subjected to the MAb-based dot-blot ELISA. The whole procedure of dot-blot ELISA took less than 2 hours. Among 873 food samples, salmonellae could be recovered from 7.4% of the samples by the bacterial isolation method (16% of fresh chicken, 8.8% of frozen chicken, 24.4% of pork, 3.6% of beef and 2% each of hen eggs and duck eggs, respectively). Salmonella derby were predominant among pork samples while S.paratyphi B biovar java predominated in chicken. The MAb-based dot-blot ELISA were positive in 19.5% of the food samples, i.e. 30% of fresh chicken, 27.6% of frozen chicken, 34.6% of pork, 21.4% of beef, 20% of shrimp, 16% of sea-mussels, 2% of hen eggs and 4% of duck eggs. The sensitivity and specificity of the MAb-based dot-blot ELISA compared to the bacterial culture method were 81.5% and 85%, respectively. The discrepancy of the data between the culture method and the dot-blot ELISA might be due to the fact that the culture method could detect only living cells at numbers that gave at least one isolated colony on the selective/differential plate while the dot-blot ELISA detects any form of Salmonella antigen. The monoclonal antibody-based dot-blot ELISA offers several advantages over the conventional bacterial culture method when it is used for screening of Salmonella contamination in foods, especially export foods. These include rapidity, cost-effectiveness and simplicity (the dot-blot ELISA does not need highly trained personnel or equipment, in contrast to the culture method). The test can be performed in field conditions and the result can be read visually. It also offers multisample analysis at one time which renders more samples of food for screening possible, thus false negative results are fewer which, in turn, assures the quality of the export food in a cost-saving, short time frame.

Latex agglutination for rapid detection of Pseudomonas pseudomallei antigen in urine of patients with melioidosis.

A latex agglutination test for the detection of Pseudomonas pseudomallei antigen in urine was evaluated for the rapid diagnosis of melioidosis. With unconcentrated urine, antigen was detected in only 18% of patients with melioidosis overall. However, when urine was concentrated 100-fold, antigen was detected in 47% overall and in 67% of patients with septicaemia or disseminated infection, in whom a rapid diagnosis is most important. The specificity of the test was 100%. These results compared favourably with an enzyme immunoassay. This latex agglutination test is a simple, rapid and highly specific method of diagnosing melioidosis, and will be particularly useful in areas with limited laboratory facilities.

Detection of Pseudomonas pseudomallei antigen in urine for the diagnosis of melioidosis.

An enzyme-linked immunosorbent assay using a fluorescein isothiocyanate (FITC)-anti-FITC amplification system, has been developed to detect Pseudomonas pseudomallei antigen in urine. The assay was evaluated in 135 patients with acute melioidosis, 194 hospitalized patients with other disorders, and 40 healthy controls. Antigen was detected in the urine of 123 (91%) patients with melioidosis. Urinary antigen was found in 85 (96%) of 89 patients with septicemic melioidosis, all six patients with P. pseudomallei urinary tract infection, and 32 (80%) of 40 patients with other localized infections. Antigen was not detected in the urine of 40 healthy individuals, but the urine of 16 (8%) of 194 hospitalized patients with diagnoses other than melioidosis gave a positive result. Of the false-positive results, 13 of 16 were associated with bacteriuria > or = 10(4) colony-forming units/ml. At a cutoff titer of 1:10, the sensitivity and specificity of the test were 81% and 96%, respectively. Enzyme immunoassay detection of urinary antigen is a valuable and rapid laboratory test for the early diagnosis of acute melioidosis.

Immunofluorescence microscopy for the rapid diagnosis of melioidosis.

A direct immunofluorescent antibody test (DIF) was developed for the rapid diagnosis of melioidosis, a potentially fatal infection caused by Pseudomonas pseudomallei. In a clinical evaluation of 369 sputum, pus, or urine specimens from 272 patients with suspected melioidosis, the DIF had a sensitivity of 73% and a specificity of 99% compared with culture. Using this DIF, a confident diagnosis of melioidosis can now be made within two hours of admission to hospital, compared with the delay of two to four days required for culture results. Consequent early institution of specific antimicrobial therapy may help to save lives.

