Primary Isolation Cultures Research Articles

Phenotypic identification of periodontal Prevotella intermedia/nigrescens group isolates validated by MALDI-TOF mass spectrometry

The accuracy of a phenotypic scheme to recognize periodontal Prevotella intermedia/nigrescens group clinical isolates on primary isolation culture plates was assessed with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 84 fresh subgingival isolates from 23 chronic periodontitis patients were presumptively recognized on anaerobically-incubated enriched Brucella blood agar primary isolation plates as P. intermedia/nigrescens based on their dark-pigmented colony morphology, brick-red autofluorescence under long-wave ultraviolet light, and a negative fluorescence test for lactose production. The presumptive P. intermedia/nigrescens clinical isolates were subjected to MALDI-TOF MS analysis using Bruker MALDI Biotyper analytic software containing mass spectra for P. intermedia and Prevotella nigrescens in its reference library of bacterial protein profiles. Using a ≥1.7 log score agreement threshold, 60 (71.4%) of the presumptive P. intermedia/nigrescens clinical isolates were confirmed as either P. intermedia (25 isolates) or P. nigrescens (35 isolates). All isolates with a <1.7 log score were also identified as P. intermedia or P. nigrescens from the top choice designated on the MALDI Biotyper most likely species identification list. These MALDI-TOF MS findings document the ability of the phenotypic scheme to correctly recognize most periodontal P. intermedia/nigrescens group clinical isolates on primary isolation culture plates.

Species distribution and antibiotic susceptibility profile of bacterial uropathogens among patients complaining urinary tract infections

BackgroundUrinary tract infection is the second most common type of infection and the problem is further compounded by the emergence of drug resistance in bacterial uropathogens. The aim of this study was to determine the spectrum of bacterial uropathogens and their drug resistant pattern.MethodsA single institutional cross-sectional study was carried out at Arsho Advanced Medical laboratory from September 2015 to May 2016. A total of 712 urine samples were collected, inoculated onto primary isolation culture media, incubated at 37 °C for 18–24 h, and significant bacteriuria was determined. Identification and the antimicrobial susceptibility testing of bacteria were determined by using the automated VITEK 2 compact system.ResultsOut of 712 urine samples processed, 256 (36%) yielded significant bacteriuria of which 208 (81.25%) were obtained from female and 48 (18.75%) from male patients. Age group of 25–44 were more affected with the infection. Of 256 bacterial isolates recovered, Escherichia coli, was the dominant bacterium. Ampicillin and trimethoprim/sulfamethoxazole were the least effective drugs while piperacillin/tazobactam was the most effective drug against Gram-negative bacteria. Erythromycin was the least effective drug while vancomycin was the most active drug against Gram-positive bacteria.ConclusionsObservation of many bacterial species causing UTI in this study warrants, a continuous epidemiological survey of UTI in health institutions across the country. High level of drug resistance to the commonly prescribed drugs necessitates a search for other options.

Cytobrush-culture method to diagnose tinea capitis

This is a comparative study to isolate the dermatophytes of tinea capitis using the cytobrush and comparing it versus the standard method. A prospective, observational, comparative trial of 178 probable cases of tinea capitis was conducted in two dermatological centers. Each patient underwent mycological tests that included direct exam with KOH and cultures with either of two methods: scraping the scalp to remove hair and cell debris, and the cytobrush. A total of 135 clinically and mycologically proven cases of tinea capitis were included; 119 were non-inflammatory and 16 inflammatory tinea. A total of 131 had a positive direct exam and subsequent primary isolation cultures were obtained in 135 cases. The main dermatophytes isolated were Microsporum canis (68%) and Trichophyton tonsurans (20%). A total of 115/135 (85.1%), were detected with the traditional method, with an average of 11.2 days until positive, while the number detected with the cytobrush was 132/135 (97.7%) with an average of 8.5 days until positive. The chi-square statistical method showed that the cytobrush culture was superior to the standard one with a chi-square of 5.078 (P = 0.025), with a statistically significant difference versus the standard method.

Phenotypic characteristics of human immunodeficiency virus type 1 subtype C isolates of Ethiopian AIDS patients.

