In vitro Comparison of Disk Diffusion and Agar Dilution Antibiotic Susceptibility Test Methods for Neisseria gonorrhoeae

At present, most Neisseria gonorrhoeae testing is done with ß-lactamase and agar dilution tests with common therapeutic agents. Generally, in bacteriological diagnosis laboratories in Argentina, study of antibiotic susceptibility of N.gonorrhoeae is based on ß-lactamase determination and agar dilution method with common therapeutic agents. The National Committee for Clinical Laboratory Standards (NCCLS) has recently described a disk diffusion test that produces results comparable to the reference agar dilution method for antibiotic susceptibility of N.gonorrhoeae, using a dispersion diagram for analyzing the correlation between both techniques. We obtained 57 gonococcal isolates from patients attending a clinic for sexually transmitted diseases in Tucumán, Argentina. Antibiotic susceptibility tests using agar dilution and disk diffusion techniques were compared. The established NCCLS interpretive criteria for both susceptibility methods appeared to be applicable to domestic gonococcal strains. The correlation between the MIC's and the zones of inhibition was studied for penicillin, ampicillin, cefoxitin, spectinomycin, cefotaxime, cephaloridine, cephalexin, tetracycline, norfloxacin and kanamycin. Dispersion diagrams showed a high correlation between both methods.

Antimalarial Drug Resistance: Surveillance and Molecular Methods for National Malaria Control Programmes

National malaria control programmes have the responsibility to develop a policy for malaria disease management based on a set of defined criteria as efficacy, side effects, costs and compliance. These will fluctuate over time and national guidelines will require periodic re-assessment and revision. Changing a drug policy is a major undertaking that can take several years before being fully operational. The standard methods on which a decision can be taken are the in vivo and the in vitro tests. The latter allow a quantitative measurement of the drug response and the assessment of several drugs at once. However, in terms of drug policy change its results might be difficult to interpret although they may be used as an early warning system for 2nd or 3rd line drugs. The new WHO 14-days in vivo test addresses mainly the problem of treatment failure and of haematological parameters changes in sick children. It gives valuable information on whether a drug still `works'. None of these methods are well suited for large-scale studies. Molecular methods based on detection of mutations in parasite molecules targeted by antimalarial drugs could be attractive tools for surveillance. However, their relationship with in vivo test results needs to be established

A comparative study on IgG-ELISA, IgM-IFT and Kato-Katz methods for epidemiological purposes in a low endemic area for schistosomiasis

The high sensitivity and the possibility of automation of the enzyme-linked-immunosorbent-assay (ELISA) has indicated this technique as one of the most useful serological test for epidemiological studies. In the present study, an ELISA for detection of IgG antibodies against adult worm antigens (IgG-ELISA) was investigated for epidemiological purposes, in a rural area of the municipality of Itariri (São Paulo, Brazil). Blood on filter paper (1,180 samples) from about 650 schoolchildren were submitted to ELISA and the data compared to the results of the parasitological method of Kato-Katz and also to the IgM-IFT (immunofluorescence test for IgM antibodies to gut associated antigens). The prevalence rates respectively of 8.5%, 43.0%, and 56.2% by the Kato-Katz, IgG-ELISA, and IgM-IFT methods suggest the poor sensitivity of the parasitological method for detection of Schistosoma mansoni eggs in individuals with low worm burden, situation commonly observed in low endemic areas. These results can partially explain the poor degree of agreement between the IgG-ELISA and the Kato-Katz, as suggested by the Kappa index of 0.170. Otherwise, the Kappa index of 0.675 showed substantial agreement between the two serological tests. Some discrepancy of results between the two serological techniques must be better investigated.

Improved Methods for the Diagnosis of African Trypanosomosis

The diagnosis of trypanosomosis in animals with low parasitaemia is hampered by low diagnostic sensitivity of traditional detection methods. An immunodiagnostic method based on a direct sandwich enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies, has been examined in a number of African laboratories for its suitability for monitoring tsetse control and eradication programmes. Generally, the direct sandwich ELISAs for the detection of trypanosomal antigens in serum samples have proved to be unsatisfactory with respect to diagnostic sensitivity when compared with traditional parasitological methods such as the dark ground/phase contrast buffy-coat technique. Consequently, antigen-detection systems exploiting various other direct, indirect and sandwich ELISA systems and sets of reagents are being developed to improve diagnosis. In addition, an existing indirect ELISA for the detection of antibodies has been improved and is being evaluated in the field in order to detect cattle that are or have been recently infected with trypanosomes. Developments and advantages of other diagnostic techniques, such as dip-stick assay and tests based on the polymerase chain reaction are also considered.

Direct methods for detecting picorna-like virus from dead and alive Triatomine insects

In this work we report four different destructive and non-destructive methods for detecting picorna-like virus particles in triatomines. The methods are based on direct observation under transmission electron microscope and they consist of four ways to prepare samples of presumable infected material. The samples are prepared processing dead or alive insect parts, or even dry or fresh insect feces. The methods can be used as analytical or preparative techniques, for quantifying virus infection and checking virus integrity as well. In this work the four methods are applied in order to detect Triatoma virus (TrV) particles in T. infestans colonies.