Antithrombin III and antivenom reversal of coagulopathy in rats envenomated with Malayan pit viper venom.

The therapeutic effects of antithrombin III (AT-III) and unrefined equine antivenom in the treatment of coagulopathy induced by Malayan pit viper (Calloselasma rhodostoma) venom were assessed in 42 adult Wistar rats. Following intramuscular venom injection (2 micrograms/g body weight), serial blood samples were taken from the femoral vein for measurement of whole blood clotting time and AT-III activity. There was progressive depletion of AT-III and blood ceased to clot a mean (S.E.) of 164 (8.3) min after venom injection. Coagulopathy was reversed by a high dose antivenom (10 micrograms/g) or a lower dose of antivenom (5 micrograms/g) in combination with AT-III (> or = 0.1 U/g; P < 0.01) but not 5 micrograms/g antivenom or AT-III alone. Following successful treatment, the mean plasma AT-III activity remained above 90%. In this animal model, systemic envenomation by the Malayan pit viper causes uncoagulable blood associated with AT-III consumption. The dose of antivenom required to reverse this coagulopathy can be reduced by half by the addition of AT-III sufficient to maintain blood concentrations within the normal range.

Albendazole stimulates outward migration of Gnathostoma spinigerum to the dermis in man.

Human gnathostomiasis is characterized by space-occupying inflammatory lesions and/or hemorrhage as a result of the migration of, very often, a single larva of Gnathostoma spinigerum. Intermittent cutaneous migratory swellings occurring over years is the most common manifestation and the rare cerebral invasion may be fatal. There are currently no effective anthelminthics for this infection. During a double-blind randomized placebo control trial evaluating the efficacy of albendazole in cutaneous gnathostomiasis at a dosage of 400 mg twice daily for two weeks, it was observed that gnathostome larvae tended to migrate outward as a result of the treatment so that they could be recovered by excisional biopsy or by picking with a needle. In the placebo-treated group (N = 40), no such migration was observed during the 8,470 patient-days of follow-up while in the albendazole-treated group (N = 41) there was one worm in an excisional biopsy done on day 16 and two worms were removed from the skin by the patients themselves on days 8 and 0. Assuming that the period of drug exposure of the gnathostomes was the 14 days of albendazole administration plus another washout period of 7 days (equivalent to 20 half-lives of the active detectable metabolite), the total patient-days of albendazole exposure was 830. The rate of outward migration of gnathostomes in the drug treated group (3 per 830 patient-days) was significantly (p < 0.0001) higher than in the placebo group (0 per 8,470 patient-days).(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic blood flow and metabolism in severe falciparum malaria: clearance of intravenously administered galactose.

1. Hypoglycaemia and lactic acidosis are important manifestations of severe falciparum malaria. To investigate hepatic gluconeogenesis in acute falciparum malaria, liver blood flow and galactose clearance were estimated in seven adult patients with moderately severe infection and seven patients with severe infection (three of whom died later). Nine patients were restudied in convalescence. 2. Liver blood flow, determined from the plasma clearance of Indocyanine Green, was lower in acute illness than in convalescence [16.1 (7.0) versus 23.9 (7.2) ml min-1 kg-1, mean (SD)], but this difference was not statistically significant (P = 0.15). There was a significant inverse correlation between admission venous plasma lactate concentrations and the liver blood flow estimated from the clearance of Indocyanine Green (rs = 0.71, P = 0.004). 3. The plasma clearance of galactose after intravenous injection was similar in the acute [15.4 (4.90) ml min-1 kg-1] and convalescent study [12.8 (2.1) ml min-1 kg-1]. The ratio of galactose clearance to Indocyanine Green clearance was significantly higher in acute disease [1.41 (0.51)] than in convalescence [0.70 (0.34)], largely because of the elevated ratios in severely ill patients [1.48 (0.50)]. 4. The rise in blood glucose concentration after galactose administration was significantly higher during acute illness [1.48 (0.72) mmol/l] than in convalescence [0.67 (0.41) mmol/l, P = 0.022], but the insulin response was similar, indicating reduced tissue insulin sensitivity. There was no significant change in the plasma concentrations of other metabolites (lactate, pyruvate, alanine and triacylglycerol) in either study. 5. These results suggest that the segment of the glycolytic pathway between galactose and glucose is unimpaired in patients with severe falciparum malaria.(ABSTRACT TRUNCATED AT 250 WORDS)

Gnathostoma larva migrans among guests of a New Year party.