It has been estimated that, to date, about 48% of all HIV-infected people in the world carry HIV-1 subtype C virus. Therefore, it is of great importance to gain better knowledge about the genetic and biological characteristics of this virus subtype. In the present study, the biological properties of HIV-1 isolates obtained from nine Ethiopian patients with AIDS were studied. DNA sequencing of the V3 loop of gp120 classified the isolates as subtype C. In primary isolation cultures, virus infection was accompanied by syncytium formation and cell lysis. Interestingly, when examining the growth in primary monocyte-macrophage cultures, initial low-level virus replication was followed by a nonproductive state, from which virus could be rescued by cocultivation with Jurkat(tat) cells. Furthermore, none of the isolates replicated in T cell lines (CEM, MT-2, HuT-78, and H9) or in the promonocytic cell line U937 clone 2. All isolates could use CCR5 as coreceptor, whereas no isolates could use CCR2b, CCR3, CCR5, CXCR4, Bonzo/STRL33, or BOB/GPR15. The genotype of the V3 region correlated with the MT-2 negative/non-syncytium-inducing (NSI) phenotype. Comparative studies revealed that the scarcity of CXCR4 usage as well as other phenotypic characteristics of subtype C isolates distinguish this subtype. On the basis of these data, we suggest that in addition, factors other than viral phenotype may govern the pathogenic potential of subtype C isolates.

Standard Conditions of Virus Isolation Reveal Biological Variability of HIV Type 1 in Different Regions of the World

HIV-1 isolates were obtained from four countries within the framework of the WHO Network for HIV Isolation and Characterization. The use of standard HIV isolation procedures allowed us to compare the biological properties of 126 HIV-1 isolates spanning five genetic subtypes. In primary isolation cultures, viruses from Uganda and Brazil appeared early and replicated without delay, whereas the replication of Thai viruses was delayed by several weeks. Regardless of genetic subtype or country of origin, blood samples collected more than 2 years after seroconversion yielded virus that replicated efficiently in the primary isolation cultures. None of the isolates obtained from Thailand or Rwanda replicated in cell lines, whereas 5 of the 13 Brazilian isolates and 7 of the 11 Ugandan isolates replicated and induced syncytia in MT-2 cells. As expected for virus isolates obtained early in HIV-1 infection (within 2 years of seroconversion), all viruses from Brazil, Rwanda, and Thailand showed a slow/low replicative pattern. For the Ugandan samples, the time from seroconversion was known precisely for a few of the samples and only in one case was less than 2 years. This may explain why the five viruses that were able to replicate in all cell lines, and thus classified as rapid/high, were of Ugandan origin. Viruses able to induce syncytia in MT-2 cells, also induced syncytia in PBMC. However, 8 slow/low viruses (out of 27) gave discordant results, inducing syncytia in PBMC but not in MT-2 cells. Furthermore, using syncytium induction as a marker, changes in virus populations during early in vitro passage in PBMC could be observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid identification of Branhamella catarrhalis a comparison of five rapid methods

Five methods for the rapid identification and differentiation of Branhamella catarrhalis from other Neisseria species in 86 respiratory specimens were compared. These tests included the 4-methylumbelliferyl butyrate (MUB), API quadFerm, B.CAT.Confirm, Gonochek II, and the Tributyrin disc. All five tests reliably and accurately identified 31 B. catarrhalis isolates. However, the MUB test was the least expensive, least labor intensive, and did not require overnight purity plates for performance. The MUB test provided same-day identification of B. catarrhalis isolates from the initial primary isolation culture.

Identification of Neisseria meningitidis from primary isolation cultures

Summary Throat cultures were taken from 251 patients attending a Department of Venereology. Isolates of Neisseria meningitidis and other pathogenic neisseriae were identified by performing a rapid carbohydrate utilization test with the growth from primary isolation cultures on modified New York City (MNYC) medium. A simple colorimetric test for β -lactamase production was done at the same time. The carrier-rate of meningococci was 47·4 per cent (119/251) while 2·8 per cent (7/251) and 2·4 per cent (6/251) of the patients harboured N. lactamica and N. gonorrhoeae respectively. Of the meningococcal isolates 70·6 per cent (84/119) were identified directly from the primary isolation culture after incubation for 24 hours. It is suggested that identification from primary isolation cultures, with the possible refinement of sero-typing by incorporation of anti-serum in the culture plates, could improve the cost-effectiveness and rapidity of meningococcal carrier surveys.

