Evaluation of a rapid culture method for tuberculosis diagnosis: a Latin American multi-center study.

SETTINGS: Tuberculosis (TB) diagnostic laboratories in Latin America. OBJECTIVES: Evaluation of thin-layer agar (TLA) compared to Löwenstein-Jensen (LJ) culture for the diagnosis of TB. DESIGN: Phase II prospective study in six laboratories. Samples included sputum and extra-pulmonary specimens from patients with a clinical diagnosis of TB. Respiratory samples were decontaminated using NaOH/ NALC; all samples were centrifuged, stained with Ziehl-Neelsen for acid-fast bacilli (AFB), cultured on LJ and TLA and identified according to recommended procedures. Sensitivity and likelihood ratios (LR), growth detection time and contamination rate were calculated for both media. RESULTS: A total of 1118 clinical specimens were studied. Cultures detected Mycobacterium tuberculosis in all AFB-positive samples, whereas for AFB-negative specimens LJ detected 3.2% and TLA 4.4%. Sensitivity was 92.6% (95%CI 87.9-95.9) and 84.7% (95%CI 78.8-89.0) for TLA and LJ, respectively. Positive and negative LRs were similar. Contamination was 5.1% for TLA and 3.0% for LJ. Median time to detection of a positive culture was 11.5 days (95%CI 9.3-15.0) for TLA and 30.5 days (95%CI 26.9-39.0) for LJ (P < 0.0001). CONCLUSION: Difference in the characteristics of the participating laboratories, the disease prevalence and the number and type of specimens processed did not affect the overall performance of TLA as compared to LJ, supporting the robustness of the method and its feasibility in different laboratory settings.

Microcolony detection in 7H11 thin layer culture is an alternative for rapid diagnosis of Mycobacterium tuberculosis infection.

SETTING: Radiometric technology and molecular biology are used in rapid diagnosis of tuberculosis in laboratories around the world. However, these technologies increase costs and are not available in laboratories where economic resources are limited. OBJECTIVE: To compare sensitivity and time for detection of positive cultures in a microcolony method, Middlebrook 7H11 thin layer agar plate (TL7H11), and a conventional culture, Lowenstein-Jensen (L-J). DESIGN: A total of 761 clinical samples were processed using acid-fast smear and culture on TL7H11 plates and L-J tubes. TL7H11 plates were checked microscopically for microcolony growth twice weekly for 4 weeks, and L-J tubes were checked once a week for 8 weeks. RESULTS: Overall positivity was 11.0%. More than 60% of the positive samples were detected within the first 10 days on TL7H11, and none on L-J. After 2 weeks, more than 80% were positive on TL7H11 compared to 10% on L-J. In paucibacillary samples, TL7H11 detected 2.18% and L-J 4.57% (P < 0.001). Microcolony morphology was 100% distinctive for Mycobacterium tuberculosis on TL7H11. The calculated cost of TL7H11 prepared in the laboratory was US$2.90 per unit. CONCLUSION: The TL7H11 method is an inexpensive, rapid and reliable alternative for diagnosing M. tuberculosis infection. It is therefore a valuable option for laboratories in low income countries.

The effects of culture-site, depth, season, and stock source on the prevalence of Marteilia refringens in cultured mussels (Mytilus galloprovincialis Lmk.) from Galicia, Spain.

Samples of a marteiliad parasite in Mytilus galloprovincialis Lmk. from NW Spain were identified as Marteilia refringens, using light and electron microscopy. The most important lesions in the mussel tissues were found in the digestive tubules at the moment of the release of the sporangia, resulting in a breakage of the epithelial cells of the digestive tubules. The plasmodia of a Marteilia-like organism were also found in the gills, accompanied by a severe host reaction in 1 of the 4,200 examined mussels. The prevalence of the M. refringens parasite was monitored for 4 yr and correlation was found with prevalence, site, and depth of mussel culture. No correlation was found with geographical origin of the mussels. Mussels collected from the inner part of the Ría de Vigo were more frequently infected (38.5%) than those from the middle (29.0%) and outer parts (5.5%). Mussels from 2-m samples showed a higher mean prevalence of M. refringens than those from the 5-m samples (13.3 and 8.3%, respectively).