An outbreak of Gnathostoma larva migrans occurred among guests of a New Year's party in Chachoengsao, Thailand. Nine people who consumed a raw fish dish called 'Hu-sae' contracted the disease. Five of them developed gastro-intestinal symptoms consisting of nausea, vomiting, abdominal cramps and diarrhea as early as within the first 24 hours, while in the other four, symptoms started on the following day. After the initial symptoms pertaining to the gut, malaise, chest discomfort, cough, myalgia, weakness, itching and migratory swellings were experienced. Eosinophilia was demonstrated in every patient with a mean (+/- SE) count of 5,516 +/- 1,010 cells/cu mm. Detection of antibody against aqueous extracts of G. spinigerum adult antigen using an enzyme-linked immunosorbent assay showed a titer of 1:1,600 or greater in every patients except one who had a titer of 1:400 (positive greater than or equal to 1:400). This outbreak illustrates the high attack rate when heavily infected fish are consumed.

Erythrocyte survival in severe falciparum malaria.

Erythrocyte survival was studied in 17 Thai patients (10 males, 7 females; aged 13-57 years) with severe falciparum malaria. To ensure radioisotopic labelling of cells before bone marrow recovery and survival analysis under near-steady state conditions, 51Cr labelling of autologous erythrocytes was performed at the time of admission (0 h) and calculation of mean cell lifespan (MCL) was based on semilogarithmic plots of corrected counts from 60 h onwards. Five patients received blood transfusions, all within 48 h of admission. The overall mean (+/- S.D.) MCL was short (44.1 +/- 21.7 days). Nontransfused patients had similar MCL values (43.6 +/- 20.4) to those of transfused patients (45.5 +/- 27.3 days, p greater than 0.8). Patients with and without palpable splenomegaly had MCL values which were not significantly different (54.1 +/- 28.8 vs. 37.2 +/- 12.3 days respectively, p greater than 0.1). There was no association between admission haematocrit or peripheral parasitaemia and MCL (p greater than 0.2 in each case), but there was an inverse correlation between total serum bilirubin and MCL (r = -0.49, p less than 0.025). There is accelerated destruction of non-parasitised erythrocytes in severe malaria resulting in a mean MCL that is half that found previously in healthy Thai volunteers (89.6 +/- 13.1 days, p less than 0.001) and significantly shorter than that reported previously in Thai patients with uncomplicated P. falciparum infections studied after parasite clearance (56.8 +/- 10.2 days, p less than 0.05).

Synergy of antithrombin III concentrate and antivenom in preventing coagulopathy in a rat model of Malayan pit viper envenoming.

The effects of unrefined equine antivenom and antithrombin III (AT-III) concentrate on the coagulopathy induced by systemic envenomation by Malayan pit viper (Calloselasma rhodostoma; MPV) venom were investigated in a rat model. 37 rats received an intramuscular injection of MPV venom and serial blood samples were taken from the femoral vein for simple whole blood clotting tests and measurement of AT-III activity. 30 min after venom injection, treatment (antivenom, AT-III or both) was given intravenously. 6 rats were untreated and all developed uncoagulable blood and AT-III depletion 90-210 (median 180) min after venom injection. A combination of high dose AT-III concentrate (0.5 units/g) and antivenom (20 micrograms/g) prevented abnormal clotting (P less than 0.001), whereas AT-III alone, antivenom alone, or a combination of low dose AT-III (0.25 units/g) and antivenom did not (P less than 0.05). These results suggest that the coagulation abnormality in MPV envenomation is secondary to activation of the coagulation cascade at several levels, and that treatment with antivenom alone may not be sufficient to reverse or prevent this phenomenon.