Identification and penicillinase testing of Neisseria gonorrhoeae from primary isolation cultures on modified New York City medium

The fluorescent-antibody test, rapid carbohydrate utilization test (RCUT), and a test for penicillinase production were performed on the bacterial growth from primary cultures on modified New York City medium. Of 134 gonococcal infections in men, 88.8% were diagnosed by the fluorescent-antibody test and 70.9% by the RCUT after incubation for 24 h; the corresponding figures for 75 infections in women were 86.7 and 54.7%, respectively. After incubation for 48 h, 100% of infections were diagnosed by the fluorescent-antibody test, wherease 88.8% of infected males and 86.7% of females were also diagnosed by the RCUT. Primary isolation cultures on modified New York City medium proved suitable for determining the ability of strains to produce penicillinase by a modified RCUT procedure. The method of isolation and identification by using primary cultures from modified New York City medium is both rapid and economical. The rapidity of diagnosis provided by the RCUT makes this a very useful diagnostic method, particularly in laboratories lacking immunofluorescence equipment. Since the penicillinase production test forms part of a routine identification procedure, no extra culture media or subcultures are required. The rapidity of this test should make it of value in tracing contacts of patients infected with penicillinase-producing strains.

Isolation Culture of 7 Strains of Mycobacterium lepraemurium and Isonicotinic Acid Hydrazide or Rifampicin Resistant Hawaiian Strain

Seven strains of Mycobacterium lepraemurium (Douglas, Fukuoka-1, Keishicho, Kurume-42, Odessa and Osaka-No. 1 strains) were isolated on the 1% Ogawa yolk medium. These colonies were white yellow, very slow growing and rough at the isolation, but were changed gradually to smooth colony by the successive cultivation. Kurume-42 and Odessa strains were strongly rough type, especially Kurume-42 strain did not change to smooth type untill the 17th generation and became extinct on account of failure of cultivation at the 18th generation. Kurume-42 strain differed from the other 7 strains of murine leprosy bacillus on the characteristic of rough type. All strains secreted coproporphyrine on the medium and coloured to red the medium surface. Many negative tubes were seen every time in the primary isolation culture and some seeded colonies did not grow in the succesive cultivation. All strains are difficult to culture.INH or rifampicin resistant strain was isolated on the 1% Ogawa yolk medium from murine leproma formed with INH or rifampicin resistant Hawaiian strain. Degree of the INH resistant is 16μg/ml and that of rifampicin resistant is 7μg/ml.Seven strains and. INH or rifampicin resistant strain of Hawaiian were cultivated successively for several generations, thereafter these strains were inoculated to mouse. These strains made murine leproma in the injection site. Many globi were observed on the smear preparation of these murine lepromata.

GRISEOFULVIN-CONTAINING MEDIUM FOR SIMPLIFIED DIAGNOSIS OF DERMATOPHYTOSIS.

By inoculating paired tubes of standard Sabouraud dextrose-chloramphenicol-cycloheximide media, one of which contained in addition 20 micrograms/ml of griseofulvin, 86 of 88 griseofulvin-sensitive dermatophytes were recognized in 226 primary isolation cultures. Most yeast, bacteria, and mold contaminants were not selectively inhibited by the media. The method facilitates the selection of patients for oral griseofulvin therapy by those with relatively little training in mycology.

Griseofulvin-Containing Medium for Simplified Diagnosis of Dermatophytosis

By inoculating paired tubes of standard Sabouraud dextrose-chloramphenicol-cycloheximide media, one of which contained in addition 20 micrograms/ml of griseofulvin, 86 of 88 griseofulvin-sensitive dermatophytes were recognized in 226 primary isolation cultures. Most yeast, bacteria, and mold contaminants were not selectively inhibited by the media. The method facilitates the selection of patients for oral griseofulvin therapy by those with relatively little training in mycology.