Analysis of genetic diversity of Trypanosoma cruzi: an application of riboprinting and gradient gel electrophoresis methods

Analysis of restriction fragment length polymorphism (RFLP) profiles derived from digestion of polymerase chain reaction (PCR) products of the ribosomal 18S from Trypanosoma cruzi yields a typical `riboprint' profile that can vary intraspecifically. A selection of 21 stocks of T. cruzi and three outgroup taxa: T. rangeli, T. conorhini and Leishmania braziliensis were analysed by riboprinting to assess divergence within and between taxa. T. rangeli, T. conorhini and L. braziliensis could be easily differentiated from each other and from T. cruzi. Phenetic analysis of PCR-RFLP profiles indicated that, with one or two exceptions, stocks of T. cruzi could be broadly partitioned into two groups that formally corresponded to T. cruzi I and T. cruzi II respectively. To test if ribosomal 18S sequences were homogeneous within each taxon, gradient gel electrophoresis methods were employed utilising either chemical or temperature gradients. Upon interpretation of the melting profiles of riboprints and a section of the 18S independently amplified by PCR, there would appear to be at least two divergent 18S types present within T. cruzi. Heterogeneity within copies of the ribosomal 18S within a single genome has therefore been demonstrated and interestingly, this dimorphic arrangement was also present in the outgroup taxa. Presumably the ancestral duplicative event that led to the divergent 18S types preceded that of speciation within this group. These divergent 18S paralogues may have, or had, different functional pressures or rates of molecular evolution. Whether or not these divergent types are equally transcriptionally active throughout the life cycle, remain to be assessed.

Analysis of parity between protein-based electrophoretic methods for the characterization of oral Candida species

Electrophoretic studies of multilocus-enzymes (MLEE) and whole-cell protein (SDS-PAGE) were carried out in order to evaluate the parity between different methods for the characterization of five Candida species commonly isolated from oral cavity of humans by numerical taxonomy methods. The obtained data revealed that sodium dodecyl sulfate polyacrylamide gel electrophoresis is more efficient in grouping strains in their respective species while MLEE has much limited resolution in organizing all strains in their respective species-specific clusters. MLEE technique must be regarded for surveys in which just one species of Candida is involved.

A comparative study on the different staining methods and number of specimens for the detection of acid fast bacilli

The presence of acid fast bacilli in multiple specimens was investigated comparatively with Ziehl-Neelsen (ZN) and fluorescence microscopy (FM) staining in order to determine sensitivity in detecting tuberculosis (TB). A total of 465 specimens obtained from 295 patients were analysed at Harran University Medical School Hospital between March 1998 and March 2000. The culture was employed as the reference method. Sixty-eight patients (23.1%) were diagnosed as having TB by culture. The ZN and FM staining sensitivities were 67.6% (46/68) and 85.2% (58/68) respectively. Two hundred and one patients (68.1%) submitted one specimen to the laboratory. TB positivity was detected in 42 (20.9%) of these patients by culture. The sensitivities of ZN and FM stains were found to be 61% and 83% in these patients. However, in 18 patients (6.1%) who submitted two specimens to the laboratory, the TB was positive in six of them (33.3%) and ZN and FM sensitivities were 66% and 83% respectively. When three specimens or more were collected from the patients (76 patients, 25.8%), TB positivity was determined in 20 of them (26.3%) and the sensitivities were 80% and 92% in the ZN- and FM-stained smears, respectively. Our data indicate that in the diagnosis of TB, FM has greater sensitivity than ZN. In particular, in the case of a single specimen, the diagnostic value of FM is quite significant. It is, therefore, possible to conclude that both ZN and FM staining can be used for the diagnosis of TB when there are more than two specimens. However, if only one or two specimens are available, FM staining is preferable.

Hepatitis C prevalence and risk factors in hemodialysis patients in Central Brazil: a survey by polymerase chain reaction and serological methods

An hemodialysis population in Central Brazil was screened by polymerase chain reaction (PCR) and serological methods to assess the prevalence of hepatitis C virus (HCV) infection and to investigate associated risk factors. All hemodialysis patients (n=428) were interviewed in eight dialysis units in Goiânia city. Blood samples were collected and serum samples screened for anti-HCV antibodies by an enzyme-linked immunosorbent assay (ELISA). Positive samples were retested for confirmation with a line immunoassay (LIA). All samples were also tested for HCV RNA by the PCR. An overall prevalence of 46.7% (CI 95%: 42-51.5) was found, ranging from 20.7% (CI 95%: 8.8-38.1) to 90.4% (CI 95%: 79.9-96.4) depending on the dialysis unit. Of the 428 patients, 185 were found to be seropositive by ELISA, and 167 were confirmed positive by LIA, resulting in an anti-HCV prevalence of 39%. A total of 131 patients were HCV RNA-positive. HCV viremia was present in 63.5% of the anti-HCV-positive patients and in 10.3% of the anti-HCV-negative patients. Univariate analysis of risk factors showed that the number of previous blood transfusions, transfusion of blood before mandatory screening for anti-HCV, length of time on hemodialysis, and treatment in multiple units were associated with HCV positivity. However, multivariate analysis revealed that blood transfusion before screening for anti-HCV and length of time on hemodialysis were significantly associated with HCV infection in this population. These data suggest that nosocomial transmission may play a role in the spread of HCV in the dialysis units studied. In addition to anti-HCV screening, HCV RNA detection is necessary for the diagnosis of HCV infection in hemodialysis patients.