Characterization of the ribosomal RNA locus of Perkinsus atlanticus and development of a polymerase chain reaction-based diagnostic assay.

The rRNA locus of Perkinsus atlanticus from the clam Ruditapes decussatus cultivated on the Atlantic coast of Spain was cloned and sequenced. Sequences of the internal transcribed spacer (ITS) from the rRNA locus were compared to sequences reported earlier for a P. atlanticus isolate from Portugal and to those from other Perkinsus species. The ITS I sequence of the Spanish P. atlanticus isolate was identical to the Portuguese P. atlanticus sequence and had 76.6% identity to the ITS1 of Perkinsus marinus. The ITS2 sequence had 99.7% identity to the Portuguese P. atlanticus ITS2, 92.5% identity to the P. marinus ITS2, and 99.5% identity to the Perkinsus olseni ITS2. We report for first the time the small subunit (SSU) and nontranscribed spacer (NTS) of P. atlanticus. The P. atlanticus SSU sequence was 99.6% identical to that of an unidentified Perkinsus species from the Australian clam Anadara trapezia and 98.0% identical to that of P. marinus. Further, our results support the proposal that P. atlanticus, P. olseni, and the Perkinsus sp. from A. trapezia constitute a subgroup of Perkinsus species distributed in the Pacific and eastern Atlantic, different from P. marinus that is distributed along the western edge of the Atlantic. Based on the NTS sequence of P. atlanticus from Spain and the differences with P. marinus NTS (62.2% identity), we developed a polymerase chain reaction (PCR)-based diagnostic assay with a lowest limit of detection of 0.01 amol of cloned NTS DNA as assessed on ethidium bromide-stained agarose gels. Specificity of the PCR-based assay was tested with samples from the clams R. decussatus, Ruditapes philippinarum, and Venerupis pullastra collected in P. atlanticus-enzootic areas of Spain. The specificity and sensitivity demonstrated for this NTS-based PCR assay validate its use as a tool for assessment of P. atlanticus in molluscs.

Nucleotide sequence variability in the nontranscribed spacer of the rRNA locus in the oyster parasite Perkinsus marinus.

We examined the sequence variability of the nontranscribed spacer (NTS) and internal-transcribed spacer (ITS1 and ITS2) domains of the rRNA locus of Perkinsus marinus from Maryland, Florida, and Louisiana. The sequence of P. marinus DNA including the 5S rRNA, NTS, small subunit (SSU) rRNA, ITSI, and ITS2 regions confirmed their contiguity in the rRNA locus and revealed differences at 28 positions with the SSU rRNA sequences published earlier. The 307-bp polymerase chain reaction (PCR)-amplified fragments from the NTS domain of the various P. marinus isolates revealed the presence of 2 distinct sequences, designated as types I and II, that differed at 6 defined nucleotide positions. Based on these differences, nested PCR and restriction enzyme digests were used to distinguish between the 2 types. Sequences of the ITS1 and ITS2 domains of samples from either NTS type I (n = 3) or type II (n = 3) showed no variation and were identical to published sequences. Frequencies of the P. marinus NTS sequence types I and II in infected oysters varied with the geographic origin of the samples. All Maryland samples examined (n = 19) corresponded to the NTS type I sequence, the type II was the most frequent in the Florida samples (n = 17), and both types were about equally represented in the Louisiana samples (n = 19), with both sequence types found in individual oyster specimens. Although it has been suggested that P. marinus is diploid, it remains to be determined if both NTS sequence types can be present in a single P. marinus trophozoite.

Species-specificity and sensitivity of a PCR-based assay for Perkinsus marinus in the eastern oyster, Crassostrea virginica: a comparison with the fluid thioglycollate assay.

We examined the species-specificity and sensitivity of a polymerase chain reaction (PCR)-based assay for Perkinsus marinus and compared its overall performance with the fluid thioglycollate medium (FTM) assay on oyster (Crassostrea virginica) hemolymph, mantle, and rectum samples. Our results indicated that the PCR-based methodology is species-specific because Perkinsus olseni, Perkinsus atlanticus, and Perkinsus spp. DNAs were not amplified with the PCR primers developed for P. marinus diagnosis. The sensitivity of the PCR method, as assessed through spike/recovery experiments, was established by the detection of as few as 1 cell of P. marinus in 30 mg of oyster tissue. Tissue samples from naturally infected oysters analyzed both by the FTM and PCR assay suggested that the latter was more sensitive for the diagnosis of P. marinus. Positive results for P. marinus infection ranged from 70% to 83% by FTM and from 92% to 100% by PCR, depending on the tissue examined. Therefore, species-specificity and sensitivity of the NTS-based PCR assay validate its use as a tool for assessment of P. marinus in mollusks.