Activation of the coagulation cascade in falciparum malaria.

The incidence and progression of coagulation abnormalities were studied in 52 patients with acute falciparum malaria. The patients were prospectively divided into 3 groups; severe (parasitaemia greater than or equal to 5% or vital organ dysfunction), 12 patients; moderate (parasitaemia 1%- less than 5% without complications), 16 patients; and mild (parasitaemia less than 1%), 24 patients. No case died or developed clinical evidence of disseminated intravascular coagulation. Conventional indices of coagulation (prothrombin time, partial thromboplastin time, fibrinogen, fibrin degradation products) were usually within the normal range but reduced plasma concentrations of antithrombin III (AT-III) levels were noted in all groups, and the incidence was significantly higher in patients with severe and moderate malaria (83% and 81%) compared with the mild group (37%; P less than 0.005). Depletion of AT-III was associated with thrombocytopenia, decreased AT-III activity and elevated plasma concentrations of thrombin-antithrombin III complexes (P less than 0.01), confirming activation of the coagulation cascade and increased clotting factor consumption. AT-III levels returned to normal coincident with clinical improvement. Activation of coagulation is a common and sensitive measure of disease activity in acute falciparum malaria. It is not a specific feature, nor is there evidence to suggest it has a primary pathological role in severe infections.

Preliminary assessment of the safety and immunogenicity of live oral cholera vaccine strain CVD 103-HgR in healthy Thai adults.

A single dose (5 x 10(8) organisms) of attenuated A- B+ Vibrio cholerae classical Inaba recombinant vaccine strain CVD 103-HgR or placebo was administered to 24 healthy young Thai adults in a randomized, placebo-controlled, double-blind trial of safety and immunogenicity. None of the volunteers experienced untoward reactions. The vaccine strain was recovered from 2 of 12 vaccines. The vibriocidal antibody response (the best immunological correlate of protection) was good: 11 of 12 vaccinees (92%) manifested significant serotype-homologous Inaba antibody rises with a peak reciprocal geometric mean titer (RGMT) postvaccination of 3,417; 9 of 12 exhibited significant serotype-heterologous Ogawa antibody rises (prevaccination RGMT, 180; peak RGMT, 2,874). Nine of 12 vaccinees had significant rises in serum antitoxin. None of the controls exhibited rises in vibriocidal or antitoxic antibody. This preliminary study further confirms the safety and immunogenicity of CVD 103-HgR live oral cholera vaccine and paves the way for larger community studies of this candidate cholera vaccine.

Comparison of the reactivities and immunogenicities of procholeragenoid and the B subunit of cholera toxin in Thai volunteers.

Procholeragenoid, a stable high-molecular-weight aggregate of cholera toxin derived by heat treatment, was evaluated for reactivity and immunogenicity in adult Thai volunteers. Since procholeragenoid is known to retain some residual activity of cholera toxin, increasing amounts were ingested until diarrhea occurred; 250 micrograms induced diarrhea, but 100 micrograms did not. Procholeragenoid and cholera toxin B subunit, both in 100-micrograms amounts, were then compared for systemic and intestinal antitoxin responses. When three peroral doses were given, these immunogens gave comparable responses. The secretory immunoglobulin A antitoxin responses to three doses of 100 micrograms of B subunit did not differ significantly from responses found in previous studies of Thai adults given 1 or 5 mg of B subunit, but serum antitoxin responses were less after 1 or 2 doses of 100 micrograms than after doses of 1 or 5 mg. Serum antitoxin levels were similar after 3 doses of B subunit. Procholeragenoid in the maximum safe dose of 100 micrograms does not offer any immunologic advantage over B subunit, although it may be less expensive and easier to produce. However, these studies suggest that higher amounts of B subunit are more immunogenic and may be preferable, if found to be sufficiently cost effective, when added to oral killed whole Vibrio cholerae vaccines.