First characterization of Candida albicans by random amplified polymorphic DNA method in Nicaragua and comparison of the diagnosis methods for vaginal candidiasis in Nicaraguan women

A total of 106 women with vaginitis in Nicaragua were studied. The positive rate for the identification of Candida species was 41% (44 positive cultures out of 106 women with vaginitis). The sensitivity of microscopic examination of wet mount with the potassium hydroxide (KOH) was 61% and 70% with Gram's stain when using the culture of vaginal fluid as gold standard for diagnosis of candidiasis. Among the 44 positives cultures, isolated species of yeast from vaginal swabs were C. albicans (59%), C. tropicalis (23%), C. glabrata (14%) and C. krusei (4%). This study reports the first characterization of 26 C. albicans stocks from Nicaragua by the random amplified polymorphic DNA method. The genetic analysis in this small C. albicans population showed the existence of linkage disequilibrium, which is consistent with the hypothesis that C. albicans undergoes a clonal propagation.

Schistosomiasis mansoni: follow-up of control program based on parasitologic and serologic methods in a Brazilian community of low endemicity

A field survey on schistosomiais was carried out in 1998, in the municipality of Pedro de Toledo, a low endemic area in the state of São Paulo, Brazil. According to the parasitologic Kato-Katz method, the prevalence rate was 1.6%, with an infection intensity of 40.9 eggs per gram of stool. By the immunofluorescence test (IFT) for detection of IgG and IgM antibodies in the serum, IgG-IFT and IgM-IFT, respectively, prevalence indices of 33.2% and 33.5% were observed. To assess the impact of the schistosomiasis control program in the area, parasitologic and serologic data obtained in 1998, analyzed according to the age, sex, and residence zone, were compared to previous data obtained in a epidemiologic study carried out in 1980, when prevalence indices were of 22.8% and 55.5%, respectively by Kato-Katz and IgG-IFT. A significant fall of the prevalence was observed, indicating that the control measures were effective. Nonetheless, residual transmission was observed, demonstrating the need for a joint effort to include new approaches for better understanding the real situation and improving the control of the disease in low endemic areas.

Use of molecular methods in identification of Candida Species and evaluation of fluconazole resistance

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.

Evaluation of four methods for detecting the beta-lactamase activity in Neisseria gonorrhoeae isolated in Cuba

Four methods (chromogenic, acidimetric, inhibition, and iodometric) for demonstration of the beta-lactamase production by 70 isolates of Neisseria gonorrhoeae, were evaluated in Cuba. There was 100% correlation between all beta-lactamase methods and the standardized penicillin dilution susceptibility test for penicillinase-non-producing N. gonorrhoeae. For penicillinase-producing N. gonorrhoeae strains, there was a perfect correlation between the chromogenic method and penicillin susceptibility testing, but one and two strains failed to give a positive result for beta-lactamase with the inhibition/acidimetric and the iodometric methods, respectively. There was a high concordance between the chromogenic method, considered as gold standard and the rest of penicillinase tests evaluated: Kappa Index (KI) = 0.98 for inhibition/acidimetric methods and KI = 0.97 for the iodometric method. The four methods evaluated were accurate, reproducible, easily readable, economical, and ease to use for screening primary isolates of N. gonorrhoeae in Cuba. We recommended the use of the inhibition method, when testing the penicillinase activity in gonococcal isolates in provincial and municipal reference laboratories.

Antimicrobial susceptibility determined by the E test, Löwenstein-Jensen proportion, and DNA sequencing methods among Mycobacterium tuberculosis isolates discrepancies, preliminary results

Mycobacterium tuberculosis strains resistant to streptomycin (SM), isoniazid (INH), and/or rifampin (RIF) as determined by the conventional Löwenstein-Jensen proportion method (LJPM) were compared with the E test, a minimum inhibitory concentration susceptibility method. Discrepant isolates were further evaluated by BACTEC and by DNA sequence analyses for mutations in genes most often associated with resistance to these drugs (rpsL, katG, inhA, and rpoB). Preliminary discordant E test results were seen in 75% of isolates resistant to SM and in 11% to INH. Discordance improved for these two drugs (63%) for SM and none for INH when isolates were re-tested but worsened for RIF (30%). Despite good agreement between phenotypic results and sequencing analyses, wild type profiles were detected on resistant strains mainly for SM and INH. It should be aware that susceptible isolates according to molecular methods might contain other mechanisms of resistance. Although reproducibility of the LJPM susceptibility method has been established, variable E test results for some M. tuberculosis isolates poses questions regarding its reproducibility particularly the impact of E test performance which may vary among laboratories despite adherence to recommended protocols. Further studies must be done to enlarge the evaluated samples and looked possible mutations outside of the hot spot sequenced gene among discrepant strains.