Fine structure of clonally propagated in vitro life stages of a Perkinsus sp. isolated from the Baltic clam Macoma balthica.

We established monoclonal in vitro cultures of a Perkinsus sp. isolated from the baltic clam Macoma balthica and compared morphological features of various life stages by light and transmission electron microscopy to those of the currently accepted Perkinsus species: Perkinsus marinus, Perkinsus olseni, Perkinsus atlanticus, and Perkinsus qugwadi. Except that trophozoites were slightly larger than those of P. marinus, and that they underwent zoosporulation in culture, observation of our isolate under light microscopy did not reveal striking differences from any Perkinsus species. Perkinsus sp. from M. balthica shared fine structural characteristics with other Perkinsus species that clearly place it within this genus. Although zoospores of Perkinsus sp. from M. balthica were slightly smaller than those from other species, the ultrastructural arrangement and appearance of the apical complex and flagella seem to be identical to those of P. marinus and P. atlanticus. Our isolate also appeared, in some sections, to have cortical alveolar expansions of the plasmalemma at regions other than the anterior end and lobulated mitochondria that were reported as unique for P. qugwadi. Little consensus exists among authors in the assignment of taxonomic weight to any particular morphological feature to designate Perkinsus species. The present study of gross morphology and ultrastructure was complemented with molecular studies reported elsewhere, which propose that Perkinsus sp. from Macoma balthica is a distinct species.

Outer membrane proteins of E. coli in the host-pathogen interaction in urinary tract infection.

Outer membrane protein patterns (Omp) of Escherichia coli obtained directly from the urine of bacteriuric patients without passage on artificial culture media (ACM) were studied by polyacrylamide gel electrophoresis (SDS-PAGE) in an effort to determine whether in vivo conditions of growth affected the expression of these bacterial surface structures. Seventeen strains studied showed two distinct Omp patterns: one protein band appeared at the level of porin proteins (40 kDa) in both patterns, but Omp A protein was at the level of 36 kDa in the first pattern and a new protein was observed at 21.5 kDa in the second pattern suggesting that it is a fragment of Omp A. High molecular weight proteins were also observed in most of the strains and this finding was related to lack of free iron when the same strains were grown under iron restricted conditions in vitro. The same strains grown in pooled urine from normal females showed the first pattern mentioned above. Comparative growth on ACM of urinary strains and E. coli strains isolated from blood, feces and wounds showed an increase in the number of porins expressed (from 1 to 2 or 3, with some variability observed between strains). Differences in osmolality between pooled urine and ACM used, plus in vitro studies varying the osmolality of culture media, showed that osmolality accounted for differences in the number of porins expressed: porin expression decreased in urine the ACM of high osmolality, suggesting that the same phenomena occurred in vivo. It is concluded that host factors including low availability of iron and high osmolality present in the urinary tract influence the expression of several E. coli surface proteins. These proteins may relate to the ability of E. coli to colonize and invade the urinary tract by regulating the physiologic and/or metabolic state of the bacterial cell favoring survival of the organism in a hostile environment. Specific immune responses directed against porins could influence the outcome of this host-parasite interaction.

Description of Perkinsus andrewsi n. sp. isolated from the Baltic clam (Macoma balthica) by characterization of the ribosomal RNA locus, and development of a species-specific PCR-based diagnostic assay.

