Xpert(®) MTB/RIF detection of Mycobacterium tuberculosis from sputum collected in molecular transport medium.

BACKGROUND: The Xpert(®) MTB/RIF assay is widely used for Mycobacterium tuberculosis detection. However, specimen transport remains a challenge. PrimeStore Molecular Transport Medium(®) (PS-MTM) inactivates specimens and stabilizes DNA/RNA at ambient temperature for subsequent molecular detection. OBJECTIVE: To compare the detection of M. tuberculosis concentrations in PS-MTM using Xpert and real-time polymerase chain reaction (RT-PCR), and smear-positive sputum specimens collected using a flocked swab. METHODS: Dilutions of M. tuberculosis in PS-MTM and phosphate buffered saline (PBS) were analyzed using the Xpert assay and commercial RT-PCR. Smear-positive (1+ to 3+) sputum specimens (n = 17) were transferred by flocked swab into PS-MTM and PBS, and were compared to standard 1.0 ml sputum Xpert analysis. RESULTS: Using the Xpert assay, cycle threshold values from high M. tuberculosis concentrations in PS-MTM (>10(3) colony forming units [cfu]/ml) were increased compared to control. In contrast, M. tuberculosis samples containing <10(3) cfu/ml, i.e., low concentrations, suspended in PS-MTM resulted in detection down to 10 cfu/ml. Xpert detection efficiency in PS-MTM treated samples (63.2%) was improved compared to PBS controls (34.9%). Xpert detected M. tuberculosis in all sputum specimens collected by flocked swabs in PS-MTM, and correlated with routine Xpert detection. CONCLUSIONS: PS-MTM enhances M. tuberculosis detection at low concentrations of M. tuberculosis, and provides a simplified and efficient collection method for Xpert detection.

Characterization of multi-drug resistant Mycobacterium tuberculosis from immigrants residing in the USA using Ion Torrent full-gene sequencing.

SUMMARY: Drug-resistant Mycobacterium tuberculosis bacterium (MTB) is spreading worldwide. Three drug-resistant isolates were detected in Burmese, Hmong, and Indian immigrants currently residing in Milwaukee, Wisconsin, USA. Ion Torrent full-gene sequencing and complete genetic analysis was performed within 5 days and compared to results from traditional drug sensitivity testing (DST). Genetic characterization of seven, full-length resistance-associated genes revealed two MDR and one highly resistant strain with important drug-resistant mutations that were confirmed by traditional DST. The rapid turnaround from sample-to-sequence underscores the public health value of Ion Torrent full-gene sequencing of MDR/XDR genes from epidemiologically significant clinical isolates.

Cryptosporidium parvum-specific antibody responses among children residing in Milwaukee during the 1993 waterborne outbreak.

A major gastroenteritis outbreak among >400,000 residents of Milwaukee, Wisconsin, in April 1993 was attributed to Cryptosporidium parvum oocysts in drinking water. Plasma specimens obtained from children (6 months to 12 years old) for routine blood lead level surveillance March-May 1993 were assayed by ELISA for levels of IgG antibody against the immunodominant Triton-17 and 27-kDa C. parvum antigens. Over a 5-week period, the seroprevalence for antibodies to the 2 antigens increased from 15% to 82% and from 17% to 87%, respectively, in samples from children living in southern ZIP code areas (n=218), whereas smaller increases (20% to 43% and 22% to 46%, respectively) were noted among samples from children living in northern ZIP code areas (n=335; P<.0001). The results demonstrate that C. parvum infection was much more widespread than previously appreciated and confirm that infection was associated with residence in the area served by the southern water treatment plant.

A massive outbreak in Milwaukee of cryptosporidium infection transmitted through the public water supply.

BACKGROUND: Early in the spring of 1993 there was a widespread outbreak of acute watery diarrhea among the residents of Milwaukee. METHODS: We investigated the two Milwaukee water-treatment plants, gathered data from clinical laboratories on the results of tests for enteric pathogens, and examined ice made during the time of the outbreak for cryptosporidium oocysts. We surveyed residents with confirmed cryptosporidium infection and a sample of those with acute watery diarrhea consistent with cryptosporidium infection. To estimate the magnitude of the outbreak, we also conducted a survey using randomly selected telephone numbers in Milwaukee and four surrounding counties. RESULTS: There were marked increases in the turbidity of treated water at the city's southern water-treatment plant from March 23 until April 9, when the plant was shut down. Cryptosporidium oocysts were identified in water from ice made in southern Milwaukee during these weeks. The rates of isolation of other enteric pathogens remained stable, but there was more than a 100-fold increase in the rate of isolation of cryptosporidium. The median duration of illness was 9 days (range, 1 to 55). The median maximal number of stools per day was 12 (range, 1 to 90). Among 285 people surveyed who had laboratory-confirmed cryptosporidiosis, the clinical manifestations included watery diarrhea (in 93 percent), abdominal cramps (in 84 percent), fever (in 57 percent), and vomiting (in 48 percent). We estimate that 403,000 people had watery diarrhea attributable to this outbreak. CONCLUSIONS: This massive outbreak of watery diarrhea was caused by cryptosporidium oocysts that passed through the filtration system of one of the city's water-treatment plants. Water-quality standards and the testing of patients for cryptosporidium were not adequate to detect this outbreak.

Microbial keratitis associated with plano tinted contact lenses.

The use of plano contact lenses for cosmetic purposes is a recent phenomenon. We report five cases of severe microbial keratitis associated with the use of these lenses. This subpopulation of patients who possess no refractive error are at risk of developing severe complications from contact lens wear. We have identified several issues which should be addressed by eye care professionals and regulatory agencies.