A Perkinsus species was isolated from the baltic clam Macoma balthica and an in vitro culture established under conditions described for P. marinus. As reported previously, morphological features remarkable enough to clearly indicate that this isolate is a distinct Perkinsus species were lacking. In this study, regions of the rRNA locus (NTS, 18S, ITS1, 5.8S, and ITS2) of this isolate were cloned, sequenced, and compared by alignment with those available for other Perkinsus species and isolates. Sequence data from the rRNA locus and species-specific PCR assays indicated not only that Perkinsus sp. from M. balthica was not P. marinus, but it was different from P. atlanticus and P. olseni. The degree of difference was comparable to or greater than differences between accepted Perkinsus species. In particular, NTS sequence and length were dramatically different from that of P. marinus and P. atlanticus. Therefore, we formally propose to designate the Perkinsus sp. from M. balthica as a separate species, P. andrewsi n. sp. Primers based on P. andrewsi NTS sequence were used to develop a PCR-based diagnostic assay that was validated for species-specificity and sensitivity. PCR-based assays specific for either P. andrewsi or P. marinus were used to test for their presence in bivalve species sympatric to M. balthica. Although isolated from M. balthica, P. andrewsi was also detected in the oyster Crassostrea virginica and clams Macoma mitchelli and Mercenaria mercenaria, and could coexist with P. marinus in all four bivalve species tested.

A multicentre, double-blind, randomised study comparing the efficacy and safety of oral levofloxacin versus ciprofloxacin in the treatment of uncomplicated skin and skin structure infections.

A multicentre, randomised, double-blind trial in Latin America compared oral levofloxacin 500 mg once daily for 7 days with oral ciprofloxacin 500 mg twice daily for 10 days in 272 patients with uncomplicated skin and skin structure infections. Among 253 subjects evaluable for clinical efficacy (129 levofloxacin, 124 ciprofloxacin), clinical success (cure and improvement) was observed in 96.1% of levofloxacin-treated patients and in 93.5% of ciprofloxacin-treated patients. Overall, bacteriological eradication rates by pathogen were 93.2% and 91.7%, respectively. Levofloxacin eradicated 94% (66/70) of Staphylococcus aureus and 94% (17/18) of Streptococcus pyogenes isolates, compared with 93% (70/75) and 92% (12/13) for ciprofloxacin. Microbiological eradication rates by subject were approximately 93% and 90% for the levofloxacin and ciprofloxacin groups, respectively. Drug-related adverse events were reported by 8.9% of those receiving levofloxacin and 8.2% of those administered ciprofloxacin. Findings support the efficacy of oral levofloxacin for uncomplicated skin and skin structure infections due to S. aureus and S. pyogenes.

Virulence of wild-type E. coli uroisolates in experimental pyelonephritis.

This study was designed to analyze the colonizing and invasive properties of wild-type bacteriuric E. coli possessing a variety of phenotypic characteristics in experimental nonobstructive pyelonephritis (P and Type 1 [T] fimbriae, hemolysin [Hly], presence of K capsules, flagella [H], serotype, biotype, human and mouse serumcidal resistance). Special emphasis was on the role of Gal-Gal adhesin (P fimbriae) of non-genetically engineered uroisolates. It was shown that organisms that are P+ or T+ or Hly+ are more likely to colonize bladders than strains negative for those parameters (P less than 0.001). Additionally, P+ strains were more often associated with kidney histopathology than P- E. coli (P less than 0.05). However, the data also indicated that fimbriae (P and Type 1) were not sole determinants of virulence since two strains devoid of fimbriae, hemolysin, K capsules and sensitive to human serumcidal activity caused incipient and acute pyelonephritis. Even among identical serotypes and biotypes, the presence/absence of fimbriae did not appear to be the critical factor in urovirulence, nor did the presence of several positive characteristics (hemolysin, K capsule, flagella, serum resistance) in a given strain enhance uropathogenicity. Therefore, these properties do not need to work together to render an E. coli urovirulent. These phenotypic characters may simply represent associated or serologic markers with the host serving as the dominant determinant of susceptibility to urinary infection. The findings emphasize the inherent limitations in relating and extrapolating colonizing and invasive properties of genetically engineered strains to those of naturally occurring, wild-type E. coli human uroisolates causing pyelonephritis.

Partial necrosis of the neck of the talus treated with arthroscopy.

An unusual case of an area of necrosis on the talar neck of a girl with a 2-year history of gradually increasing pain following an ankle sprain is presented. Initial radiographs were normal but follow-up radiography showed irregularity in the anterior talar neck. A definitive diagnosis was made by magnetic resonance imaging. Arthroscopic removal of a necrotic fragment from the anterior talar neck that was impinging on the tibia relieved her symptoms. Two years after the operation the patient has normal radiographs and full activity.