Filter-culture technique using amoeba saline transport medium for the noninvasive diagnosis of Acanthamoeba keratitis.

Acanthamoeba, a common free-living amoeba, is increasingly incriminated as a cause of keratitis and corneal ulceration. Between March 1986 and July 1988, specimens from seven patients submitted by ophthalmologists to the City of Milwaukee Health Department's Bureau of Laboratories were culture positive for Acanthamoeba. All patients were contact lens wearers. The specimens were transported at ambient temperature in amoebasaline (5.0 mL) and filtered through 13 mm 0.22 microns cellulose filters. The filters were then plated in cocultivation with Escherichia coli on nonnutrient agar and had positive results for Acanthamoeba in two to five days. Contact lens cases were culture positive for Acanthamoeba in three instances. These results indicate that corneal scraping in amoeba saline transport medium can provide an effective way to diagnose Acanthamoeba keratitis when direct culture of such specimens is not possible.

Comparison of the QuadFERM+ 2-hr identification system with conventional carbohydrate degradation tests for confirmatory identification of Neisseria gonorrhoeae.

The QuadFERM+ (Analytab Products, Plainview, NY) rapid identification system was evaluated for its ability to identify correctly Neisseria gonorhoeae isolates from specimens obtained at a sexually transmitted disease clinic. One hundred eighty-five isolates (115 N. gonorrhoeae, 45 Neisseria meningitidis, 16 Neisseria species, and nine Branhamella catarrhalis; fresh isolates, frozen stock cultures, and cultures referred from local health agencies) were tested with the QuadFERM+ system and conventional biochemical tests. The two discrepant results were obtained with QuadFERM+, for a lactose-positive isolate of Neisseria sicca and a maltose-positive N. meningitidis. Both were negative by conventional sugar degradation tests. The N. sicca was negative when repeated in the QuadFERM+, and the N. meningitidis reverted from maltose-positive to maltose-negative after 3 hr. Twelve beta-lactamase positive organisms (six N. gonorrhoeae plus six B. catarrhalis) and 173 beta-lactamase-negative organisms showed 100% agreement between the acidometric QuadFERM+ beta-lactamase test and the conventional starch-iodine method. Thus the QuadFERM+ is a rapid and acceptable alternative for the identification of N. gonorrhoeae in a sexually transmitted disease clinic.

Shigellosis winter trends in Milwaukee.

Shigellosis cases reported to the Milwaukee Health Department have been highest during the months November, December, and January for eight of the past 11 years (1978 to 1988). Shigellosis in the United States has classically been described as a summer or late summer-early fall disease.

An isolation method of DNA from Pneumocystis carinii: a quantitative comparison to known parasitic protozoan DNA.

1. A method for isolating DNA from Pneumocystis carinii is described. 2. The DNA content per nucleus is 0.22-0.34 pg. 3. This finding is consistent with other parasitic protozoa DNA content per nuclei.

An improved method of isolating Pneumocystis carinii from infected rat lungs.

A new method of isolating Pneumocystis carinii from infected lungs of cortisonized rats is described. Clumping of parasites and host lung material was diminished by suspension of macerated Pneumocystis-laden rat lung in a modified calcium, magnesium-free Hanks' balanced salts solution at physiologic pH and osmolality, containing the wetting agent G-acid. After washing, this material was suspended in a second buffer system for digestion. The digestion step was done in the same buffer but with the addition of calcium, magnesium, collagenase, hyaluronidase and deoxyribonuclease. These innovations allowed enumeration of trophozoites as well as cysts. Following digestion, the parasites were separated from particulate host lung debris by Percoll density gradients designed to pellet the debris, leaving parasites in the gradient. Density studies done prior to this step revealed that trophozoites and non-nucleated cysts had similar densities, 1.028 g/ml, whereas nucleated cysts were heavier at 1.030 g/ml. Particulate host lung debris could be removed due to its heavier density, 1.040 g/ml. The significance of this study includes: relatively clump-free suspensions of infected rat lung, enumeration of trophozoites as well as cysts, and characterization of nucleated cysts.

Evaluation of the BIOGRAM antimicrobial susceptibility test system.

BIOGRAM is an antimicrobial susceptibility test system for the determination of MICs from the standard disk diffusion test zone diameters. The system was challenged with 511 recent clinical isolates of members of the family Enterobacteriaceae, nonfermentative gram-negative bacteria, staphylococci, and enterococci. Results were compared with those obtained with the broth microdilution method. Appropriate control organisms were included with each test series. A total of 10,085 organism-drug combinations were evaluated. BIOGRAM demonstrated an overall correlation of 95.9% with the reference broth microdilution method.

Electrophoretic analysis of soluble proteins and esterase, superoxide dismutase and acid phosphatase isoenzymes of members of the protozoan family trichomonadidae.

Polyacrylamide gel electrophoresis was used to compare the proteins and isoenzymes of esterase, superoxide dismutase, and acid phosphatase in soluble, whole-cell extracts of four strains of Trichomonas vaginalis, two strains of Trichomonas gallinae, and one strain each of Tritrichomonas foetus, Tritrichomonas augusta, Tetratrichomonas gallinarum, and Pentatrichomonas hominis. Intraspecific, interspecific, and intergeneric differences were found in protein and isoenzyme profiles. At least four to seven isoenzymes were detected among the ten strains for each of the three enzymes studied. Each strain usually contained one or two isoenzymes of both esterase and acid phosphatase, and two or three isoenzymes of superoxide dismutase.